Table 4.

PMCA seeding activity in atypical scrapie passaged in tg338

PMCA seeds			PrPres positive PMCA reactions		Seeding activity, SA50/mL
Case	Origin		TgBov substrate	TgARQ substrate	TgBov substrate	TgARQ substrate
AS 25	Sheep		0/12	1/12	0 (0–101.86)*	101.40
	First passage in tg338 (neat)	Mouse 1	2/12	1/12	101.72	101.40
	Second passage in tg338 (neat)	Mouse 1	2/12	1/12	101.72	101.40
	End-point titration in tg338 (10−6 dilution)	Mouse 1	2/12	3/12	101.72	101.92
		Mouse 2	1/12	3/12	101.40	101.92
		Mouse 3	2/12	2/12	101.72	101.72
AS 26	Sheep		1/12	1/12	101.40	101.40
	First passage in tg338 (neat)	Mouse 1	2/12	0/12	101.72	0 (0–101.86) *
	Second passage in tg338 (neat)	Mouse 1	2/12	0/12	101.72	0 (0–101.86) *
	End-point titration in tg338 (10−6 dilution)	Mouse 1	2/12	1/12	101.72	101.40
		Mouse 2	1/12	1/12	101.40	101.40
	Noninoculated tg338	Mouse 1	0/12	0/12	—	—
		Mouse 2	0/12	0/12	—	—
		Mouse 3	0/12	0/12	—	—

Two sheep atypical scrapie (AS) isolates were selected. The 10% w/vol brain homogenates were inoculated into tg338 mice (2 iterative passages). Groups of 6 tg338 mice were inoculated intracerebrally with 20 µL of serial 10-fold dilutions of the same homogenates. Transmission was observed in 3 (AS 25) and 2 (AS 26) mice inoculated with 10⁻⁶ brain homogenate. No transmission was observed at lower dilutions. PMCA reactions (12 replicates) were seeded with 1/50 diluted brain homogenate (10% w/vol) from 1) the original sheep, 2) the second passage tg338 mice (pool of brains), and 3) individual brain from positive tg338 in the endpoint titration experiment. Brain homogenates (10% w/vol) from age-matched, noninoculated tg338 mice were also used as seeds (1/50 diluted). Two different PMCA substrates were used. The first one was prepared using brains from transgenic mice overexpressing the ARQ variant of the sheep prion protein. The second was prepared using brains from transgenic mice overexpressing the bovine prion protein (tgBov). Reactions were subjected to up to 4 amplification rounds. After each round, reaction products (1 volume) were mixed with fresh substrate (9 volumes) to seed the following round. PMCA reaction products were analyzed by Western blot for the presence of PrP^res. The number of PrP^res Western blot-positive reactions/total number of reactions are reported. Seeding activity titers were estimated using the Spearman–Karber’s limiting dilution titration method (most likely value) or, when no positive reaction was observed, by the Poisson’s probabilistic model. Titers are given as the number of PMCA SA₅₀ per milliliter of 10% brain homogenate.

^*Most likely value and IC 95% as described by Brown et al. (66).