Research
Persisting atypical and cystic forms of Borrelia burgdorferi and
local inflammation in Lyme neuroborreliosis
Judith Miklossy1 , Sandor Kasas2 , Anne D Zurn3 , Sherman McCall4 ,
Sheng Yu1 and Patrick L McGeer1

1Kinsmen Laboratory of Neurological Research, University of British
Columbia, 2255 Wesbrook Mall, Vancouver, B.C. V6T1Z3, Canada

2Laboratoire de Physique de la Matière Vivante, Ecole Polytechnique
Fédérale de Lausanne, 1015 Lausanne, Switzerland and Département de
Biologie Cellulaire et de Morphologie, Université de Lausanne, 1005
Lausanne, Switzerland

3Department of Experimental Surgery, Lausanne University Hospital, CH-
1011 Lausanne, Switzerland

4Pathology Laboratory, U.S. Army Medical Research Institute of
Infectious Diseases (USAMRIID), 1425 Porter St., Ft. Detrick, MD
21702-5011, USA

author email corresponding author email

Journal of Neuroinflammation 2008, 5:40doi:10.1186/1742-2094-5-40

The electronic version of this article is the complete one and can be
found online at: http://www.jneuroinflammation.com/content/5/1/40

Received: 15 April 2008
Accepted: 25 September 2008
Published: 25 September 2008

© 2008 Miklossy et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the
Creative Commons Attribution License
(http://creativecommons.org/licenses/by/2.0), which permits
unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly cited.

Abstract
Background
The long latent stage seen in syphilis, followed by chronic central
nervous system infection and inflammation, can be explained by the
persistence of atypical cystic and granular forms of Treponema
pallidum. We investigated whether a similar situation may occur in
Lyme neuroborreliosis.

Method
Atypical forms of Borrelia burgdorferi spirochetes were induced
exposing cultures of Borrelia burgdorferi (strains B31 and ADB1) to
such unfavorable conditions as osmotic and heat shock, and exposure
to the binding agents Thioflavin S and Congo red. We also analyzed
whether these forms may be induced in vitro, following infection of
primary chicken and rat neurons, as well as rat and human astrocytes.
We further analyzed whether atypical forms similar to those induced
in vitro may also occur in vivo, in brains of three patients with
Lyme neuroborreliosis. We used immunohistochemical methods to detect
evidence of neuroinflammation in the form of reactive microglia and
astrocytes.

Results
Under these conditions we observed atypical cystic, rolled and
granular forms of these spirochetes. We characterized these abnormal
forms by histochemical, immunohistochemical, dark field and atomic
force microscopy (AFM) methods. The atypical and cystic forms found
in the brains of three patients with neuropathologically confirmed
Lyme neuroborreliosis were identical to those induced in vitro. We
also observed nuclear fragmentation of the infected astrocytes using
the TUNEL method. Abundant HLA-DR positive microglia and GFAP
positive reactive astrocytes were present in the cerebral cortex.

Conclusion
The results indicate that atypical extra- and intracellular
pleomorphic and cystic forms of Borrelia burgdorferi and local
neuroinflammation occur in the brain in chronic Lyme
neuroborreliosis. The persistence of these more resistant spirochete
forms, and their intracellular location in neurons and glial cells,
may explain the long latent stage and persistence of Borrelia
infection. The results also suggest that Borrelia burgdorferi may
induce cellular dysfunction and apoptosis. The detection and
recognition of atypical, cystic and granular forms in infected
tissues is essential for the diagnosis and the treatment as they can
occur in the absence of the typical spiral Borrelia form.

Background
The similarity of clinical and pathological manifestations of
syphilis caused by Treponema pallidum [1] and Lyme disease caused by
Borrelia burgdorferi [2] is well established. In analogy to Treponema
pallidum, Borrelia burgdorferi persists in the brain in chronic Lyme
neuroborreliosis [3]. How Borrelia burgdorferi is able to survive in
infected tissues for years or decades is not well understood. Ways
for long term survival may be through transformation into more
resistant atypical forms and through intracellular localization.

As early as 1905 it was suspected that the classical spiral
(vegetative) form was not the only one that spirochetes could assume
[1,4]. Transformation of various types of spirochetes into cystic
forms through end knob, loop, ring-shaped and spherule formation has
since been repeatedly reported [5-10]. Agglomeration of spirochetes
into colonies [11-14], enclosing numerous cystic forms, has been
observed both in vitro and in vivo [12].

Treponema pallidum and Borrelia burgdorferi produce vesicular budding
from the membrane, which may become detached. In Borrelia burgdorferi
these free vesicular or granular structures contain spirochetal
surface proteins and linear and circular DNA [15,16].

Granular disintegration of spirochetes resulting in a chain of fine
granules also occurs under adverse conditions [17-22]. Minute
granules are liberated from the periplasmic sheath through budding
and extrusion, which may multiply and may be transmissible [23-31].
Their presence in syphilitic patients was regarded as confirmatory of
the syphilitic nature of the lesions even in the absence of classical
spiral forms [26,27,30]. These spore-like minute granules (0.1–0.3 μm
in diameter) may pass the 0.2 μm "China" filter (32) and can grow
into young spirochetes [6,19,25-38]. The newly formed spirochetes are
delicate L or metacyclic forms [25,32,39].

These various atypical forms were suggested to be part of a complex
developmental cycle, a form of resistance to adverse conditions, and
a source for reproduction under more favorable conditions.
Reconversion of cystic Borrelia burgdorferi into the typical spiral
form has been demonstrated in vitro and in vivo [8,10,31,40].

The occurrence of pleomorphic forms of Treponema pallidum in the
brain in general paresis and their abundance in juvenile paresis is
well documented [6,18,26,41,42].

Treponema pallidum may invade virtually all parenchymal and
mesenchymal cells, including plasma cells, macrophages, neurons and
glial cells [39,40,43]. Atypical and cystic forms of Treponema
pallidum have been observed both extra- and intracellularly [30]. It
has also been described in other spirochetal infections [e.g. [44-
46]].

Only limited data are available on the occurrence of atypical, cystic
or granular forms of Borrelia burgdorferi in infected tissues. Their
occurrence has been reported in skin lesions [14], in an ex vivo
system in tonsil tissue [47] and on silver stained hippocampus
section in a patient with concurrent Alzheimer disease (AD) and Lyme
neuroborreliosis [48]. Intracellular localization of Borrelia
burgdorferi was observed in macrophages and keratinocytes in the skin
[14] and in neurons and glial cells in vitro and in vivo [3,49-51].

The goal of the present study was to compare whether atypical and
cystic forms of Borrelia burgdorferi spirochetes induced in vitro are
similar to those occurring in vivo. Three patients with chronic Lyme
neuroborreliosis were used in the study. Immunodetection of reactive
microglia and astrocytes was also performed to detect
neuroinflammation.

Methods
Cultivation of Borrelia burgdorferi spirochetes in BSK II medium
Borrelia burgdorferi spirochetes strains B31 and ADB1 [3,51-53] were
cultivated in BSK II medium [54]. To 500 ml BSK medium (Sigma B 3528)
containing 6% rabbit serum (Sigma R-7136) and 7% gelatin (Difco 0143-
15-1), 6 mg acetyl muramic acid (Sigma A 3007) and 0.2 g N-acetyl
glucosamine (Sigma A8625), Rimactan (Novartis, 420 ul) and Fosfocin
(Boehringer Mannheim, 300 ul) were added. The spirochetes were
cultivated at 32°C. The pH of BSK II medium was adjusted to pH 7.

To induce atypical spirochete forms 5 ml of cultivated Borrelia
burgdorferi spirochetes (5 × 105/ml) were exposed to various harmful
conditions. Spirochetes were exposed to strong acidic and basic
conditions by adjusting the pH of the BSK II medium to pH2 and pH10
using sterile 1 M HCl or 1 M NaOH. The harmful effect of alcohol was
analyzed by adding 1 ml of either 70% or 95% ethanol to 5 ml
cultivated spirochetes. Heat shock was produced by cultivating
spirochetes at 45°C.

Spirochetes are known to bind Congo red and Thioflavin S [55,56],
both of which are widely used to stain amyloid. To analyze whether
they may induce atypical Borrelia forms, 1 mg or 5 mg of Congo red,
or 1 mg or 5 mg Thioflavin S were directly added to 5 ml of
spirochete culture. The same amounts dissolved in 2 ml of 70% alcohol
were also used to induce atypical spirochetes. The effect of acridin
orange, another fluorochrom which binds to spirochetes, was also
analyzed by adding 1 mg or 5 mg acridin orange powder to 5 ml of
cultivated spirochetes.

Following 1 hour, 6 hours, and 1 week exposure times, 50 μl samples
were taken and put on glass slides, cover-slipped, and then examined
by dark field microscopy. Series of 50 μl samples were used to
prepare smears for histochemical and immunohistochemical
investigation. Additional 500 μl samples were removed and fixed in
glutaraldehyde for atomic force microscopy (AFM) analysis.
Spirochetes cultivated at 32°C at pH 7 for the same periods of time
were used as controls. An exception with respect to the exposure
times was induction of osmotic shock by cold H2O. Two ml sterile cold
H2O was added to 5 ml cultured spirochetes that had been collected by
centrifugation at 1000 rpm for 5 minutes. Here the samples were
examined following 1 and 6 hours of exposure.

In order to analyze whether the typical spiral Borrelia form may be
resuscitated, at the end of each experiments 200 μl samples were
reinoculated in BSK II medium at Ph7 and following one week of
culture at room temperature, 30 μl samples were analyzed by dark
field microscopy.

Infection of cell cultures with Borrelia burgdorferi
Superior cervical ganglia from 8- to 12-day-old chicken embryos were
dissociated as described previously [57]. Briefly, neurons were
separated from non-neuronal cells using a density gradient formed
with Percoll. The sympathetic neurons were then grown for 3–4 weeks
in serum containing medium on a polyornithine substrate pre-coated
with heart-conditioned medium [57]. Neurons dissociated from the
telencephalon of 21-day-old rat were cultured either on collagen or
polylysine substrate in a serum-containing medium [58]. Rat primary
astrocytes (106) were prepared as described earlier [59]. Following
the characterization of the primary astrocytic cell cultures using
anti-GFAP antibody (Dako, Z334) more than 95% of the cells were GFAP
reactive (not shown here). The cells were cultured in 2 well chambers
(177429 Lab-Tek, Christschurch, New Zealand) or in six well clusters
(3506, Costar, Acton, Maryland) in a humidified CO2 (6%) incubator at
37°C.

To infect neurons and astrocytes, Borrelia spirochetes of the
virulent strains B31 and ADB1, the latter having been cultured from
the brain of a patient with concurrent Lyme neuroborreliosis and AD
[3], were employed. Equal volumes of medium for the given primary
cells and for spirochetes (BSK II) were used as the culture medium.
The final concentration of spirochetes in the infected medium
corresponded to 5 × 105/ml. Before exposure to spirochetes, the cells
were tested with 4',6-diamidine-2'-phenylindole dihydrochloride
(DAPI, 236 276, Boehringer Mannheim, Germany) to exclude Mycoplasma
infection. Parallel cultures not infected with spirochetes were
always used as controls.

After 1 week exposure, the medium was removed and the cells were
rinsed with PBS (2 ml, 2 × 3 minutes). To analyze the morphology of
free floating spirochetes, 50 μl samples of culture medium were taken
and analyzed by dark field microscopy. Smears were also prepared for
histochemical and immunohistochemical analyses.

After 1 week exposure, 200 μl samples from all infected cell cultures
were also re-inoculated in BSK II medium and were cultivated at room
temperature, Ph 7, for one week. Then 30 μl samples were analyzed by
dark field microscopy.

Detection of apoptosis by deoxynucleotidyltransferase (TdT)-mediated
dUTP nick end labeling (TUNEL)
Cells in 6 wells chambers were fixed with 4% paraformaldehyde for 10
minutes in room temperature. Following an incubation with proteinase
K (20 μg/ml) in TRIS HCL (pH 7.4) for 15 minutes at 37°C the cells
were rinsed with 2 ml PBS (2 × 3 minutes). Then cells were treated
with a permeabilisation solution containing 0.1% Triton X100, in 0.1%
sodium citrate, for 2 minutes on ice followed by a rinse with PBS (2
× 3 minutes).

The cells were incubated with a freshly prepared TUNEL reaction
mixture kept on ice containing 45 μl of TUNEL label solution
(1767291, Boehringer) containing unlabeled dNTPs and fluorescein
isothiocyanate tagged dUTP (FITC-dUTP) and 5 μl of TUNEL enzyme
(Terminal deoxynucleotidyl Transferase (TdT) (1767305, Boehringer)
for 1 hour and 30 minutes at 37°C in a humidified chamber. For a
negative control the TUNEL enzyme was omitted from the TUNEL reaction
mixture and 50 μl TUNEL label solution alone was used.

Detection of pleomorphic Borrelia forms in vivo
Brains of three patients with pathologically and serologically
confirmed Lyme neuroborreliosis and concurrent AD were analyzed [3].
From the brains of these three patients, aged 74, 78, and 86 years,
spirochetes were successfully cultivated in BSK II medium. In two of
them (strains ADB1 and ADB2) 16SrRNA gene sequence analysis
identified the spirochetes as Borrelia burgdorferi sensu stricto (s.
s.).

To detect whether atypical and cystic forms of Borrelia burgdorferi
spirochetes are present in the brains of these patients, frozen
sections of the hippocampus, frontal, temporal and parietal cortex
were analyzed using dark field microscopy, as well as histochemical
and immunohistochemical techniques. Before immunostaining the
sections were fixed in acetone for 10 minutes at 4°C. Acetone fixed
frozen sections which were cut from samples taken from identical
brain areas in three control patients without neurological symptoms
and without brain lesions were also processed and analyzed in the
same fashion.

Paraffin sections (20 μm thick) cut from various cortical samples
(from archival material of the Armed Forces Institute of Pathology,
USA) of two patients (31 and 52 year old males) with clinically,
serologically and pathologically confirmed general paresis, were also
analyzed for the presence of Treponema pallidum. The goal was to
compare atypical spirochetal forms of Borrelia burgdorferi in Lyme
neuroborreliosis with those of Treponema pallidum in general paresis.

Dark field microscopy, histochemical and immunohistochemical analysis
of spirochetes
From cultivated Borrelia spirochetes exposed to various harmful
conditions, 50 μl samples were used as wet preparation for dark field
microscopy analysis. Additional samples (50 μl) were used to prepare
smears for the histochemical and immunohistochemical analyses. In
order to analyze free floating spirochetes in Borrelia infected cell
cultures, 50 μl samples of the co-culture medium were analyzed.

Smears and brain sections were stained with the Warthin-Starry and
Bosma-Steiner silver techniques as described for the detection of
spirochetes. Spirochetal DNA was detected in smear preparations of
cultivated spirochetes exposed to various adverse conditions, in
infected cells, and in unfixed frozen brain sections by staining with
DAPI (236 276, Boehringer Mannheim, Germany) following instructions
of the manufacturer. The same preparations were also used for
immunohistochemical analysis. Smears prepared on glass slides from
cultivated spirochetes, from medium of infected cells, and from
cryostat cut brain sections were fixed in acetone for 10 minutes at 4
°C prior to immunostaining. Infected cells in six wells, where
acetone cannot be used, were fixed in 4% paraformaldehyde for 5
minutes. Before immunostaining, frozen brain sections following
acetone fixation were incubated in 0.1% amylase for 5 min at 37°C.
The following anti-Borrelia burgdorferi antibodies were used:
monoclonal anti-OspA (H5332, H3T5, Symbicom, 1:50) and anti-flagellin
(G 9724, H605, Symbicom, 1:50), polyclonal B65302R (Biodesign, 1:100)
and BB-1017 (1:500) [3] antibodies. The specificity of these mono-
and polyclonal antibodies was previously tested by Western blot
analysis [3].

For immunostaining, the avidin-biotin-peroxidase technique was used.
Following 24, 48 or 72 hours incubation with a primary antibody at 4°
C, the sections were incubated with the appropriate secondary
antibodies. For the monoclonal antibodies, a biotinylated F(ab)
fragment of affinity isolated rabbit anti-mouse immunoglobulin (Dako,
E413) was used. The immunoreaction was revealed by diaminobenzidine
(DAB) alone, or with nickel-ammonium sulfate as described previously
[60]. Frozen sections immunostained in the absence of a primary
antibody or with an irrelevant mono- or polyclonal antibody were used
as negative controls. Immunostaining was also performed with various
anti-Borrelia burgdorferi antibodies using FITC tagged anti-mouse or
anti-rabbit secondary antibody depending on the primary antibody
used. The green fluorescence of Borrelia burgdorferi spirochetes was
analyzed with a Zeiss fluorescent microscope. A monoclonal antibody
(Biogenesis 7263-1006 or Chemicon MAB995, dil.1: 200) for the
analysis of the presence of bacterial peptidoglycan, a bacterial cell
wall component of virtually all Eubacteria, including spirochetes,
was also used as previously described in detail [61].

Floating paraffin sections (20 μm thick) of the cerebral cortex of
the two patients with general paresis were immunostained with a
polyclonal anti-Treponema pallidum antibody (Biodesign, B65210R).

Detection of neuroinflammation
Paraffin sections of the hippocampus, frontal and parietal cortices
of the three patients with Lyme neuroborreliosis were also used for
the immunohistochemical detection of reactive microglia and
astrocytes. Anti-HLA-DR (clone CR3/43, M775, Dako) and anti-CD68
(clone KP1, M814, Dako) monoclonal antibodies were used to visualize
microglia and a polyclonal anti-GFAP antibody (Z334, Dako) to detect
astrocytes. Paraffin sections of the same cortical areas of a female
patient (aged 59 years) without brain lesion were used as controls.
In addition, sections of the three patients with Lyme
neuroborreliosis were also immunostained with the omission of these
primary antibodies.

Atomic force microscopy (AFM) analysis
To 500 μl samples of cultivated Borrelia spirochetes exposed to
various harmful conditions 500 μl of 2.5% buffered glutaraldehyde was
added. Samples were then stored at 4°C until used for the atomic
force microscopy (AFM) analysis. 20–50 μl samples were put on the
surface of a Nucleopore® filter of 2 μm hole size and were dried at
room temperature in air, as previously described [62]. The filters
were fixed on metallic discs or on glass slides using a double face
rubber strip, and were imaged with a Bioscope I atomic force
microscope (AFM) and a Nanoscope® III atomic force microscope (AFM)
equipped with a J-scanner. All the images were taken in the tapping
mode at room temperature in air. The scanning rate varied from 0.1 to
5 Hz. Images were obtained in both the constant force mode providing
true height, and the amplitude mode, for highlighting sharp contours.
The images were processed and the measurements were done using the
Nanoscope III image processing software.

The human brains analyzed were from the University Institute of
Pathology, Lausanne, Switzerland. The study adhered to the tenets of
the Helsinki Declaration. Animal experimentation conformed to the
Guide for the Care and Use of Laboratory Animals, formulated by the
National Research Council, 1996, and the Swiss law on animal
protection.

Results
Figure 1 illustrates the typical morphology and colony-like formation
of strains B31 (A-D) and ADB1 (E-H). Panels A and B show the regular
coiled morphology and colony-like aggregates of spirochetes (B31
strain) as observed by dark field microscopy analysis. Regularly
coiled OspA-immunoreactive spirochetes of the same strain are seen
following immunostaining with a monoclonal anti-OspA antibody in
panel C. An atomic force microscopy (AFM) image of a regularly coiled
fragment of a Borrelia spirochete (strain B31) is visible in panel D.
The typical coiled morphology of spirochetes of the ADB1 strain,
which were cultivated from the brain of a patient with chronic Lyme
neuroborreliosis, is illustrated in panels E and F by dark field
microscopy and in panel G by immunohistochemistry using a polyclonal
anti-Borrelia burgdorferi antibody (Biodesign, B65302R). The primary
anti-Borrelia burgdorferi antibody was revealed with FITC-tagged
secondary antibody showing green fluorescence. Panel H illustrates
the typical spiral appearance of spirochetes (strain ADB1) by silver
impregnation using the Bosma-Steiner microwave technique.

The typical spiral forms of Borrelia spirochetes were converted to
atypical and cystic forms when exposed to various unfavorable culture
conditions. Atypical and cystic forms were seen at 1 hour exposure.
Their numbers increased following 6 hours exposure and was highest at
1 week, where the majority of spirochetes showed atypical morphology.
Osmotic shock induced by cold sterile distilled water induced
atypical and cyst forms of the majority of spirochetes following 1
and 6 hour exposure. The atypical and cyst forms retained an affinity
for silver and were immunoreactive with the various anti-Borrelia
burgdorferi antibodies.

Figure 1. Characteristic morphology of Borrelia burgdorferi seen by
various techniques following one week of culture in BSKII medium. A
and B: Dark field microscopy images of Borrelia burgdorferi strain
B31 showing the usual spiral form of spirochetes (A) and their
agglomeration into colony-like masses (B). Similar spiral morphology
of strain B31 is illustrated by OspA immunoreactivity (C) and by
atomic force microscopy (AFM) imaging (D). E and F: Dark field
microscopy images showing the typical spiral form (E) and colony
formation (F) of Borrelia burgdorferi strain ADB1. G: Similar spiral
morphology of strain ADB1 shown by immunostaining with a polyclonal
anti-Borrelia burgdorferi antibody (Biodesign, B65302R). The green
fluorescent immunoreaction was revealed with an FITC tagged secondary
antibody. H: Similar morphology of strain ADB1 revealed by silver
impregnation with the Bosma Steiner microwave technique. Bars: A, C =
10 μm; B = 30 μm; D = 1 μm; E, G, H = 8 μm; F = 25 μm.

The morphological changes were similar in both the B31 and ADB1
strains, whether induced by osmotic shock, heat shock, alcohol,
acidic or basic pH. Atypical and cystic forms were also seen
following Congo red and Thioflavin S exposures.

A stronger effect was observed when Thioflavin S and Congo red were
dissolved in ethanol before addition to the culture.

Atypical forms included knob, ring-shaped and loop formations, uni-
or multi-spirochetal cysts, bleb formation, granular disintegration
and colony-like masses enclosing numerous cystic forms. Following 1
week exposure, free minute granules and re-growing of slender motile
L forms of young spirochetes along injured spirochetal cells were
observed.

Figure 2 illustrates rolled spirochetes forming ring-shaped and
globular structures. Panel A-D show large aggregates of multiple
rolled, ring-shaped and cystic spirochetes in response to osmotic
shock generated by cold sterile distilled water. Panels A and C
illustrate these atypical forms as seen by dark field microscopy at
lower (A) and higher (C) magnifications. Panels B and D illustrate
similar results following immunostaining with a monoclonal anti-OspA
antibody. Atomic force microscopy (AFM) images of loop and ring
formations of Borrelia spirochetes are illustrated in panels E and F.

Figure 2. Atypical forms of Borrelia burgdorferi (B31 strain)
spirochetes induced by harmful culture conditions. A-D: Large
agglomerates of atypical ring shaped and spherule forms of Borrelia
burgdorferi after one week of BSKII culture followed by 5 minutes of
osmotic shock generated by cold distilled water. A and C: Low and
high power fields as revealed by dark field microscopy. B and D:
Similar morphology in low and high power fields as revealed by
immunohistochemistry using the anti-OspA monoclonal antibody. E and
F: Low and high power atomic force microscopy (AFM) images showing
similar morphology. In this case the inducing agent was 1 mg of
Thioflavin S added to the medium at the commencement of one week of
culture. See materials and methods for details. Bars: A, B = 30 μm, C-
D = 20 μm; E = 5 μm, F = 1 μm.

Figure 3A illustrates atypical and cystic forms of Borrelia
burgdorferi induced by Thioflavin S (1 mg dissolved in 2 ml 70%
ethanol). The agglomeration of spirochetes exhibits green
fluorescence due to the binding of Thioflavin S. Numerous ring-shaped
and globular cystic forms (arrows) were observed in the periphery of
the spirochetal mass. Similar rolled and cystic Borrelia forms (B, C)
induced by Thioflavin S are illustrated by dark field microscopy.
Panels D and E show spirochetes with similar morphology, which were
immunostained with anti-OspA antibody. Panels F-H show the results of
atomic force microscopy (AFM) analysis. The atomic force microscopy
(AFM) images reveal rolled spirochetes inside of cysts. Cystic forms
entirely covered by a thickened external membrane masking the content
were also observed. One of them is illustrated in panel H. It is
similar to those observed by dark field microscopy as well as with an
anti-OspA antibody (compare H with C, D and E).

Figure 3. Rolled and cystic forms of Borrelia burgdoferi spirochetes
observed after one week of culture in medium to which Thioflavin S
had been added. A: Observation by Thioflavin S fluorescence. Arrows
point to rolled cystic forms at the periphery of an agglomerated mass
of spirochetes from strain B31. Rolled (B) and cystic (C) forms
observed by dark field microscopy (strain B31). D and E: Cyst forms
of Borrelia burgdorferi (strains ADB1 and B31, respectively)
following immunostaining with the monoclonal anti-OspA antibody. F-H:
Atomic force microscopy (AFM) images of Borrelia cysts. Rolled
spirochetes are clearly visible in F (strain B31) and G (strain
ADB1). Arrow in G shows that the cyst is formed by two spirochetes
rolled together. H: The cystic form is entirely covered by a
thickened external membrane masking the content of the cyst (strain
B31). Bars: A-D = 6 μm; E = 5 μm; F = 1 μm; G = 2.5 μm; H =
0.5 μm.

Atypical cystic, granular forms and colony-like aggregation of
spirochetes into large masses enclosing cystic forms were also
observed following 1 week infection of primary neurons and astrocytes
with Borrelia spirochetes. The morphological changes observed were
similar in chicken sympathetic neurons, rat telencephalic neurons and
also in rat and human primary astrocytes. Both Borrelia strains (B31
and ADB1) showed identical morphological changes. Following 1 week
exposure of the cells to Borrelia burgdorferi, it is difficult to
find spirochetes which have preserved the typical spiral form.

Figure 4 illustrates atypical forms of spirochetes following 1 week
exposure of chicken primary sympathetic neurons (A, C-G) and rat
astrocytes (B and H) to Borrelia burgdorferi, strain ADB1. Large
colony-like aggregates are illustrated in infected neuronal culture
(A) as seen by dark field microscopy and in rat primary astrocytic
cultures (B) immunostained with an anti-Borrelia burgdorferi
antibody. Borrelia spirochetes surrounding neuronal perikarya are
seen in panel C. Panels D and E illustrate OspA immunoreactive
intracytoplasmic atypical filamentous, ring shaped (arrow) forms of
Borrelia burgdorferi. Some extracellular spirochetes showing ring-
shaped formation are also present (E, arrow). Panel F shows an OspA
immunoreactive atypical spirochete with a double ring-shaped
formation at one end and some OspA positive granules along the
injured spirochete. A small colony like mass is illustrated in panel
G in which the majority of spirochetes show atypical ring-shaped
cystic formations. Regularly coiled spirochetes are not present. OspA
immunoreactive ring shaped forms and spherules of Borrelia
spirochetes in rat astrocytic culture are illustrated in panel H.

Figure 4. Atypical and cystic Borrelia forms following 1 week
exposure of primary neuronal and astrocytic cultures to Borrelia
burgdorferi. Panels A, C-G illustrate atypical Borrelia forms in
primary chicken neuron cultures and panels B and H in rat astrocytic
cultures. A is by dark field microscopy; B-H are by anti-OspA
immunostaining. A: Formation of large colony like aggregates in a
neuronal culture as observed by dark field microscopy (strain B31)
and in astrocytic culture as visualized by anti-OspA immunostaining
(strain ADB1). C: OspA positive Borrelia spirochetes closely
surrounding neurons (strain B31). D: Atypical filamentous and ring-
shaped cystic, apparently intra-cellular spirochetes in a neuron
(strain B31). E: Filamentous and granular forms are seen in the
cytoplasm in one neuron. Some extracellular spirochetes show ring-
shaped atypical forms (strain ADB1). F: Immunoreactive ring-shaped
spherules are seen at one end of a spirochete with some small minute
granules along the injured cell (strain B31). G: A small colony like
mass is seen in which numerous ring-shaped spherules are visible in
the absence of typical coiled spirochetes (strain B31). In H ring-
shaped and cystic forms in infected rat astrocytic culture are
visible (strain ADB1). Bars: A = 40 μm; B = 30 μm; C = 60 μm; D,
E =
10 μm; F-H = 5

Atypical forms, including ring-shaped, uni- or multi-spirochetal
cystic and granular forms also occurred free floating in the medium
of primary neuronal and glial cell cultures. Figure 5 illustrates
these atypical forms following 1 week exposure to Borrelia
spirochetes. Dark field microscopy images of ring-shaped and cystic
forms are seen in panels A and B. Arrows in B and C point to bleb
formations still attached with thin stalks to the surface of the
spirochete cell. Panels D-G show OspA immunoreactive multiple ring-
shaped (D and E) and cystic (F-G) Borrelia forms. The cysts may
sometimes be formed by multiple spirochetes. For example, in panel G
an anti-Ospa immunoreactive cyst formed by two spirochetes is
visible. Panels H-J illustrate spirochetal cysts as visualized by
dark field microscopy (H), by immunofluorescence using an anti-OspA
antibody (I) and by DAPI staining (J). OspA immunoreactive large,
thick, elongated bodies were also observed as seen in panel K. Panel
H shows dark field microscopy image of a cyst where the free end
(arrow) of the rolled spirochete is visible.

Figure 5. Atypical cystic spirochetes in the medium of neuronal and
astrocytic cultures following 1 week exposure to Borrelia
burgdorferi. A-C and H are dark field microscopy images. Panels D-G
illustrate immunostaining with anti-OspA antibody. A: In addition to
typical spiral-shaped spirochetes, several rolled, looped, and ring-
shaped forms are seen. B: Atypical spirochetes showing ring shaped
forms, blebs still attached to the spirochetes (arrows) as well as
some minute granules. C: Arrow points to a bleb still attached to the
surface of the spirochete. Multiple ring-shaped (D, E) and cystic
forms (F, G) are visible. Notice that in G the cyst is formed by two
spirochetes. H-J: Borrelia cysts as visualized by dark field
microscopy; the arrow points to the end of the spirochete forming the
cyst. Cyst form as seen by immunofluorescence using anti-OspA
antibody (I) and DAPI-DNA staining (J). K: OspA immunoreactive thick,
elongated bodies were also observed. Panels A-G correspond to strain
ADB1 and H-K to strain B31. Bars: A, B = 10 μm; C = 4 μm; D = 8
μm;
E, F = 6 μm; G = 5 μm; H-K = 4 μm.

When spirochetes were re-cultured from various harmful conditions and
from infected cell cultures in BSK II medium in optimal condition,
the typical spiral form of Borrelia spirochetes was recovered. A dark
field microscopic image of the vegetative form of Borrelia
spirochetes recovered from Thioflavin S (5 mg) treated cultures is
illustrated in Figure 6A. Classical spiral Borrelia spirochetes
recovered from infected rat astrocytes cultured for 1 week are
illustrated in Figure 6B by their immunoreaction to anti-Borrelia
antibody BB-1017.

Figure 6. Recovery of the typical vegetative form of spirochetes re-
cultured in BSK II medium and nuclear fragmentation of rat primary
astrocytes exposed to Borrelia burgdorferi. A: Dark field microscopy
image of numerous Borrelia burgdorferi spirochetes (B31 strain)
exhibiting the regular spiral form, re-covered in BSK-II medium
following 1 week exposure to 5 mg Thioflavin S. B: Typical vegetative
form re-covered from rat astrocyte culture exposed to Borrelia
burgdorferi (ADB1) for 1 week, as revealed with a rabbit polyclonal
anti-Borrelia burgdorferi antibody (BB-1017). Compare the regular
spiral morphology of these spirochetes with those seen in Fig. 4H,
where virtually all spirochetes showed atypical forms. C: Green
fluorescent apoptotic nuclei of rat astrocytes as visualized with the
TUNEL technique using FITC tagged dUTP. D: Uninfected primary
astrocytes cultivated in parallel for 1 week did not show nuclear
fragmentation. Bars: A, B: 25 μm; C, D: 50 μm.

Figure 6C illustrates nuclear fragmentation in astrocytes following 1
week exposure to Borrelia burgdorferi as visualized by the TUNEL
method using FITC tagged dUTPs. Nuclear fragmentation was not
observed in control cultures, which were not infected with Borrelia
(D).

Identical atypical and cystic forms were observed in the cerebral
cortex of the three patients with pathologically confirmed chronic
Lyme neuroborreliosis. Figure 7 illustrates these atypical and cystic
forms. OspA immunoreactive colony-like agglomeration of spirochetes
is seen in panel A. In such "colonies" or agglomerates of
spirochetes, atypical, stretched filamentous forms, as well as
numerous ring-shaped forms and spherules are frequently present.
Panel B shows, in the periphery of such agglomerates, atypical ring
shaped structures and spherules (asterisks), which are identical to
those observed in vitro. Ring shaped spirochetes showing a positive
immunoreaction with anti-Borrelia burgdorferi antibody in the
cerebral cortex in a case of parenchymatous Lyme neuroborreliosis are
seen in panel C. These ring-shaped forms are similar to those of
Treponema pallidum (arrows in D) as illustrated in the cerebral
cortex of a patient with general paresis using a polyclonal anti-
Treponema pallidum antibody (Biodesign, B65210R). Arrows point to
helical (E) and ring-shaped OspA immunoreactive forms (F) accumulated
in the cytoplasm of cortical neurons. Rolled spirochetes forming
large rings in the cerebral cortex (G) and in the cytoplasm of an
epithelial cell of the choroid plexus (H) are seen in panels G and H,
as visualized by anti-OspA and antibacterial peptidoglycan
antibodies, respectively. Panel I shows similar atypical rolled forms
as visualized with Thioflavin S in the brain of the same patient. In
addition to some filamentous spirochete forms with more regular
spirals (arrow) cystic forms were also observed in the cerebral
cortex (asterisks in J and K). The atypical and cystic spirochetes
observed in the brain of the patient from which ADB1 strain was
cultivated were identical to those induced when the spirochetes of
this strain were cultivated under various harmful conditions, or when
primary astrocytes or neurons were infected by these spirochetes.
Cortical sections of control cases immunostained with various anti-
Borrelia burgdorferi antibodies were negative.

Figure 7. Extra- and intracellular atypical and cystic forms of
spirochetes in the cerebral cortex of a patient with pathologically
and serologically confirmed chronic Lyme neuroborreliosis where
Borrelia burgdorferi sensu stricto was cultivated from the brain. A:
Colony-like agglomeration of spirochetes as revealed by monoclonal
anti-OspA antibody in the cerebral cortex. B: A close up of the
central part of the mass seen in A. In addition to a few helically
shaped spirochetes (arrow) numerous ring-shaped forms and spherules
(asterisk) are visible, which are identical to those observed in
vitro following 1 week Borrelia exposure of primary neurons (compare
with Fig. 4G). C: Spirochetes showing loop or ring-shaped formations
(arrows) in the cerebral cortex immunostained with a polyclonal anti-
Borrelia burgdorferi antibody (Biodesign, B65302R). They are similar
to those of Treponema pallidum (arrows in D) observed in the cerebral
cortex of a patient with general paresis. Immunostaining was
performed using a polyclonal anti-Treponema pallidum antibody
(Biodesign, B65210R). E: Helically shaped OspA immunoreactive
spirochetes in the cytoplasm of a cortical pyramidal neuron. In
addition to one more typical form (arrow), fine OspA positive minute
granules along filamentous forms are seen. F: Intracellular ring-
shaped forms (arrow) showing positive immunoreaction with a
polyclonal anti-Borrelia burgdorferi antibody (BB1017). They are
identical to those observed in chicken primary neurons infected with
Borrelia (compare F with Figure 4D). Near the asterisk a large
strongly immunoreactive cyst form is visible. Spirochete forming loop
in the cerebral cortex (G) and in the cytoplasm of an epithelial cell
of the choroid plexus (H) are seen as visualized by anti-OspA and
anti-bacterial peptidoglycan antibodies, respectively. I: A similar
atypical spirochete forming loops in the cerebral cortex as
visualized with Thioflavin S. J: In an area with colony-like
spirochete aggregation in addition to some typical, regularly coiled
Borrelia spirochetes (arrow) OspA positive cystic forms (asterisk)
are seen. K: In the cerebral cortex near the colony-like spirochetal
agglomerate a spirochete cyst (asterisk) similar to that observed in
vitro is visible (compare it with Figure 5 G-J). Immunostaining was
performed using a monoclonal anti-OspA antibody. Bars: A = 20 μm; B-J
= 10 μm, K = 5 μm. Panels C and E were reprinted from panels F and D
of Figure 5 of Mikossy et al., 2004 [3], with permission from IOS
Press.

There was no apparent lympho-plasmocytic infiltrates on Hematoxylin
and Eosin-stained sections in the brains of the three patients with
Lyme neuroborreliosis (not shown). However on sections immunostained
with monoclonal antibodies for HLA-DR and CD68, abundant reactive
microglia, frequently forming clumps, were observed in the cerebral
cortex. Accumulation of GFAP positive reactive astrocytes was also
present. Figure 8 illustrates HLA-DR and CD68 immunoreactive
microglia and GFAP-positive reactive astrocytes in the frontal cortex
in one patient, where Borrelia burgdorferi (ADB1 strain) was
cultivated from the brain. Some HLA-DR reactive resting microglial
cells were observed in the frontal cortex of the control patient. No
apparent immunostaining was observed with the anti-CD68 antibody. On
GFAP-immunostained sections some astrocytes with poor cytoplasm and
thin processes without signs of hyperplasia or hypertrophy were
visible. Brain sections immunostained with the omission of the
primary antibodies were negative.

Figure 8. Chronic neuroinflammation in the frontal cortex of a
patient with Lyme neuroborreliosis. First column (A, D and G):
Accumulation of HLA-DR (A) and CD68 (D) immunoreactive microglia
forming clumps, and GFAP (G) positive large reactive astrocytes in
the frontal cortex of a patient with Lyme neuroborreliosis. Second
column (B, E, H) : On frontal sections of the control patient,
activated microglia or astrocytes are not visible. Some resting
microglia showing weak HLA-DR immunostaining (B), absence of CD68
immunoreaction (E) and weak GFAP immunostaining of non reactive
astrocytes and astrocytic processes (H) are visible. C, F and I:
Absence of immunoreaction on sections of a patient with Lyme
neuroborreliosis where immunostaining was performed with omission of
the anti-HLA-DR (C), anti-CD68 (F) and anti-GFAP (I) antibodies.
Bars: A, B, F, H, I = 150 μm; C, D, E = 120 μm; G = 100 μm.

Discussion
Treponema pallidum and Borrelia burgdorferi are associated with
various chronic neuro-psychiatric disorders. Treponema pallidum
persists in the brain and causes various neuropsychiatric disorders
including dementia, cortical atrophy and amyloid deposition years or
decades following the primary infection [63-65]. The persistence of
more resistant atypical cystic and granular forms of Treponema
pallidum, which are less sensitive to chemicals and antibiotics, are
responsible for the long latent stage in chronic syphilis and for the
infectivity of tissues devoid of the demonstrable vegetative form of
spirochetes. The intracellular localization of Treponema pallidum is
another way of evading from destruction by the host immune system
[30,39]. Virtually all types of mammalian cells can be invaded by
Treponema pallidum resulting ultimately in functional cell damage and
cell destruction.

Recently we reported evidence that Borrelia burgdorferi can also
persist in the brain in chronic Lyme neuroborreliosis and, in analogy
to Treponema pallidum, may cause dementia, cortical atrophy and
amyloid deposition [3,49,51]. Only limited data have previously been
available on the presence of atypical, cystic forms of spirochetes in
the brain in chronic Lyme neuroborreliosis. Whether such forms may
eventually cause functional damage and cell death is still not
certain.

Here we analyzed atypical, cystic forms of Borrelia burgdorferi
induced by unfavorable culture conditions and compared these with
forms observed following 1 week of infection of primary chicken and
rat neurons, as well as primary rat and human astrocytes. We also
analyzed whether similar atypical and cystic forms may occur in vivo
in brains of patients with pathologically and serologically confirmed
Lyme neuroborreliosis and compared them to the atypical forms of
Treponema pallidum in brains of patients with general paresis. The
results show that under harmful culture conditions, the typical forms
of Borrelia spirochetes are replaced by atypical forms varying from
ring-shaped and cystic forms to fine single granules of almost
submicroscopic size. These results are in harmony with previous
observations [8,55,66]. The effect of osmotic shock induced with cold
distilled water or heat shock was identical to those previously
observed in other spirochetes [25,67]

Thioflavine S and Congo red had a similar effect. The mechanism of
the harmful effect of these dyes is not known. They may act by
binding to the outer sheath of Borrelia spirochetes [e.g. [56]].
Thioflavin S and Congo red are widely used to detect amyloid deposits
in affected tissues. Several observations suggested that Borrelia
burgdorferi possesses amyloidogenic proteins [51,68,69]. Peptides
derived from the OspA single-layer beta-sheet showed fibrillary
amyloid formation, which may be an explanation of the binding of
Thioflavin S and Congo red to the outer surface of Borrelia
burgdorferi.

Atomic force microscopy (AFM) analysis showed rolled Borrelia
spirochetes inside of a cyst covered by a thin outer membrane. This
has also been observed in various types of spirochetes [e.g.
[11,38,70]] including Borrelia burgdorferi [8] by transmission
electron microscopy analyses. Uni- or multi-spirochetal cysts may be
formed. We illustrated by atomic force microscopy (AFM) rolling of
two Borrelia spirochetes to form a cyst. The size of such cysts
depends on the number of spirochetes packed inside of the cyst [8].
We observed bleb formation, connected to Borrelia spirochetes by a
fine stalk, in both Borrelia strains. Thin newly formed spirochetes
attached to spirochete cells, and to free minute granules were also
observed.

Similar atypical, cystic and granular forms were observed in primary
neuronal and astrocytic cell cultures exposed for 1 week to the
Borrelia burgdorferi strains B31 and ADB1. Nuclear fragmentation of a
subset of infected cells as revealed by TUNEL suggests that Borrelia
burgdorferi can cause functional damage and cell death. The
intracellular localization of filamentous, ring-shaped, cystic and
granular forms suggests that such intracellular Borrelia spirochetes
can be protected from destruction by the host immune system.

Identical atypical and cystic forms were also observed in the
cerebral cortex of the three patients with chronic Lyme
neuroborreliosis with concurrent AD. This indicates that Borrelia
burgdorferi spirochetes can form resistant cystic forms, which may
persist in the brain. Numerous colonies were also observed in vitro
and in vivo. In the brain they were restricted to the cerebral
cortex. These spirochetal masses included numerous cystic forms as
has been described for Treponema pallidum and other spirochetes. Like
for Treponema pallidum in neurosyphilis, atypical and cystic forms of
Borrelia burgdorferi were also observed intracellularly in the brains
of these patients, as it has previously been documented [3,51]. These
results suggest that Borrelia burgdorferi, in analogy to Treponema
pallidum may also invade neurons and glial cells and cause cell
dysfunction and progressive cell death.

The results also showed that atypical Borrelia forms may be present
in the absence of typical coiled forms, indicating that detection of
atypical forms in infected tissues may be of diagnostic value.
Treponema pallidum and Borrelia burgdorferi can persist in infected
tissues, even in the absence of an apparent lymphoplasmacytic
infiltration. Consequently, when the clinical and histopathologic
features suggest syphilis or Lyme disease, the detection of these
spirochetes in infected body fluids and tissues may be of diagnostic
importance [55,71].

Borrelia burgdorferi cultured in harmful conditions and in infected
cell cultures where virtually all spirochetes showed pleomorphic and
cystic forms were resuscitated under appropriate conditions in BSK-II
medium, where apparently all spirochetes showed the typical spiral
morphology. This suggests that these atypical forms may be viable
Borrelia forms. Despite that, under the present experimental
conditions, we cannot exclude whether such growth may represent
propagation of some residual spiral forms, previous observations (7,
8) showing that cystic forms of spirochetes can revert into
vegetative form suggest that at least part of the pleomorphic forms
observed may revert into vegetative form. That Borrelia burgdorferi
was successfully cultivated from brains of the three patients with
Lyme neuroborreliosis in BSK-II medium where pleomorphic and cystic
forms were observed in the brain [3,51,52,72,73] suggests that at
least part of the persisting spirochetes are viable. The typical
spiral form of these cultivated Borrelia burgdorferi spirochetes in
addition to the present Figure 1 E-H was previously illustrated (52,
73). Whether the polymorphic forms observed in the brains of these
three patients may correspond to living, degenerating or "dormant"
spirochetes remains to be determined. Further studies will be
necessary to analyze whether individual Borrelia cysts taken from the
affected brain may revert into vegetative form.

That the ADB1 strain invades neurons and astrocytes in vitro
indicates that these surviving cultivatable spirochetes are still
virulent.

The accumulation of immunocompetent HLA-DR positive microglia and
reactive astrocytes in the cerebral cortex of these patients clearly
indicates the presence of chronic inflammation as previously
suggested [51]. Indeed, Treponema pallidum, Borrelia burgdorferi and
their lipoproteins evoke cytokine responses in cells of the
monocytes/macrophage lineage as well as initiate complement
activation. The response elicited by the major membrane lipoproteins
of Treponema pallidum and Borrelia burgdorferi was analogous to that
observed with whole bacteria. The vegetative and cystic forms
including the vesicular blubs and free vesicular structures of
Borrelia burgdorferi all contain the biologically active spirochetal
surface proteins indicating that they all elicit inflammatory
responses including complement activation [reviewed in [73]].

The clinical and the pathological hallmarks of Alzheimer's disease,
including beta-amyloid deposition are also present in the atrophic
form of general paresis and in tertiary Lyme neuroborreliosis
[3,63,72-74]. The facts that Borrelia burgdorferi spirochetes were
cultivated from the brains of these patients, that Borrelia antigens
and genes were co-localized with beta-amyloid deposits in cortical
spirochetal colonies, and that the serology of these patients was
positive for Borrelia burgdorferi are evidences that the present
cases correspond to the atrophic parenchymatous form of late Lyme
neuroborreliosis.

Conclusion
Dark field microscopy, histochemical, immunohistochemical and atomic
force microscopy (AFM) analyses revealed that pleomorphic and cystic
Borrelia forms were induced by the various unfavorable conditions
that were employed. Extra- and intracellular atypical and cystic
forms were observed in neuronal and astrocytic cultures following 1
week of exposure to Borrelia burgdorferi (B31 and ADB1). Identical
extra- and intracellular atypical and cystic Borrelia forms were also
observed in the brains of all three patients with Lyme
neuroborreliosis, which were also similar to the atypical forms of
Treponema pallidum in the brains of patients with general paresis.
Astrocytes infected with Borrelia burgdorferi exhibited nuclear
fragmentation.

Our results suggest that pleomorphic forms, including cystic forms of
Borrelia burgdorferi may persist in the brain and may explain the
long latent stage and persisting infection in Lyme neuroborreliosis.
The identification of these extra- or intracellular atypical, cystic
and granular forms of Borrelia burgdorferi is essential for the
histopathological diagnosis of Lyme disease as they may indicate
chronic Borrelia infection, even in cases where the typical coiled
spirochetes are apparently absent. In analogy to Treponema pallidum,
Borrelia burgdorferi can persist in the brain in Lyme
neuroborreliosis and may initiate and sustain chronic inflammation
and tissue damage.

Competing interests
The authors declare that they have no competing interests.

Authors' contributions
JM contributed to the direction of the investigation, data
interpretation and to the writing of the manuscript. SK contributed
to the AFM analysis, ADZ contributed in experiments of neuronal cell
cultures, SMC contributed in the analysis of syphilitic brains and in
organizing the interaction of several laboratories, as well as in
data interpretation, SY contributed to the immunohistochemical
analysis, PLMG contributed to the writing of the manuscript and data
interpretation. All authors read and approved the final manuscript.

Acknowledgements
We would like to express our particular thanks for P. Darekar who has
done the major part of the cell culture, histology and
immunohistochemical analyses. It was a tremendous work. We are
grateful for her. We are also grateful for P. Hansma. The AFM work
has been done in his laboratory where the atmosphere, rigor and
enthusiasm were excellent and unforgettable. We would like to thank
all those colleagues and friends who strongly supported this work. We
are grateful for J. and L. Krasinsky. Their generous help contributed
to the realization of this work. The work was supported by grants
from the Societe Academique Vaudoise. We are grateful for the support
of the Institute of Histology and Embryology, University of Fribourg,
Switzerland. The work was also supported by the Pacific Alzheimer
Foundation, Vancouver, Canada

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