Neuroinfectiology of an atypical anthrax-causing pathogen in wild chimpanzees

Reviewer #1 (Public Review):

Summary:

Gräßle et al. provide a series of four post-mortem cases of chimpanzees with PCR-proven Bacillus cereus biovar anthracis (Bcbva), who reportedly died of this infection. One control case is also provided. Compelling post-mortem Magnetic resonance imaging scans of the highest technical standards are presented. Last, the authors provide some histopathology of the brains aiming at showing the neuroinfective potential of Bcbva.

Strengths:

The merits of this study are highly acknowledged. This reviewer deems it very important to implement the latest methodology in such veterinary observational studies, in order to investigate what is going on in wildlife regarding zoonoses. The scans of five whole post-mortem chimpanzee brains with exquisite MRI technology (extremely good scan quality) represent such an implementation.

Weaknesses:

The conclusions from the necropsies are, unfortunately, on weak grounds:

(1) The authors claim that all 4 infected individuals have suffered from meningitis. However, I do not see evidence for that, neither in the gross macroscopical images provided in Figure 1. The authors claim congestion of superficial veins, at least in cases 1-3, and interpret this as pointing towards meningitis. I do not see major superficial vein congestion in any of the cases. Furthermore, vessel congestion here would rather indicate brain swelling and subsequent inhibition of venous blood outflow from the skull, which would relate to brain edema. Bacterial meningitis would itself display as clouding of the meninges, while the meninges presented in all 4 cases are perfectly translucent and gracile.

(2) The authors show a bacterial overgrowth, of brains, which was most severe in cases 1 and 2, less so in case 3, and least in case 4 (Table 1). This correlates very well with post-mortem intervals (Supplementary Table 1). The amount of bacteria is remarkable, while there is practically no brain inflammation, only moderate microglia activation. Also, the authors do not convincingly prove the proposed meningitis at the histological level, since Figure 6 does not show it in a convincing manner. Also, moderate superficial gliosis shown in Figure 6 g+h is for me not evidence of meningitis. I would expect masses of granulocytes and lymphocytes, given the amount of bacteria shown.

(3) The pattern of bacterial invasion, i.e. first confined to vessels as in case 4 with short post-mortem interval, and then overgrowing the brain with practically no glial or inflammatory reaction, is very typical of post-mortem putrefication. It is conceivable that the chimpanzees had severe bacteremia, which, after death, quickly led to bacterial invasion into the brain parenchyma. While authors state the post-mortem intervals in hours, they do not state whether bodies were immediately cooled after death.

(4) I find it difficult to see evidence of superficial siderosis in any of the images. In particular, case 2 in Figure 1 does not convincingly display leptomeningeal hemorrhage. Dark granules, e.g. shown in Figure 4 e, are very typical of so-called formalin pigment. If that would be hemosiderin or some other form of iron, it would be expected that it displays much stronger in the DAB-enhanced perls stain (Figure 4 c).

Reviewer #2 (Public Review):

In "Neuroinfectiology of an atypical anthrax-causing pathogen in wild chimpanzees" Tobias et.al. provide a detailed histologic characterization of B.cereus biovar anthracis in the brain of four wild chimpanzees in comparison to an uninfected age-matched chimp. The authors present a combination of special stains, radiography (MRI), bacterial culture, and immunohistochemistry including some quantitative image analysis to support the assessment of the neuropathogenicity of Bcbva. However, the study has major limitations that detract from the conclusions presented regarding the neurovirulence of this strain. Namely, there is a near complete lack of traditional histopathological and radiographic interpretation by qualified experts in which to frame the detailed tissue studies. The authors mention that facultative anaerobes are capable of post-mortem replication. Pathologists use comprehensive pathological assessments to determine the extent of disease caused by the primary infection, none of which is mentioned in this study (spleen, heart, lungs), which makes it difficult to determine if the findings in the brain align with the rest of the post-mortem assessment. If these were not included due to severe post-mortem autolysis, it heightens the risk of post-mortem bacterial replication in the CNS. The most important limitation is the fact that the meninges were removed and were not available for assessment therefore any comparisons with existing data on neuropathogenicity of B. anthracis is not possible. An advantage of the study is the inclusion of the control age-matched chimp, but the controls are not shown for many of the IHC and special stains - limiting interpretations. In general, the article is difficult to follow with the figures since many panels are only discussed and interpreted in the figure legends and not the text. In some cases, the results are overly technical with limited clinical insight which makes the article less easy to interpret next to human clinical reports.

Author response:

We are thankful to the expert reviewers and the editorial team for their assessment of our manuscript and valuable comments, which will help us to improve our manuscript. While Reviewer #1 appreciated the comprehensive assessment using advanced methods, Reviewer #2 asked for an extension of traditional neuropathological and neuroradiological assessments. Both reviewers identified limitations of the study like the inability to provide direct histopathological evidence for meningitis due to missing meninges tissue, resulting in the conclusions being based on indirect evidence. The reviewers raised concerns about potential post mortem penetration of bacteria into the brain parenchyma. Reviewer #1 also questioned the evidence for cortical siderosis based on the intensity of histological stains.

We agree with both reviewers and the editorial comment that a traditional neuropathological assessment of meningeal status would have strongly boosted the study's conclusions. Please note that the opportunistic sampling approach after a wild animal’s “natural” death, which is the only ethical method to study infection biology in great apes, is intrinsically accompanied by some limitations such as the lack of standardized post mortem intervals or incomplete sampling. In the revised version of the manuscript, we will complement the advanced MRI and histology already presented by extended traditional neuroradiological and neuropathological assessments as recommended by Reviewer #2, including a report on the status of other organs. However, it is important to note that the interpretation of post mortem MRI of brain material collected in the field differs substantially from conventional in vivo MRI and requires tailored analysis and interpretation. Below we comment on three aspects addressed by reviewers:

* Missing meninges *: The meninges and associated vessels had to be removed to reduce blood-related artifacts in previously performed MRI measurements. We are aware that this poses a major limitation of this study, and thus rely on the evidence derived from the material at hand. Neuropathological assessment is in agreement with the reviewer's comments that no overt acute bacterial meningitis with e.g. turbid appearance, purulent exudates or frank hemorrhages is apparent in the macroscopic inspection of the presented material. However, the macroscopic changes should be evaluated in the light of the brief time interval between bacterial colonization and death. Meningeal bacterial invasion was visualized on a few meningeal residues we found in case 1, proofing the invasion of the subarachnoid space. Based on the reviewer's suggestions, the microscopic neuropathological evaluation will be expanded with the aim to identify further regions with meningeal residues to include more regions to 1) reduce potential sampling bias and 2) to better characterize the leptomeningeal infiltrates focusing on early inflammatory markers.
However, an extensive assessment of the histopathological inflammatory status must be clarified in future studies on specimens with remaining meninges.

*Putrefaction/Post mortem bacterial proliferation*:
Reviewers raised important points by remarking that the tissue alterations could be due to putrefaction/post mortem effects. Classical bacterial putrefaction is unlikely, since no mixed flora of opportunistic bacteria was detected, suggesting that time before fixation was sufficient to prevent secondary bacterial invasion in the presented specimens. Moreover, it has been shown that for the post mortem interval of <24 hours bacterial invasion of the brain is rare even at higher temperatures (Ith et al 2011, https://doi.org/10.1002/nbm.1623). The possibility of post mortem tissue propagation of Bcbva must be considered, since there is a lack of experimental data on the pathogen’s growth after host death, which has been discussed by us in the "Limitations" section in the original manuscript. Although it seems plausible that post mortem multiplication in the brain does occur to a certain extent, several observations suggest that this is not the only mechanism at play in the presented cases. We observed early microglial activation and astrogliosis indicating a beginning inflammatory reaction in the brain parenchyma. Taken together, the data presented suggest a short time interval between bacterial colonization and death. Under this premise, further analyses for the revision of the manuscript will more closely investigate pathological in vivo tissue alterations.

*Siderosis* Signs of cortical siderosis were evident in the MRI images of all adult cases (1, 3, and 4), appearing as a hyperintense rim in quantitative R2* maps, indicating substantially elevated levels of iron on the brain surface. These findings were confirmed by Perls’s stain for iron. Such rims in R2* are a typical sign of cortical iron deposition due to siderosis, as observed in conditions like angiopathies. Meningeal bleedings are the most probable source of the elevated iron levels in the cortex. Importantly, such signs were never observed in the post mortem brains of chimpanzees not infected with Anthrax (about 30 cases analyzed so far). Reviewer #1 noted that the intensity of the Perls’s stain seemed too low for siderosis. However, this intensity can vary depending on staining procedure and may be lower for the acute and short disease course of Bcbva-induced Anthrax compared to the chronic human cases Reviewer #1 may be referring to. Taken together, we believe that the evidence of cortical siderosis is compelling, speaking in favor of pre mortem meningeal hemorrhage.

In summary, in the revised version of the manuscript, we plan to: (1) add a traditional neuroradiological assessment of all scans; (2) present an extended traditional neuropathological assessment of all cases; (3) report results on the status of early inflammatory markers; and (4) discuss the limitations of the study in more detail.