Molecular typing of protease-resistant prion protein in transmissible spongiform encephalopathies of small ruminants, France, 2002-2009

Abstract

The agent that causes bovine spongiform encephalopathy (BSE) may be infecting small ruminants, which could have serious implications for human health. To distinguish BSE from scrapie and to examine the molecular characteristics of the protease-resistant prion protein (PrP(res)), we used a specifically designed Western blot method to test isolates from 648 sheep and 53 goats. During 2002-2009, classical non-Nor98 transmissible spongiform encephalopathy had been confirmed among ≈1.7 million small ruminants in France. Five sheep and 2 goats that showed a PrP(res) pattern consistent with BSE, or with the CH1641 experimental scrapie source, were identified. Later, bioassays confirmed infection by the BSE agent in 1 of the 2 goats. Western blot testing of the 6 other isolates showed an additional C-terminally cleaved PrP(res) product, with an unglycosylated band at ≈14 kDa, similar to that found in the CH1641 experimental scrapie isolate and different from the BSE isolate.

Figure 1

Immunoblots obtained for reference brain samples by discriminatory Western blot method. The first membrane (A, B) was probed with Bar233 antibody. The second membrane (C, D) was probed with monoclonal antibody P4. The 2 immunoblots were loaded with a natural classical bovine spongiform encephalopathy (BSE) isolate (lane 1); an isolate from a sheep experimentally infected with classical BSE 4 (SB1, lanes 2, 6); 2 sheep-passaged scrapie isolates (SSBP/1, lanes 3, 5; CH1641, lane 4); and an isolate from a goat experimentally infected with classical BSE (CH41x76, lane 7).

Figure 2

Molecular mass obtained for the di-, mono-, and unglycosylated protein bands (A–C) and the glycoform proportions (D–F) between the diglycosylated band and the monoglycosylated band of the protease-resistant prion protein of the reference transmissible spongiform encephalopathies isolates (A, D), CH1641-like isolates in sheep (B, E), and unusual isolates in goats (C, F). Results were obtained from immunoblots detected by Bar233 antibody. BSE, bovine spongiform encephalopathy.

Figure 3

Differences in molecular mass observed between protease-resistant prion protein in cattle bovine spongiform encephalopathy (BSE) and usual transmissible spongiform encephalopathy cases in small ruminants. Differential molecular mass was obtained by subtracting the molecular mass of the unglycosylated band of the cattle BSE control to that of the natural small ruminant isolate from an immunoblot detected by Bar233 antibody.

Figure 4

Western blot analysis of protease-resistant prion protein in 2 CH1641-like sheep isolates (06-017, lane 3; 06-287, lane 4) detected by Bar233 (A), P4 (B), and SAF84 (C) antibodies. These samples were compared with 2 sheep-passaged scrapie isolates (SSBP/1, lane 1; CH1641, lane 5) and an isolate from a sheep experimentally infected with classical spongiform encephalopathy (SB1, lane 2). Samples in panel C were deglycosylated with peptide N-glycosidase F before Western blot analysis.

Figure 5

Western blot analysis of protease-resistant prion protein in 2 goat isolates (CH636, lane 3; 08-357, lane 4) detected by Bar233 (A), P4 (B), and SAF84 (C) antibodies. These samples were compared with an isolate from a goat naturally infected with scrapie (lane 1); an isolate from a goat experimentally infected with classical BSE (CH41x76, lane 2); and a sheep-passaged scrapie isolate (CH1641, lane 5). Samples in panel C were deglycosylated with peptide N-glycosidase F before Western blot analysis.