Figure. . Western blot detection, using the serial protein misfolding cyclic amplification technique, of the abnormal (disease-associated) form of the prion protein (PrP^Sc) in concentrated saliva samples from 3 cows experimentally infected by inoculation with the agent of bovine spongiform encephalopathy: cows 5413 (A), 5444 (B), and 5437 (C). PrP^Sc was detected in saliva samples at the initial clinical and terminal stages of the disease (A, B). PrP^Sc was also detected in a saliva sample, after 3 rounds of amplification, obtained 2 months before the onset of clinical symptoms in 1 of the 3 cows (C). All saliva samples were concentrated by using the sodium phosphotungstic acid precipitation method. After protein misfolding cyclic amplification, extra bands with a molecular weight higher than that for PrP^Sc were occasionally observed, likely corresponding to prion protein aggregates or to residue of the normal isoform of prion protein resulting from incomplete proteinase K digestion. Molecular mass markers (in kDa) are shown on the right. R1–R4, rounds 1–4 of amplification; Ns, no seed control; mpi, months postinoculation.