Tongue involvement by prion spreading was shown to be a common outcome after oral or intracranial experimental challenge with scrapie and transmissible mink encephalopathy sources in rodent models. It is also known that bovine spongiform encephalopathy, which is pathogenic for humans, is experimentally transmissible to sheep and can lead to a disease indistinguishable from scrapie. A recent European Food Safety Authority opinion recommended research into PrP sc accumulation in the tongues of ruminants. We report on the detection of PrP sc in the tongues of seven scrapie-infected sheep by immunohistochemistry and Western blotting.Recent studies on rodent models (1,8,10), have reported the detection of PrP sc in tongue tissue after oral or intracranial experimental challenge with scrapie and transmissible mink encephalopathy sources. Unlike bovine spongiform encephalopathy, scrapie is not considered a risk to humans health (2), but it has been demonstrated that under experimental conditions sheep are easily infected by the bovine spongiform encephalopathy agent and that they carry abundant amounts of infectivity throughout most body tissues (6). A European Food Safety Authority opinion (3) recommends testing for PrP sc presence and accumulation in ruminant tongues in order to facilitate risk quantification and assessment. In this study, we report on the presence of PrP sc in the tongues of sheep infected with naturally occurring scrapie.We studied 10 negative, regularly slaughtered adult sheep and 10 adult sheep positive by rapid test (Prionics-Check Western) coming from two different affected flocks in Piedmont and Tuscany, respectively (flock A, eight sheep of the Biellese breed, aged 20 months to 7 years; flock B, two sheep of the Sarda breed, aged 20 months and 2.5 years). The disease was confirmed by histology, immunohistochemistry (IHC), and Western blotting (WB) in the brainstem. Both sheep from flock B and four from flock A showed clinical signs of scrapie (tremors, emaciation, falling). No relevant differences in the amount of PrP sc in the obex were found between animals with or without clinical signs. All animals had an ARQ/ARQ PrP genotype.The tongue of each animal was cut into two halves, of which one was fixed in buffered formalin and the other was frozen. Two specular areas of each half were examined independently: one at the level of the apex and one from the corpus linguae. For IHC, 10 serial tissue sections, 5 to 6 m thick, were obtained and numbered: sections numbered 1, 4, and 7 were stained to detect PrP sc immunoreactivity; sections numbered 2, 5, and 8 and 3, 6, and 9, respectively, were stained using PGP 9.5 and neurofilament to investigate the possible involvement of cells and nerve fibers. All tissue sections were dewaxed and rehydrated by routine methods and then subjected to an antigen retrieval procedure (5). The sections were immersed in 98% formic acid for 20 min, washed in distilled water, and then autoclaved for 20 min at 121°C in distilled water. Endogenous peroxidase activity was...