Archival search for historical atypical scrapie in sheep reveals evidence for mixed infections

Abstract

Natural scrapie in sheep occurs in classical and atypical forms, which may be distinguished on the basis of the associated neuropathology and properties of the disease-associated prion protein on Western blots. First detected in 1998, atypical scrapie is known to have occurred in UK sheep since the 1980s. However, its aetiology remains unclear and it is often considered as a sporadic, non-contagious disease unlike classical scrapie which is naturally transmissible. Although atypical scrapie tends to occur in sheep of prion protein (PRNP) genotypes that are different from those found predominantly in classical scrapie, there is some overlap so that there are genotypes in which both scrapie forms can occur. In this search for early atypical scrapie cases, we made use of an archive of fixed and frozen sheep samples, from both scrapie-affected and healthy animals (∼1850 individuals), dating back to the 1960s. Using a selection process based primarily on PRNP genotyping, but also on contemporaneous records of unusual clinical signs or pathology, candidate sheep samples were screened by Western blot, immunohistochemistry and strain-typing methods using tg338 mice. We identified, from early time points in the archive, three atypical scrapie cases, including one sheep which died in 1972 and two which showed evidence of mixed infection with classical scrapie. Cases with both forms of scrapie in the same animal as recognizable entities suggest that mixed infections have been around for a long time and may potentially contribute to the variety of scrapie strains.

Fig. 1.

(a–i) Western blots of PrP^Sc from sheep brain of unusual scrapie cases and controls using antibodies P4 (a, b, e, g, h) and 6H4 (c, d, f, i). M, molecular mass markers (kDa). (a, c) Lanes 1–4, L4823, serial 1 : 3 dilutions; lanes 5 and 6, L4824, serial 1 : 3 dilutions. (b, d) Lanes 1–3, H800, serial 1 : 3 dilutions. (e) Lane 1, 13 × 85; lanes 2 and 3, two concurrent natural scrapie sheep; lane 4, 13 × 85, 1 : 3 dilution; lanes 5 and 6, concurrent natural scrapie sheep, 1 : 3 dilutions. (f) Lane 1, unrelated sample; lane 2, SSBP/1; lane 3, 13 × 85. (g) Lanes 1, 3 and 5, natural scrapie controls; lane 2, 13 × 69; lane 4, 13 × 85. (h, i) Lanes 1 and 4, CH1641; lanes 2 and 5, SSBP/1; lane 3, unrelated sample.

Fig. 2.

Lesion profiles of unusual scrapie cases and controls in brain of clinically affected tg338 mice. Patterns of severity of vacuolation (with se bars) in nine grey matter areas (G1–G9: medulla, cerebellum, superior colliculus, hypothalamus, thalamus, hippocampus, septum, retrosplenial cortex, and cingulate and motor cortex) and three white matter areas (W1–W3: cerebellum, superior cerebral peduncle and basal cerebral peduncle). (a) Classical scrapie 68 × 81, (b) SSBP/1, (c) CH1641, (d) atypical scrapie Scr2, (e) atypical scrapie 51 × 45, (f) L4824, (g) H800, (h) 13 × 85 and (i) vacuolation damage in two individual clinically positive mice inoculated with L4823 [mouse 4 (L4823/4) and mouse 9 (L4823/9)].

Fig. 3.

Immunohistochemical PrP^d features of tg338 mice. Thalamus from mice inoculated with (a) atypical scrapie (Scr2), (b) classical scrapie (68 × 81), (c) L4824, (d) H800, (e) L4823/9 and (f) dorsal motor nucleus of the vagus from mouse inoculated with SSBP/1. IHC with 2G11 or (f only) SAF84 with haematoxylin counterstaining.

Fig. 4.

Immunohistochemical PrP^d features of tg338 mice inoculated with sheep brain. (a, b) Thalamus from mice inoculated with 13 × 85, (c) corpus striatum and (d) deep cerebellar nuclei from mouse L4823/9. IHC with 2G11 and haematoxylin counterstaining.

Fig. 5.

(a–i) Western blots of PrP^Sc from brain of tg338 mice inoculated with unusual or control scrapie sheep brain using antibodies P4 (a, b, e, f, g) and 6H4 (c, d, h, i). M, molecular mass markers (kDa). Inocula: (a, c) lanes 1, 6 and 9, natural scrapie control (68 × 81); lane 2, L4824; lane 3, atypical scrapie control (51 × 45); lanes 4 and 8, CH1641(J2916); lane 5, 13 × 85; lane 7, H800; (b, d) lanes 1–3, 1 : 3 dilutions of atypical scrapie Scr2; lanes 4–6, 1 : 3 dilutions of SSBP/1; (e) lane 1, atypical scrapie (51 × 45); lane 2, 13 × 85 loaded × 6; (f) lanes 1 and 3, natural scrapie controls; lane 2, 13 × 85 loaded × 6; (g, h) lanes 1, 4 and 11, natural scrapie control (68 × 81); lanes 2 and 6, CH1641 (J2916); lanes 3, 7 and 8, single mouse L4823/4; lanes 5 and 10, single mouse L4823/9; lane 9, atypical scrapie control (51 × 45).