Detection of PrPsc in blood from sheep infected with the scrapie and bovine spongiform encephalopathy agents.

The role of blood in the iatrogenic transmission of transmissible spongiform encephalopathy (TSE) or prion disease has become an increasing concern since the reports of variant Creutzfeldt-Jakob disease (vCJD) transmission through blood transfusion from humans with subclinical infection. The development of highly sensitive rapid assays to screen for prion infection in blood is of high priority in order to facilitate the prevention of transmission via blood and blood products. In the present study we show that PrP(sc), a surrogate marker for TSE infection, can be detected in cells isolated from the blood from naturally and experimentally infected sheep by using a rapid ligand-based immunoassay. In sheep with clinical disease, PrP(sc) was detected in the blood of 55% of scrapie agent-infected animals (n = 80) and 71% of animals with bovine spongiform encephalopathy (n = 7). PrP(sc) was also detected several months before the onset of clinical signs in a subset of scrapie agent-infected sheep, followed from 3 months of age to clinical disease. This study confirms that PrP(sc) is associated with the cellular component of blood and can be detected in preclinical sheep by an immunoassay in the absence of in vitro or in vivo amplification.

Atypical prion protein in sheep brain collected during the British scrapie-surveillance programme.

Scrapie of sheep and goats is the most common prion disease (or transmissible spongiform encephalopathy, TSE) of mammals and aggregates of abnormal, proteinase-resistant prion protein (PrP(Sc)) are found in all naturally occurring prion diseases. During active surveillance of British sheep for TSEs, 29 201 sheep brain stem samples were collected from abattoirs and analysed for the presence of PrP(Sc). Of these samples, 54 were found to be positive by using an ELISA screening test, but 28 of these could not be confirmed initially by immunohistochemistry. These unconfirmed or atypical cases were generally found in PrP genotypes normally associated with relative resistance to clinical scrapie and further biochemical analysis revealed that they contained forms of PrP(Sc) with a relatively protease-sensitive amyloid core, some resembling those of Nor98 scrapie. The presence of these atypical forms of protease-resistant PrP raises concerns that some TSE disorders of PrP metabolism previously may have escaped identification in the British sheep population.

Improved spectrophotometric assay for beta-lactam residues in kidney tissue.

This paper describes a detection system for beta-lactams using a commercially prepared carboxypeptidase enzyme (CPase) and a substrate system in which lactic acid is cleaved from a synthetic peptide, N alpha-N epsilon-diacetyl-L-lysyl-d-alanyl-d-lactic acid. The lactate is itself oxidized by lactate dehydrogenase to form NADH. Oxidized NAD+ is regenerated by diaphorase with the simultaneous reduction of the colourless 2-(p-iodophenyl)-3-(p-nitrophenyl)-5-phenyl tetrazolium chloride hydrate (INT) indicator substrate to produce a red-mauve colour that is proportional to CPase activity. The presence of beta-lactams decreases the intensity of colour produced. The lower limit of detection for benzyl penicillin (Pen G) by this system is 20 ng g-1 compared with 50 ng g-1 by the same assay but using a R-d-ala-d-ala substrate from a commercial kit.