Table 6. 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.

	Brain*		D4 WBC				D5 WBC				D6 WBC				D7 WBC
			Fresh		PFA fixed		Fresh		PFA fixed		Fresh		PFA fixed		Fresh		PFA fixed
Dilution	Pos mice	dpi	Pos mice	dpi	Pos mice	dpi	Pos mice	dpi	Pos mice	dpi	Pos mice	dpi	Pos mice	dpi	Pos mice	dpi	Pos mice	dpi
neat	6/6	64±4	6/6	90±1	6/6	92±3	6/6	100±11	6/6	106±6	6/6	97±4	6/6	95±3	6/6	87±3	6/6	85±2
10−1	6/6	76±3	5/6	107±11	5/6	106±8	ND		ND		ND		ND		4/6	108±13	5/6	117±19
10−2	6/6	87±2	2/6	111,115†	2/6	108,110†									1/6	111†	0/6	>200
10−3	6/6	97±5	0/6	>200	0/6	>200								-	0/6	>200	0/6	>200
10−4	3/6	110±4
10−5	2/6	117–121†
10−6	0/6	>200
10−7	0/6	>200
Infectious titer	107 (106.4–107.6) ID50/g		18.6 (6–57.5) ID50/mL		18.6 (6–57.5) ID50/mL		NA		NA		NA		NA		8.6 (2.8–26.7) ID50/mL		8.6 (4.3–17.4) ID50/mL

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 (tg338) were intracerebrally (20 µL) inoculated with successive 1/10 dilutions of this homogenate. These data were already used in a previous publication [16]. 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 (Table 5) 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 [19]. 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.