TABLE 4.

Intracerebral inoculation of tg338 mice with WBC and cell-sorted mononuclear cell subpopulations prepared from VRQ/VRQ sheep orally inoculated with PG127 scrapie^a

Cell type	Infection characteristicsb
	D1				D2				D3				D4
	Fraction purity (%)	tg338 transmission		Infectious titerc	Fraction purity (%)	tg338 transmission		Infectious titerc	Fraction purity (%)	tg338 transmission		Infectious titerc	Fraction purity (%)	tg338 transmission		Infectious titerc
		No. positive/total no.	Incubation period (dpi)			No. positive/total no.	Incubation period (dpi)			No. positive/total no.	Incubation period (dpi)			No. positive/total no.	Incubation period (dpi)
PBMC		6/6	110 ± 12	102.28		6/6	92 ± 5	103.8		6/6	90 ± 2	103.8		6/6	103 ± 7	102.79
CD14+	98.2	6/6	110 ± 9	102.27	96.4	6/6	96 ± 10	103.47	95.1	6/6	107 ± 9	103.47	95.2	4/6	118 ± 9	101.67
CD4+/8+	97.1	6/6	90 ± 5	104	96.1	6/6	90 ± 2	103.95	95.3	6/6	106 ± 5	103.95	95.5	5/6	111 ± 17	102.22
CD45R+	95.3	5/6	121 ± 7	101.56	98.2	6/6	116 ± 12	101.91	99.2	3/6	100, 104, 106*	101.91	97.3	2/6	110, 117	101.62

^aFour susceptible VRQ/VRQ sheep aged between 6 and 10 months (identified as D1, D2, D3, and D4) were orally challenged with 2 g of brain homogenate (10^6.6 ID₅₀/g intracranially in tg338 mice). These animals were sampled at 180 dpi. The donor sheep were euthanized at 207, 219, 217, and 196 dpi, respectively.

^bClassical scrapie occurrence was confirmed by histopathology (vacuolar change in central nervous system) and detection of abnormal PrP deposits in central nervous system and lymphoid tissues. A fraction of the collected whole blood (150 ml) was used to prepare PBMC. CD14⁺ (FITC-labeled VPM65), CD4⁺/8⁺ (FITC-labeled SBU/T4 and SBU/T8), and CD45R⁺ (FITC-labeled 20.29) subpopulations were sorted using anti-FITC-coated magnetic beads and two passages on magnetically activated cell sorting minicolumns. The purity of sorted cells was assessed by flow cytometry. After their preparation, cells were counted and aliquots of 5 × 10⁶ cells were homogenized in 200 μl of a 5% glucose solution. Each homogenate was inoculated intracerebrally into tg338 mice (n = 6; 20 μl per mouse, i.e., 5 × 10⁵ cells per mouse). Mice then were observed until the occurrence of clinical signs or were killed after 200 dpi. Mice were considered positive when abnormal PrP deposition was detected in brain of prion disease mice. Incubation periods are presented as means ± standard deviations, except for the dilution with which less than 50% of mice were found to be prion disease positive. In that case (indicated by an asterisk), incubation times of the positive mice are individually presented.

^cIndividual incubation periods were used to estimate the infectious titers (ID₅₀ per ml of cell homogenate) in each cell population according to the method developed by Arnold et al. (22).