scholarly journals PrPSc detection and infectivity in semen from scrapie-infected sheep

2012 ◽  
Vol 93 (6) ◽  
pp. 1375-1383 ◽  
Author(s):  
Richard Rubenstein ◽  
Marie S. Bulgin ◽  
Binggong Chang ◽  
Sharon Sorensen-Melson ◽  
Robert B. Petersen ◽  
...  
Keyword(s):  
Incubation Time ◽  
Protein Misfolding ◽  
Infected Sheep ◽  
Transmissible Spongiform Encephalopathies ◽  
Animal Testing ◽  
Live Animal ◽  
Transgenic Mouse Line ◽  
Protein Misfolding Cyclic Amplification ◽  
Scrapie Strain ◽  
Short Incubation Time

A scrapie-positive ewe was found in a flock that had been scrapie-free for 13 years, but housed adjacent to scrapie-positive animals, separated by a wire fence. Live animal testing of the entire flock of 24 animals revealed seven more subclinical scrapie-positive ewes. We hypothesized that they may have contracted the disease from scrapie-positive rams used for breeding 4 months prior, possibly through the semen. The genotypes of the ewe flock were highly scrapie-susceptible and the rams were infected with the ‘Caine’ scrapie strain having a short incubation time of 4.3–14.6 months in sheep with 136/171 VQ/VQ and AQ/VQ genotypes. PrPSc accumulates in a variety of tissues in addition to the central nervous system. Although transmission of prion diseases, or transmissible spongiform encephalopathies, has been achieved via peripheral organ or tissue homogenates as well as by blood transfusion, neither infectivity nor PrPSc have been found in semen from scrapie-infected animals. Using serial protein misfolding cyclic amplification followed by a surround optical fibre immunoassay, we demonstrate that semen from rams infected with a short-incubation-time scrapie strain contains prion disease-associated-seeding activity that generated PrPSc in sPMCA (serial protein misfolding cyclic amplification). Injection of the ovinized transgenic mouse line TgSShpPrP with semen from scrapie-infected sheep resulted in PrPSc-seeding activity in clinical and, probably as a result of the low titre, non-clinical mouse brain. These results suggest that the transmissible agent, or at least the seeding activity, for sheep scrapie is present in semen. This may be a strain-specific phenomenon.

scholarly journals Hyperefficient PrPScamplification of mouse-adapted BSE and scrapie strain by protein misfolding cyclic amplification technique

FEBS Journal ◽  
2009 ◽  
Vol 276 (10) ◽  
pp. 2841-2848 ◽  
Author(s):  
Aiko Fujihara ◽  
Ryuichiro Atarashi ◽  
Takayuki Fuse ◽  
Kaori Ubagai ◽  
Takehiro Nakagaki ◽  
...  
Keyword(s):  
Protein Misfolding ◽  
Protein Misfolding Cyclic Amplification ◽  
Scrapie Strain ◽  
Amplification Technique

scholarly journals Preclinical Diagnosis of Scrapie by Immunohistochemistry of Third Eyelid Lymphoid Tissue

2000 ◽  
Vol 38 (9) ◽  
pp. 3254-3259 ◽  
Author(s):  
K. I. O'Rourke ◽  
T. V. Baszler ◽  
T. E. Besser ◽  
J. M. Miller ◽  
R. C. Cutlip ◽  
...  
Keyword(s):  
Lymphoid Tissue ◽  
Mammalian Species ◽  
Clinical Disease ◽  
Transmissible Spongiform Encephalopathies ◽  
Lymphoid Tissues ◽  
Animal Testing ◽  
Live Animal ◽  
Spongiform Encephalopathies ◽  
Clinically Normal ◽  
Neurologic Disorders

Ovine scrapie is a member of the transmissible spongiform encephalopathies (TSEs), a heterogeneous family of fatal neurologic disorders characterized by deposition of an abnormal isoform (prion protein [PrP] PrP-Sc) of a cellular sialoglycoprotein in neural tissue. PrP-Sc is detectable in some lymphoid tissues of infected sheep months or years before development of clinical disease. Detection of PrP-Sc in these tissues is the basis for live-animal testing. In this study, we characterize the performance of a preclinical diagnostic test for ovine scrapie based on a monoclonal antibody (MAb)-based immunohistochemistry assay of nictitating membrane (“third eyelid”)-associated lymphoid tissue. The results of third eyelid immunohistochemistry assay agreed with the scrapie status of the sheep for 41 of 42 clinical suspects with confirmed scrapie and 174 of 175 sheep without scrapie. Third eyelid sampling agreed with the scrapie status for 36 of 41 clinically normal sheep positive for PrP-Sc immunostaining of brain tissue, including 27 sheep with positive biopsy specimens that progressed to clinical disease with confirmed scrapie 3 to 20 months after biopsy. The assay used MAb F89/160.1.5, which binds to residues 142 to 145 of ovine PrP. This antibody can be used in combination with MAb F99/97.6.1, which binds to residues 220 to 225. One or both MAbs in this cocktail recognize PrP sequences conserved in most mammalian species in which natural TSEs have been reported. Immunohistochemistry assay of routinely formalin-fixed lymphoid tissues with a cocktail of pan-specific MAbs is a practical, readily standardized live-animal and preclinical test for ovine scrapie.

scholarly journals Sensitive protein misfolding cyclic amplification of sporadic Creutzfeldt–Jakob disease prions is strongly seed and substrate dependent

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maxime Bélondrade ◽  
Simon Nicot ◽  
Charly Mayran ◽  
Lilian Bruyere-Ostells ◽  
Florian Almela ◽  
...  
Keyword(s):  
Prion Protein ◽  
Protein Misfolding ◽  
Protein Misfolding Cyclic Amplification ◽  
Bank Voles ◽  
Substrate Dependence ◽  
Brain Extracts ◽  
Jakob Disease ◽  
Human Prion Protein

AbstractUnlike variant Creutzfeldt–Jakob disease prions, sporadic Creutzfeldt–Jakob disease prions have been shown to be difficult to amplify in vitro by protein misfolding cyclic amplification (PMCA). We assessed PMCA of pathological prion protein (PrPTSE) from 14 human sCJD brain samples in 3 substrates: 2 from transgenic mice expressing human prion protein (PrP) with either methionine (M) or valine (V) at position 129, and 1 from bank voles. Brain extracts representing the 5 major clinicopathological sCJD subtypes (MM1/MV1, MM2, MV2, VV1, and VV2) all triggered seeded PrPTSE amplification during serial PMCA with strong seed- and substrate-dependence. Remarkably, bank vole PrP substrate allowed the propagation of all sCJD subtypes with preservation of the initial molecular PrPTSE type. In contrast, PMCA in human PrP substrates was accompanied by a PrPTSE molecular shift during heterologous (M/V129) PMCA reactions, with increased permissiveness of V129 PrP substrate to in vitro sCJD prion amplification compared to M129 PrP substrate. Combining PMCA amplification sensitivities with PrPTSE electrophoretic profiles obtained in the different substrates confirmed the classification of 4 distinct major sCJD prion strains (M1, M2, V1, and V2). Finally, the level of sensitivity required to detect VV2 sCJD prions in cerebrospinal fluid was achieved.

scholarly journals Evaluation of a Preclinical Blood Test for Scrapie in Sheep Using Immunocapillary Electrophoresis

2006 ◽  
Vol 89 (3) ◽  
pp. 720-727 ◽  
Author(s):  
Roy Jackman ◽  
David J Everest ◽  
Mary Jo Schmerr ◽  
Mohammed Khawaja ◽  
Pat Keep ◽  
...  
Keyword(s):  
Capillary Electrophoresis ◽  
Prion Protein ◽  
Clinical Signs ◽  
Test System ◽  
Buffy Coat ◽  
Infected Sheep ◽  
Test Method ◽  
Peptide Sequence ◽  
Transmissible Spongiform Encephalopathies ◽  
Blood Samples

Abstract An analytical method is described for detection of endogenous disease-associated prion protein in the buffy coat fraction from the blood of sheep infected with scrapie. The method has been improved and evaluated for its performance in the preclinical diagnosis of ovine transmissible spongiform encephalopathies. The test system uses a protocol for sample preparation that includes extraction and concentration and a test method that uses a liquid-phase competitive immunoassay for prion protein. Antibodies directed to a peptide sequence at the C-terminus of the prion protein (PrP) and a fluorescein-labeled peptide conjugate are used in the assay. Free zone capillary electrophoresis with laser-induced fluorescence for detection is used to separate the antibody-bound fluorescently labeled peptide and free labeled peptide. In this assay, the PrP competes with the fluorescently labeled peptide for limited antibody binding sites, which results in a reduction of the peak representing the immunocomplex of the antibody bound to the fluorescently labeled peptide. When blood samples from scrapie-infected sheep aged 712 months and of the scrapie-susceptible PrP genotypes VRQ/VRQ and VRQ/ARQ were analyzed, the abnormal PrP was found in blood samples. These results correlated with the post-mortem diagnosis of scrapie. The sheep were preclinical and appeared normal at the time of testing but later died with clinical disease approximately 12 months after testing. In older animals, and those with clinical signs, a smaller percentage of animals tested positive. This study has demonstrated that this technology can be used as a sensitive, rapid preclinical test to detect the disease-associated PrP in the blood of scrapie-infected sheep. Improvements in the extraction protocol and capillary electrophoresis conditions will enhance the robustness of this test.

scholarly journals Protein misfolding cyclic amplification of infectious prions

2012 ◽  
Vol 7 (7) ◽  
pp. 1397-1409 ◽  
Author(s):  
Rodrigo Morales ◽  
Claudia Duran-Aniotz ◽  
Rodrigo Diaz-Espinoza ◽  
Manuel V Camacho ◽  
Claudio Soto
Keyword(s):  
Protein Misfolding ◽  
Protein Misfolding Cyclic Amplification

Optimal Geometric Control Applied to the Protein Misfolding Cyclic Amplification Process

2014 ◽  
Vol 135 (1) ◽  
pp. 145-173 ◽  
Author(s):  
Monique Chyba ◽  
Jean-Michel Coron ◽  
Pierre Gabriel ◽  
Alain Jacquemard ◽  
Geoff Patterson ◽  
...  
Keyword(s):  
Protein Misfolding ◽  
Geometric Control ◽  
Protein Misfolding Cyclic Amplification ◽  
Amplification Process
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