A lymphofollicular microenvironment is required for pathological prion protein deposition in chronically inflamed tissues from scrapie-affected sheep

In sheep scrapie, pathological prion protein (PrPSc) deposition occurs in the lymphoreticular and central nervous systems. We investigated PrPSc distribution in scrapie- affected sheep showing simultaneous evidence of chronic lymphofollicular, lymphoproliferative/non-lymphofollicular,and/or granulomatous inflammations in their mammary gland, lung, and ileum. To do this, PrPSc detection was carried out via immunohistochemistry and Western Blotting techniques, as well as through inflammatory cell immunophenotyping. Expression studies of gene coding for biological factors modulating the host’s inflammatory response were also carried out. We demonstrated that ectopic PrPSc deposition occurs exclusively in the context of lymphofollicular inflammatory sites, inside newly formed and well-organized lymphoid follicles harboring follicular dendritic cells. On the contrary, no PrPSc deposition was detected in granulomas, even when they were closely located to newly formed lymphoid follicles. A significantly more consistent expression of lymphotoxin α and β mRNA was detected in lymphofollicular inflammation compared to the other two types, with lymphotoxin α and β signaling new lymphoid follicles’ formation and, likely, the occurrence of ectopic PrPSc deposition inside them. Our findings suggest that, in sheep co-affected by scrapie and chronic inflammatory conditions, only newly formed lymphoid follicles provide a suitable micro-environment that supports the scrapie agent’s replication in inflammatory sites, with an increased risk of prion shedding through body secretions/excretions.[...

Immunohistochemistry, Immunofluorescence and Confocal Laser Microscopy: an Insight into the Enteric Nervous System During Sheep Infection.

P03.133 Immunohistochemistry, Immunofluorescence and Confocal Laser Microscopy: An Insight into the Enteric Nervous System During Sheep Scrapie Infection Di Guardo, G1; Marruchella, G1; Ligios, C2; Baffoni, M1; Cancedda, GM2; Macciocu, S2; Gioia, L1; Lalatta Costerbosa, G3; Chiocchetti, R3; Clavenzani, P3; De Grossi, L4; Agrimi, U5; Aguzzi, A6 1Faculty of Veterinary Medicine, University of Teramo, Dept Comparative Biomedical Sciences, Italy; 2Istituto Zooprofilattico Sperimentale della Sardegna, Italy; 3Faculty of Veterinary Medicine, University of Bologna, Dept Vet Morphophysiology and Animal Productions, Italy; 4Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana, Italy; 5Istituto Superiore di Sanit\ue0, Dept Food Safety and Veterinary Public Health, Italy; 6University Hospital of Zurich, Institute of Neuropathology, Switzerland The enteric nervous system (ENS) is likely to play a role in the early pathogenesis of sheep scrapie, but little is known about the ENS cell types involved. We investigated the ileal myenteric plexi (MPs) and submucosal plexi (SMPs) of 4 natural and 4 oral experimental scrapie-affected ARQ/ARQ Sarda breed sheep, as well as of 12 healthy sheep carrying different PRNP genotypes. In all control animals, as well as in all scrapie-affected sheep, which were euthanized at the end stage of clinical disease, detailed laboratory investigations were carried out by means of immunohistochemistry (IHC), as well as by indirect immunofluorescence (IIF) and confocal laser microscopy (CLM) techniques. All the above 8 scrapie-affected animals showed IHC evidence of PrPSc deposition within both MPs and SMPs, with IIF and CLM studies allowing us to identify enteroglial cells (EGCs) and, for the first time, also calbindin (CALB)-immunoreactive (IR) and neuronal nitric oxide synthase (nNOS)-IR neurons as the ENS cytotypes involved in PrPSc accumulation and plausibly, thereby, in the subsequent process of \u201cneuroinvasion\u201d. In conclusion, IHC, IIF and CLM proved to be in our hands three highly valuable and complementary laboratory techniques for investigating the