TSE Transmission to Sea Bream Figure 8. Morphology of the abnormal deposition in the brains of the BSE-challenged fish 24 months p.i.. Sagittal 4 mm-thick brain sections where stained with hematoxylin without a counterstain. pH variation renders the deposits visible by light microscopy. The panels on the left show diffuse ‘‘pre-mature’’ deposits in A, diencephalon; D, optic tectum; G, cerebellum; J, brainstem. The middle panels display examples of compact ‘‘mature’’ deposition in B, diencephalon; E, optic tectum; H, cerebellum; K, telencephalon. The panels at the right show intermediate stage- abnormal deposition within fiber bundles that disrupts synapses in C, diencephalon; F, optic tectum; I, valvula cerebelli; L, telencephalon. S, spicule- like projections; CM, condensed material. Scale bars, 10 mm. doi:10.1371/journal.pone.0006175.g008 [42]. In the present study it may have contributed to the ‘‘eclipse- like’’ temporal appearance of the abnormal deposition, as well as to the lack of clinical symptoms in our BSE-challenged fish. Despite the positive IHC results, western blotting failed to detect PK-resistant PrP isoforms in the TSE-challenged fish brains (Fig. S5), possibly because the whole brain homogenates used did not have a high enough concentration of PK-resistant PrP to allow detection. In fact, it is clear from the IHC results that even at 24 months p.i., the brain regions associated with the abnormal deposits constitute only a small percentage of the whole brain mass. The results of this TSE transmission study with gilthead sea bream indicate the development of a CNS histopathology in the brains of the fish challenged with the TSE-inocula. This neuropathology displays characteristics resembling a novel fish amyloidosis more than a classical TSE. Specifically, while the fish PLoS ONE | www.plosone.org 9 July 2009 | Volume 4 | Issue 7 | e6175