Differentiating Ovine BSE from CH1641 Scrapie by Serial Protein Misfolding Cyclic Amplification

Taema, Maged M.; Maddison, Ben C.; Thorne, Leigh; Bishop, Keith; Owen, Jonathan P.; Hunter, Nora; Baker, Claire A.; Terry, Linda A.; Gough, Kevin C.

doi:10.1007/s12033-011-9460-0

Cited by 10 publications

(16 citation statements)

References 45 publications

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“…In a blind trial, we analyzed a total of 216 TSE samples (108 BSE-scrapie mixes and 108 scrapie-only samples) ( Table 1). Samples were prepared as 10% (wt/vol) brain homogenates, as previously described (15).…”

Section: Methodsmentioning

confidence: 99%

“…sPMCA was carried out as previously described (15). Ten percent (wt/vol) brain homogenate substrates were prepared from scrapie-free sheep from a flock with extremely high levels of biosecurity and no history of classical scrapie.…”

Section: Methodsmentioning

confidence: 99%

“…Reaction products were digested with proteinase K (PK) and then analyzed by Western blotting using the monoclonal antibody SHa31 as described previously (15). Briefly, samples were digested with 50 g/ml PK in the presence of 0.045% (wt/vol) SDS for 1 h at 37°C.…”

Section: Methodsmentioning

confidence: 99%

“…We previously detailed the selective in vitro amplification of BSE PrP Sc using specific brain substrates prepared from healthy sheep (15). Here, this methodology was modified for the specific amplification of BSE PrP Sc within BSE/scrapie agent mixes, a scenario which may mimic brain tissue within mixed prion infections.…”

Section: A Standard Methodology For the Differentiation Of Bse From Smentioning

confidence: 99%

“…BSE prions are readily amplified under a set of defined conditions in which all scrapie isolates tested were refractory to amplification irrespective of the PRNP genotype or scrapie strain (15).…”

mentioning

confidence: 99%

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Highly Sensitive Detection of Small Ruminant Bovine Spongiform Encephalopathy within Transmissible Spongiform Encephalopathy Mixes by Serial Protein Misfolding Cyclic Amplification

2014

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bIt is assumed that sheep and goats consumed the same bovine spongiform encephalopathy (BSE)-contaminated meat and bone meal that was fed to cattle and precipitated the BSE epidemic in the United Kingdom that peaked more than 20 years ago. Despite intensive surveillance for cases of BSE within the small ruminant populations of the United Kingdom and European Union, no instances of BSE have been detected in sheep, and in only two instances has BSE been discovered in goats. If BSE is present within the small ruminant populations, it may be at subclinical levels, may manifest as scrapie, or may be masked by coinfection with scrapie. To determine whether BSE is potentially circulating at low levels within the European small ruminant populations, highly sensitive assays that can specifically detect BSE, even within the presence of scrapie prion protein, are required. Here, we present a novel assay based on the specific amplification of BSE PrP Sc using the serial protein misfolding cyclic amplification assay (sPMCA), which specifically amplified small amounts of ovine and caprine BSE agent which had been mixed into a range of scrapie-positive brain homogenates. We detected the BSE prion protein within a large excess of classical, atypical, and CH1641 scrapie isolates. In a blind trial, this sPMCA-based assay specifically amplified BSE PrP Sc within brain mixes with 100% specificity and 97% sensitivity when BSE agent was diluted into scrapie-infected brain homogenates at 1% (vol/vol).

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: A Standard Methodology For the Differentiation Of Bse From Smentioning

confidence: 99%

mentioning

confidence: 99%

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Highly Sensitive Detection of Small Ruminant Bovine Spongiform Encephalopathy within Transmissible Spongiform Encephalopathy Mixes by Serial Protein Misfolding Cyclic Amplification

2014

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In vitro amplification of H-type atypical bovine spongiform encephalopathy by protein misfolding cyclic amplification

O’Connor

Bishop

Workman

et al. 2017

Prion

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The in vitro amplification of prions by serial protein misfolding cyclic amplification has been shown to detect PrPSc to levels at least as sensitive as rodent bioassay but in a fraction of the time. Bovine spongiform encephalopathy is a zoonotic prion disease in cattle and has been shown to occur in 3 distinct forms, classical BSE (C-BSE) and 2 atypical BSE forms (L-BSE and H-BSE). Atypical forms are usually detected in asymptomatic, older cattle and are suggested to be spontaneous forms of the disease. Here, we show the development of a serial protein misfolding cyclic amplification method for the detection of H-BSE. The assay could detect PrPSc from 3 distinct experimental isolates of H-BSE, could detect PrPSc in as little as 1×10−12 g of brain material and was highly specific. Additionally, the product of serial protein misfolding cyclic amplification at all dilutions of seed analyzed could be readily distinguished from L-BSE, which did not amplify, and C-BSE, which had PrPSc with distinct protease K-resistance and protease K-resistant PrPSc molecular weights.

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Ovine recombinant PrP as an inhibitor of ruminant prion propagation in vitro

Workman

Maddison

Gough

2017

Prion

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Prion diseases are fatal and incurable neurodegenerative diseases of humans and animals. Despite years of research, no therapeutic agents have been developed that can effectively manage or reverse disease progression. Recently it has been identified that recombinant prion proteins (rPrP) expressed in bacteria can act as inhibitors of prion replication within the in vitro prion replication system protein misfolding cyclic amplification (PMCA). Here, within PMCA reactions amplifying a range of ruminant prions including distinct Prnp genotypes/host species and distinct prion strains, recombinant ovine VRQ PrP displayed consistent inhibition of prion replication and produced IC50 values of 122 and 171 nM for ovine scrapie and bovine BSE replication, respectively. These findings illustrate the therapeutic potential of rPrPs with distinct TSE diseases.

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Differentiating Ovine BSE from CH1641 Scrapie by Serial Protein Misfolding Cyclic Amplification

Cited by 10 publications

References 45 publications

Highly Sensitive Detection of Small Ruminant Bovine Spongiform Encephalopathy within Transmissible Spongiform Encephalopathy Mixes by Serial Protein Misfolding Cyclic Amplification

Highly Sensitive Detection of Small Ruminant Bovine Spongiform Encephalopathy within Transmissible Spongiform Encephalopathy Mixes by Serial Protein Misfolding Cyclic Amplification

In vitro amplification of H-type atypical bovine spongiform encephalopathy by protein misfolding cyclic amplification

Ovine recombinant PrP as an inhibitor of ruminant prion propagation in vitro

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