Linda A. Terry

In vitro amplification of PrPSc derived from the brain and blood of sheep infected with scrapie

Scrapie is a fatal, naturally transmissible, neurodegenerative prion disease that affects sheep and goats and is characterized by the accumulation of a misfolded protein, PrPSc, converted from host-encoded PrPc, in the central nervous system of affected animals. Highly efficient in vitro conversion of host PrPc to PrPSc has been achieved in models of scrapie and in natural prion diseases by protein misfolding cyclic amplification (PMCA). Here, we demonstrate amplification, by serial PMCA, of PrPSc from individual sources of scrapie-infected sheep. Efficiency of amplification was affected by the pairing of the source of PrPSc with the control brain substrate of different genotypes of PrP. In line with previous studies, efficiency of amplification was greatly enhanced with the addition of a synthetic polyanion, polyadenylic acid (PolyA), facilitating rapid detection of low levels of PrPSc from body fluids such as blood. To this end PrPSc was amplified, in a 3 day PMCA assay, from blood leukocyte preparations from VRQ/VRQ scrapie-affected sheep at clinical end point. While PolyA-assisted PMCA resulted in spontaneous conversion of PrPc, we were able to distinguish blood samples from unaffected and affected sheep under controlled conditions. This study demonstrates that highly efficient amplification of PrPSc can be achieved for ovine scrapie from both brain and blood from naturally infected sheep and shows potential applications for improvements in current diagnostics and pre-mortem testing.

Prions Are Secreted into the Oral Cavity in Sheep with Preclinical Scrapie

A major concern in prion disease transmission is the spread of the disease agent by means of secretions and excretions. We analyzed buccal swab samples obtained from preclinical scrapie-infected sheep by concentrating the collected prions on silicon dioxide, followed by amplification by serial protein misfolding cyclic amplification. Data clearly demonstrate that prions are present in buccal swab samples from sheep with a VRQ/VRQ PRNP genotype during preclinical scrapie infection. These data describe for the first time to our knowledge the secretion of prions into the oral cavity of sheep, a finding with implications for the transmission of ovine scrapie and very likely other prion diseases.

Isolation of Prion with BSE Properties from Farmed Goat

BSE can infect small ruminants and could be misdiagnosed as scrapie.

Environmental Sources of Scrapie Prions

ABSTRACT Ovine scrapie and cervine chronic wasting disease show considerable horizontal transmission. Here we report that a scrapie-affected sheep farm has a widespread environmental contamination with prions. Prions were amplified by protein-misfolding cyclic amplification (sPMCA) from seven of nine environmental swab samples taken, including those from metal, plastic, and wooden surfaces. Sheep had been removed from the areas from which the swabs were taken up to 20 days prior to sampling, indicating that prions persist for at least that long. These data implicate inanimate objects as environmental reservoirs for prion infectivity that are likely to contribute to facile disease transmission.

Prions Are Secreted in Milk from Clinically Normal Scrapie-Exposed Sheep

ABSTRACT The potential spread of prion infectivity in secreta is a crucial concern for prion disease transmission. Here, serial protein misfolding cyclic amplification (sPMCA) allowed the detection of prions in milk from clinically affected animals as well as scrapie-exposed sheep at least 20 months before clinical onset of disease, irrespective of the immunohistochemical detection of protease-resistant PrPSc within lymphoreticular and central nervous system tissues. These data indicate the secretion of prions within milk during the early stages of disease progression and a role for milk in prion transmission. Furthermore, the application of sPMCA to milk samples offers a noninvasive methodology to detect scrapie during preclinical/subclinical disease.

Detection of prion protein particles in blood plasma of scrapie infected sheep.

Prion diseases are transmissible neurodegenerative diseases affecting humans and animals. The agent of the disease is the prion consisting mainly, if not solely, of a misfolded and aggregated isoform of the host-encoded prion protein (PrP). Transmission of prions can occur naturally but also accidentally, e.g. by blood transfusion, which has raised serious concerns about blood product safety and emphasized the need for a reliable diagnostic test. In this report we present a method based on surface-FIDA (fluorescence intensity distribution analysis), that exploits the high state of molecular aggregation of PrP as an unequivocal diagnostic marker of the disease, and show that it can detect infection in blood. To prepare PrP aggregates from blood plasma we introduced a detergent and lipase treatment to separate PrP from blood lipophilic components. Prion protein aggregates were subsequently precipitated by phosphotungstic acid, immobilized on a glass surface by covalently bound capture antibodies, and finally labeled with fluorescent antibody probes. Individual PrP aggregates were visualized by laser scanning microscopy where signal intensity was proportional to aggregate size. After signal processing to remove the background from low fluorescence particles, fluorescence intensities of all remaining PrP particles were summed. We detected PrP aggregates in plasma samples from six out of ten scrapie-positive sheep with no false positives from uninfected sheep. Applying simultaneous intensity and size discrimination, ten out of ten samples from scrapie sheep could be differentiated from uninfected sheep. The implications for ante mortem diagnosis of prion diseases are discussed.

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.

Molecular Profiling of Ovine Prion Diseases by Using Thermolysin-Resistant PrPSc and Endogenous C2 PrP Fragments

ABSTRACT Disease-associated PrP fragments produced upon in vitro or in vivo proteolysis can provide significant insight into the causal strain of prion disease. Here we describe a novel molecular strain typing assay that used thermolysin digestion of caudal medulla samples to produce PrPres signatures on Western blots that readily distinguished experimental sheep bovine spongiform encephalopathy (BSE) from classical scrapie. Furthermore, the accumulation of such PrPres species within the cerebellum also appeared to be dependent upon the transmissible spongiform encephalopathy (TSE) strain, allowing discrimination between two experimental strains of scrapie and grouping of natural scrapie isolates into two profiles. The occurrence of endogenously produced PrP fragments, namely, glycosylated and unglycosylated C2, within different central nervous system (CNS) regions is also described; this is the first detailed description of such scrapie-associated fragments within a natural host. The advent of C2 fragments within defined CNS regions, compared between BSE and scrapie cases and also between two experimental scrapie strains, appeared to be largely dependent upon the TSE strain. The combined analyses of C2 fragments and thermolysin-resistant PrP species within caudal medulla, cerebellum, and spinal cord samples allowed natural scrapie isolates to be separated into four distinct molecular profiles: most isolates produced C2 and PrPres in all CNS regions, a second group lacked detectable cerebellar C2 fragments, one isolate lacked both cerebellar PrPres and C2, and a further isolate lacked detectable C2 within all three CNS regions and also lacked cerebellar PrPres. This CNS region-specific deposition of disease-associated PrP species may reflect the natural heterogeneity of scrapie strains in the sheep population in the United Kingdom.

Experimental oral transmission of atypical scrapie to sheep.

Such transmission results in peripheral tissue infectivity that is not detectable by current surveillance screening methods.