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. 2008 Sep 25;5:40.
doi: 10.1186/1742-2094-5-40.

Persisting Atypical and Cystic Forms of Borrelia Burgdorferi and Local Inflammation in Lyme Neuroborreliosis

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Persisting Atypical and Cystic Forms of Borrelia Burgdorferi and Local Inflammation in Lyme Neuroborreliosis

Judith Miklossy et al. J Neuroinflammation. .
Free PMC article

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.

Figures

Figure 1
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.
Figure 2
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 3
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.
Figure 4
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
Figure 5
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.
Figure 6
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 7
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.
Figure 8
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.

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