Disease phenotype in sheep after infection with cloned murine scrapie strains

Figures & data

Table 1. Summary of the experimental design, attack rates (AR), survival times (ST) and global immunohistochemical results on the central nervous system (CNS), peripheral nervous system (PNS), gut-associated lymphoid tissues (GALT) and non-GALT (nGALT).

Strain	Host	Route	Recipients	AR	ST	CNS	PNS	GALT	nGALT
ME7	Suff AA	Oral	3	2/3^R	763, 1209	8.4	4.6	0.4	1.8
		Combined	3	3/3^R	365 ± 256	7.6	3.2	12	3.8
	Chev VV	Oral	5	4/4	1146 ± 193	11.6	6.6	9.1	35.7
		Combined	3	3/3	659 ± 100	9.1	3.1	4.7	19.2
	Chev AA	Oral	5^ID1	4/4	1388 ± 12	11	5	2.5	10.2
		Combined	3	3/3	624 ± 60	5.7	0.9	0	2.2
79A	Suff AA	Oral	3	0/3^R, *	-	-	-	-	-
		Combined	3	3/3^R	653 ± 335	7.7	1	0	0.4
	Chev VV	Oral	3	1/3	481	6.9	3.1	1	5.4
		Combined	3^ID1	2/2	671, 743	8.8	0.7	4.1	12.4
	Chev AA	Oral	3	1/3*	2450	7.5	0.5	0	1.7
		Combined	3^ID1	2/2	647, 1043	5.5	0.2	0	1.7
22A	Suff AA	Oral	3	2/3*	2274*	0.5	0	0	0
		Combined	3	3/3	592 ± 575	7.3	1.3	3.7	16.4
	Chev VV	Oral	3^ID2	1/2	1865	12.3	2.1	1.3	14.4
		Combined	3^ID1	2/2	259, 479	8.9	1.5	3.1	18.3
	Chev AA	Oral	3	3/3	2107 ± 296	6.3	1	4	13.5
		Combined	3	3/3	687 ± 270	7.2	0.4	4.9	16.9
87V	Suff AA	Oral	3	0/3	-	-	-	-	-
		Combined	3	3/3^R	991 ± 607	9.1	0	0	0
	Chev VV	Oral	3	0/3	-	-	-	-	-
		Combined	3	3/3	687 ± 156	14.4	0	0	0
	Chev AA	Oral	3	0.3	-	-	-	-	-
		Combined	3	3/3	509 ± 85	10.1	0	0	0

Standard deviations are given for the ST (in days) to highlight individual variation. Scores for the different compartments indicate the sum of the averaged magnitude of PrP^d in each individual tissue or area contained in each compartment. The combined route (in italic) includes the oral, sub-cutaneous and intracerebral routes. All groups, except for the oral ME7 Cheviot sheep, which had a total of five VRQ and five ARQ sheep, initially contained a total of three sheep. However, some sheep died of intercurrent deaths before the first positive animal of that group did and are excluded from the table. The superscript (^ID) followed by a number in the column of recipients indicates the number of sheep that died with intercurrent deaths. The superscript R indicates that one animal in that group was polymorphic at codon M112T (^R). An asterix (*) designates culled at the end of the experiment. Survival times (in days), which are defined as the interval of days between the inoculation date and the post-mortem, are only given for sheep that were demonstrably infected, and are given as individual values rather than as an average if groups are smaller than three sheep.

Figure 1. Influence of the host breed and genotype on the PrP^d profiles when inoculated with ME7, 79A, 22A and 87V by the combined route. The x axis of the above graph refers to different morphological types of PrP^d labeling identified by immunohistochemistry. PrP^d types are: ITNR, intraneuronal; ITAS, intrastrocytic; ITMG, intramicroglial; STEL, stellate; SBPL, subpial; SBEP, subependymal; PRVS, perivascular; PVAC, perivacuolar; PART, fine particulate-coalescing; LINR, linear; PNER, perineuronal; EPEN, ependymal; NVPL, non vascular plaques; VASC, vascular plaques.

Table 2. Summary of the PrP^d cell distribution according to strain and host.

Strain	Host	ITNR	ITGL	GLAS	NRPL	EPEN	NVPL	VSPL
ME7	Suff AA	23 ± 2	10 ± 2	19 ± 0	44 ± 5	1 ± 2	0.3 ± 0	2 ± 2
	Chev VV	14 ± 9	23 ± 5	29 ± 8	31 ± 18	0.0	0.0	2 ± 2
	Chev AA	23 ± 16	18 ± 4	23 ± 2	35 ± 21	0.0	0.0	0.0
79A	Suff AA	12	32	21	35	3	0.0	0.0
	Chev VV	4 ± 3	37 ± 10	35 ± 13	25 ± 0	0.0	0.0	0.0
	Chev AA	18 ± 15	23 ± 20	21 ± 23	37 ± 13	0.0	0.0	0.0
22A	Suff AA	22	23	23	29	2	0.0	0.0
	ChevVV	19 ± 6	26 ± 8	32 ± 1	10 ± 5	13 ± 19	0.0	0.0
	Chev AA	5 ± 5	4 ± 2	34 ± 12	44 ± 15	4 ± 6	11 ± 15	0.0
87A	Suff AA	44	29	1	26	0.0	0.0	0.0
	Chev VV	45	36	0.5	18	0.0	0.0	0.0
	Chev AA	52	37	1	10	0.0	0.0	0.0

The scores shown are average short PrPd profiles grouped according to the host and to the strain but not the route. The intraneuronal (ITNR) score (in percentage) is the amount of PrPd found within the perikarya of neurons relative to that found elsewhere. The intraglial (ITGL) type includes the intra-astrocytic and intra-microglial. The glia-associated PrPd (GLAS) includes the stellate, subpial, subependymal, perivascular and perivacuolar PrPd types. The neuropil-associated PrPd includes the particulate, linear and perineuronal. Plaque-like PrPd was scored as associated with plaques (vascular plaques [VSPL]) or not (non-vascular plaques [NVPL]). The host was either Suffolk (Suff) or Cheviot (Chev) sheep and of ARQ/ARQ (AA) or VRQ/VRQ (VV) Prnp genotype

Figure 2. Main PrP^d features in brains of sheep infected with ME7, 79A, 22A, or 87V. In ME7 infections (A-C), neuropil-associated PrP^d types predominated, like-wise perineuronal, coalescing or diffuse punctate. A prominent characteristic of the 22A agent in sheep (D-F) was the glia-associated PrP^d types like the perivascular and perivacuolar in the white matter with some plaque-like depositions (arrows) in particular in Cheviots of the ARQ/ARQ genotype. 79A infections (G-I) resulted in a combination of intraglial-coalescing pattern (G) with stellate-like labeling (H, I). Sheep infected with 87V (J-L) unequivocally showed most PrP^d associated with neurons or glial cells. Immunohistochemistry for PrP^d using R145 antibody. Magnifications: x10 (D-F, H and I); x20 (A-C, G, J-L).

Figure 3. Epitope mapping results for the 87V infections in the dorsal motor nucleus of the vagus nerve of sheep. Note that Suffolk ARQ/ARQ sheep displayed intraneuronal immunolabelling with C-terminal antibodies such as Bar224 (A) and N-terminal antibodies such as P4 (B). Instead, Cheviot VRQ/VRQ (C, D) and ARQ/ARQ (E, F) sheep displayed the intraneuronal type with Bar224 (C, E) but not with P4 (D, F), a feature that is characteristic of strains like BSE and CH1641 when experimentally passaged in sheep. Immunohistochemistry for PrP^d using Bar224 or P4 antibodies. Magnifications: x10 (A-D), x20 (E, F).

Figure 4. Triplot analyses from brains of sheep infected with cloned murine strains. Colors denote strain whereas symbol denotes breed and genotype. This plot shows total number of animals infected either by a single oral dose or a combined challenge. Eighteen sheep infected with ME7 (green) gave a positive result in Western-blot. Nine did with 79A (blue). Eleven sheep infected with 22A (red) gave a positive result in WB. All nine sheep infected with 87V (yellow) displayed strong signals of PrP^res in their brains. Note that the PrP^res associated with ovine ME7 showed higher amounts of the di-glycosylated band, followed by intermediate amounts of the mono-glycosylated band and low amounts of the a-glycosylated band. The characteristics of PrP^res in ovine 22A infections are similar to ME7 ones but with a relatively lower and higher amount of the di- and a-glycosylated bands, respectively. The mono- and the a-glycosylated bands of PrP^res are significantly higher after 79A infections. This pattern was similar to that observed for the PrP^res associated with the 87V strain in Cheviot sheep of the VRQ/VRQ (triangles) and ARQ/ARQ (squares) genotypes, but not in the Suffolk sheep of the ARQ/ARQ genotype (circles), in which, the di-glycosylated band predominated.