Concentration of Disease-Associated Prion Protein with Silicon Dioxide

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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.

mentioning

confidence: 99%

Environmental Sources of Scrapie Prions

Maddison

Baker

Terry

et al. 2010

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“…Four swabs were collected from each animal at single time points and, as such, provide a snapshot of the likely presence of prions in the oral cavity. Two buccal swab samples were processed using a silicon dioxide (SiO 2 ) enrichment step as previously described (17,22). The SiO 2 -extracted material was then centrifuged for 3 min at 16,000 ϫ g, and the supernatant was diluted 1:10 in PMCA brain homogenate substrate (10% [wt/vol]) from a VRQ/ VRQ PRNP genotype animal (in phosphate-buffered saline [PBS]), 150 mmol/liter NaCl, 4 mmol/liter EDTA, pH 8.0, 1% (wt/vol) Triton X-100, and mini-protease inhibitor (Roche) to a final volume of 100 l. All buccal samples were amplified in this single substrate, as positive buccal swab extracts were amplified most efficiently within VRQ/VRQ substrate rather than the homologous heterozygous substrate (data not presented).…”

Section: Methodsmentioning

confidence: 99%

“…Amplified samples were digested with 50 g/ml proteinase K (PK) and 0.045% (wt/vol) SDS for 1 h at 37°C before Western blot analysis using 12% (wt/vol) NuPAGE precast Bis-Tris gels as described previously (22). A reaction was scored as positive if there was a defined PK-resistant triplet (ϳ18 to 27 kDa) visible on the Western blot; in each instance, positive signals were determined to be at least 3 times the signal for the negative samples run on that particular Western blot, as determined by Quantiscan software.…”

Section: Methodsmentioning

confidence: 99%

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The Oral Secretion of Infectious Scrapie Prions Occurs in Preclinical Sheep with a Range of PRNP Genotypes

Gough

Baker

Rees

et al. 2012

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Preclinical sheep with the highly scrapie-susceptible VRQ/VRQ PRNP genotype secrete prions from the oral cavity. In order to further understand the significance of orally available prions, buccal swabs were taken from sheep with a range of PRNP genotypes and analyzed by serial protein misfolding cyclic amplification (sPMCA). Prions were detected in buccal swabs from scrapie-exposed sheep of genotypes linked to high (VRQ/VRQ and ARQ/VRQ) and low (ARR/VRQ and AHQ/VRQ) lymphoreticular system involvement in scrapie pathogenesis. For both groups, the level of prion detection was significantly higher than that for scrapie-resistant ARR/ARR sheep which were kept in the same farm environment and acted as sentinel controls for prions derived from the environment which might contaminate the oral cavity. In addition, sheep with no exposure to the scrapie agent did not contain any measurable prions within the oral cavity. Furthermore, prions were detected in sheep over a wide age range representing various stages of preclinical disease. These data demonstrate that orally available scrapie prions may be a common feature in sheep incubating scrapie, regardless of the PRNP genotype and any associated high-level accumulation of PrP Sc within lymphoreticular tissues. PrP Sc was present in buccal swabs from a large proportion of sheep with PRNP genotypes associated with relatively low disease penetrance, indicating that subclinical scrapie infection is likely to be a common occurrence. The significance of positive sPMCA reactions was confirmed by the transmission of infectivity in buccal swab extracts to Tg338 mice, illustrating the likely importance of orally available prions in the horizontal transmission of scrapie.

“…4B). Other metals, minerals, and resins have been found to interact with normal and disease-associated prion protein (2,12,16,25 (Fig. 5A).…”

Section: Prpmentioning

confidence: 99%

Superparamagnetic Nanoparticle Capture of Prions for Amplification

Miller

Supattapone

2011

Prion diseases are associated with the presence of PrPSc , a disease-associated misfolded conformer of the prion protein. We report that superparamagnetic nanoparticles bind PrP Sc molecules efficiently and specifically, permitting magnetic separation of prions from a sample mixture. Captured PrP Sc molecules retain the activity to seed protein misfolding cyclic amplification (PMCA) reactions, enabling the rapid concentration of dilute prions to improve detection. Furthermore, superparamagnetic nanoparticles clear contaminated solutions of PrP Sc . Our findings suggest that coupling magnetic nanoparticle capture with PMCA could accelerate and improve prion detection. Magnetic nanoparticles may also be useful for developing a nontoxic prion decontamination method for biologically derived products.Bovine spongiform encephalopathy, Creutzfeldt-Jakob disease, and other prion diseases are caused by an infectious agent that contains PrP Sc , a misfolded conformer of the normal cellular prion protein (PrP C ) (24). Low-abundance sources of prions, such as blood, may still transmit disease (17,23). Prion diseases currently have no therapy, nor can prions be specifically removed from contaminated material.Inoculation bioassay serves as the gold standard for specific detection of prion infectivity. For sensitive detection, protein misfolding cyclic amplification (PMCA) has emerged as a rapid alternative to bioassay. PMCA exploits prion multiplication mechanisms to amplify PrP Sc in vitro using PrP C substrate (1, 5). Analogous to amplification of DNA sequence template by PCR, PrP Sc template seeds the conversion of PrP C substrate in PMCA, resulting in propagation and amplification of the PrP Sc conformation. Each serial PMCA round increases the detection sensitivity exponentially but requires ϳ72 h (7). The application of PMCA is also limited by prion propagation inhibitors present in blood and other biological solutions (6). An effective method to concentrate prions would improve subsequent PMCA sensitivity and utility.Nanotechnology presents many opportunities for fine control of molecular events. Certain iron oxide crystals less than ϳ25 nm in diameter exhibit superparamagnetism, with a net magnetization only occurring in the presence of an external magnetic field (15). MagnaBind and Dynal superparamagnetic beads contain many iron oxide crystals, dispersed such that no permanent magnetic order can form. This enables the whole particles to be superparamagnetic, allowing them to be rapidly attracted to a magnet and to lose magnetic interactions upon removal of the magnet (28). In molecular biology, superparamagnetic beads are often conjugated to specifically bind a target molecule.Using superparamagnetic nanoparticles, we have identified a novel binding interaction with PrPSc . Magnetic capture of PrP Sc may be applied to prion detection and prion decontamination. MATERIALS AND METHODSPreparation of scrapie-infected and uninfected brain homogenate. CD-1 mouse (prion strains RML, Me7, and 301C) and Syrian hamster (prion ...