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Persisting atypical and cystic forms of Borrelia burgdorferi and local inflammation in Lyme neuroborreliosis Miklossy, Judith; Kasas, Sandor; Zurn, Anne D; McCall, Sherman; Yu, Sheng; McGeer, Patrick L Sep 25, 2008

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ralssBioMed CentJournal of NeuroinflammationOpen AcceResearchPersisting atypical and cystic forms of Borrelia burgdorferi and local inflammation in Lyme neuroborreliosisJudith Miklossy*1, Sandor Kasas2, Anne D Zurn3, Sherman McCall4, Sheng Yu1 and Patrick L McGeer1Address: 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 and 4Pathology Laboratory, U.S. Army Medical Research Institute of Infectious Diseases (USAMRIID), 1425 Porter St., Ft. Detrick, MD 21702-5011, USAEmail: Judith Miklossy* -; Sandor Kasas -; Anne D Zurn -; Sherman McCall -; Sheng Yu -; Patrick L McGeer -* Corresponding author    AbstractBackground: The long latent stage seen in syphilis, followed by chronic central nervous system infectionand inflammation, can be explained by the persistence of atypical cystic and granular forms of Treponemapallidum. We investigated whether a similar situation may occur in Lyme neuroborreliosis.Method: Atypical forms of Borrelia burgdorferi spirochetes were induced exposing cultures of Borreliaburgdorferi (strains B31 and ADB1) to such unfavorable conditions as osmotic and heat shock, andexposure to the binding agents Thioflavin S and Congo red. We also analyzed whether these forms maybe induced in vitro, following infection of primary chicken and rat neurons, as well as rat and humanastrocytes. We further analyzed whether atypical forms similar to those induced in vitro may also occur invivo, in brains of three patients with Lyme neuroborreliosis. We used immunohistochemical methods todetect evidence of neuroinflammation in the form of reactive microglia and astrocytes.Results: Under these conditions we observed atypical cystic, rolled and granular forms of thesespirochetes. We characterized these abnormal forms by histochemical, immunohistochemical, dark fieldand atomic force microscopy (AFM) methods. The atypical and cystic forms found in the brains of threepatients with neuropathologically confirmed Lyme neuroborreliosis were identical to those induced invitro. 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 cerebralcortex.Conclusion: The results indicate that atypical extra- and intracellular pleomorphic and cystic forms ofBorrelia burgdorferi and local neuroinflammation occur in the brain in chronic Lyme neuroborreliosis. Thepersistence of these more resistant spirochete forms, and their intracellular location in neurons and glialcells, may explain the long latent stage and persistence of Borrelia infection. The results also suggest thatBorrelia 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 canPublished: 25 September 2008Journal of Neuroinflammation 2008, 5:40 doi:10.1186/1742-2094-5-40Received: 15 April 2008Accepted: 25 September 2008This article is available from:© 2008 Miklossy et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.Page 1 of 18(page number not for citation purposes)occur in the absence of the typical spiral Borrelia form.Journal of Neuroinflammation 2008, 5:40 similarity of clinical and pathological manifestationsof syphilis caused by Treponema pallidum [1] and Lyme dis-ease caused by Borrelia burgdorferi [2] is well established.In analogy to Treponema pallidum, Borrelia burgdorferi per-sists in the brain in chronic Lyme neuroborreliosis [3].How Borrelia burgdorferi is able to survive in infected tis-sues for years or decades is not well understood. Ways forlong term survival may be through transformation intomore resistant atypical forms and through intracellularlocalization.As early as 1905 it was suspected that the classical spiral(vegetative) form was not the only one that spirochetescould assume [1,4]. Transformation of various types ofspirochetes into cystic forms through end knob, loop,ring-shaped and spherule formation has since beenrepeatedly reported [5-10]. Agglomeration of spirochetesinto colonies [11-14], enclosing numerous cystic forms,has been observed both in vitro and in vivo [12].Treponema pallidum and Borrelia burgdorferi produce vesic-ular budding from the membrane, which may becomedetached. In Borrelia burgdorferi these free vesicular orgranular structures contain spirochetal surface proteinsand linear and circular DNA [15,16].Granular disintegration of spirochetes resulting in a chainof fine granules also occurs under adverse conditions [17-22]. Minute granules are liberated from the periplasmicsheath through budding and extrusion, which may multi-ply and may be transmissible [23-31]. Their presence insyphilitic patients was regarded as confirmatory of thesyphilitic nature of the lesions even in the absence of clas-sical spiral forms [26,27,30]. These spore-like minutegranules (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 Lor metacyclic forms [25,32,39].These various atypical forms were suggested to be part ofa complex developmental cycle, a form of resistance toadverse conditions, and a source for reproduction undermore favorable conditions. Reconversion of cystic Borreliaburgdorferi into the typical spiral form has been demon-strated in vitro and in vivo [8,10,31,40].The occurrence of pleomorphic forms of Treponema palli-dum in the brain in general paresis and their abundance injuvenile paresis is well documented [6,18,26,41,42].Treponema pallidum may invade virtually all parenchymaland mesenchymal cells, including plasma cells, macro-both extra- and intracellularly [30]. It has also beendescribed in other spirochetal infections [e.g. [44-46]].Only limited data are available on the occurrence of atyp-ical, cystic or granular forms of Borrelia burgdorferi ininfected tissues. Their occurrence has been reported inskin lesions [14], in an ex vivo system in tonsil tissue [47]and on silver stained hippocampus section in a patientwith concurrent Alzheimer disease (AD) and Lyme neu-roborreliosis [48]. Intracellular localization of Borreliaburgdorferi was observed in macrophages and keratinoc-ytes in the skin [14] and in neurons and glial cells in vitroand in vivo [3,49-51].The goal of the present study was to compare whetheratypical and cystic forms of Borrelia burgdorferi spirochetesinduced in vitro are similar to those occurring in vivo.Three patients with chronic Lyme neuroborreliosis wereused in the study. Immunodetection of reactive microgliaand astrocytes was also performed to detect neuroinflam-mation.MethodsCultivation of Borrelia burgdorferi spirochetes in BSK II mediumBorrelia burgdorferi spirochetes strains B31 and ADB1[3,51-53] were cultivated in BSK II medium [54]. To 500ml BSK medium (Sigma B 3528) containing 6% rabbitserum (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. ThepH of BSK II medium was adjusted to pH 7.To induce atypical spirochete forms 5 ml of cultivated Bor-relia burgdorferi spirochetes (5 × 105/ml) were exposed tovarious harmful conditions. Spirochetes were exposed tostrong acidic and basic conditions by adjusting the pH ofthe BSK II medium to pH2 and pH10 using sterile 1 MHCl or 1 M NaOH. The harmful effect of alcohol was ana-lyzed by adding 1 ml of either 70% or 95% ethanol to 5ml cultivated spirochetes. Heat shock was produced bycultivating spirochetes at 45°C.Spirochetes are known to bind Congo red and ThioflavinS [55,56], both of which are widely used to stain amyloid.To analyze whether they may induce atypical Borreliaforms, 1 mg or 5 mg of Congo red, or 1 mg or 5 mg Thio-flavin S were directly added to 5 ml of spirochete culture.The same amounts dissolved in 2 ml of 70% alcohol werealso used to induce atypical spirochetes. The effect of acri-din orange, another fluorochrom which binds to spiro-Page 2 of 18(page number not for citation purposes)phages, neurons and glial cells [39,40,43]. Atypical andcystic forms of Treponema pallidum have been observedchetes, was also analyzed by adding 1 mg or 5 mg acridinorange powder to 5 ml of cultivated spirochetes.Journal of Neuroinflammation 2008, 5:40 1 hour, 6 hours, and 1 week exposure times, 50μl 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 forhistochemical and immunohistochemical investigation.Additional 500 μl samples were removed and fixed in glu-taraldehyde for atomic force microscopy (AFM) analysis.Spirochetes cultivated at 32°C at pH 7 for the same peri-ods of time were used as controls. An exception withrespect to the exposure times was induction of osmoticshock by cold H2O. Two ml sterile cold H2O was addedto 5 ml cultured spirochetes that had been collected bycentrifugation at 1000 rpm for 5 minutes. Here the sam-ples were examined following 1 and 6 hours of exposure.In order to analyze whether the typical spiral Borreliaform may be resuscitated, at the end of each experiments200 μl samples were reinoculated in BSK II medium atPh7 and following one week of culture at room tempera-ture, 30 μl samples were analyzed by dark field micros-copy.Infection of cell cultures with Borrelia burgdorferiSuperior cervical ganglia from 8- to 12-day-old chickenembryos were dissociated as described previously [57].Briefly, neurons were separated from non-neuronal cellsusing a density gradient formed with Percoll. The sympa-thetic neurons were then grown for 3–4 weeks in serumcontaining medium on a polyornithine substrate pre-coated with heart-conditioned medium [57]. Neuronsdissociated from the telencephalon of 21-day-old rat werecultured either on collagen or polylysine substrate in aserum-containing medium [58]. Rat primary astrocytes(106) were prepared as described earlier [59]. Followingthe characterization of the primary astrocytic cell culturesusing anti-GFAP antibody (Dako, Z334) more than 95%of the cells were GFAP reactive (not shown here). The cellswere 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%) incu-bator at 37°C.To infect neurons and astrocytes, Borrelia spirochetes ofthe virulent strains B31 and ADB1, the latter having beencultured from the brain of a patient with concurrent Lymeneuroborreliosis and AD [3], were employed. Equal vol-umes of medium for the given primary cells and for spiro-chetes (BSK II) were used as the culture medium. The finalconcentration of spirochetes in the infected medium cor-responded to 5 × 105/ml. Before exposure to spirochetes,the cells were tested with 4',6-diamidine-2'-phenylindoledihydrochloride (DAPI, 236 276, Boehringer Mannheim,Germany) to exclude Mycoplasma infection. Parallel cul-After 1 week exposure, the medium was removed and thecells were rinsed with PBS (2 ml, 2 × 3 minutes). To ana-lyze the morphology of free floating spirochetes, 50 μlsamples of culture medium were taken and analyzed bydark field microscopy. Smears were also prepared for his-tochemical and immunohistochemical analyses.After 1 week exposure, 200 μl samples from all infectedcell cultures were also re-inoculated in BSK II medium andwere cultivated at room temperature, Ph 7, for one week.Then 30 μl samples were analyzed by dark field micros-copy.Detection of apoptosis by deoxynucleotidyltransferase (TdT)-mediated dUTP nick end labeling (TUNEL)Cells in 6 wells chambers were fixed with 4% paraformal-dehyde for 10 minutes in room temperature. Followingan incubation with proteinase K (20 μg/ml) in TRIS HCL(pH 7.4) for 15 minutes at 37°C the cells were rinsed with2 ml PBS (2 × 3 minutes). Then cells were treated with apermeabilisation solution containing 0.1% Triton X100,in 0.1% sodium citrate, for 2 minutes on ice followed bya rinse with PBS (2 × 3 minutes).The cells were incubated with a freshly prepared TUNELreaction mixture kept on ice containing 45 μl of TUNELlabel solution (1767291, Boehringer) containing unla-beled dNTPs and fluorescein isothiocyanate tagged dUTP(FITC-dUTP) and 5 μl of TUNEL enzyme (Terminal deox-ynucleotidyl Transferase (TdT) (1767305, Boehringer) for1 hour and 30 minutes at 37°C in a humidified chamber.For a negative control the TUNEL enzyme was omittedfrom the TUNEL reaction mixture and 50 μl TUNEL labelsolution alone was used.Detection of pleomorphic Borrelia forms in vivoBrains of three patients with pathologically and serologi-cally confirmed Lyme neuroborreliosis and concurrentAD were analyzed [3]. From the brains of these threepatients, aged 74, 78, and 86 years, spirochetes were suc-cessfully cultivated in BSK II medium. In two of them(strains ADB1 and ADB2) 16SrRNA gene sequence analy-sis identified the spirochetes as Borrelia burgdorferi sensustricto (s. s.).To detect whether atypical and cystic forms of Borreliaburgdorferi spirochetes are present in the brains of thesepatients, frozen sections of the hippocampus, frontal,temporal and parietal cortex were analyzed using darkfield microscopy, as well as histochemical and immuno-histochemical techniques. Before immunostaining thesections were fixed in acetone for 10 minutes at 4°C. Ace-tone fixed frozen sections which were cut from samplesPage 3 of 18(page number not for citation purposes)tures not infected with spirochetes were always used ascontrols.taken from identical brain areas in three control patientswithout neurological symptoms and without brainJournal of Neuroinflammation 2008, 5:40 were also processed and analyzed in the samefashion.Paraffin sections (20 μm thick) cut from various corticalsamples (from archival material of the Armed Forces Insti-tute of Pathology, USA) of two patients (31 and 52 yearold males) with clinically, serologically and pathologi-cally confirmed general paresis, were also analyzed for thepresence of Treponema pallidum. The goal was to compareatypical spirochetal forms of Borrelia burgdorferi in Lymeneuroborreliosis with those of Treponema pallidum in gen-eral paresis.Dark field microscopy, histochemical and immunohistochemical analysis of spirochetesFrom cultivated Borrelia spirochetes exposed to variousharmful conditions, 50 μl samples were used as wet prep-aration for dark field microscopy analysis. Additionalsamples (50 μl) were used to prepare smears for the histo-chemical and immunohistochemical analyses. In order toanalyze free floating spirochetes in Borrelia infected cellcultures, 50 μl samples of the co-culture medium wereanalyzed.Smears and brain sections were stained with the Warthin-Starry and Bosma-Steiner silver techniques as describedfor the detection of spirochetes. Spirochetal DNA wasdetected in smear preparations of cultivated spirochetesexposed to various adverse conditions, in infected cells,and in unfixed frozen brain sections by staining withDAPI (236 276, Boehringer Mannheim, Germany) fol-lowing instructions of the manufacturer. The same prepa-rations were also used for immunohistochemical analysis.Smears prepared on glass slides from cultivated spiro-chetes, from medium of infected cells, and from cryostatcut brain sections were fixed in acetone for 10 minutes at4 °C prior to immunostaining. Infected cells in six wells,where acetone cannot be used, were fixed in 4% parafor-maldehyde for 5 minutes. Before immunostaining, frozenbrain sections following acetone fixation were incubatedin 0.1% amylase for 5 min at 37°C. The following anti-Borrelia burgdorferi antibodies were used: monoclonalanti-OspA (H5332, H3T5, Symbicom, 1:50) and anti-flag-ellin (G 9724, H605, Symbicom, 1:50), polyclonalB65302R (Biodesign, 1:100) and BB-1017 (1:500) [3]antibodies. The specificity of these mono- and polyclonalantibodies was previously tested by Western blot analysis[3].For immunostaining, the avidin-biotin-peroxidase tech-nique was used. Following 24, 48 or 72 hours incubationwith a primary antibody at 4°C, the sections were incu-bated with the appropriate secondary antibodies. For the(Dako, E413) was used. The immunoreaction wasrevealed by diaminobenzidine (DAB) alone, or withnickel-ammonium sulfate as described previously [60].Frozen sections immunostained in the absence of a pri-mary antibody or with an irrelevant mono- or polyclonalantibody were used as negative controls. Immunostainingwas also performed with various anti-Borrelia burgdorferiantibodies using FITC tagged anti-mouse or anti-rabbitsecondary antibody depending on the primary antibodyused. The green fluorescence of Borrelia burgdorferi spiro-chetes was analyzed with a Zeiss fluorescent microscope.A monoclonal antibody (Biogenesis 7263-1006 orChemicon MAB995, dil.1: 200) for the analysis of thepresence of bacterial peptidoglycan, a bacterial cell wallcomponent of virtually all Eubacteria, including spiro-chetes, was also used as previously described in detail[61].Floating paraffin sections (20 μm thick) of the cerebralcortex of the two patients with general paresis were immu-nostained with a polyclonal anti-Treponema pallidum anti-body (Biodesign, B65210R).Detection of neuroinflammationParaffin sections of the hippocampus, frontal and parietalcortices of the three patients with Lyme neuroborreliosiswere also used for the immunohistochemical detection ofreactive microglia and astrocytes. Anti-HLA-DR (cloneCR3/43, M775, Dako) and anti-CD68 (clone KP1, M814,Dako) monoclonal antibodies were used to visualizemicroglia and a polyclonal anti-GFAP antibody (Z334,Dako) to detect astrocytes. Paraffin sections of the samecortical areas of a female patient (aged 59 years) withoutbrain lesion were used as controls. In addition, sections ofthe three patients with Lyme neuroborreliosis were alsoimmunostained with the omission of these primary anti-bodies.Atomic force microscopy (AFM) analysisTo 500 μl samples of cultivated Borrelia spirochetesexposed to various harmful conditions 500 μl of 2.5%buffered glutaraldehyde was added. Samples were thenstored at 4°C until used for the atomic force microscopy(AFM) analysis. 20–50 μl samples were put on the surfaceof a Nucleopore® filter of 2 μm hole size and were dried atroom temperature in air, as previously described [62]. Thefilters were fixed on metallic discs or on glass slides usinga double face rubber strip, and were imaged with a Bio-scope 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 atroom temperature in air. The scanning rate varied from0.1 to 5 Hz. Images were obtained in both the constantPage 4 of 18(page number not for citation purposes)monoclonal antibodies, a biotinylated F(ab) fragment ofaffinity isolated rabbit anti-mouse immunoglobulinforce mode providing true height, and the amplitudemode, for highlighting sharp contours. The images wereJournal of Neuroinflammation 2008, 5:40 and the measurements were done using theNanoscope III image processing software.The human brains analyzed were from the UniversityInstitute of Pathology, Lausanne, Switzerland. The studyadhered to the tenets of the Helsinki Declaration. Animalexperimentation conformed to the Guide for the Care andUse of Laboratory Animals, formulated by the NationalResearch Council, 1996, and the Swiss law on animal pro-tection.ResultsFigure 1 illustrates the typical morphology and colony-like formation of strains B31 (A-D) and ADB1 (E-H). Pan-els A and B show the regular coiled morphology and col-ony-like aggregates of spirochetes (B31 strain) as observedby dark field microscopy analysis. Regularly coiled OspA-immunoreactive spirochetes of the same strain are seenfollowing immunostaining with a monoclonal anti-OspAantibody in panel C. An atomic force microscopy (AFM)image of a regularly coiled fragment of a Borrelia spiro-chete (strain B31) is visible in panel D. The typical coiledmorphology of spirochetes of the ADB1 strain, whichwere cultivated from the brain of a patient with chronicLyme neuroborreliosis, is illustrated in panels E and F bydark field microscopy and in panel G by immunohisto-chemistry using a polyclonal anti-Borrelia burgdorferi anti-body (Biodesign, B65302R). The primary anti-Borreliaburgdorferi antibody was revealed with FITC-tagged sec-ondary antibody showing green fluorescence. Panel Hillustrates 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 con-verted to atypical and cystic forms when exposed to vari-ous unfavorable culture conditions. Atypical and cysticforms were seen at 1 hour exposure. Their numbersincreased following 6 hours exposure and was highest at1 week, where the majority of spirochetes showed atypicalmorphology. Osmotic shock induced by cold sterile dis-tilled water induced atypical and cyst forms of the major-ity of spirochetes following 1 and 6 hour exposure. Theatypical and cyst forms retained an affinity for silver andwere immunoreactive with the various anti-Borrelia burg-dorferi antibodies.The morphological changes were similar in both the B31and ADB1 strains, whether induced by osmotic shock,heat shock, alcohol, acidic or basic pH. Atypical and cysticforms were also seen following Congo red and ThioflavinS exposures.A stronger effect was observed when Thioflavin S andCongo red were dissolved in ethanol before addition tothe culture.Atypical forms included knob, ring-shaped and loop for-mations, uni- or multi-spirochetal cysts, bleb formation,granular disintegration and colony-like masses enclosingnumerous cystic forms. Following 1 week exposure, freeminute granules and re-growing of slender motile L formsof young spirochetes along injured spirochetal cells wereobserved.Figure 2 illustrates rolled spirochetes forming ring-shapedand globular structures. Panel A-D show large aggregatesof multiple rolled, ring-shaped and cystic spirochetes inresponse to osmotic shock generated by cold sterile dis-tilled water. Panels A and C illustrate these atypical formsas seen by dark field microscopy at lower (A) and higher(C) magnifications. Panels B and D illustrate similarresults following immunostaining with a monoclonalanti-OspA antibody. Atomic force microscopy (AFM)images of loop and ring formations of Borrelia spirochetesare illustrated in panels E and F.Figure 3A illustrates atypical and cystic forms of Borreliaburgdorferi induced by Thioflavin S (1 mg dissolved in 2ml 70% ethanol). The agglomeration of spirochetesexhibits green fluorescence due to the binding of Thiofla-vin S. Numerous ring-shaped and globular cystic forms(arrows) were observed in the periphery of the spirochetalmass. Similar rolled and cystic Borrelia forms (B, C)induced by Thioflavin S are illustrated by dark field micro-scopy. Panels D and E show spirochetes with similar mor-phology, which were immunostained with anti-OspAantibody. Panels F-H show the results of atomic forcemicroscopy (AFM) analysis. The atomic force microscopy(AFM) images reveal rolled spirochetes inside of cysts.Cystic forms entirely covered by a thickened externalmembrane masking the content were also observed. Oneof them is illustrated in panel H. It is similar to thoseobserved by dark field microscopy as well as with an anti-OspA antibody (compare H with C, D and E).Atypical cystic, granular forms and colony-like aggrega-tion of spirochetes into large masses enclosing cysticforms were also observed following 1 week infection ofprimary neurons and astrocytes with Borrelia spirochetes.The morphological changes observed were similar inchicken sympathetic neurons, rat telencephalic neuronsand also in rat and human primary astrocytes. Both Borre-lia strains (B31 and ADB1) showed identical morpholog-ical changes. Following 1 week exposure of the cells toBorrelia burgdorferi, it is difficult to find spirochetes whichPage 5 of 18(page number not for citation purposes)have preserved the typical spiral form.Journal of Neuroinflammation 2008, 5:40 6 of 18(page number not for citation purposes)Characteristic morphology of Borrelia burgdorferi seen by various techniques following one week of culture in BSKII mediumFigure 1Characteristic 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 spi-rochetes (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 sil-ver 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.Journal of Neuroinflammation 2008, 5:40 7 of 18(page number not for citation purposes)Atypical forms of Borrelia burgdorferi (B31 strain) spirochetes induced by harmful culture conditionsFigure 2Atypical 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.Journal of Neuroinflammation 2008, 5:40 8 of 18(page number not for citation purposes)Rolled and cystic forms of Borrelia burgdoferi spirochetes observed after one week of culture in medium to which Thioflavin S had bee  addedFigure 3Rolled 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 immunos-taining with the monoclonal anti-OspA antibody. F-H: Atomic force microscopy (AFM) images of Borrelia cysts. Rolled spiro-chetes 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.Journal of Neuroinflammation 2008, 5:40 4 illustrates atypical forms of spirochetes following1 week exposure of chicken primary sympathetic neurons(A, C-G) and rat astrocytes (B and H) to Borrelia burgdor-feri, strain ADB1. Large colony-like aggregates are illus-trated in infected neuronal culture (A) as seen by darkfield microscopy and in rat primary astrocytic cultures (B)immunostained with an anti-Borrelia burgdorferi antibody.Borrelia spirochetes surrounding neuronal perikarya areseen in panel C. Panels D and E illustrate OspA immuno-reactive intracytoplasmic atypical filamentous, ringshaped (arrow) forms of Borrelia burgdorferi. Some extra-cellular spirochetes showing ring-shaped formation arealso present (E, arrow). Panel F shows an OspA immuno-reactive atypical spirochete with a double ring-shaped for-mation at one end and some OspA positive granulesalong the injured spirochete. A small colony like mass isillustrated in panel G in which the majority of spirochetesshow atypical ring-shaped cystic formations. Regularlycoiled spirochetes are not present. OspA immunoreactivering shaped forms and spherules of Borrelia spirochetes inrat astrocytic culture are illustrated in panel H.Atypical forms, including ring-shaped, uni- or multi-spi-rochetal cystic and granular forms also occurred free float-ing in the medium of primary neuronal and glial cellcultures. Figure 5 illustrates these atypical forms following1 week exposure to Borrelia spirochetes. Dark field micro-scopy images of ring-shaped and cystic forms are seen inpanels A and B. Arrows in B and C point to bleb forma-tions still attached with thin stalks to the surface of thespirochete cell. Panels D-G show OspA immunoreactivemultiple ring-shaped (D and E) and cystic (F-G) Borreliaforms. The cysts may sometimes be formed by multiplespirochetes. For example, in panel G an anti-Ospa immu-noreactive cyst formed by two spirochetes is visible. Pan-els H-J illustrate spirochetal cysts as visualized by darkfield microscopy (H), by immunofluorescence using ananti-OspA antibody (I) and by DAPI staining (J). OspAimmunoreactive large, thick, elongated bodies were alsoobserved as seen in panel K. Panel H shows dark fieldmicroscopy image of a cyst where the free end (arrow) ofthe rolled spirochete is visible.When spirochetes were re-cultured from various harmfulconditions and from infected cell cultures in BSK IImedium in optimal condition, the typical spiral form ofBorrelia spirochetes was recovered. A dark field micro-scopic image of the vegetative form of Borrelia spirochetesrecovered from Thioflavin S (5 mg) treated cultures isillustrated in Figure 6A. Classical spiral Borrelia spiro-chetes recovered from infected rat astrocytes cultured for 1week are illustrated in Figure 6B by their immunoreactionto anti-Borrelia antibody BB-1017.Figure 6C illustrates nuclear fragmentation in astrocytesfollowing 1 week exposure to Borrelia burgdorferi as visual-ized by the TUNEL method using FITC tagged dUTPs.Nuclear fragmentation was not observed in control cul-tures, which were not infected with Borrelia (D).Identical atypical and cystic forms were observed in thecerebral cortex of the three patients with pathologicallyconfirmed chronic Lyme neuroborreliosis. Figure 7 illus-trates these atypical and cystic forms. OspA immunoreac-tive colony-like agglomeration of spirochetes is seen inpanel A. In such "colonies" or agglomerates of spiro-chetes, atypical, stretched filamentous forms, as well asnumerous ring-shaped forms and spherules are frequentlypresent. Panel B shows, in the periphery of such agglom-erates, atypical ring shaped structures and spherules(asterisks), which are identical to those observed in vitro.Ring shaped spirochetes showing a positive immunoreac-tion with anti-Borrelia burgdorferi antibody in the cerebralcortex in a case of parenchymatous Lyme neuroborreliosisare seen in panel C. These ring-shaped forms are similar tothose of Treponema pallidum (arrows in D) as illustrated inthe cerebral cortex of a patient with general paresis usinga polyclonal anti-Treponema pallidum antibody (Biode-sign, B65210R). Arrows point to helical (E) and ring-shaped OspA immunoreactive forms (F) accumulated inthe cytoplasm of cortical neurons. Rolled spirochetesforming large rings in the cerebral cortex (G) and in thecytoplasm of an epithelial cell of the choroid plexus (H)are seen in panels G and H, as visualized by anti-OspAand antibacterial peptidoglycan antibodies, respectively.Panel I shows similar atypical rolled forms as visualizedwith Thioflavin S in the brain of the same patient. In addi-tion to some filamentous spirochete forms with more reg-ular spirals (arrow) cystic forms were also observed in thecerebral cortex (asterisks in J and K). The atypical andcystic spirochetes observed in the brain of the patientfrom which ADB1 strain was cultivated were identical tothose induced when the spirochetes of this strain were cul-tivated under various harmful conditions, or when pri-mary astrocytes or neurons were infected by thesespirochetes. Cortical sections of control cases immunos-tained with various anti-Borrelia burgdorferi antibodieswere negative.There was no apparent lympho-plasmocytic infiltrates onHematoxylin and Eosin-stained sections in the brains ofthe three patients with Lyme neuroborreliosis (notshown). However on sections immunostained with mon-oclonal antibodies for HLA-DR and CD68, abundant reac-tive microglia, frequently forming clumps, were observedin the cerebral cortex. Accumulation of GFAP positivereactive astrocytes was also present. Figure 8 illustratesPage 9 of 18(page number not for citation purposes)HLA-DR and CD68 immunoreactive microglia and GFAP-positive reactive astrocytes in the frontal cortex in oneJournal of Neuroinflammation 2008, 5:40 4 (see legend on next page)Page 10 of 18(page number not for citation purposes)Journal of Neuroinflammation 2008, 5:40, where Borrelia burgdorferi (ADB1 strain) was culti-vated from the brain. Some HLA-DR reactive restingmicroglial cells were observed in the frontal cortex of thecontrol patient. No apparent immunostaining wasobserved with the anti-CD68 antibody. On GFAP-immu-nostained sections some astrocytes with poor cytoplasmand thin processes without signs of hyperplasia or hyper-trophy were visible. Brain sections immunostained withthe omission of the primary antibodies were negative.DiscussionTreponema pallidum and Borrelia burgdorferi are associatedwith various chronic neuro-psychiatric disorders.Treponema pallidum persists in the brain and causes vari-ous neuropsychiatric disorders including dementia, corti-cal atrophy and amyloid deposition years or decadesfollowing the primary infection [63-65]. The persistenceof more resistant atypical cystic and granular forms ofTreponema pallidum, which are less sensitive to chemicalsand antibiotics, are responsible for the long latent stage inchronic syphilis and for the infectivity of tissues devoid ofthe demonstrable vegetative form of spirochetes. Theintracellular localization of Treponema pallidum is anotherway of evading from destruction by the host immune sys-tem [30,39]. Virtually all types of mammalian cells can beinvaded by Treponema pallidum resulting ultimately infunctional cell damage and cell destruction.Recently we reported evidence that Borrelia burgdorferi canalso persist in the brain in chronic Lyme neuroborreliosisand, in analogy to Treponema pallidum, may cause demen-tia, cortical atrophy and amyloid deposition [3,49,51].Only limited data have previously been available on thepresence of atypical, cystic forms of spirochetes in thebrain in chronic Lyme neuroborreliosis. Whether suchforms may eventually cause functional damage and celldeath is still not certain.pared these with forms observed following 1 week ofinfection of primary chicken and rat neurons, as well asprimary rat and human astrocytes. We also analyzedwhether similar atypical and cystic forms may occur in vivoin brains of patients with pathologically and serologicallyconfirmed Lyme neuroborreliosis and compared them tothe atypical forms of Treponema pallidum in brains ofpatients with general paresis. The results show that underharmful culture conditions, the typical forms of Borreliaspirochetes are replaced by atypical forms varying fromring-shaped and cystic forms to fine single granules ofalmost submicroscopic size. These results are in harmonywith previous observations [8,55,66]. The effect ofosmotic shock induced with cold distilled water or heatshock was identical to those previously observed in otherspirochetes [25,67]Thioflavine S and Congo red had a similar effect. Themechanism of the harmful effect of these dyes is notknown. They may act by binding to the outer sheath ofBorrelia spirochetes [e.g. [56]]. Thioflavin S and Congored are widely used to detect amyloid deposits in affectedtissues. Several observations suggested that Borrelia burg-dorferi possesses amyloidogenic proteins [51,68,69]. Pep-tides derived from the OspA single-layer beta-sheetshowed fibrillary amyloid formation, which may be anexplanation of the binding of Thioflavin S and Congo redto the outer surface of Borrelia burgdorferi.Atomic force microscopy (AFM) analysis showed rolledBorrelia spirochetes inside of a cyst covered by a thin outermembrane. This has also been observed in various typesof spirochetes [e.g. [11,38,70]] including Borrelia burgdor-feri [8] by transmission electron microscopy analyses.Uni- or multi-spirochetal cysts may be formed. We illus-trated by atomic force microscopy (AFM) rolling of twoBorrelia spirochetes to form a cyst. The size of such cystsdepends on the number of spirochetes packed inside ofAtypical and cystic Borrelia forms following 1 week exposure of primary neuronal and astrocytic cultures to Borrelia burgdorferiFigure 4 (see previous page)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 = 5Here we analyzed atypical, cystic forms of Borrelia burgdor- the cyst [8]. We observed bleb formation, connected toPage 11 of 18(page number not for citation purposes)feri induced by unfavorable culture conditions and com- Borrelia spirochetes by a fine stalk, in both BorreliaJournal of Neuroinflammation 2008, 5:40 12 of 18(page number not for citation purposes)Atypical cystic spirochetes in the medium of neuronal and astrocytic cultures following 1 week exposure to Borrelia burgdorferiFigure 5Atypical 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 anti-body. A: In addition to typical spiral-shaped spirochetes, several rolled, looped, and ring-shaped forms are seen. B: Atypical spi-rochetes 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 anti-body (I) and DAPI-DNA staining (J). K: OspA immunoreactive thick, elongated bodies were also observed. Panels A-G corre-spond 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.Journal of Neuroinflammation 2008, 5:40 Thin newly formed spirochetes attached to spiro-chete cells, and to free minute granules were alsoobserved.Similar atypical, cystic and granular forms were observedin primary neuronal and astrocytic cell cultures exposedfor 1 week to the Borrelia burgdorferi strains B31 andADB1. Nuclear fragmentation of a subset of infected cellsas revealed by TUNEL suggests that Borrelia burgdorferi cancause functional damage and cell death. The intracellularlocalization of filamentous, ring-shaped, cystic and gran-ular forms suggests that such intracellular Borrelia spiro-Identical atypical and cystic forms were also observed inthe cerebral cortex of the three patients with chronic Lymeneuroborreliosis with concurrent AD. This indicates thatBorrelia burgdorferi spirochetes can form resistant cysticforms, which may persist in the brain. Numerous colonieswere also observed in vitro and in vivo. In the brain theywere restricted to the cerebral cortex. These spirochetalmasses included numerous cystic forms as has beendescribed for Treponema pallidum and other spirochetes.Like for Treponema pallidum in neurosyphilis, atypical andcystic forms of Borrelia burgdorferi were also observedintracellularly in the brains of these patients, as it has pre-Recovery of the typical vegetative form of spirochetes re-cultured in BSK II medium and nuclear fragmentation of rat primary astr cytes exposed to Borr li  burgdorferiFigur  6Recovery of the typical vegetative form of spirochetes re-cultured in BSK II medium and nuclear fragmenta-tion 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 mor-phology of these spirochetes with those seen in Fig. 4H, where virtually all spirochetes showed atypical forms. C: Green fluo-rescent 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.Page 13 of 18(page number not for citation purposes)chetes can be protected from destruction by the hostimmune system.viously been documented [3,51]. These results suggestthat Borrelia burgdorferi, in analogy to Treponema pallidumJournal of Neuroinflammation 2008, 5:40 7 (see legend on next page)Page 14 of 18(page number not for citation purposes)Journal of Neuroinflammation 2008, 5:40 also invade neurons and glial cells and cause cell dys-function and progressive cell death.The results also showed that atypical Borrelia forms maybe present in the absence of typical coiled forms, indicat-ing that detection of atypical forms in infected tissues maybe of diagnostic value. Treponema pallidum and Borreliaburgdorferi can persist in infected tissues, even in theabsence of an apparent lymphoplasmacytic infiltration.Consequently, when the clinical and histopathologic fea-tures suggest syphilis or Lyme disease, the detection ofthese spirochetes in infected body fluids and tissues maybe of diagnostic importance [55,71].Borrelia burgdorferi cultured in harmful conditions and ininfected cell cultures where virtually all spirochetesshowed pleomorphic and cystic forms were resuscitatedunder appropriate conditions in BSK-II medium, whereapparently all spirochetes showed the typical spiral mor-phology. This suggests that these atypical forms may beviable Borrelia forms. Despite that, under the presentexperimental conditions, we cannot exclude whether suchgrowth may represent propagation of some residual spiralforms, previous observations (7, 8) showing that cysticforms of spirochetes can revert into vegetative form sug-gest that at least part of the pleomorphic forms observedmay revert into vegetative form. That Borrelia burgdorferiwas successfully cultivated from brains of the threepatients with Lyme neuroborreliosis in BSK-II mediumwhere pleomorphic and cystic forms were observed in thethese cultivated Borrelia burgdorferi spirochetes in additionto the present Figure 1 E-H was previously illustrated (52,73). Whether the polymorphic forms observed in thebrains of these three patients may correspond to living,degenerating or "dormant" spirochetes remains to bedetermined. Further studies will be necessary to analyzewhether individual Borrelia cysts taken from the affectedbrain may revert into vegetative form.That the ADB1 strain invades neurons and astrocytes invitro indicates that these surviving cultivatable spirochetesare still virulent.The accumulation of immunocompetent HLA-DR posi-tive microglia and reactive astrocytes in the cerebral cortexof these patients clearly indicates the presence of chronicinflammation as previously suggested [51]. Indeed,Treponema pallidum, Borrelia burgdorferi and their lipopro-teins evoke cytokine responses in cells of the monocytes/macrophage lineage as well as initiate complement activa-tion. The response elicited by the major membrane lipo-proteins of Treponema pallidum and Borrelia burgdorferi wasanalogous to that observed with whole bacteria. The veg-etative and cystic forms including the vesicular blubs andfree vesicular structures of Borrelia burgdorferi all containthe biologically active spirochetal surface proteins indicat-ing that they all elicit inflammatory responses includingcomplement activation [reviewed in [73]].The clinical and the pathological hallmarks of Alzheimer'sExtra- and intracellular atypical and cystic forms of spirochetes in the cerebral cortex of a patient with pathologically and sero-logic lly confi med ch onic Lyme neuroborreliosis where Borrel a burgdo feri sensu stricto was cultivated from the brainFi ure 7 (see pr vious page)Extra- and intracellular atypical and cystic forms of spirochetes in the cerebral cortex of a patient with patho-logically 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 follow-ing 1 week Borrelia exposure of primary neurons (compare with Fig. 4G). C: Spirochetes showing loop or ring-shaped forma-tions (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) show-ing 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 immunoreac-tive 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 anti-body. 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.brain [3,51,52,72,73] suggests that at least part of the per- disease, including beta-amyloid deposition are alsoPage 15 of 18(page number not for citation purposes)sisting spirochetes are viable. The typical spiral form of present in the atrophic form of general paresis and in ter-Journal of Neuroinflammation 2008, 5:40 Lyme neuroborreliosis [3,63,72-74]. The facts thatBorrelia burgdorferi spirochetes were cultivated from thebrains of these patients, that Borrelia antigens and geneswere co-localized with beta-amyloid deposits in corticalspirochetal colonies, and that the serology of thesepatients was positive for Borrelia burgdorferi are evidencesthat the present cases correspond to the atrophic paren-chymatous form of late Lyme neuroborreliosis.ConclusionDark field microscopy, histochemical, immunohisto-chemical and atomic force microscopy (AFM) analysesrevealed that pleomorphic and cystic Borrelia forms wereforms were observed in neuronal and astrocytic culturesfollowing 1 week of exposure to Borrelia burgdorferi (B31and ADB1). Identical extra- and intracellular atypical andcystic Borrelia forms were also observed in the brains of allthree patients with Lyme neuroborreliosis, which werealso similar to the atypical forms of Treponema pallidum inthe brains of patients with general paresis. Astrocytesinfected with Borrelia burgdorferi exhibited nuclear frag-mentation.Our results suggest that pleomorphic forms, includingcystic forms of Borrelia burgdorferi may persist in the brainand may explain the long latent stage and persisting infec-Chronic neuroinflammation in the frontal cortex of a patient with Lyme neuroborreliosisFigure 8Chronic 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 reac-tive 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 immunos-taining (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.Page 16 of 18(page number not for citation purposes)induced by the various unfavorable conditions that wereemployed. Extra- and intracellular atypical and cystiction in Lyme neuroborreliosis. The identification of theseextra- or intracellular atypical, cystic and granular forms ofJournal of Neuroinflammation 2008, 5:40 burgdorferi is essential for the histopathologicaldiagnosis of Lyme disease as they may indicate chronicBorrelia infection, even in cases where the typical coiledspirochetes are apparently absent. In analogy toTreponema pallidum, Borrelia burgdorferi can persist in thebrain in Lyme neuroborreliosis and may initiate and sus-tain chronic inflammation and tissue damage.Competing interestsThe authors declare that they have no competing interests.Authors' contributionsJM contributed to the direction of the investigation, datainterpretation and to the writing of the manuscript. SKcontributed to the AFM analysis, ADZ contributed inexperiments of neuronal cell cultures, SMC contributed inthe analysis of syphilitic brains and in organizing theinteraction of several laboratories, as well as in data inter-pretation, SY contributed to the immunohistochemicalanalysis, PLMG contributed to the writing of the manu-script and data interpretation. All authors read andapproved the final manuscript.AcknowledgementsWe 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 unforget-table. We would like to thank all those colleagues and friends who strongly supported this work. We are grateful for J. and L. Krasinsky. Their gener-ous help contributed to the realization of this work. The work was sup-ported 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 Alzhe-imer Foundation, Vancouver, CanadaReferences1. Schaudinn F, Hoffman S: Über Spirochaeta pallida bei Syphilisund die Unterschiede dieser Form gegenuber anderen Artendieser Gattung.  Berlin Klin Wochschr 1905, 42:673-675.2. Burgdorfer W, Barbour AG, Hayes SF, Benach JL, Grunwaldt E, DavisJP: Lyme disease-a tick-borne spirochetosis?  Science 1982,216:1317-1319.3. 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