End-point titration in tg338 mice of a 10% brain homogenate and white blood cells samples, collected in VRQ/VRQ sheep orally inoculated with PG127 scrapie.

<p>10% weight/volume homogenate (*) was prepared using posterior brainstem from VRQ/VRQ sheep inoculated with PG127 scrapie isolate and at the terminal stage of disease. Groups of 6 mice that over-express the VRQ ovine PrP (tg<i>338</i>) were intracerebrally (20 µL) inoculated with successive 1/10 dilutions of this homogenate. These data were already used in a previous publication <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002782#ppat.1002782-Andreoletti1" target="_blank">[16]</a>. In parallel, four susceptible VRQ/VRQ sheep (identified as D4, D5, D6 and D7) were orally challenged with PG127 classical scrapie isolate (total dose 10^6.7 ID₅₀ IC in tg338 mice). Scrapie incubation period in sheep were respectively 226 days, 238 days, 228 days and 242 days post inoculation (dpi). Aliquot of the same fresh and PFA 2% fixed WBC than those IV administrated sheep (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002782#ppat-1002782-t005" target="_blank">Table 5</a>) were homogenised in 5% glucose before intracerebral inoculation in tg338 mice (n = 6 per sample, 20 µL per mice). Each mouse received a quantity of WBC that is equivalent to 2.5 mL of starting whole blood. Mice were observed till occurrence of clinical signs compatible with a transmissible spongiform encephalopathy and considered positive when abnormal PrP deposition was detected in brain. Incubation periods (days post inoculation: dpi) in mice are presented as mean +/−SD except for those dilutions with which less than 50% of mice were found positive. In that case individual incubation period are reported (†). Infectious titers were estimated by the Spearman-Karber method <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1002782#ppat.1002782-Markus1" target="_blank">[19]</a>. Infectious titer is expressed as number of ID₅₀ per mL of whole blood. For each samples, the most likely value and, in parentheses, the lower and upper value of the 95% confidence interval are reported.