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Prion diseases are characterized by a long incubation period. In scrapie, sheep may incubate and spread the infection for several years before clinical signs evolve. We have previously studied the occurrence of subclinical infection in the brain. Now, we have studied the occurrence of subclinical infection in the brain and several lymphoid tissues in two scrapie-affected Icelandic sheep flocks by immunohistochemistry for PrP(Sc), a molecular marker for infectivity, and correlated this with results of PrP genotyping. At culling, one flock had one confirmed scrapie case, while the other flock had two. Analysis of 106 asymptomatic sheep by immunostaining for PrP(Sc) revealed that the incidence of subclinical infection was 58.3% in one flock and 42.5% in the other. PrP(Sc) was only detected in lymphoid tissues. The youngest positive sheep were 4 months old. PrP genotyping showed that over 90% of the sheep were of a genotype which is moderately sensitive to infection and may delay neuroinvasion. Our results show that asymptomatic sheep may spread the infection during the long incubation period of several years, which constitutes an important obstacle in the eradication of scrapie. Our findings indicate that contamination of the environment plays an important part in sustaining the infection.