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

2009 ◽  
Vol 83 (23) ◽  
pp. 12552-12558 ◽  
Author(s):  
L. A. Terry ◽  
L. Howells ◽  
J. Hawthorn ◽  
J. C. Edwards ◽  
S. J. Moore ◽  
...  
Keyword(s):  
Bovine Spongiform Encephalopathy ◽  
Transmissible Spongiform Encephalopathy ◽  
Clinical Signs ◽  
Surrogate Marker ◽  
Infected Sheep ◽  
Clinical Disease ◽  
Spongiform Encephalopathy ◽  
Scrapie Agent

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.

scholarly journals PrP P102L and Nearby Lysine Mutations Promote Spontaneous In Vitro Formation of Transmissible Prions

2017 ◽  
Vol 91 (21) ◽  
Author(s):  
Allison Kraus ◽  
Gregory J. Raymond ◽  
Brent Race ◽  
Katrina J. Campbell ◽  
Andrew G. Hughson ◽  
...  
Keyword(s):  
Prion Protein ◽  
Prion Diseases ◽  
Transmissible Spongiform Encephalopathy ◽  
Clinical Signs ◽  
Protein Aggregates ◽  
Structural Features ◽  
Spongiform Encephalopathy ◽  
Specific Sequence

ABSTRACT Accumulation of fibrillar protein aggregates is a hallmark of many diseases. While numerous proteins form fibrils by prion-like seeded polymerization in vitro, only some are transmissible and pathogenic in vivo. To probe the structural features that confer transmissibility to prion protein (PrP) fibrils, we have analyzed synthetic PrP amyloids with or without the human prion disease-associated P102L mutation. The formation of infectious prions from PrP molecules in vitro has required cofactors and/or unphysiological denaturing conditions. Here, we demonstrate that, under physiologically compatible conditions without cofactors, the P102L mutation in recombinant hamster PrP promoted prion formation when seeded by minute amounts of scrapie prions in vitro. Surprisingly, combination of the P102L mutation with charge-neutralizing substitutions of four nearby lysines promoted spontaneous prion formation. When inoculated into hamsters, both of these types of synthetic prions initiated substantial accumulation of prion seeding activity and protease-resistant PrP without transmissible spongiform encephalopathy (TSE) clinical signs or notable glial activation. Our evidence suggests that PrP's centrally located proline and lysine residues act as conformational switches in the in vitro formation of transmissible PrP amyloids. IMPORTANCE Many diseases involve the damaging accumulation of specific misfolded proteins in thread-like aggregates. These threads (fibrils) are capable of growing on the ends by seeding the refolding and incorporation of the normal form of the given protein. In many cases such aggregates can be infectious and propagate like prions when transmitted from one individual host to another. Some transmitted aggregates can cause fatal disease, as with human iatrogenic prion diseases, while other aggregates appear to be relatively innocuous. The factors that distinguish infectious and pathogenic protein aggregates from more innocuous ones are poorly understood. Here we have compared the combined effects of prion seeding and mutations of prion protein (PrP) on the structure and transmission properties of synthetic PrP aggregates. Our results highlight the influence of specific sequence features in the normally unstructured region of PrP that influence the infectious and neuropathogenic properties of PrP-derived aggregates.

scholarly journals Prion Type-Dependent Deposition ofPRNPAllelic Products in Heterozygous Sheep

2015 ◽  
Vol 90 (2) ◽  
pp. 805-812 ◽  
Author(s):  
J. P. M. Langeveld ◽  
J. G. Jacobs ◽  
N. Hunter ◽  
L. J. M. van Keulen ◽  
F. Lantier ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Bovine Spongiform Encephalopathy ◽  
Prion Protein ◽  
Transmissible Spongiform Encephalopathy ◽  
Infected Sheep ◽  
Transmissible Spongiform Encephalopathies ◽  
Spongiform Encephalopathy ◽  
Sequence Variants ◽  
Spongiform Encephalopathies

ABSTRACTSusceptibility or resistance to prion infection in humans and animals depends on single prion protein (PrP) amino acid substitutions in the host, but the agent's modulating role has not been well investigated. Compared to disease incubation times in wild-type homozygous ARQ/ARQ (where each triplet represents the amino acids at codons 136, 154, and 171, respectively) sheep, scrapie susceptibility is reduced to near resistance in ARR/ARR animals while it is strongly enhanced in VRQ/VRQ carriers. Heterozygous ARR/VRQ animals exhibit delayed incubation periods. In bovine spongiform encephalopathy (BSE) infection, the polymorphism effect is quite different although the ARR allotype remains the least susceptible. In this study, PrP allotype composition in protease-resistant prion protein (PrPres) from brain of heterozygous ARR/VRQ scrapie-infected sheep was compared with that of BSE-infected sheep with a similar genotype. A triplex Western blotting technique was used to estimate the two allotype PrP fractions in PrPresmaterial from BSE-infected ARR/VRQ sheep. PrPresin BSE contained equimolar amounts of VRQ- and ARR-PrP, which contrasts with the excess (>95%) VRQ-PrP fraction found in PrP in scrapie. This is evidence that transmissible spongiform encephalopathy (TSE) agent properties alone, perhaps structural aspects of prions (such as PrP amino acid sequence variants and PrP conformational state), determine the polymorphic dependence of the PrPresaccumulation process in prion formation as well as the disease-associated phenotypic expressions in the host.IMPORTANCETransmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative and transmissible diseases caused by prions. Amino acid sequence variants of the prion protein (PrP) determine transmissibility in the hosts, as has been shown for classical scrapie in sheep. Each individual produces a separate PrP molecule from its two PrP gene copies. Heterozygous scrapie-infected sheep that produce two PrP variants associated with opposite scrapie susceptibilities (136V-PrP variant, high; 171R-PrP variant, very low) contain in their prion material over 95% of the 136V PrP variant. However, when these sheep are infected with prions from cattle (bovine spongiform encephalopathy [BSE]), both PrP variants occur in equal ratios. This shows that the infecting prion type determines the accumulating PrP variant ratio in the heterozygous host. While the host's PrP is considered a determining factor, these results emphasize that prion structure plays a role during host infection and that PrP variant involvement in prions of heterozygous carriers is a critical field for understanding prion formation.

scholarly journals Does the Presence of Scrapie Affect the Ability of Current Statutory Discriminatory Tests To Detect the Presence of Bovine Spongiform Encephalopathy?

2015 ◽  
Vol 53 (8) ◽  
pp. 2593-2604 ◽  
Author(s):  
M. M. Simmons ◽  
M. J. Chaplin ◽  
C. M. Vickery ◽  
S. Simon ◽  
L. Davis ◽  
...  
Keyword(s):  
Bovine Spongiform Encephalopathy ◽  
Protein Misfolding ◽  
Transmissible Spongiform Encephalopathy ◽  
Cost Effective ◽  
Phenotypic Characterization ◽  
Enzyme Linked Immunosorbent Assay ◽  
Spongiform Encephalopathy ◽  
Cost Effective Alternative ◽  
Variable Success

Current European Commission (EC) surveillance regulations require discriminatory testing of all transmissible spongiform encephalopathy (TSE)-positive small ruminant (SR) samples in order to classify them as bovine spongiform encephalopathy (BSE) or non-BSE. This requires a range of tests, including characterization by bioassay in mouse models. Since 2005, naturally occurring BSE has been identified in two goats. It has also been demonstrated that more than one distinct TSE strain can coinfect a single animal in natural field situations. This study assesses the ability of the statutory methods as listed in the regulation to identify BSE in a blinded series of brain samples, in which ovine BSE and distinct isolates of scrapie are mixed at various ratios ranging from 99% to 1%. Additionally, these current statutory tests were compared with a newin vitrodiscriminatory method, which uses serial protein misfolding cyclic amplification (sPMCA). Western blotting consistently detected 50% BSE within a mixture, but at higher dilutions it had variable success. The enzyme-linked immunosorbent assay (ELISA) method consistently detected BSE only when it was present as 99% of the mixture, with variable success at higher dilutions. Bioassay and sPMCA reported BSE in all samples where it was present, down to 1%. sPMCA also consistently detected the presence of BSE in mixtures at 0.1%. While bioassay is the only validated method that allows comprehensive phenotypic characterization of an unknown TSE isolate, the sPMCA assay appears to offer a fast and cost-effective alternative for the screening of unknown isolates when the purpose of the investigation was solely to determine the presence or absence of BSE.

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

2007 ◽  
Vol 81 (19) ◽  
pp. 10532-10539 ◽  
Author(s):  
Jonathan P. Owen ◽  
Helen C. Rees ◽  
Ben C. Maddison ◽  
Linda A. Terry ◽  
Leigh Thorne ◽  
...  
Keyword(s):  
Prion Diseases ◽  
Transmissible Spongiform Encephalopathy ◽  
Spongiform Encephalopathy ◽  
Western Blots ◽  
Sheep Population ◽  
Natural Scrapie ◽  
Caudal Medulla ◽  
Scrapie Strains

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.

Rapid recontamination of a farm building occurs after attempted prion removal

2019 ◽  
Vol 184 (3) ◽  
pp. 97-97 ◽  
Author(s):  
Kevin Christopher Gough ◽  
Claire Alison Baker ◽  
Steve Hawkins ◽  
Hugh Simmons ◽  
Timm Konold ◽  
...  
Keyword(s):  
Health Management ◽  
Transmissible Spongiform Encephalopathy ◽  
Immunohistochemical Analysis ◽  
Spongiform Encephalopathy ◽  
Wasting Disease ◽  
Replication Assay ◽  
Scrapie Agent ◽  
Farm Buildings ◽  
Protein Genotype

The transmissible spongiform encephalopathy scrapie of sheep/goats and chronic wasting disease of cervids are associated with environmental reservoirs of infectivity. Preventing environmental prions acting as a source of infectivity to healthy animals is of major concern to farms that have had outbreaks of scrapie and also to the health management of wild and farmed cervids. Here, an efficient scrapie decontamination protocol was applied to a farm with high levels of environmental contamination with the scrapie agent. Post-decontamination, no prion material was detected within samples taken from the farm buildings as determined using a sensitive in vitro replication assay (sPMCA). A bioassay consisting of 25 newborn lambs of highly susceptible prion protein genotype VRQ/VRQ introduced into this decontaminated barn was carried out in addition to sampling and analysis of dust samples that were collected during the bioassay. Twenty-four of the animals examined by immunohistochemical analysis of lymphatic tissues were scrapie-positive during the bioassay, samples of dust collected within the barn were positive by month 3. The data illustrates the difficulty in decontaminating farm buildings from scrapie, and demonstrates the likely contribution of farm dust to the recontamination of these environments to levels that are capable of causing disease.

scholarly journals Ultrasensitive detection of PrPSc in the cerebrospinal fluid and blood of macaques infected with bovine spongiform encephalopathy prion

2014 ◽  
Vol 95 (11) ◽  
pp. 2576-2588 ◽  
Author(s):  
Yuichi Murayama ◽  
Kentaro Masujin ◽  
Morikazu Imamura ◽  
Fumiko Ono ◽  
Hiroaki Shibata ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Bovine Spongiform Encephalopathy ◽  
White Blood Cells ◽  
Clinical Signs ◽  
Cynomolgus Macaque ◽  
Cellular Protein ◽  
Spongiform Encephalopathy ◽  
Peripheral Tissues ◽  
Human Primate

Prion diseases are characterized by the prominent accumulation of the misfolded form of a normal cellular protein (PrPSc) in the central nervous system. The pathological features and biochemical properties of PrPSc in macaque monkeys infected with the bovine spongiform encephalopathy (BSE) prion have been found to be similar to those of human subjects with variant Creutzfeldt–Jakob disease (vCJD). Non-human primate models are thus ideally suited for performing valid diagnostic tests and determining the efficacy of potential therapeutic agents. In the current study, we developed a highly efficient method for in vitro amplification of cynomolgus macaque BSE PrPSc. This method involves amplifying PrPSc by protein misfolding cyclic amplification (PMCA) using mouse brain homogenate as a PrPC substrate in the presence of sulfated dextran compounds. This method is capable of amplifying very small amounts of PrPSc contained in the cerebrospinal fluid (CSF) and white blood cells (WBCs), as well as in the peripheral tissues of macaques that have been intracerebrally inoculated with the BSE prion. After clinical signs of the disease appeared in three macaques, we detected PrPSc in the CSF by serial PMCA, and the CSF levels of PrPSc tended to increase with disease progression. In addition, PrPSc was detectable in WBCs at the clinical phases of the disease in two of the three macaques. Thus, our highly sensitive, novel method may be useful for furthering the understanding of the tissue distribution of PrPSc in non-human primate models of CJD.

scholarly journals T2-Weighted MRI Signal Alterations in the Early-Clinical Phase of Transmissible Spongiform Encephalopathy in a Scrapie Rodent Model

2009 ◽  
Vol 3 ◽  
pp. MRI.S2550
Author(s):  
G. Carpinelli ◽  
R. Canese ◽  
V. Vetrugno ◽  
M.A. Di Bari ◽  
F. Santoro ◽  
...  
Keyword(s):  
Transmissible Spongiform Encephalopathy ◽  
Brain Mri ◽  
Clinical Signs ◽  
Rodent Model ◽  
Spongiform Encephalopathy ◽  
Thalamic Nuclei ◽  
Clinical Phase ◽  
Longitudinal Mri ◽  
Scrapie Strain

Background and Purpose Transmissible spongiform encephalopathy (TSE) diseases are fatal, progressive neurodegenerative disorders affecting both humans and animals. Clinical signs typically appear after years and even decades of silent disease progression. This study was aimed at investigating whether altered brain MRI patterns may precede clinical signs in a TSE rodent model. Methods In vivo T2-weighted (T2W) MRI examinations (4.7 T) were performed on Golden Syrian hamsters (GSH) intracerebrally, orally, or intraperitoneally (i.p.) infected with the 263K scrapie strain. Histopathological analyses were performed on i.p. infected GSH at the end of one-day or longitudinal MRI sessions. Results T2W-MRI hyperintensity was detected in the thalamic nuclei of GSH with clinical signs, irrespective of the infection route. Hyperintensity in the thalamus was also observed in pre-clinical animals, between 106 and 121 days post-infection (dpi), while normal T2W intensity was detected in four animals examined between 72 and 96 dpi. Pathological prion protein deposition (but no astrogliosis and only occasionally, weak spongiosis) was detected between 106 and 121 dpi. Conclusions The altered T2W-MRI pattern detected in the thalamus of asymptomatic i.p. infected GSH provides a useful basis for evaluating the effectiveness of possible therapeutic approaches at early stages of TSE disease.

scholarly journals Characterization and polyanion-binding properties of purified recombinant prion protein

1999 ◽  
Vol 342 (3) ◽  
pp. 605-613 ◽  
Author(s):  
Debbie B. BRIMACOMBE ◽  
Alan D. BENNETT ◽  
Fred S. WUSTEMAN ◽  
Andrew C. GILL ◽  
Janine C. DANN ◽  
...  
Keyword(s):  
Prion Protein ◽  
Mammalian Cells ◽  
Metal Ion ◽  
Transmissible Spongiform Encephalopathy ◽  
Spongiform Encephalopathy ◽  
Divalent Metal Ions ◽  
Dissociation Kinetics ◽  
Recombinant Prion Protein

Certain polysulphated polyanions have been shown to have prophylactic effects on the progression of transmissible spongiform encephalopathy disease, presumably because they bind to prion protein (PrP). Until now, the difficulty of obtaining large quantities of native PrP has precluded detailed studies of these interactions. We have over-expressed murine recombinant PrP (recPrP), lacking its glycophosphoinositol membrane anchor, in modified mammalian cells. Milligram quantities of secreted, soluble and partially glycosylated protein were purified under non-denaturing conditions and the identities of mature-length aglycosyl recPrP and two cleavage fragments were determined by electrospray MS. Binding was assessed by surface plasmon resonance techniques using both direct and competitive ligand-binding approaches. recPrP binding to immobilized polyanions was enhanced by divalent metal ions. Polyanion binding was strong and showed complex association and dissociation kinetics that were consistent with ligand-directed recPrP aggregation. The differences in the binding strengths of recPrP to pentosan polysulphate and to other sulphated polyanions were found to parallel their in vivo anti-scrapie and in vitro anti-scrapie-specific PrP formation potencies. When recPrP was immobilized by capture on metal-ion chelates it was found, contrary to expectation, that the addition of polyanions promoted the dissociation of the protein.

scholarly journals Prion Strain- and Species-Dependent Effects of Antiprion Molecules in Primary Neuronal Cultures

2007 ◽  
Vol 81 (24) ◽  
pp. 13794-13800 ◽  
Author(s):  
Sabrina Cronier ◽  
Vincent Beringue ◽  
Anne Bellon ◽  
Jean-Michel Peyrin ◽  
Hubert Laude
Keyword(s):  
Transmissible Spongiform Encephalopathy ◽  
Cell System ◽  
Transmissible Spongiform Encephalopathies ◽  
Spongiform Encephalopathy ◽  
Neuronal Cultures ◽  
Primary Neuronal Cultures ◽  
Spongiform Encephalopathies ◽  
The Central Nervous System

ABSTRACT Transmissible spongiform encephalopathies (TSE) arise as a consequence of infection of the central nervous system by prions and are incurable. To date, most antiprion compounds identified by in vitro screening failed to exhibit therapeutic activity in animals, thus calling for new assays that could more accurately predict their in vivo potency. Primary nerve cell cultures are routinely used to assess neurotoxicity of chemical compounds. Here, we report that prion strains from different species can propagate in primary neuronal cultures derived from transgenic mouse lines overexpressing ovine, murine, hamster, or human prion protein. Using this newly developed cell system, the activity of three generic compounds known to cure prion-infected cell lines was evaluated. We show that the antiprion activity observed in neuronal cultures is species or strain dependent and recapitulates to some extent the activity reported in vivo in rodent models. Therefore, infected primary neuronal cultures may be a relevant system in which to investigate the efficacy and mode of action of antiprion drugs, including toward human transmissible spongiform encephalopathy agents.

156 INVESTIGATION OF BOVINE HERPESVIRUS IN CUMULUS - OOCYTE COMPLEXES AND FOLLICULAR FLUID

2008 ◽  
Vol 20 (1) ◽  
pp. 158
Author(s):  
A. P. Oliveira ◽  
R. C. Leite ◽  
M. B. Heinmam ◽  
L. G. B. Siqueira ◽  
A. Maciel ◽  
...  
Keyword(s):  
Follicular Fluid ◽  
Stress Condition ◽  
Clinical Signs ◽  
Negative Control ◽  
In Vivo Model ◽  
Spongiform Encephalopathy ◽  
Vitro Fertilization ◽  
Virus Serotype

The world market for bovine embryos has increased in the past few years. However, sanitary problems such as foot and mouth disease in Brazil, vesicular stomatitis in South America, and bovine spongiform encephalopathy (BSE) in North America and Europe have increased concerns regarding the risk of introducing exotic diseases and/or more virulent serotypes of endemic diseases by embryo transfer. Many countries are trying to develop and/or improve new techniques for infectious disease detection, with the scientific basis to support the import and export of animal germplasm. Therefore, the epidemiology of the diseases and the interaction between pathogens and cumulus–oocyte complexes (COCs), embryos, and semen must be investigated. Despite the many studies that have been carried out to evaluate the possibility of transmission of infectious agents by the embryo, few data are available regarding COC susceptibility (Tsuboi et al. 1992 J. Vet. Med. Sci. 54, 1179–1181). The aim of this study was to evaluate the presence of bovine herpes virus serotype 1 (BHV-1) in COCs and follicular fluid (FF) collected from naturally infected animals in a low stress condition. Blood samples of non-lactating Gyr breed (Bos indicus) cows were collected and evaluated for BHV-1 antibodies by the serum neutralization microplate test, performed as described in the Manual for Standards for Diagnostic Tests and Vaccines (OIE, 1992). The cows were diagnosed as serologically positive (n = 38) or serologically negative (n = 8), and kept under grazing in Brachiaria decumbens pasture with mineral supplementation. The cows considered as positive showed titers greater than 1/4. COCs and follicular fluid (FF) were obtained by ovum pick-up (OPU) using sterile and disposable materials for each animal. Virus detection was performed by the PCR technique. PCR sensitivity was made using COCs and FF recovered from eight BHV-1 serologically negative animals. These samples were either artificially infected on plates with 106.5 TCID in 50 µL of IBR Colorado 1 reference serotype (ATCC, VR-864) or used as a negative control. The PCR analitical sensitivity was 100.5 TCID. The presence of BHV-1 in COCs and FF was not detected in any of the animals, despite the high sensitivity of the PCR technique. In the present in vivo model, results show that COCs collected from serologically BHV-1 positive cows presenting no clinical signs of the illness and managed in a low stress condition could be used as donors for in vitro fertilization procedures with minimal sanitary risks. Also, the absence of the virus in COCs and FF cannot be used as a predictor of BHV-1 infection status in bovine herds.

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