non-ruminant livestock [20]. The European Commission’s TSE risk-reducing measures include a total EU-wide ban on the use of all processed animal protein in livestock and aquaculture feeds. Any consideration of lifting this ban requires a scientific assessment of the TSE transmission risk through fishmeal. Another issue to be addressed is the rising concern that pigs, poultry or fish bred for human consumption and inadvertently fed with TSE-contaminat- ed feed could eventually either develop clinical TSE or serve as reservoirs of infectivity without ever displaying clinical disease themselves. Such an assessment should consider the risk from TSE-contaminated feed being fed to farmed fish [18,21]. In aquaculture, a rapidly growing industry of economic importance in several EU countries, the farmed fish receive commercial feed containing 40–55% protein during the 12–20 months they generally spend in aquaculture facilities. Although remote, the possibility that some of this feed might be contaminated with mammalian prion cannot be excluded. In the present work, we evaluated the potential transmission of TSEs to gilthead sea bream, a commercially important fish species. After force-feeding with multiple doses of brain homogenate prepared from either healthy or naturally BSE- or scrapie-infected cow or sheep, the fish were monitored for 2 years for evidence of disease development by clinical, histopathological and immuno- histochemical criteria. None of the fish examined, showed symptoms of clinical disease. However, signs of neurodegeneration were often present and abnormal deposition was detected in the brains of both the scrapie-challenged and the BSE-challenged fish by 24 months post inoculation. Results and Discussion To evaluate the clinical state of the fish, we monitored control and TSE–challenged populations on a daily basis. Since locomotor deficits are often a major feature of the clinical presentation of prion diseases in a variety of hosts, we used the swimming behavior of the challenged fish as an indicator of their general activity and exploratory behavior. No clinical symptoms, including erratic swimming or behavioral abnormalities, were observed in any of the groups monitored. Although unusual in prion disease, a similar absence of clinical symptoms upon interspecies challenge has been reported for both the first passage of sheep scrapie and hamster prion transmission to mice [2]. In these cases, subsequent passage of brain material from the challenged individuals to additional mice did produce clinical disease, thereby demonstrating that asymptomatic animals can harbour high levels of infectious prions in their brains. Additionally, it is important to note that while certain experimentally or virally induced neurodegenerative effects do modify swimming parameters in fish, such as the swimming distance and orientation, the mean velocity, the turning angle and the equilibrium [22–24], this is not always the case. For instance, while both sea bass and sea bream can be infected with nodavirus, a naturally occurring piscine virus that causes brain lesions in both species, sea bream, in contrast to sea bass, show no clinical symptoms of disease [25,26]. To facilitate our evaluation we generated polyclonal antibodies against four different fish PrPs. The specificity of each antiserum was confirmed by both western blot (Fig. S1A) and immunohis- tochemistry (IHC) (Fig. S2A–C) with normal sea bream brain. Furthermore, anti-mammalian PrP antibodies (6H4, 12F10) did not stain sea bream brain, nor did our anti-fish PrP antisera recognize mammalian PrPSc (Fig. S1B). Moreover, absorption of our SaurPrP1 (Sparus aurata PrP1) antisera with recombinant sea bream PrP-1 protein [6], against which it was raised, resulted in a TSE Transmission to Sea Bream complete loss of its specific immunostaining in control fish (Fig. S2D). Initially, in order to determine the distribution of normal endogenous PrP in the central nervous system of gilthead sea bream, we used our polyclonal antisera to perform a detailed immunohistochemical evaluation on brain sections from control fish. Regions displaying abundant PrP included the optic tectum (Fig. 1A), valvula cerebelli (Fig. 1B) and corpus cerebelli (Fig. 1C), while strong PrP-immunopositivity was generally observed in the nerve fibers (Fig. 1D). The most prominently stained regions of the optic tectum, homologue to the superior colliculus in mammals [22], were striatum fibrosum marginale (Fig. 1A and Fig. S2A, B) and striatum fibrosum profundum (Fig. S2A, B). Less intense labeling was observed in the striatum griseum centrale and striatum plexiform fibrosum externum layers of the optic tectum (Fig. 1A and Fig. S2A, B). Cerebellar PrP-immunopositivity was detected mainly in the molecular layer, between Purkinje cell dendrites, and in the granular layer, in the matrix surrounding the granule cells (Fig. 1C). The valvula cerebelli, a rostral protrusion of the cerebellum in the midbrain ventricle that has no counterpart in mammals, showed significant PrP-immunopositivity, similar to that observed in the molecular and granular layers of cerebellum (Fig. 1B and Fig. S2A, B). In cerebral regions, including thalamus, medulla oblongata, diencephalon and the lateral telencephalic pallium, proposed to be a homologue of the mammalian hippocampus [22], we observed intense labeling of fiber bundles (Fig. 1D), consisting primarily of dendritic and axonal prolonga- tions in the neuropil. The same general PrPC expression pattern was observed in challenged and control populations, with no variation detected over time. The remarkable similarity of the overall immunolabeling pattern obtained with our SaurPrP1 antiserum in sea bream brain to the PrP-immunostaining profile in the mammalian brain [27,28] provided further assurance of the specificity of our antibody for piscine PrP. At the intracellular level, staining outlining the neuronal body was present in most of the neuronal populations observed, e.g. in the large neurons of the brainstem (Figure 1E). Axons displayed intense staining, while diffuse staining was observed inside some of these neuronal somata, suggesting a degree of PrP-immunoposi- tivity within cell departments, e.g. the Golgi complex (Fig. 1E). These findings suggest that the intracellular localization of PrP in fish brain is comparable to the neuronal intracellular localization of mammalian PrP [27,29]. To test for pathology in selected peripheral tissues, we examined intestines and spleens from TSE-challenged fish, sampled at different timepoints. No lesions or any other abnormalities were revealed in comparison to the control individuals. Intestinal PrP- immunoreactivity was evident in the serosa, myenteric plexus and submucous plexus (Fig. 1F). At all timepoints, PrP-immunolabel- ing in spleen and intestinal tissue was similar in both TSE- challenged and control fish and revealed no PK-resistance and no lesions or any other abnormalities. Detailed examination of brain sections revealed no histopath- ological evidence of disease in scrapie–challenged sea bream through 18 months post inoculation (p.i.). At 24 months, however, 2 out of 5 fish showed limited abnormal, PrP-immunoreactive, PK-sensitive, extracellular deposits (Table S3B) in the neuropil of brainstem, diencephalon, corpus cerebelli, valvula cerebelli, optic tectum and telencephalon (Fig. 2). Whilst the number of animals where plaques were found was too small to reach statistical significance, based on the high likelihood that the fish examined developed no plaques, we believe that the observation of aggregates in 2 out of 5 fish at the final time point could be considered as an important event of qualitative (and not PLoS ONE | www.plosone.org 2 July 2009 | Volume 4 | Issue 7 | e6175