Molecular analysis of cases of Italian sheep scrapie and comparison with cases of bovine spongiform encephalopathy (BSE) and experimental BSE in sheep

Abstract

Concerns have been raised about the possibility that the bovine spongiform encephalopathy (BSE) agent could have been transmitted to sheep populations via contaminated feedstuffs. The objective of our study was to investigate the suitability of molecular strain typing methods as a surveillance tool for studying scrapie strain variations and for differentiating PrP(Sc) from sheep scrapie, BSE, and sheep BSE. We studied 38 Italian sheep scrapie cases from 13 outbreaks, along with a British scrapie case, an experimental ovine BSE, and 3 BSE cases, by analyzing the glycoform patterns and the apparent molecular masses of the nonglycosylated forms of semipurified, proteinase-treated PrP(Sc). Both criteria were able to clearly differentiate sheep scrapie from BSE and ovine experimental BSE. PrP(Sc) from BSE and sheep BSE showed a higher glycoform ratio and a lower molecular mass of the nonglycosylated form compared to scrapie PrP(Sc). Scrapie cases displayed homogeneous PrP(Sc) features regardless of breed, flock, and geographic origin. The glycoform patterns observed varied with the antibody used, but either a monoclonal antibody (MAb) (F99/97.6.1) or a polyclonal antibody (P7-7) was able to distinguish scrapie from BSE PrP(Sc). While more extensive surveys are needed to further corroborate these findings, our results suggest that large-scale molecular screening of sheep populations for BSE surveillance may be eventually possible.

FIG. 1.

Western blot of proteinase K-treated PrP^Sc from scrapie cases and experimental BSE in sheep using MAb F99.97.6.1. (A) Representative immunoblot showing sheep BSE (lane 2) and scrapie cases (lanes 1, 3, 5, 6, 8, and 12, Sarda sheep; lanes 4, 9, and 11, Comisana sheep; lanes 7 and 10, Massese sheep). The positions of molecular mass markers (kilodaltons) are indicated. (B) Representative blot scans from the experiment seen in panel A. Shown are results for sheep BSE and the scrapie case in lanes 2 and 12, respectively, of panel A. The optical densities were normalized by taking the maximum value in each scan as 100% and the minimum as 0%.

FIG. 2.

Scattergraph of proportions of proteinase K-treated diglycosylated and monoglycosylated PrP^Sc in sheep scrapie, BSE, and an experimental BSE in sheep. (A) Comparison among Italian scrapie cases (n = 38), United Kingdom scrapie (n = 1), BSE (n = 3), and a sheep experimentally infected with BSE, obtained with MAb F99.97.6.1. Error bars represent standard deviations of at least three independent determinations. Scrapie cases appear as a separate group not overlapping with BSE. (B) Comparison of Italian scrapie cases in different sheep breeds (MAb F99.97.6.1). Error bars are omitted. (C) Comparison of scrapie cases in outbreaks related or not with an accidentally contaminated vaccine (MAb F99.97.6.1). Error bars are omitted. (D) Comparison of Italian scrapie cases (n = 19), BSE from the United Kingdom (n = 1), and experimental sheep BSE (n = 1), obtained with the polyclonal antibody P7-7.

FIG. 3.

Mean molecular masses of proteinase K-treated, nonglycosylated PrP^Sc bands in Italian scrapie cases (n = 38), United Kingdom scrapie (n = 1), BSE (n = 3), and experimental sheep BSE (n = 1). Error bars represent standard deviations of at least three independent determinations.

FIG. 4.

Western blots of proteinase K-treated PrP^Sc using MAb F99/97.6.1. (A) Comparison of a United Kingdom BSE case (lanes 1 and 2), sheep BSE (lanes 3 and 4), and an Italian scrapie case (lanes 4 and 5), either deglycosylated (lanes 2, 4, and 6) or not (lanes 1, 3, and 5). (B) Comparison of the sheep BSE (lanes 1 and 4), an Italian scrapie case (lanes 2 and 5), and an Italian BSE case (lanes 3 and 6), either deglycosylated (lanes 4, 5, and 6) or not (lanes 1, 2, and 3). (C) Immunoblot of PrP^Sc from sheep BSE (lanes 1 to 4) and United Kingdom BSE (lanes 5 to 8). Proteinase K-treated diglycosylated and monoglycosylated PrP^Sc fragments were eluted from gel, deglycosylated as described in Materials and Methods, and then loaded at three consecutive dilutions (lanes 2 to 4, sheep BSE; lanes 6 to 8, United Kingdom BSE) for direct comparison with nonglycosylated PrP^Sc bands in untreated samples (lanes 1 and 5, sheep BSE and United Kingdom BSE, respectively). Deglycosylated PrP^Sc obtained from the United Kingdom BSE is higher than its respective nonglycosylated fragment.