TSE Transmission to Sea Bream Figure 1. Normal PrPC distribution in CNS and peripheral tissues. Sagittal, 4 mm-thick brain and intestine sections from control fish were treated with SaurPrP1 (1:2000 and 1:250, respectively) and normal endogenous PrP labeling in different anatomical regions was examined. A, Optic tectum; B, Valvula cerebelli; C, Corpus cerebelli; D, Nerve fibers in diencephalon; E, Neurons in brainstem; F, PrP-immunoreactive areas in the intestine. Rectangles indicate areas of magnification shown in the panel directly below. Arrowheads show positively stained regions. SGC, striatum griseum centrale; SPFE, striatum plexiforme et fibrosum externum; SFM, striatum fibrosum marginale; ML, molecular layer; GL, granular layer, m, plasma membrane; a, axon; c, cytoplasm. se, serosa; mp, myenteric plexus; sp, submucous plexus. Scale bars, 100 mm. doi:10.1371/journal.pone.0006175.g001 quantitative) value (Text S1). No lesions were detected in the control fish force-fed normal sheep brain homogenate (Fig. S3 and Table S3B). Plaque-like deposits were also observed in the brains of the BSE- challenged fish, beginning at earlier timepoints. Initially, at 8 months p.i., the majority of these aggregates were localized in brainstem, less in diencephalon and optic tectum, and even fewer in valvula cerebelli, cerebellum and telencephalon (Figure 3D–F). Just 10% of the deposits were PK-resistant and these had a mean diameter of 5 mm. Subsequently, we observed a general progression in their distribution, size, PK-resistance and morpho- logical features. The incidence of the abnormal deposition was higher in fish sacrificed at earlier time points than at intermediate time points. However, the highest levels were measured at later time points, evocative of the phenomenon described in other prion cross-species transmission studies as an ‘‘eclipse’’ period [30]. Further analysis of the spatial and temporal progression revealed that with increasing time p.i., the deposition became more prominent in rostral brain regions, although caudal regions continued to be affected. By 24 months p.i., deposition in the brains of the BSE-challenged sea bream presented a striking picture, in which three out of five fish showed 500–800 deposits each, 70–85% of which were PK-resistant with a mean diameter of 30 mm. With regard to the remaining two fish, one displayed approximately 150 deposits, 93% of which were PK-resistant and the other showed only limited signs of abnormal aggregation. While deposits continued to be distributed throughout the brain at 24 months p.i., in the three highly affected fish the greatest increases in deposit numbers occurred in brainstem and diencephalon. The progression of the abnormal deposition is apparent in Fig. 3. and summarized in Figs. 4 and 5 and Tables S3A and S4. In contrast to the BSE-challenged fish, no aggregates were detected at any time in the brains of the control fish fed with normal bovine brain homogenate (Fig. S4 and Table S3A). Notably, none of the brain tissues positive for abnormal deposition showed evidence of neuronal body degeneration. Finally, no residual mammalian PrPSc was detected using 12F10 and 6H4 PLoS ONE | www.plosone.org 3 July 2009 | Volume 4 | Issue 7 | e6175