Author Contributions
Designed experiments: R.S.K., H.I., A.M., J.E.G., and E.S. Performed experiments: R.S.K., H.I., J.P.T., M.O., G.G., and A.M. Analyzed the data: A.M., S.M., K.B.L., J.E.G., and E.S. Pathologic analysis: J.L. and J.E.G. Contributed reagents/materials/analysis tools: J.P.T., M.O., S.M., G.G., and A.M. Wrote and edited the manuscript: K.B.L., K.E., J.L., J.E.G., and E.S.
Figure 1.
Representative pathological images of CNS. (a) A section through the frontal leptomeninges shows meningeal thickening and chronic inflammatory infiltrates. There is an infiltrate surrounding a penetrating vessel (arrow). (b) A leptomeningeal vessel that has been occluded, showing small zones of recanalization. (c) Deep frontal white matter contains infarcts, one illustrated here (*). (d) A blood vessel in the frontal deep white matter shows a mixture of lymphocytes and plasma cells. (e) An infarct in the frontal cortex (*) near a blood vessel with a chronic inflammatory infiltrate (arrow). All sections stained with H&E. Scale bars: (a) 100 μm, (b) 25 μm, (c) 100 μm, (d) 25 μm, and (e) 200 μm.
Figure 1.
Representative pathological images of CNS. (a) A section through the frontal leptomeninges shows meningeal thickening and chronic inflammatory infiltrates. There is an infiltrate surrounding a penetrating vessel (arrow). (b) A leptomeningeal vessel that has been occluded, showing small zones of recanalization. (c) Deep frontal white matter contains infarcts, one illustrated here (*). (d) A blood vessel in the frontal deep white matter shows a mixture of lymphocytes and plasma cells. (e) An infarct in the frontal cortex (*) near a blood vessel with a chronic inflammatory infiltrate (arrow). All sections stained with H&E. Scale bars: (a) 100 μm, (b) 25 μm, (c) 100 μm, (d) 25 μm, and (e) 200 μm.
Figure 2.
Representative pathological images of CNS and PNS. (a) A section of frontal cortex and leptomeninges (L) shows thickened, inflamed meninges and adjacent cortex with infarcts (*). (b) A large perivascular chronic inflammatory infiltrate in the putamen (arrow). (c) An infarct in the cerebellar white matter and cortex (*). There is a vessel with a surrounding chronic inflammatory infiltrate at the left edge of the infarct. (d) A Bielschowsky silver stain of an entire transverse section of the spinal cord shows severe, unilateral corticospinal tract degeneration, both lateral (**) and anterior (*) tracts (Bielshowsky silver stain). (e) A section of a cranial nerve shows a chronic inflammatory infiltrate associated with small vessels in the perineurium. The degree of myelination of surrounding axons appears normal. Autopsy tissues are stained with standard H&E histological stains. Scale bars: (a‒c) 200 μm, (e) 25 μm.
Figure 2.
Representative pathological images of CNS and PNS. (a) A section of frontal cortex and leptomeninges (L) shows thickened, inflamed meninges and adjacent cortex with infarcts (*). (b) A large perivascular chronic inflammatory infiltrate in the putamen (arrow). (c) An infarct in the cerebellar white matter and cortex (*). There is a vessel with a surrounding chronic inflammatory infiltrate at the left edge of the infarct. (d) A Bielschowsky silver stain of an entire transverse section of the spinal cord shows severe, unilateral corticospinal tract degeneration, both lateral (**) and anterior (*) tracts (Bielshowsky silver stain). (e) A section of a cranial nerve shows a chronic inflammatory infiltrate associated with small vessels in the perineurium. The degree of myelination of surrounding axons appears normal. Autopsy tissues are stained with standard H&E histological stains. Scale bars: (a‒c) 200 μm, (e) 25 μm.
Figure 3.
Representative pathological images of the kidney. All sections stained with standard H&E histological stains. (a) A section of the left ventricle with extensive fibrous scarring (*). Scale bar: 200 μm. (b) A section of kidney shows inflammatory infiltrates (*) scattered among glomeruli and tubules and areas of fibrous scarring (**). Scale bar: 100 μm. (c) A section of liver with inflammation in the portal zone (*). Scale bar: 100 μm.
Figure 3.
Representative pathological images of the kidney. All sections stained with standard H&E histological stains. (a) A section of the left ventricle with extensive fibrous scarring (*). Scale bar: 200 μm. (b) A section of kidney shows inflammatory infiltrates (*) scattered among glomeruli and tubules and areas of fibrous scarring (**). Scale bar: 100 μm. (c) A section of liver with inflammation in the portal zone (*). Scale bar: 100 μm.
Figure 4.
Representative images of CD3 staining in CNS and kidney tissue section. (a) A brain section immunostained for T cells (CD3) with a perivascular infiltrate and scattered T cells in the parenchyma. (b) A kidney section immunostained for T cells (CD3) shows a T cell aggregate and scattered T cells in the tissue. Scale bars: 100 μm.
Figure 4.
Representative images of CD3 staining in CNS and kidney tissue section. (a) A brain section immunostained for T cells (CD3) with a perivascular infiltrate and scattered T cells in the parenchyma. (b) A kidney section immunostained for T cells (CD3) shows a T cell aggregate and scattered T cells in the tissue. Scale bars: 100 μm.
Figure 5.
Representative images of IHC staining of autopsy tissues (heart, kidney, liver, brain) with Borrelia- and alginate-specific antibodies. Aggregates stained positive with Borrelia- (green staining: A,E,I,M) and alginate-specific antibodies (red staining: B,F,J,N) respectively. A nonspecific IgG antibody was used as an additional negative control for the primary antibodies using the sequential tissue section (C,G,K,O). Differential interference microscopy (DIC) showing the size and tissue morphology (D,H,L,P). Scale bar: 200 μm.
Figure 5.
Representative images of IHC staining of autopsy tissues (heart, kidney, liver, brain) with Borrelia- and alginate-specific antibodies. Aggregates stained positive with Borrelia- (green staining: A,E,I,M) and alginate-specific antibodies (red staining: B,F,J,N) respectively. A nonspecific IgG antibody was used as an additional negative control for the primary antibodies using the sequential tissue section (C,G,K,O). Differential interference microscopy (DIC) showing the size and tissue morphology (D,H,L,P). Scale bar: 200 μm.
Figure 6.
A representative three-dimensional (3D) confocal image of a Borrelia spirochete and Borrelia biofilm-positive heart autopsy tissue. Heart autopsy tissue section was immunostained with antibodies for Borrelia (green) and alginate (blue) and analyzed by confocal microscopy using individual z-stacks to obtain a composite 3D view (Image J) of the spatial distribution of Borrelia spirochetes (green arrows) and Borrelia aggregates with alginate on the surface (blue arrow) in the infected heart tissue. Scale bars: 20 μm.
Figure 6.
A representative three-dimensional (3D) confocal image of a Borrelia spirochete and Borrelia biofilm-positive heart autopsy tissue. Heart autopsy tissue section was immunostained with antibodies for Borrelia (green) and alginate (blue) and analyzed by confocal microscopy using individual z-stacks to obtain a composite 3D view (Image J) of the spatial distribution of Borrelia spirochetes (green arrows) and Borrelia aggregates with alginate on the surface (blue arrow) in the infected heart tissue. Scale bars: 20 μm.
Figure 7.
Three-dimensional (3D) analyses of a Borrelia biofilm in infected liver tissue. (A,B) Fluorescent microscopy images of a liver tissue section positively immunostained with antibodies against Borrelia (green) and alginate (blue) antigens. Scale bar: 100 µm. Confocal microscopy analyses of the same tissues section were performed using individual z-stacks to form a composite 3D image (Image J) to illustrate the spatial distribution of Borrelia biofilm (C) and Borrelia biofilm with surface alginate (D). Scale bar: 100 µm.
Figure 7.
Three-dimensional (3D) analyses of a Borrelia biofilm in infected liver tissue. (A,B) Fluorescent microscopy images of a liver tissue section positively immunostained with antibodies against Borrelia (green) and alginate (blue) antigens. Scale bar: 100 µm. Confocal microscopy analyses of the same tissues section were performed using individual z-stacks to form a composite 3D image (Image J) to illustrate the spatial distribution of Borrelia biofilm (C) and Borrelia biofilm with surface alginate (D). Scale bar: 100 µm.
Figure 8.
Representative images of presence of Borrelia biofilm DNA using fluorescent in situ hybridization (FISH) combined with alginate IHC in brain, heart, kidney, and liver autopsy tissues. The FISH results showed positive staining for Borrelia DNA in all four organs (A,G,M,S; green arrow) using Borrelia-specific 16S rDNA probes. The Borrelia-positive aggregates, but not the spirochetes (G: small green arrowhead), are also stained with alginate antibody (B,H,N,T; blue staining). Differential interference contrast (DIC) microscopy (C,I,O,U) was used to visualize the morphology of the biofilm structure and the surrounding tissue. For negative controls of all FISH experiments, competing oligonucleotide probes (D,J,P,V), DNase I-treated samples (E,K,Q,Y), and a random oligonucleotide probe (F,L,R,Z), were used to test the specificity of the 16S rDNA probes. Scale bars: 200 μm.
Figure 8.
Representative images of presence of Borrelia biofilm DNA using fluorescent in situ hybridization (FISH) combined with alginate IHC in brain, heart, kidney, and liver autopsy tissues. The FISH results showed positive staining for Borrelia DNA in all four organs (A,G,M,S; green arrow) using Borrelia-specific 16S rDNA probes. The Borrelia-positive aggregates, but not the spirochetes (G: small green arrowhead), are also stained with alginate antibody (B,H,N,T; blue staining). Differential interference contrast (DIC) microscopy (C,I,O,U) was used to visualize the morphology of the biofilm structure and the surrounding tissue. For negative controls of all FISH experiments, competing oligonucleotide probes (D,J,P,V), DNase I-treated samples (E,K,Q,Y), and a random oligonucleotide probe (F,L,R,Z), were used to test the specificity of the 16S rDNA probes. Scale bars: 200 μm.
Figure 9.
Representative IHC images of Borrelia, alginate, and CD3+ T lymphocytes staining in infected brain, heart, kidney, and liver autopsy tissue sections. Tissues sections that had positive staining for Borrelia (green staining: A,E,I,M) and alginate (red staining: B,F,J,N) were subjected to additional IHC analyses by immunostaining the sequential sections with a T cell marker, CD3-specific antibody (brown staining: D,H,L,P). Fluorescent images were taken at 100x magnification to illustrate a larger section of the tissue. CD3-positive lymphocytes surrounded these aggregates, as depicted with brown staining in the brain, kidney, and liver tissues (D,L,P). There was no presence of CD3-positive lymphocytes in the heart tissues (H). Nonspecific IgG antibody was used as a negative control for the primary antibodies (C,G,K,O). Scale bar: 100 μm.
Figure 9.
Representative IHC images of Borrelia, alginate, and CD3+ T lymphocytes staining in infected brain, heart, kidney, and liver autopsy tissue sections. Tissues sections that had positive staining for Borrelia (green staining: A,E,I,M) and alginate (red staining: B,F,J,N) were subjected to additional IHC analyses by immunostaining the sequential sections with a T cell marker, CD3-specific antibody (brown staining: D,H,L,P). Fluorescent images were taken at 100x magnification to illustrate a larger section of the tissue. CD3-positive lymphocytes surrounded these aggregates, as depicted with brown staining in the brain, kidney, and liver tissues (D,L,P). There was no presence of CD3-positive lymphocytes in the heart tissues (H). Nonspecific IgG antibody was used as a negative control for the primary antibodies (C,G,K,O). Scale bar: 100 μm.
Table 1.
Quantitative analysis of the IHC experiments on Borrelia biofilms in brain, heart, kidney, and liver tissues. SD = standard deviation.
Table 1.
Quantitative analysis of the IHC experiments on Borrelia biofilms in brain, heart, kidney, and liver tissues. SD = standard deviation.
Organ | Number of IHC Stained Slides | Number of Biofilms per Slide ± SD | Size of the Biofilm (µm) |
---|
Brain | 250 | 0–4 ± 1.2 | 20–150 |
Heart | 155 | 0–6 ± 1.5 | 20–100 |
Kidney | 165 | 0–4 ± 1.1 | 20–200 |
Liver | 180 | 0–7 ± 1.6 | 20–300 |
Table 2.
Quantitative analysis of the FISH experiments on Borrelia biofilms in brain, heart, kidney, and liver tissues.
Table 2.
Quantitative analysis of the FISH experiments on Borrelia biofilms in brain, heart, kidney, and liver tissues.
Organ | Number of IHC Stained Slides | Number of Biofilms per Slide ± SD | Size of the Biofilm (µm) |
---|
Brain | 210 | 0–3 ± 1.1 | 20–150 |
Heart | 130 | 0–4 ± 1.2 | 20–100 |
Kidney | 145 | 0–4 ± 1.1 | 20–100 |
Liver | 150 | 0–6 ± 1.5 | 20–300 |
Table 3.
The actual sequences, the gene/genomic regions, and the percentage of coverage/identity with E values. There were an additional 20 sequencing reads out of 517 reads which were matches for other Borrelia strains with >90% identity and coverage, but they also had similar identities to other bacterial species (data not shown). For the control liver, eight sequences came through the metagenomics analyses pipeline for B. burgdorferi, but they did not match to any Borrelia species.
Table 3.
The actual sequences, the gene/genomic regions, and the percentage of coverage/identity with E values. There were an additional 20 sequencing reads out of 517 reads which were matches for other Borrelia strains with >90% identity and coverage, but they also had similar identities to other bacterial species (data not shown). For the control liver, eight sequences came through the metagenomics analyses pipeline for B. burgdorferi, but they did not match to any Borrelia species.
Whole Genome Sequencing Reads For Borrelia Burgdorferi Sensu Stricto Strains | Gene/Region | Coverage | Identity | E value |
---|
AGCTTTGCTATCTCAAATGTCAAAGACTCTATCTCTTCTTGAGAAAGATACTTAAACACTTTAGAAGAGATTTCAGAACCTATTGAAACCAACAAAATAG | Flagella switch protein (fliG) | 100% | 100% | 3e−42 |
AAAACTATTAAAATTACCCTTAACAATTGCAATGTAAACTTTATTTGTTCTTTTATCTTTAAACTGCTGAGCTAAAAATCTTAAGGTGCTAATGTTTTTT | Helicase protein(Yfi) | 100% | 99% | 1e−40 |
AAGGTCTTATGCCAATAAAAATCCAATCACAGAATACAAAGAAGAGGGATTTTCAATATTTAGCGAGCTTATTAAAGATATTAAAGTTTCTACCATAAGG | SecA protein | 100% | 100% | 3e−42 |
AAGAAAAGATTTTCCTATTTTAAATAAAAAATTTGACAATAAGTATATAATTTACTTTGATAATGCAGCAACCTCTCAAAAGCCCAAAAACGTAATTTAT | M11p aminotransferase (nifS) | 100% | 100% | 3e−35 |
ATTACAGCGTTACTGTTTTAATGAAGCAATTGCCATACTATCAAAACCAATTAGCATTTATCATGAAAGATGTGCTTAGTCGATATAAAGTTGATAGTTC | Left subtelomeric chromosomal region | 100% | 100% | 3e−42 |
TCATTTCAAAAACATGTATTTCTGAAAGCAAAAAATACAACAGCAAAAAAACTACTACCAAACTGCTTGTAAATCCAATAATTTCATTATAAGCTCTTGT | Left subtelomeric chromosomal region | 100% | 100% | 3e−42 |
TTGAATATTTTGAAATAACTTATGAGGCTTATGCTCCTTATGGAGTGGCTCTAATGATTAAATGCTTAACGGATAATAAAAACAGAACCTCTAGCGATGT | Intergenic region | 100% | 100% | 3e−42 |
AAGAAGAATTAGAAGTTTGCGAGCTAAATGGAAAAGATTGGACATTAAAATTTAAAAAACCGCTAAAAGCATATAAATTCTTAAAATCCGTAGGAAG | Intergenic region | 99% | 100% | 1e−40 |
TTACTAAAACTTCAGAAGAGCCCCTAATGCTTGTTTTAATGATAGGCATTATTTCTTTGGCCTGTTGATAGTCTATGTTTGTGTATGTATTGTTATTCAT | Intergenic region | 100% | 99% | 1e−40 |
AATCTTAAAATTAAAAGATAACGACAAATTTAAATTTGGTATTCTTGGAGAAAAAAACATTTACCACTGCATTTACAAAAAAGATAAAAAACTATTTTTC | Intergenic region | 100% | 100% | 3e−42 |
TAAGTTATAATTGAGGAATAATAGCAAATATTTTAACTTTTTGGTATAAATTACTACTAGATTTATATGTTAAGTTTTGCGAGGTATTTAAATGGCAGTA | Intergenic region | 100% | 99% | 1e−40 |
CAAGAGTTAGTATTGGCCTTAAAAAACGATAAAGTTGATTATATATATGGTGATTGCAAGACTTTACATTATATTGCAAATAACTTTTTAAGTGA | Intergenic region | 100% | 100% | 1e−39 |
CTTGAGGGATTTAAAGAAGTTAAGCCTGTAGTATTCTCTTCAGTTTATCCGTTGATGCTAATCAATATGATGATCTTTTAAGGGCAATGGATAGATTAA | Intergenic region | 100% | 99% | 1e−40 |
TTTATACTAATAAACTTTCAATTTCTTTTGTGAAGATATTGAAAGAAATCCATGTCTGTTGAGAAAATTTTTCTTTTATCTTTTAATACTGCTTTATAGC | Intergenic region | 100% | 100% | 3e−42 |