Assessment of bacterial load and perineuronal nets’ integrity in different brain areas. The semiquantitative assessment was performed for iv=intravascular and ev=extravascular compartments on the following scale:; + very sparse; ++ sparse; +++ moderate; ++++ dense ; +++++ very dense and was validated by quantitative analysis on the occipital cortex. Numerical density of neurons with perineuronal nets decreased with the increase of Bcbva bacterial load in the primary visual cortex. All infected individuals showed a significantly lower PNN density compared to the control case, as demonstrated by an unpaired Student’s t-test, with p<.001 after family-wise-error correction.

Postmortem formalin-fixed brains of all four Bcbva cases. The superficial vessels were congested to different degrees (most apparent in cases 1-3) pointing towards inflammatory meningeal involvement (inserts). For case 2, the photograph taken during brain extraction before fixation also shows focal meningeal haemorrhage (insert).

Chromosomal maximum likelihood SNP phylogeny of Bacillus cereus biovar anthracis (n = 90). The tree is midpoint rooted and internal branches with transfer bootstrap values below 0.7 are plotted in non-bold. Tip points indicate the isolates of case 3 and case 4, which fall within the diversity of previously published isolates. The isolation sources of all sequences are illustrated by a coloured strip and the scale bar indicates the substitutions per site. No evidence of altered (neuro)pathogenicity within the isolated strains was found, suggesting that the study cases are representative for Bcbva’s neuroinvasive capabilities.

Quantitative MRI of chimpanzee brains from four confirmed cases of Bcbva infection (four lower rows) compared to a brain of a control individual (upper row). Three quantitative MRI parameters [magnetisation transfer saturation (MTsat), longitudinal relaxation rate (R1) and effective transverse relaxation rate (R2*)], presented in three columns, are sensitive to the tissue microstructure such as macromolecular and iron content. The signs of heterolytic damage in deep white matter (white arrows) and on the brain surface (yellow arrows) are visible in MTsat and R1 maps. The heterolytic lesions were most prominent in the cases with a higher intraparenchymal bacterial load (case 1 and case 2, see following sections and Table 1), forming large liquid-filled cavities. R2* maps show a hyperintense rim near the brain surface (blue arrows), indicating iron leaked into the tissue, putatively due to meningeal haemorrhage and superficial cortical siderosis, confirmed by Perls’s stain (Fig. 4).

Signs of cortical siderosis caused by Bcbva infection in a representative case 3 revealed by quantitative R2* map (a), and histology using DAB-enhanced Perls’s stain for iron (b). R2* maps show a hyperintense double-banded rim running parallel to the brain surface, indicating iron leaked into the tissue, possibly due to meningeal bleedings causing cortical siderosis, confirmed by Perls’s stain. The characteristic double band sign in the Perls’s stain corresponds with an altered tissue structure in the Nissl stain (c,d). In high magnification the superficial cortical siderosis with dark granules in cortical layer I and II are clearly visible (e).

Intravascular and parenchymal distribution and density of Bcbva. In all cases bacteria were found intravascularly (red arrows) with moderate to high density. Invasion into the brain parenchyma could be confirmed in all cases (green arrows), however, with different densities.a Severe bacterial load was found in case 1, a moderate load in case 2, while cases 3 and 4 were mildly affected.

Histological evidence of Bcbva’s invasion of and effects on the central nervous system (case 1 shown): (a,b) Pia mater adjacent to the frontal cortical surface with leukocytes, indicating meningitis, visualised by H/E stain in (a) and aggregated bacilli (arrows) visualised by Nissl stain (b); (c) Superficial cortical siderosis in the frontal cortex layer I-II with haemosiderin particles and dense conglomerates of bacilli on the cerebral surcface (H/E stain), (d) Blood vessels with Bcbva infiltration into the parenchyma through a broken blood brain barrier (Nissl), (e) No morphological signs of microglia activation was detected (rabbit anti-Iba1 antibody, DAB, Nissl), (f) Also, astrocytes were regularly distributed and appeared morphologically unaltered (rabbit anti-GFAP, DAB, Nissl), (g,h) However, enhanced glial fibrillary acidic protein (GFAP) immunoreactivity was apparent in the glia limitans in the outer cortical layers and close to walls of penetrating blood vessels, revealing early stages of astroglial activation (rabbit anti-Iba1 antibody, DAB, Nissl) (i); MPO+ leucocytes were mostly detected inside vessels and rarely entered the parenchyma (arrows) (rabbit anti-MPO antibody, Nissl).

Laminar distribution of the major extracellular matrix (ECM) protein aggrecan (acan) in the occipital lobe (chimpanzee analogue of Brodmann Area 17, primary visual cortex V1) of Bcbva infected cases and one control. (a) Acan as an established marker for perineuronal nets indicated many perineuronal nets (PNNs) in layer III, IVa and V. Fewer PNNs were seen in layer VI, whereas layer I, II and IVc are virtually devoid of acan positive PNNs. (a, a’) This pattern reveals the expected distribution of PNNs in a healthy hominoid brain with regularly structured PNNs. (b,c,d,e) A severe reduction of PNNs is visible in severely affected cases 1 and 2. Less reduction was observed for Cases 3 and 4 with lower bacteria load. Scale bars (a)-(e): 200µm, (a’)-(e’): 20µm.

Case descriptions of four chimpanzees with confirmed infection of Bcvba and one control case. PMI - post mortem interval before fixation. F=female, M=Male. *Age estimate.

qPCR primers for Bcbva detection.

Primary antibodies used for staining of specific compartments.

Superficial cortical siderosis and iron-rich double-banded rim was observed in three out of four cases. Superficial cortical siderosis with dark granules in cortical layers I and II is present in cases 1, 3 and 4 (a, c, d), but not in most regions of case 2 (b). The iron-rich double-banded rim visible in R2* maps also appears in the Perls’s stain in cases 1, 3 and 4 (e, g, h). Case 2 reveals an overall low iron level due to the young age of the specimen and does not show the rim in both R2* maps and Perls’s stain (f).

Visualisation of Bcbva in case 1 with (a) Nissl (cresyl violet) stain, (b) haematoxylin/eosin stain and (c) validation by immunohistochemistry with rabbit anti-B. anthracis protein (whole cell protein). Characteristic elongated chains of the pathogen were visible in H/E stain and Nissl stain and corresponded well in length, thickness and distribution to the gold standard immunohistochemical stain.

Distribution patterns and density of Bcbva in the most severely affected case 1. Bcbva chains were detected across the entire brain: (a) frontal cortex, layer I-III (b) frontal cortex, layer V (c) frontal lobe, white matter (d) temporal cortex, layer I-III (e) temporal cortex, layer V (f) temporal lobe, white matter (g) occipital lobe, V1, layer I-III (h) occipital lobe, V1, layer IVa, IVb (i) occipital lobe, white matter (k) cerebellum, molecular layer (l) cerebellum, granular layer (m) cerebellum, arbor vitae (n) striatum (o) brainstem, inferior olivary nucleus (p) brainstem, pons.

Glial reaction and MPO+ leukocyte invasion for all cases. Enhanced GFAP immunoreactivity is present in all cases in the glia limitans on the brain surface as well as around penetrating blood vessels (a - d; rabbit anti-GFAP, Ni-DAB). White matter astrocytes are regularly distributed and do not reveal any morphological changes, indicating no signs of activation (e - h; rabbit anti-GFAP, Ni-DAB). Microglia appears regularly shaped and distributed (i - m; rabbit anti-Iba1 antibody, DAB). MPO+ leucocytes were found in blood vessels and rarely infiltrated in the brain tissue (n - q; rabbit anti-MPO antibody, Nissl; MPO+ leukocytes outside vessels are indicated by arrows).