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. 2020 Mar 12;10:4554. doi: 10.1038/s41598-020-61483-4

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Experimental Design. (A,B) Mice were orally infected with a low dose of T. muris infective eggs (~20/mouse) and serum collected 35 d later. (A) Comparison of IFN-γ concentrations in the serum of T. muris infected mice and uninfected (uninf.) control mice. Each point represents data from an individual mouse (n = 5–6 mice/group). **P < 0.01, Student’s t-test. Horizontal bars, median. (B) Analysis of T. muris E/S antigen-specific IgG1 (closed circles) and IgG2c (closed squares) levels in the sera of T. muris infected mice. Each point represents the mean OD 405 nm ± SEM, n = 3 mice/group. (C) Experiment design. Cartoon shows the relative timings of the individual oral T. muris infections in relation to the ongoing prion infection in the CNS. Dpi, d post IC prion infection; green arrows, d on which mice were orally infected with a single low dose of T. muris. The green triangles represent the relative magnitude of the T. muris-specific immune response, with the peak response occurring by ~35 d and gradually declining afterwards. (D,E) Mice were injected IC with ME7 scrapie prions directly into the CNS and 84, 112 and 140 d later brains were collected and the neuropathology compared by histopathology. (D) The severity of the spongiform pathology (vacuolation) within each brain was scored on a scale of 1–5 in nine grey matter and three white matter areas: G1, dorsal medulla; G2, cerebellar cortex; G3, superior colliculus; G4, hypothalamus; G5, thalamus; G6, hippocampus; G7, septum; G8, retrosplenial and adjacent motor cortex; G9, cingulate and adjacent motor cortex; W1, inferior and middle cerebellar peduncles; W2, decussation of superior cerebellar peduncles; and W3, cerebellar peduncles. Each point represents the mean vacuolation score ± SEM, n = 4 mice/group. Scores from mice with terminal ME7 prion disease are included for comparison. (E) Histopathological comparison of the spongiform pathology (H&E, upper row), PrP^d accumulation (brown, second row), reactive astrocytes expressing GFAP (brown, third row) and active microglia expressing AIF-1 (brown, bottom row) in the brains of mice at times indicated after IC injection with prions. Haematoxylin was used as a nuclear counterstain (blue). n = 4 mice/group.