Figure. . Western blot detection, using the serial protein misfolding cyclic amplification technique, of the abnormal (disease-associated) form of the prion protein (PrPSc) 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). PrPSc was detected in saliva samples at the initial clinical and terminal stages of the disease (A, B). PrPSc 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 PrPSc 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.