As part of a large scrapie surveillance in Great Britain, 70 sheep tested positive by BioRad but negative by IHC, making their TSE status unclassifiable. Significantly, 10 of the unclassified results were obtained from samples originating from ARR/ARR sheep, a genotype previously considered to be resistant to scrapie, and a further 25 from AHQ/AHQ genotypes which rarely, if ever, develop scrapie.
Whether the unclassified samples correspond to the detection of non-infectious but PK resistant forms of PrPC, a novel isoform or low levels of infectious PrPSc not detectable by IHC, will need to be addressed through various routes of investigation.
This project meets the requirements of the expert panel and EFSA by focusing on the investigation of a possible correlation between Prnp genetics and the unclassified samples. Sequencing of the PrP coding region could identify rare polymorphisms that affect the conformation or cellular location of the PrP protein which in turn could influence its resistance to proteinase K digestion, disease susceptibility or incubation period. Polymorphisms located in the promoter region of PrP could affect the expression level of PrPC or its distribution in different tissues.
Each of the alleles,found in the unclassified cases,is also found in the true negative cases, therefore, they do not in themselves confer any specific conformation that might affect detection using the BioRad system. However the BioRad unclassified cases may be associated with a further codon change in the open reading frame (ORF) that alters conformation, resulting in either increased resistance to PK digestion yielding a false positive BioRad result. Alternatively, these cases could represent increased susceptibility to TSE disease or a specific strain, yielding a true positive BioRad result that is not detected by IHC due to altered distribution or conformation.
This genetics-based project will complement other investigations to understand the basis of the unclassified scrapie cases in order that the implications to the NSP and scrapie surveillance can be more fully understood.VLA will undertake this project with full consultation of Drs Wilfred Goldmann and Nora Hunter of IAH Edinburgh.