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Journal of Clinical Microbiology, March 2004, p. 972-980, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.972-980.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Discrimination between Scrapie and Bovine Spongiform Encephalopathy in Sheep by Molecular Size, Immunoreactivity, and Glycoprofile of Prion Protein

C. M. A. Thuring,1,2,{dagger} J. H. F. Erkens,1 J. G. Jacobs,1 A. Bossers,1 L. J. M. Van Keulen,1 G. J. Garssen,1 F. G. Van Zijderveld,1 S. J. Ryder,3 M. H. Groschup,4 T. Sweeney,2 and J. P. M. Langeveld1*

Central Institute for Animal Disease Control, 8203 AA 2004 Lelystad, The Netherlands,1 Pre-Clinical Division, Faculty of Veterinary Medicine and Conway Institute, University College Dublin, Belfield, Dublin 4, Ireland,2 Veterinary Laboratories Agency—Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom,3 Institute of Novel and Emerging Infectious Diseases, Federal Research Centre for Virus Diseases of Animals, Boddenblick 5a17498 Insel Riems, Germany4

Received 29 August 2003/ Returned for modification 12 November 2003/ Accepted 3 December 2003

A procedure for discrimination between scrapie and bovine spongiform encephalopathy (BSE) in sheep is of importance for establishing whether BSE has entered the sheep population. Since BSE has not yet been found in sheep at the farm level, such discrimination procedures can be developed only with experimental sheep BSE. Two distinctive molecular features of the prion protein (PrP)—molecular size and glycosylation profile—in proteinase K digests of brain stem tissue from sheep were used here; upon Western blotting, these features led to an unequivocal discrimination among natural scrapie, experimental scrapie, and experimental BSE. The higher electrophoretic mobility of PrP in sheep BSE could be best observed after deglycosylation treatment with N-glycosidase F. A simpler method for confirmation of this size difference involved comparison of the ratios for the binding of two monoclonal antibodies: P4 and 66.94b4. Based on epitope mapping studies with P4 and peptides, it appeared that N-terminal amino acid sequence WGQGGSH was intact only in sheep scrapie digests. Another feature typical for PrP in sheep BSE was the large fraction of diglycosylated PrP (70% or more). These data were obtained for a large group of positive sheep, consisting of 7 sheep with experimental BSE infection (genotypes: six ARQ/ARQ and one AHQ/AHQ), 48 sheep naturally infected with scrapie (six different genotypes), and 3 sheep with primary experimental scrapie infection. Routine tests of slaughter material serve well for the initial detection of both BSE and scrapie. With Western blotting as a rapid follow-up test, a 66.94b4/P4 antibody binding ratio above 1.5 is a practical indicator for serious suspicion of BSE infection in sheep.

* Corresponding author. Mailing address: Central Institute for Animal Disease Control, P.O. Box 2004, 8203 AA Lelystad, The Netherlands. Phone: 31 320 237217. Fax: 31 320 238153. E-mail: jan.langeveld{at}wur.nl.

{dagger} Present address: State University of Groningen, 9713 AZ Groningen, The Netherlands.

Journal of Clinical Microbiology, March 2004, p. 972-980, Vol. 42, No. 3
0095-1137/04/$08.00+0     DOI: 10.1128/JCM.42.3.972-980.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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