in our study showed no brain spongiosis and no clinical abnormalities, we did find numerous plaque-like deposits in the brains of a significant proportion of the BSE-challenged fish, especially. Although much of the PrP associated with these deposits is PK-sensitive, this should not be taken as an indicator of low potential infectivity, as instances of clinical prion disease, and even infectivity, associated with extremely low levels of detectable PK-resistant PrP have been reported [43–45]. In light of the serious ramifications that would follow an unequivocal demonstration of prion disease transmission to fish, it must be emphasized here that the abnormal deposition we observed in the brains of the TSE-challenged fish could possibly have resulted from pathogenic factors other than the prions they were fed. Despite the fact that no such naturally occurring, cross- species infections from mammals to fish have ever been reported [46], we cannot completely rule out this possibility. Thus, however unlikely, one must consider the possibility that the brains used to prepare the inocula for the TSE challenge were infected with an undetected virus or bacteria in addition to the scrapie or BSE present. Together, the time course of brain lesion appearance, i.e. months not days, the ability of the agent to survive the oral challenge route, the absence of brain histopathology in any of the control groups and the production of novel histological lesions in both the BSE- and the scrapie-challenged fish, in the absence of inflammation, however, make this possibility a remote one. A more plausible alternate explanation would be that the amyloido- genic nature of the TSE-inocula might have contributed to the development of a novel fish brain amyloidosis. Infectivity and transmissibility are crucial issues that still need to be addressed. From a public health standpoint, the transmissibility of each prion strain and the relative ease with which it crosses species barriers, are its most significant characteristics. The spectrum of prionopathies, which has broadened in recent years, includes prion diseases that are not readily transmissible (e.g. some GSS cases), prion strains often associated with negligible clinical symptoms (e.g. the Nor98 scrapie strain), and even some without detectable PrPSc (e.g. PSPr) [44,47,48]. It is clear, then, that the evaluation and identification of both unusual prion diseases and prion diseases affecting unusual hosts is a complex task, requiring lengthy studies of pathogenesis, infectivity and transmissibility [49]. Until ongoing transmission studies using ‘‘bovinized’’ transgenic mice are completed, the possibility that the affected sea bream brain tissue might be infectious, must be taken seriously in any consideration to lift EU feed bans, especially those related to farmed fish. Materials and Methods Ethics Statement All fish used in the experiments described in this work were treated in accordance with EU Council Directive 86/609/EEC for the protection of animals used for experimental and other scientific purposes. Fish Sixteen hundred gilthead sea bream of approximately 20 g weight were purchased from a commercial farm (Interfish, Greece). At the commercial farm, before purchase, fish were fed commercial pellets (Biomar), none of which contained protein sources derived from land animal products. After transportation to the laboratory (Fisheries Research Institute, Kavala, Greece), they were maintained at 18uC in temperature-controlled recirculating water tanks. After a two week adaptation period, the fish were divided into groups of 200 in separate tanks. The fish were allowed TSE Transmission to Sea Bream a further three weeks of acclimatization before experimental manipulations were initiated. Preparation of inocula For the force feeding of the fish, 10% (w/v) brain homogenates from scrapie-infected sheep, healthy control sheep, BSE-infected cow and healthy control cow were prepared in PBS (pH 7.4). For the sheep brain homogenates both cerebellum and brainstem from two animals were used (kindly provided by Dr. P. Toumazos, Veterinary Services, Cypriot Ministry of Agriculture), while the bovine brain homogenates were each prepared from the brainstem of a single animal. The BSE sample (RBSE 21028), taken in 1991 from a female Fresian two months after disease onset, was kindly provided by Dr. Ian Dexter, Pathology Department, Veterinary Laboratories Agency, Weybridge, UK. As healthy herdmate tissue was not available, the healthy control bovine brainstem was taken from a local Greek cow in 2002. All brain samples were stored at 280uC prior to use. Challenge and maintenance For inoculations, fish were removed from the tanks and mildly anaesthetized with 0.3% ethylene glycol monophenyl ether. Following anaesthesia, each fish was force-fed 100 ml brain homogenate. In total, 2 groups of 400 fish each, were each treated with scrapie-infected or control sheep brain homogenate and 2 groups of 200 fish each were each treated with BSE-infected or control bovine brain homogenate. For both the experimental and control groups, the force-feeding procedure was repeated fortnightly for a total of five treatments, so that the cumulative inoculum for each fish was 50 mg brain equivalents. Following the inoculation period all fish were kept on a maintenance diet with commercially available chow to prevent excessive growth and overcrowding during the multiyear study period. Data regarding maintenance of the fish, mortality due to technical and natural causes and sampling are shown in Table S2. Clinical examination Clinical evaluation of the fish in each tank was made on a daily basis, checking especially for any behavioral or swimming abnormalities. Histopathological evaluation Individuals from each group (5 TSE-treated and 5 controls) were sacrificed at regular selected time points post inoculation (3, 6, 8, 10, 12, 14, 16, 18, 24 months) and tissue samples, including brain, spleen and intestine, were taken. Tissues were fixed in buffered formalin (pH 7.4), embedded in paraffin wax and finally 4 mm-thick serial sections were subjected to conventional staining with a variety of staining techniques including H&E, PAS, Alcian blue, von Kossa and Klu¨ ver-Barrera. The resulting sections were examined histologically using light microscopy (Axioplan 2 Imaging System, Zeiss). Tissue pictures were taken using the Nikon Digital Sight DS-SMc visualizing system. Congo red staining For the identification of possible amyloid-like structures, 10 mm- thick brain sections were deparaffinized, stained with 0.5% Congo red (Merck, Darmstadt, Germany) alcohol solution for 15 minutes, destained in 0.2% KOH, subsequently counterstained with Mayer’s hematoxyline, and after a short dehydration, they were finally cleared in xylene. The stained sections were observed microscopically under both normal and polarized light (Axiolab PLoS ONE | www.plosone.org 10 July 2009 | Volume 4 | Issue 7 | e6175