TSE Transmission to Sea Bream Figure 2. Progression of abnormal deposition in 2 scrapie-challenged fish. Sagittal brain sections from scrapie-challenged fish at 12 and 24 months p.i. were stained with H&E (A, D), or treated with SaurPrP1 (1:2000) without PK-digestion (B, E) and with PK-digestion (C, F). Images show diencephalon. The mean number of deposits (per section of fish containing deposits) observed in different brain regions without PK-treatment is indicated by the fill-type in the schematic drawings at the far left. CCe, corpus cerebelli; Di, diencephalon; Hyp, hypothalamus; LI, lobi inferioris; MO, medulla oblongata; OB, olfactory bulb; OC, optic chiasm; OlN, olfactory nerve; OT, optic tectum; P, pituitary; POA, preoptic area; SC, spinal cord; Tel, telencephalon; TL, torus longitudinalis; VCe, valvula cerebelli. The following areas were not examined: OB; OIN; OC; P. Rectangles indicate areas of magnification shown in the panels directly below. Arrowheads indicate the abnormal aggregates. Scale bars, 100 mm. doi:10.1371/journal.pone.0006175.g002 monoclonal antibodies (data not shown). Overall, these data suggest that while both TSE strains resulted in similar abnormal brain pathology, the brains of BSE-challenged individuals were more rapidly and severely affected than those of scrapie- challenged fish. BSE, known to be a zoonotic TSE, may represent a thermodynamically favored PrPSc conformation that is permis- sive for PrP expressed in a wide range of mammalian species [31]. Despite this permissibility, however, attempts to orally transmit BSE to pigs and chickens have failed [32,33]. To characterize the nature of the deposition, we employed a variety of conventional staining techniques. Congo red-stained deposits in BSE-challenged sea bream brains at 24 months p.i. were congophilic (Figs. 6A and 7B) and birefringent under polarized light (Fig. 6B), suggesting an amyloid or amyloid-like component [34]. No Congo red birefringence was observed in either the control tissues or the scrapie-challenged fish brains (data not shown). While the plaque-like aggregates were prominent with hematoxyline and eosin (H&E) (Fig. 7A), Klu¨ ver-Barrera staining for myelin structures and von Kossa staining for calcium deposition both gave negative reactions (data not shown). Deposits were also PAS positive (Fig. 7C) but Alcian blue negative (Fig. 7E), indicating the presence of carbohydrates and the absence of acidic glycosaminoglycans, respectively. Finally, our four anti-fish PrP antisera positively labeled the deposits (see Fig. 7D for SaurPrP1), whereas the 12F10 and 6H4 antibodies did not (data not shown). Two main types of plaque-like deposits were identified in the brains of the BSE-challenged fish: fibrous, diffusely stained aggregates (Fig. 8A, D, G, J), and those that were more amorphous and dense (Fig. 8B, E, H, K). At 8 months, small aggregates, generally in close proximity to neurofibrils, were detected, whereas the majority of the adjacent fiber bundles remained intact (Fig. 3D–F). At 10 and 12 months the first signs of neurodegen- eration appeared as a primitive disorganization of dendrites and axons. By 16 and 18 months, the distention of neurites, mostly in grey matter, was exacerbated. The extensive deconstruction of microfilaments within the axons and the loss of their coherence, especially at 18 months, were detected histopathologically. Aggregates of dystrophic neurites were immunostained with SaurPrP1, exhibiting a diffuse PrP-immunolabeling with some marginal spicule-like projections (data not shown). At 24 months the diffusely immunolabeled aggregates of dystrophic neurites (Fig. 8A, D, G, J) coexisted with deposits that appeared more amorphous, condensed and flocculated and therefore were more intensely stained with all the techniques used (Fig. 8B, E, H, K). PLoS ONE | www.plosone.org 4 July 2009 | Volume 4 | Issue 7 | e6175