Sally J. Everest

Detection of prions in the faeces of sheep naturally infected with classical scrapie

Classical scrapie is a naturally transmitted prion disease of sheep and goats. Contaminated environments may contribute to the spread of disease and evidence from animal models has implicated urine, blood, saliva, placenta and faeces as possible sources of the infection. Here we sought to determine whether sheep naturally infected with classical scrapie shed prions in their faeces. We used serial protein misfolding cyclic amplification (sPMCA) along with two extraction methods to examine faeces from sheep during both the clinical and preclinical phases of the disease and showed amplification of PrPSc in 7 of 15 and 14 of 14 sheep respectively. However PrPSc was not amplified from the faeces of 25 sheep not exposed to scrapie. These data represent the first demonstration of prion shedding in faeces from a naturally infected host and thus a likely source of prion contamination in the environment.

Detection of PrPsc in Blood from Sheep Infected with the Scrapie and Bovine Spongiform Encephalopathy Agents

ABSTRACT 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 PrPsc, 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, PrPsc was detected in the blood of 55% of scrapie agent-infected animals (n = 80) and 71% of animals with bovine spongiform encephalopathy (n = 7). PrPsc 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 PrPsc 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.

First case of H-type bovine spongiform encephalopathy identified in Great Britain

have been recognised that differ in their PrPres profiles from those typically found. These cases have been detected in Europe (Biacabe and others 2004, Casalone and others 2004, Buschmann and others 2006), Japan (Yamakawa and others 2003) and the USA (Richt and others 2007). Colloquially referred to as ‘atypical BSE’, these cases fall into two types based on the molecular mass of the unglycosylated PrPres band relative to that of classical BSE, one of higher molecular mass (H-type) and the other lower molecular mass (L-type). A transmission study of an H-type BSE agent of French origin to mice has shown that it has biological characteristics distinct from those of the classical BSE agent (Beringue and others 2006). This short communication describes the first case of BSE in Great Britain showing an H-type molecular profile that was indistinguishable from a case previously identified in France. Frozen brainstem from five cases of BSE in cattle and one sheep scrapie case were tested by immunoblotting (TeSeE Western blot kit; Bio-Rad Laboratories) (Arsac and others 2007) according to the manufacturer’s instructions. The samples were diluted, in order to balance the PrPres in each lane, before loading on to a 12 per cent Bis-Tris SDS gel (Bio-Rad

Detection and Localisation of PrPSc in the Liver of Sheep Infected with Scrapie and Bovine Spongiform Encephalopathy

Prions are largely contained within the nervous and lymphoid tissue of transmissible spongiform encephalopathy (TSE) infected animals. However, following advances in diagnostic sensitivity, PrPSc, a marker for prion disease, can now be located in a wide range of viscera and body fluids including muscle, saliva, blood, urine and milk, raising concerns that exposure to these materials could contribute to the spread of disease in humans and animals. Previously we demonstrated low levels of infectivity in the liver of sheep experimentally challenged with bovine spongiform encephalopathy. In this study we show that PrPSc accumulated in the liver of 89% of sheep naturally infected with scrapie and 100% of sheep challenged with BSE, at both clinical and preclinical stages of the disease. PrPSc was demonstrated in the absence of obvious inflammatory foci and was restricted to isolated resident cells, most likely Kupffer cells.

Improved spectrophotometric assay for β-lactam residues in kidney tissue

This paper describes a detection system for β-lactams using a commercially prepared carboxypeptidase enzyme (CPase) and a substrate system in which lactic acid is cleaved from a synthetic peptide, Nα-NIµ-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 β-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 an R-d-ala-d-ala substrate from a commercial kit.