Classical natural ovine scrapie prions detected in practical volumes of blood by lamb and transgenic mouse bioassays

Scrapie is diagnosed antemortem in sheep by detecting misfolded isoforms of prion protein (PrP(Sc)) in lymphoid follicles of the rectal mucosa and nictitating membranes. Assay sensitivity is limited if (a) the biopsy is collected early during disease development, (b) an insufficient number of follicles is collected, or (c) peripheral accumulation of PrP(Sc) is reduced or delayed. A blood test would be convenient for mass live animal scrapie testing. Currently approved techniques, however, have their own detection limits. Novel detection methods may soon offer a non-animal-based, rapid platform with detection sensitivities that rival the prion bioassay. In anticipation, we sought to determine if diseased animals could be routinely identified with a bioassay using B lymphocytes isolated from blood sample volumes commonly collected for diagnostic purposes in small ruminants. Scrapie transmission was detected in five of six recipient lambs intravenously transfused with B lymphocytes isolated from 5~10 mL of blood from a naturally scrapie-infected sheep. Additionally, scrapie transmission was observed in 18 ovinized transgenic Tg338 mice intracerebrally inoculated with B lymphocytes isolated from 5~10 mL of blood from two naturally scrapie-infected sheep. Based on our findings, we anticipate that these blood sample volumes should be of diagnostic value.

Immunohistochemical detection of Prion protein (PrP-Sc) and epidemiological study of BSE in Korea

Though the aetiology of transmissible spongiform encephalopathies (TSEs) remains uncertain, proteinase resistant prion protein (PrP-Sc), a converted form of the normal cellular prion protein (PrP-C), accumulates in the lysosome of cells of the nervous systems of animals with TSEs. In this study, clinical and epidemiological examinations of bovine spongiform encephalopathy (BSE) were conducted in Korea. During the investigated period, none of the cattle exhibited typical clinical signs of BSE, such as behavioral disturbances, high sensitivity, and abnormal locomotion. Immunohistochemical analysis and western immunoblotting were established to detect PrP-Sc in the brain tissue using monoclonal antibody (MAb) F89/160.1.5, produced by immunizing mice with a synthetic peptide which corresponds to bovine PrP residues 146-159, NH2-SRPLIHFGSDYEDRC-COOH. Although some BSE-like spongiform changes were observed in bovine brains randomly collected from Korean slaughterhouses from 1996 to 1999, no PrP-Sc was detected in those brains with the established immunohistochemistry and western immunoblotting assay. Also, no positive reaction was observed in bovine brains infected with rabies. These immunohistochemical and western immunoblotting methods using MAbs, specifically reactive with conserved epitopes on ruminant PrP, can be used for postmortem diagnosis of BSE. Further, the method can be applied to antemortem and the preclinical diagnosis of ovine scrapie by detecting PrP-Sc in lymphoid tissues, such as the tonsils, third eyelid or peripheral lymph nodes.