Unraveling the key to the resistance of canids to prion diseases
A & B. Rounds (R1-R10) of serial PMCA using brain homogenates from two different breeds of dog as substrates: Cocker Spaniel and German Wirehaired Pointer. Several procedures to propagate different inocula over dog species were tested. A1: Standard 1–10% seeded PMCA with serial 1:10 dilution rounds. B1: 30–50% seeded PMCA with serial 1:10 dilution rounds. B2: same as B1 with the addition of zirconia-silica beads . C1: 10% seeded PMCA with serial 1:2 dilution rounds. D1: 30–50% seeded PMCA with serial 1:2 dilution rounds. Different inocula or mix of inocula were used as seed. Mouse strain pool: Pool of mouse PrPSc strains containing equal amounts of ME7, RML, 22F, 22L, 87V, 22A, 79A, 139A and other spontaneously in vitro obtained strains. Sheep strain pool: Pool of sheep PrPSc strains containing equal amounts of 7 different scrapie isolates. Cervid strain pool: Pool of cervid PrPSc strains containing equal amounts of 2 mule deer and 2 elk isolates. BSE-C: Three different isolates of classical BSE (isolate 1, isolate 2 and isolate 3). BSE-L: Atypical L-type BSE. sBSE: Sheep BSE. * Twelve unseeded tubes were extended to 20 rounds for each set of experiments. Dog (Cocker Spaniel)-BSE obtained after the propagation of BSE-C (isolate 3) was shown in Vidal et al. 2013 . C. One tube of round 10 of each PK-resistant sample was selected to show the biochemical analysis of BSE-C (1; isolate 1 and 2; isolate 2) and sheep BSE (sBSE) seeded material generated by PMCA compared to brain-derived BSE-C and RML. Samples were digested with 85 μg/ml PK and analyzed by Western blot using monoclonal antibody D18 (1:5,000). PK: Protease-K. Mo: Mouse. Co: Cow. Do: Dog. Dog brain: negative control; undigested dog whole brain homogenate. Mw: Molecular weight.