Genetic and Pathological Follow-Up Study of Goats Experimentally and Naturally Exposed to a Sheep Scrapie Isolate

PubMed Central

Maestrale, Caterina; Cancedda, Maria G.; Pintus, Davide; Masia, Mariangela; Nonno, Romolo; Ru, Giuseppe; Carta, Antonello; Demontis, Francesca; Santucciu, Cinzia

2015-01-01

ABSTRACT Thirty-seven goats carrying different prion protein genotypes (PRNP) were orally infected with a classical scrapie brain homogenate from wild-type (ARQ/ARQ) sheep and then mated to obtain 2 additional generations of offspring, which were kept in the same environment and allowed to be naturally exposed to scrapie. Occurrence of clinical or subclinical scrapie was observed in the experimentally infected goats (F0) and in only one (F1b) of the naturally exposed offspring groups. In both groups (F0 and F1b), goats carrying the R154H, H154H, R211Q, and P168Q-P240P dimorphisms died of scrapie after a longer incubation period than wild-type, G37V, Q168Q-P240P, and S240P goats. In contrast, D145D and Q222K goats were resistant to infection. The immunobiochemical signature of the scrapie isolate and its pathological aspects observed in the sheep donors were substantially maintained over 2 goat generations, i.e., after experimental and natural transmission. This demonstrates that the prion protein gene sequence, which is shared by sheep and goats, is more powerful than any possible but unknown species-related factors in determining scrapie phenotypes. With regard to genetics, our study confirms that the K222 mutation protects goats even against ovine scrapie isolates, and for the first time, a possible association of D145 mutation with scrapie resistance is shown. In addition, it is possible that the sole diverse frequencies of these genetic variants might, at least in part, shape the prevalence of scrapie among naturally exposed progenies in affected herds. IMPORTANCE This study was aimed at investigating the genetic and pathological features characterizing sheep-to-goat transmission of scrapie. We show that in goats with different prion protein gene mutations, the K222 genetic variant is associated with scrapie resistance after natural and experimental exposure to ovine prion infectivity. In addition, we observed for the first time a protective effect of the D145 goat variant against scrapie. Importantly, our results demonstrate that the phenotypic characteristic of the wild-type sheep scrapie isolate is substantially preserved in goats carrying different susceptible PRNP gene variants, thus indicating that the prion protein gene sequence, which is shared by sheep and goats, plays a fundamental role in determining scrapie phenotypes. PMID:26202249

Using mass spectrometry to determine the relative susceptibility of PrP polymorphisms to atypical scrapie

USDA-ARS?s Scientific Manuscript database

A novel form of scrapie was described in 1998 and referred to as Nor98 for the country of origin and date of its discovery. Since then it has been found in numerous countries, including New Zealand and Australia, and has been renamed atypical scrapie. Unlike classical scrapie, the epidemiology of th...

Design and Implementation of Distributed Crawler System Based on Scrapy

NASA Astrophysics Data System (ADS)

Fan, Yuhao

2018-01-01

At present, some large-scale search engines at home and abroad only provide users with non-custom search services, and a single-machine web crawler cannot sovle the difficult task. In this paper, Through the study and research of the original Scrapy framework, the original Scrapy framework is improved by combining Scrapy and Redis, a distributed crawler system based on Web information Scrapy framework is designed and implemented, and Bloom Filter algorithm is applied to dupefilter modul to reduce memory consumption. The movie information captured from douban is stored in MongoDB, so that the data can be processed and analyzed. The results show that distributed crawler system based on Scrapy framework is more efficient and stable than the single-machine web crawler system.

Skin-derived dendritic cells acquire and degrade the scrapie agent following in vitro exposure

PubMed Central

Mohan, Joanne; Hopkins, John; Mabbott, Neil A

2005-01-01

The accumulation of the scrapie agent in lymphoid tissues following inoculation via the skin is critical for efficient neuroinvasion, but how the agent is initially transported from the skin to the draining lymph node is not known. Langerhans cells (LCs) are specialized antigen-presenting cells that continually sample their microenvironment within the epidermis and transport captured antigens to draining lymph nodes. We considered LCs probable candidates to acquire and transport the scrapie agent after inoculation via the skin. XS106 cells are dendritic cells (DCs) isolated from mouse epidermis with characteristics of mature LC cells. To investigate the potential interaction of LCs with the scrapie agent XS106 cells were exposed to the scrapie agent in vitro. We show that XS106 cells rapidly acquire the scrapie agent following in vitro exposure. In addition, XS106 cells partially degrade the scrapie agent following extended cultivation. These data suggest that LCs might acquire and degrade the scrapie agent after inoculation via the skin, but data from additional experiments demonstrate that this ability could be lost in the presence of lipopolysaccharide or other immunostimulatory molecules. Our studies also imply that LCs would not undergo maturation following uptake of the scrapie agent in the skin, as the expression of surface antigens associated with LC maturation were unaltered following exposure. In conclusion, although LCs or DCs have the potential to acquire the scrapie agent within the epidermis our data suggest it is unlikely that they become activated and stimulated to transport the agent to the draining lymph node. PMID:16108824

Goats singly heterozygous for PRNP S146 or K222 orally inoculated with classical scrapie at birth show no disease at ages well beyond six years

USDA-ARS?s Scientific Manuscript database

Scrapie is a transmissible spongiform encephalopathy of sheep and goats, and scrapie eradication programs in many parts of the world rely on strong genetic resistance to classical scrapie in sheep. However, the utility of putative resistance alleles in goats has been a focus of research because goat...

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

PubMed Central

Gough, Kevin C.; Baker, Claire A.; Rees, Helen C.; Terry, Linda A.; Spiropoulos, John; Thorne, Leigh

2012-01-01

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 PrPSc within lymphoreticular tissues. PrPSc 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. PMID:22013047

Evidence of scrapie transmission to sheep via goat milk.

PubMed

Konold, Timm; Thorne, Leigh; Simmons, Hugh A; Hawkins, Steve A C; Simmons, Marion M; González, Lorenzo

2016-09-17

Previous studies confirmed that classical scrapie can be transmitted via milk in sheep. The current study aimed to investigate whether scrapie can also be transmitted via goat milk using in vivo (new-born lambs fed milk from scrapie-affected goats due to the unavailability of goat kids from guaranteed scrapie-free herds) and in vitro methods (serial protein misfolding cyclic amplification [sPMCA] on milk samples). In an initial pilot study, new-born lambs of two different prion protein gene (PRNP) genotypes (six VRQ/VRQ and five ARQ/ARQ) were orally challenged with 5 g brain homogenate from two scrapie-affected goats to determine susceptibility of sheep to goat scrapie. All sheep challenged with goat scrapie brain became infected based on the immunohistochemical detection of disease-associated PrP (PrP(sc)) in lymphoid tissue, with an ARQ/ARQ sheep being the first to succumb. Subsequent feeding of milk to eight pairs of new-born ARQ/ARQ lambs, with each pair receiving milk from a different scrapie-affected goat, resulted in scrapie in the six pairs that received the largest volume of milk (38-87 litres per lamb), whereas two pairs fed 8-9 litres per lamb, and an environmental control group raised on sheep milk from healthy ewes, did not show evidence of infection when culled at up to 1882 days of age. Infection in those 12 milk recipients occurred regardless of the clinical status, PrP(sc) distribution, caprine arthritis-encephalitis virus infection status and PRNP polymorphisms at codon 142 (II or IM) of the donor goats, but survival time was influenced by PRNP polymorphisms at codon 141. Serial PMCA applied to a total of 32 milk samples (four each from the eight donor goats collected throughout lactation) detected PrP(sc) in one sample each from two goats. The scrapie agent was present in the milk from infected goats and was able to transmit to susceptible species even at early preclinical stage of infection, when PrP(sc) was undetectable in the brain of the donor goats. Serial PMCA as a PrP(sc) detection method to assess the risk of scrapie transmission via milk in goats proved inefficient compared to the bioassay.

PRNP genetic variability and molecular typing of natural goat scrapie isolates in a high number of infected flocks

PubMed Central

2011-01-01

One hundred and four scrapie positive and 77 negative goats from 34 Greek mixed flocks were analysed by prion protein gene sequencing and 17 caprine scrapie isolates from 11 flocks were submitted to molecular isolate typing. For the first time, the protective S146 variant was reported in Greece, while the protective K222 variant was detected in negative but also in five scrapie positive goats from heavily infected flocks. By immunoblotting six isolates, including two goat flockmates carrying the K222 variant, showed molecular features slightly different from all other Greek and Italian isolates co-analysed, possibly suggesting the presence of different scrapie strains in Greece. PMID:21961834

PRNP genetic variability and molecular typing of natural goat scrapie isolates in a high number of infected flocks.

PubMed

Fragkiadaki, Eirini G; Vaccari, Gabriele; Ekateriniadou, Loukia V; Agrimi, Umberto; Giadinis, Nektarios D; Chiappini, Barbara; Esposito, Elena; Conte, Michela; Nonno, Romolo

2011-09-30

One hundred and four scrapie positive and 77 negative goats from 34 Greek mixed flocks were analysed by prion protein gene sequencing and 17 caprine scrapie isolates from 11 flocks were submitted to molecular isolate typing. For the first time, the protective S146 variant was reported in Greece, while the protective K222 variant was detected in negative but also in five scrapie positive goats from heavily infected flocks. By immunoblotting six isolates, including two goat flockmates carrying the K222 variant, showed molecular features slightly different from all other Greek and Italian isolates co-analysed, possibly suggesting the presence of different scrapie strains in Greece.

Cloning of a Gene Whose Expression is Increased in Scrapie and in Senile Plaques in Human Brain

NASA Astrophysics Data System (ADS)

Wietgrefe, S.; Zupancic, M.; Haase, A.; Chesebro, B.; Race, R.; Frey, W.; Rustan, T.; Friedman, R. L.

1985-12-01

A complementary DNA library was constructed from messenger RNA's extracted from the brains of mice infected with the scrapie agent. The library was differentially screened with the objectives of finding clones that might be used as markers of infection and finding clones of genes whose increased expression might be correlated with the pathological changes common to scrapie and Alzheimer's disease. A gene was identified whose expression is increased in scrapie. The complementary DNA corresponding to this gene hybridized preferentially and focally to cells in the brains of scrapie-infected animals. The cloned DNA also hybridized to the neuritic plaques found with increased frequency in brains of patients with Alzheimer's disease.

A cross-sectional study of PRNP gene in two native Sicilian goat populations in Italy: a relation between prion gene polymorphisms and scrapie incidence.

PubMed

Migliore, Sergio; Agnello, Stefano; D'Avola, Salvatore; Goldmann, Wilfred; Di Marco Lo Presti, Vincenzo; Vitale, Maria

2017-06-01

Transmissible spongiform encephalopathies (TSEs) are a group of neurodegenerative diseases affecting humans and animals, and scrapie in small ruminants is considered the archetype of TSEs. Derivata di Siria is a native dairy goat of Sicily (south Italy), which is related to Syrian goat breeds. Scrapie disease is considered endemic in Sicily since 1997, following the administration of an infected vaccine.Derivata di Siria goatswere involved in six of 66 scrapie-infected flocks in Sicily. Prion protein gene (PRNP) analysis revealed that none of the scrapie cases carried the p.Gln222Lys variant. Sequencing of PRNP in this goat population showed a high frequency (15%) of p.Gln222Lys variant confirming its association with scrapie resistance. PRNP polymorphisms were also analysed in the population of Pantelleria, a small Sicilian Island, where scrapie has never been reported. The native goat breed 'Pantesca' was maintained up to almost 80 years and the size of the sheep population on this island has historically been very low. Currently, a crossbreed goat population of 253 heads is present on the island. PRNP genotyping of Pantelleria goats showed genetic variation, with low presence of wild-type goats and the lack of protective alleles. These data reinforce the association between PRNP polymorphisms in small ruminants and scrapie incidence.

The bank vole (Myodes glareolus) as a sensitive bioassay for sheep scrapie.

PubMed

Di Bari, Michele Angelo; Chianini, Francesca; Vaccari, Gabriele; Esposito, Elena; Conte, Michela; Eaton, Samantha L; Hamilton, Scott; Finlayson, Jeanie; Steele, Philip J; Dagleish, Mark P; Reid, Hugh W; Bruce, Moira; Jeffrey, Martin; Agrimi, Umberto; Nonno, Romolo

2008-12-01

Despite intensive studies on sheep scrapie, a number of questions remain unanswered, such as the natural mode of transmission and the amount of infectivity which accumulates in edible tissues at different stages of scrapie infection. Studies using the mouse model proved to be useful for recognizing scrapie strain diversity, but the low sensitivity of mice to some natural scrapie isolates hampered further investigations. To investigate the sensitivity of bank voles (Myodes glareolus) to scrapie, we performed end-point titrations from two unrelated scrapie sources. Similar titres [10(5.5) ID50 U g(-1) and 10(5.8) ID50 U g(-1), both intracerebrally (i.c.)] were obtained, showing that voles can detect infectivity up to 3-4 orders of magnitude lower when compared with laboratory mice. We further investigated the relationships between PrPSc molecular characteristics, strain and prion titre in the brain and tonsil of the same scrapie-affected sheep. We found that protease-resistant PrPSc fragments (PrPres) from brain and tonsil had different molecular features, but induced identical disease phenotypes in voles. The infectivity titre of the tonsil estimated by incubation time assay was 10(4.8) i.c. ID50 U g(-1), i.e. fivefold less than the brain. This compared well with the relative PrPres content, which was 8.8-fold less in tonsil than in brain. Our results suggest that brain and tonsil harboured the same prion strain showing different glycoprofiles in relation to the different cellular/tissue types in which it replicated, and that a PrPSc-based estimate of scrapie infectivity in sheep tissues could be achieved by combining sensitive PrPres detection methods and bioassay in voles.

9 CFR 79.4 - Designation of scrapie-positive animals, high-risk animals, exposed animals, suspect animals...

Code of Federal Regulations, 2011 CFR

2011-01-01

... (INCLUDING POULTRY) AND ANIMAL PRODUCTS SCRAPIE IN SHEEP AND GOATS § 79.4 Designation of scrapie-positive... breeding records that indicate the flock meets the criteria of the relevant definition in § 79.1. (i) A...

9 CFR 79.4 - Designation of scrapie-positive animals, high-risk animals, exposed animals, suspect animals...

Code of Federal Regulations, 2010 CFR

2010-01-01

... (INCLUDING POULTRY) AND ANIMAL PRODUCTS SCRAPIE IN SHEEP AND GOATS § 79.4 Designation of scrapie-positive... breeding records that indicate the flock meets the criteria of the relevant definition in § 79.1. (i) A...

Genetic, histochemical and biochemical studies on goat TSE cases from Cyprus.

PubMed

Niedermeyer, Susanne; Eiden, Martin; Toumazos, Pavlos; Papasavva-Stylianou, Penelope; Ioannou, Ioannis; Sklaviadis, Theodoros; Panagiotidis, Cynthia; Langeveld, Jan; Bossers, Alex; Kuczius, Thorsten; Kaatz, Martin; Groschup, Martin H; Fast, Christine

2016-10-06

Scrapie and bovine spongiform encephalopathy (BSE) are transmissible spongiform encephalopathies (TSE's) affecting sheep and goats. Susceptibility of goats to scrapie is influenced by polymorphisms of the prion protein gene (PRNP) of the host. Five polymorphisms are associated with reduced susceptibility to TSE's. In the study presented here caprine samples from a scrapie eradication program on Cyprus were genotyped and further characterized using BioRad TeSeE rapid test, histological, immunohistochemical and biochemical methods. In total 42 goats from 20 flocks were necropsied from which 25 goats showed a positive result in the rapid test, a spongiform encephalopathy and an accumulation of pathological prion protein (PrP Sc ) in the obex. PrP Sc deposits were demonstrated in the placenta, peripheral nervous and lymphoreticular system. Two animals showed PrP Sc -accumulations in peripheral tissues only. By discriminatory immunoblots a scrapie infection could be confirmed for all cases. Nevertheless, slight deviations in the glycosylation pattern might indicate the presence of different scrapie strains. Furthermore scrapie samples from goats in the current study demonstrated less long term resistance to proteinase K than ovine or caprine BSE control samples. Reduced scrapie susceptibility according to the PRNP genotype was demonstrated (Fishers Exact test, p < 0.05) for the goats with at least one polymorphism (p = 0.023) at the six codons examined and in particular for those with polymorphisms at codon 146 (p = 0.016). This work characterizes scrapie in goats having implications for breeding and surveillance strategies.

Atypical/Nor98 Scrapie Infectivity in Sheep Peripheral Tissues

PubMed Central

Andréoletti, Olivier; Orge, Leonor; Benestad, Sylvie L.; Beringue, Vincent; Litaise, Claire; Simon, Stéphanie; Le Dur, Annick; Laude, Hubert; Simmons, Hugh; Lugan, Séverine; Corbière, Fabien; Costes, Pierrette; Morel, Nathalie; Schelcher, François; Lacroux, Caroline

2011-01-01

Atypical/Nor98 scrapie was first identified in 1998 in Norway. It is now considered as a worldwide disease of small ruminants and currently represents a significant part of the detected transmissible spongiform encephalopathies (TSE) cases in Europe. Atypical/Nor98 scrapie cases were reported in ARR/ARR sheep, which are highly resistant to BSE and other small ruminants TSE agents. The biology and pathogenesis of the Atypical/Nor98 scrapie agent in its natural host is still poorly understood. However, based on the absence of detectable abnormal PrP in peripheral tissues of affected individuals, human and animal exposure risk to this specific TSE agent has been considered low. In this study we demonstrate that infectivity can accumulate, even if no abnormal PrP is detectable, in lymphoid tissues, nerves, and muscles from natural and/or experimental Atypical/Nor98 scrapie cases. Evidence is provided that, in comparison to other TSE agents, samples containing Atypical/Nor98 scrapie infectivity could remain PrPSc negative. This feature will impact detection of Atypical/Nor98 scrapie cases in the field, and highlights the need to review current evaluations of the disease prevalence and potential transmissibility. Finally, an estimate is made of the infectivity loads accumulating in peripheral tissues in both Atypical/Nor98 and classical scrapie cases that currently enter the food chain. The results obtained indicate that dietary exposure risk to small ruminants TSE agents may be higher than commonly believed. PMID:21347349

Fluorescence spectroscopy of the retina from scrapie-infected mice

USDA-ARS?s Scientific Manuscript database

Recently, we have proposed that the fluorescence spectra of sheep retina can be well correlated to the presence or absence of scrapie. Scrapie is the most widespread TSE (transmissible spongiform encephalopathy) affecting sheep and goats worldwide. Mice eyes have been previously reported as a model ...

Characterization of a U.S. Sheep Scrapie Isolate with Short Incubation Time

USDA-ARS?s Scientific Manuscript database

Scrapie is a naturally occurring fatal neurodegenerative disease of sheep and goats. Susceptibility to the disease is partly dependent upon the genetic makeup of the host. In a previous study it was shown that sheep intracerebrally inoculated with US scrapie inoculum (No. 13-7) developed terminal di...

9 CFR 54.7 - Procedures for destruction of animals.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Procedures for destruction of animals. 54.7 Section 54.7 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT... SCRAPIE Scrapie Indemnification Program § 54.7 Procedures for destruction of animals. (a) Scrapie-positive...

9 CFR 54.7 - Procedures for destruction of animals.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Procedures for destruction of animals. 54.7 Section 54.7 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT... SCRAPIE Scrapie Indemnification Program § 54.7 Procedures for destruction of animals. (a) Scrapie-positive...

9 CFR 54.7 - Procedures for destruction of animals.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Procedures for destruction of animals. 54.7 Section 54.7 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT... SCRAPIE Scrapie Indemnification Program § 54.7 Procedures for destruction of animals. (a) Scrapie-positive...

9 CFR 54.7 - Procedures for destruction of animals.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Procedures for destruction of animals. 54.7 Section 54.7 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT... SCRAPIE Scrapie Indemnification Program § 54.7 Procedures for destruction of animals. (a) Scrapie-positive...

9 CFR 54.7 - Procedures for destruction of animals.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Procedures for destruction of animals. 54.7 Section 54.7 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT... SCRAPIE Scrapie Indemnification Program § 54.7 Procedures for destruction of animals. (a) Scrapie-positive...

9 CFR 79.6 - Standards for States to qualify as Consistent States.

Code of Federal Regulations, 2011 CFR

2011-01-01

... suspected of having scrapie and test results for any animals tested for scrapie to State or Federal animal... that ensure later movement to slaughter, for destruction, or for research. Scrapie-positive and suspect animals may be moved only for transport to an approved research facility or for purposes of destruction...

9 CFR 79.6 - Standards for States to qualify as Consistent States.

Code of Federal Regulations, 2013 CFR

2013-01-01

... suspected of having scrapie and test results for any animals tested for scrapie to State or Federal animal... that ensure later movement to slaughter, for destruction, or for research. Scrapie-positive and suspect animals may be moved only for transport to an approved research facility or for purposes of destruction...

9 CFR 79.6 - Standards for States to qualify as Consistent States.

Code of Federal Regulations, 2010 CFR

2010-01-01

... suspected of having scrapie and test results for any animals tested for scrapie to State or Federal animal... that ensure later movement to slaughter, for destruction, or for research. Scrapie-positive and suspect animals may be moved only for transport to an approved research facility or for purposes of destruction...

9 CFR 79.6 - Standards for States to qualify as Consistent States.

Code of Federal Regulations, 2014 CFR

2014-01-01

... suspected of having scrapie and test results for any animals tested for scrapie to State or Federal animal... that ensure later movement to slaughter, for destruction, or for research. Scrapie-positive and suspect animals may be moved only for transport to an approved research facility or for purposes of destruction...

9 CFR 79.6 - Standards for States to qualify as Consistent States.

Code of Federal Regulations, 2012 CFR

2012-01-01

... suspected of having scrapie and test results for any animals tested for scrapie to State or Federal animal... that ensure later movement to slaughter, for destruction, or for research. Scrapie-positive and suspect animals may be moved only for transport to an approved research facility or for purposes of destruction...

Classical natural ovine scrapie prions are detected in practical volumes of blood by lamb and transgenic mouse bioassay

USDA-ARS?s Scientific Manuscript database

In vitro ligand-based immunoassay studies revealed abnormal isoforms of prion protein (PrP-Sc) are primarily associated with B lymphocytes of scrapie-infected sheep. Our recent study also demonstrated efficient transmission of scrapie to lambs following a transfusion of B lymphocytes isolated from 5...

Scrapie resistant goats: Keep your herd healthy and help your business with a new gene test

USDA-ARS?s Scientific Manuscript database

Classical scrapie is an infectious disease of goats and sheep that causes slowly progressive but ultimately fatal degeneration of the brain, with advanced cases often hindering their ability to move properly. Currently, a single diagnosis of classical scrapie results in permanent quarantine or eutha...

Classical scrapie prions are associated with peripheral blood monocytes and T-lymphocytes from naturally infected sheep

USDA-ARS?s Scientific Manuscript database

Classical scrapie is a transmissible spongiform encephalopathy that affects sheep and goats. As detected by enzyme-linked immunoassay, previous studies suggested scrapie prions in the blood of sheep might be associated with B lymphocytes but not with monocytes or T lymphocytes. The association of sc...

Post-mortem immunodiagnosis of scrapie and bovine spongiform encephalopathy.

PubMed

Farquhar, C F; Somerville, R A; Ritchie, L A

1989-01-01

Two polyclonal antisera were raised in rabbits against the scrapie-associated fibril protein (PrP) prepared from sheep and mice which were terminally infected with experimental scrapie. The anti-mouse PrP serum identifies the proteins of scrapie-associated fibrils (SAF) from all the host species studied (mouse, hamster, sheep and goat) and bovine spongiform encephalopathy (BSE) fibrils from cow. The anti-sheep PrP serum displays species restricted immunoreactivity. While it identifies several PrP polypeptides from terminally affected sheep, goat and cow material, only the highest molecular weight band is recognised from hamster and there is no detection of mouse PrP. The use of these antisera in routine laboratory testing at post mortem provides a highly sensitive test for scrapie and BSE and may allow the identification of infected animals prior to the onset of clinical signs.

Selective propagation of mouse-passaged scrapie prions with long incubation period from a mixed prion population using GT1-7 cells

PubMed Central

Masujin, Kentaro; Okada, Hiroyuki; Ushiki-Kaku, Yuko; Matsuura, Yuichi; Yokoyama, Takashi

2017-01-01

In our previous study, we demonstrated the propagation of mouse-passaged scrapie isolates with long incubation periods (L-type) derived from natural Japanese sheep scrapie cases in murine hypothalamic GT1-7 cells, along with disease-associated prion protein (PrPSc) accumulation. We here analyzed the susceptibility of GT1-7 cells to scrapie prions by exposure to infected mouse brains at different passages, following interspecies transmission. Wild-type mice challenged with a natural sheep scrapie case (Kanagawa) exhibited heterogeneity of transmitted scrapie prions in early passages, and this mixed population converged upon one with a short incubation period (S-type) following subsequent passages. However, when GT1-7 cells were challenged with these heterologous samples, L-type prions became dominant. This study demonstrated that the susceptibility of GT1-7 cells to L-type prions was at least 105 times higher than that to S-type prions and that L-type prion-specific biological characteristics remained unchanged after serial passages in GT1-7 cells. This suggests that a GT1-7 cell culture model would be more useful for the economical and stable amplification of L-type prions at the laboratory level. Furthermore, this cell culture model might be used to selectively propagate L-type scrapie prions from a mixed prion population. PMID:28636656

Selective propagation of mouse-passaged scrapie prions with long incubation period from a mixed prion population using GT1-7 cells.

PubMed

Miyazawa, Kohtaro; Masujin, Kentaro; Okada, Hiroyuki; Ushiki-Kaku, Yuko; Matsuura, Yuichi; Yokoyama, Takashi

2017-01-01

In our previous study, we demonstrated the propagation of mouse-passaged scrapie isolates with long incubation periods (L-type) derived from natural Japanese sheep scrapie cases in murine hypothalamic GT1-7 cells, along with disease-associated prion protein (PrPSc) accumulation. We here analyzed the susceptibility of GT1-7 cells to scrapie prions by exposure to infected mouse brains at different passages, following interspecies transmission. Wild-type mice challenged with a natural sheep scrapie case (Kanagawa) exhibited heterogeneity of transmitted scrapie prions in early passages, and this mixed population converged upon one with a short incubation period (S-type) following subsequent passages. However, when GT1-7 cells were challenged with these heterologous samples, L-type prions became dominant. This study demonstrated that the susceptibility of GT1-7 cells to L-type prions was at least 105 times higher than that to S-type prions and that L-type prion-specific biological characteristics remained unchanged after serial passages in GT1-7 cells. This suggests that a GT1-7 cell culture model would be more useful for the economical and stable amplification of L-type prions at the laboratory level. Furthermore, this cell culture model might be used to selectively propagate L-type scrapie prions from a mixed prion population.

Quantitating PrP polymorphisms present in prions from heterozygous scrapie-infected sheep

USDA-ARS?s Scientific Manuscript database

Scrapie is a prion (PrPSc) disease of sheep. The incubation period of sheep scrapie is strongly influenced by polymorphisms at positions 136, 154, and 171 of a sheep’s normal cellular prion protein (PrPC). Chymotrypsin was used to digest sheep recombinant PrP to identify a set of characteristic pept...

Lack of prion accumulation in lymphoid tissues of PRNP ARQ/ARR sheep intracranially inoculated with the agent of scrapie

USDA-ARS?s Scientific Manuscript database

Sheep scrapie is a transmissible spongiform encephalopathy that can be transmitted horizontally. The prion protein gene (PRNP) profoundly influences the susceptibility of sheep to the scrapie agent and the tissue levels and distribution of PrPSc in affected sheep. The purpose of this study was to co...

The placenta shed from goats with classical scrapie is infectious to goat kids and lambs

USDA-ARS?s Scientific Manuscript database

Classical scrapie is a natural prion disease of sheep in which the immediate postpartum period and, in particular, the placenta have long been known to play key roles in natural horizontal transmission. Goats, too, are a natural host of classical scrapie and are frequently raised with sheep; but the...

Lack of Prion Accumulation in Lymphoid Tissues of Scrapie-affected Sheep with the AA136, QR171 Prion Protein Genotype

USDA-ARS?s Scientific Manuscript database

Background: Sheep scrapie is a transmissible spongiform encephalopathy which can be transmitted horizontally through the shedding of an infectious conformer (PrP**Sc) of the normal cellular prion protein (PrP**c). Genetics profoundly influence the susceptibility of sheep to scrapie. PrP**c amino-aci...

PrP genotype frequencies and risk evaluation for scrapie in dairy sheep breeds from southern Italy.

PubMed

Martemucci, Giovanni; Iamartino, Daniela; Blasi, Michele; D'Alessandro, Angela Gabriella

2015-12-01

Concerns regarding scrapie in sheep breeding have increased in the last few decades. The present study was carried out in dairy sheep breeds from southern Italy. In order to find breeding animals resistant to scrapie, the PrP genes of 1,205 animals from entire flocks of dairy native Apulian Leccese and Altamurana breeds, and Sicilian Comisana breed, were analysed for polymorphisms at codons 136, 154, and 171 related to scrapie resistance/susceptibility. The Altamurana breed was considered as two populations (Alt-Cav and Alt-Cra-Zoe), based on presumed cross-breeding. A total of five alleles and ten different genotypes were found. The ARQ allele was predominant for all breeds followed by ARR, the most resistant allele to scrapie, which was highly prevalent in Comisana (50%) and in native Alt-Cav (42.4%). The VRQ allele, associated with the highest susceptibility to scrapie, was detected at not negligeable levels in allocthonous Comisana (3.5%), at a low frequency (0.2%) in native Leccese and Alt-Cra-Zoe, while it was absent in Alt-Cav. The frequencies of PrP genotypes with a very low susceptibility risk to scrapie (R1) was higher in Comisana and Alt-Cav. The most susceptible genotype, ARQ/VRQ, was found only in Comisana. Within the Altamurana breed, there were notable differences between Alt-Cav and Alt-Cra-Zoe sheep. The Alt-Cav was characterised by the absence of VRQ and AHQ alleles and by the higher frequency of the ARR/ARR genotype (18.7%). Breeding programs, mainly in endangered breeds such as Altamurana, should be conducted gradually, combining resistance to scrapie, maintenance of genetic variability, and production. Copyright © 2015 Elsevier B.V. All rights reserved.

Low fraction of the 222K PrP variant in the protease-resistant moiety of PrPres in heterozygous scrapie positive goats.

PubMed

Mazza, Maria; Guglielmetti, Chiara; Ingravalle, Francesco; Brusadore, Sonia; Langeveld, Jan P M; Ekateriniadou, Loukia V; Andréoletti, Olivier; Casalone, Cristina; Acutis, Pier Luigi

2017-07-01

The presence of lysine (K) at codon 222 has been associated with resistance to classical scrapie in goats, but few scrapie cases have been identified in 222Q/K animals. To investigate the contribution of the 222K variant to PrPres formation in natural and experimental Q/K scrapie cases, we applied an immunoblotting method based on the use of two different monoclonal antibodies, F99/97.6.1 and SAF84, chosen for their different affinities to 222K and 222Q PrP variants. Our finding that PrPres seems to be formed nearly totally by the 222Q variant provides evidence that the 222K PrP variant confers resistance to conversion to PrPres formation and reinforces the view that this mutation has a protective role against classical scrapie in goats.

Low fraction of the 222K PrP variant in the protease-resistant moiety of PrPres in heterozygous scrapie positive goats

PubMed Central

Guglielmetti, Chiara; Ingravalle, Francesco; Brusadore, Sonia; Langeveld, Jan P. M.; Ekateriniadou, Loukia V.; Andréoletti, Olivier; Casalone, Cristina; Acutis, Pier Luigi

2017-01-01

The presence of lysine (K) at codon 222 has been associated with resistance to classical scrapie in goats, but few scrapie cases have been identified in 222Q/K animals. To investigate the contribution of the 222K variant to PrPres formation in natural and experimental Q/K scrapie cases, we applied an immunoblotting method based on the use of two different monoclonal antibodies, F99/97.6.1 and SAF84, chosen for their different affinities to 222K and 222Q PrP variants. Our finding that PrPres seems to be formed nearly totally by the 222Q variant provides evidence that the 222K PrP variant confers resistance to conversion to PrPres formation and reinforces the view that this mutation has a protective role against classical scrapie in goats. PMID:28691895

Transmission of scrapie prions to primate after an extended silent incubation period.

PubMed

Comoy, Emmanuel E; Mikol, Jacqueline; Luccantoni-Freire, Sophie; Correia, Evelyne; Lescoutra-Etchegaray, Nathalie; Durand, Valérie; Dehen, Capucine; Andreoletti, Olivier; Casalone, Cristina; Richt, Juergen A; Greenlee, Justin J; Baron, Thierry; Benestad, Sylvie L; Brown, Paul; Deslys, Jean-Philippe

2015-06-30

Classical bovine spongiform encephalopathy (c-BSE) is the only animal prion disease reputed to be zoonotic, causing variant Creutzfeldt-Jakob disease (vCJD) in humans and having guided protective measures for animal and human health against animal prion diseases. Recently, partial transmissions to humanized mice showed that the zoonotic potential of scrapie might be similar to c-BSE. We here report the direct transmission of a natural classical scrapie isolate to cynomolgus macaque, a highly relevant model for human prion diseases, after a 10-year silent incubation period, with features similar to those reported for human cases of sporadic CJD. Scrapie is thus actually transmissible to primates with incubation periods compatible with their life expectancy, although fourfold longer than BSE. Long-term experimental transmission studies are necessary to better assess the zoonotic potential of other prion diseases with high prevalence, notably Chronic Wasting Disease of deer and elk and atypical/Nor98 scrapie.

Transmission of scrapie prions to primate after an extended silent incubation period

PubMed Central

Comoy, Emmanuel E.; Mikol, Jacqueline; Luccantoni-Freire, Sophie; Correia, Evelyne; Lescoutra-Etchegaray, Nathalie; Durand, Valérie; Dehen, Capucine; Andreoletti, Olivier; Casalone, Cristina; Richt, Juergen A.; Greenlee, Justin J.; Baron, Thierry; Benestad, Sylvie L.; Brown, Paul; Deslys, Jean-Philippe

2015-01-01

Classical bovine spongiform encephalopathy (c-BSE) is the only animal prion disease reputed to be zoonotic, causing variant Creutzfeldt-Jakob disease (vCJD) in humans and having guided protective measures for animal and human health against animal prion diseases. Recently, partial transmissions to humanized mice showed that the zoonotic potential of scrapie might be similar to c-BSE. We here report the direct transmission of a natural classical scrapie isolate to cynomolgus macaque, a highly relevant model for human prion diseases, after a 10-year silent incubation period, with features similar to those reported for human cases of sporadic CJD. Scrapie is thus actually transmissible to primates with incubation periods compatible with their life expectancy, although fourfold longer than BSE. Long-term experimental transmission studies are necessary to better assess the zoonotic potential of other prion diseases with high prevalence, notably Chronic Wasting Disease of deer and elk and atypical/Nor98 scrapie. PMID:26123044

Caprine PrP variants harboring Asp-146, His-154 and Gln-211 alleles display reduced convertibility upon interaction with pathogenic murine prion protein in scrapie infected cells.

PubMed

Kanata, Eirini; Arsenakis, Minas; Sklaviadis, Theodoros

2016-09-02

Scrapie, the prion disease of sheep and goats, is a devastating malady of small ruminants. Due to its infectious nature, epidemic outbreaks may occur in flocks/herds consisting of highly susceptible animals. Field studies identified scrapie-protective caprine PrP variants, harboring specific single amino acid changes (Met-142, Arg-143, Asp-146, Ser-146, His-154, Gln-211 and Lys-222). Their effects are under further evaluation, and aim to determine the most protective allele. We assessed some of these variants (Asp-146, His-154, Gln-211 and Lys-222), after their exogenous expression as murine-caprine chimeras in a scrapie- infected murine cell line. We report that exogenously expressed PrPs undergo conformational conversion upon interaction with the endogenous pathological murine prion protein (PrP SC ), which results in the detection of goat-specific and partially PK-resistant moieties. These moieties display a PK-resistance pattern distinct from the one detected in natural goat scrapie cases. Within this cellular model, distinct conformational conversion potentials were assigned to the tested variants. Molecules carrying the Asp-146, His-154 and Gln-211 alleles showed significantly lower conversion levels compared to wild type, confirming their protective effects against scrapie. Although we utilized a heterologous conversion system, this is to our knowledge, the first study of caprine PrP variants in a cellular context of scrapie, that confirms the protective effects of some of the studied alleles.

Oral inoculation of neonatal Suffolk sheep with the agent of classical scrapie results in PrPSc accumulation in sheep with the PRNP ARQ/ARQ but not the ARQ/ARR genotype

USDA-ARS?s Scientific Manuscript database

Background Scrapie is a transmissible spongiform encephalopathy that can be transmitted amongst susceptible sheep. The prion protein gene (PRNP) profoundly influences the susceptibility of sheep to the scrapie agent. Findings This study reports the failure to detect PrPSc in nervous or lymphoid tis...

Codon 141 polymorphisms of the ovine prion protein gene affect the phenotype of classical scrapie transmitted from goats to sheep.

PubMed

Konold, Timm; Phelan, Laura J; Donnachie, Ben R; Chaplin, Melanie J; Cawthraw, Saira; González, Lorenzo

2017-05-04

A study to investigate transmission of classical scrapie via goat milk was carried out in sheep: firstly, lambs were challenged orally with goat scrapie brain homogenate to confirm transmission of scrapie from goats to sheep. In the second study phase, milk from scrapie-infected goats was fed to lambs. Lambs were selected according to their prion protein gene (PRNP) genotype, which was either VRQ/VRQ or ARQ/ARQ, with or without additional polymorphisms at codon 141 (FF 141 , LF 141 or LL 141 ) of the ovine PRNP. This report describes the clinical, pathological and molecular phenotype of goat scrapie in those sheep that progressed to clinical end-stage. Ten sheep (six VRQ/VRQ and four ARQ/ARQ, of which three FF 141 and one LL 141 ) challenged with one of two scrapie brain homogenates, and six pairs of sheep (ARQ, of which five LL 141 and seven LF 141 ) fed milk from six different goats, developed clinical disease, which was characterised by a pruritic (all VRQ/VRQ and LL 141 sheep) or a non-pruritic form (all LF 141 and FF 141 sheep). Immunohistochemical (IHC) examination revealed that the pattern of intra- and extracellular accumulation of disease-associated prion protein in the brain was also dependent on PRNP polymorphisms at codon 141, which was similar in VRQ and LL 141 sheep but different from LF 141 and FF 141 sheep. The influence of codon 141 was also seen in discriminatory Western blot (WB), with LF 141 and FF 141 sheep showing a bovine spongiform encephalopathy-like profile (diminished reactivity with P4 antibody) on brain tissue. However, discriminatory WB in lymphoid tissues, and IHC pattern and profile both in lymphoid and brain tissue was consistent with classical scrapie in all sheep. This study provided further evidence that the clinical presentation and the pathological and molecular phenotypes of scrapie in sheep are influenced by PRNP polymorphisms, particularly at codon 141. Differences in the truncation of disease-associated prion protein between LL 141 sheep and those carrying the F 141 allele may be responsible for these observations.

Atypical scrapie prions from sheep and lack of disease in transgenic mice overexpressing human prion protein.

PubMed

Wadsworth, Jonathan D F; Joiner, Susan; Linehan, Jacqueline M; Balkema-Buschmann, Anne; Spiropoulos, John; Simmons, Marion M; Griffiths, Peter C; Groschup, Martin H; Hope, James; Brandner, Sebastian; Asante, Emmanuel A; Collinge, John

2013-11-01

Public and animal health controls to limit human exposure to animal prions are focused on bovine spongiform encephalopathy (BSE), but other prion strains in ruminants may also have zoonotic potential. One example is atypical/Nor98 scrapie, which evaded statutory diagnostic methods worldwide until the early 2000s. To investigate whether sheep infected with scrapie prions could be another source of infection, we inoculated transgenic mice that overexpressed human prion protein with brain tissue from sheep with natural field cases of classical and atypical scrapie, sheep with experimental BSE, and cattle with BSE. We found that these mice were susceptible to BSE prions, but disease did not develop after prolonged postinoculation periods when mice were inoculated with classical or atypical scrapie prions. These data are consistent with the conclusion that prion disease is less likely to develop in humans after exposure to naturally occurring prions of sheep than after exposure to epizootic BSE prions of ruminants.

A Case–Control Study on the Origin of Atypical Scrapie in Sheep, France

PubMed Central

Morignat, Eric; Ducrot, Christian; Calavas, Didier

2009-01-01

A matched case–control study (95 cases and 220 controls) was designed to study risk factors for atypical scrapie in sheep in France. We analyzed contacts with animals from other flocks, lambing and feeding practices, and exposure to toxic substances. Data on the prnp genotype were collected for some case and control animals and included in a complementary analysis. Sheep dairy farms had a higher risk for scrapie (odds ratio [OR] 15.1, 95% confidence interval [CI] 3.3–69.7). Lower risk was associated with organic farms (OR 0.15, 95% CI 0.02–1.26), feeding corn silage (OR 0.16, 95% CI 0.05–0.53), and feeding vitamin and mineral supplements (OR 0.6, 95% CI 0.32–1.14). Genetic effects were quantitatively important but only marginally changed estimates of other variables. We did not find any risk factor associated with an infectious origin of scrapie. Atypical scrapie could be a spontaneous disease influenced by genetic and metabolic factors. PMID:19402956

Atypical Scrapie Prions from Sheep and Lack of Disease in Transgenic Mice Overexpressing Human Prion Protein

PubMed Central

Joiner, Susan; Linehan, Jacqueline M.; Balkema-Buschmann, Anne; Spiropoulos, John; Simmons, Marion M.; Griffiths, Peter C.; Groschup, Martin H.; Hope, James; Brandner, Sebastian; Asante, Emmanuel A.; Collinge, John

2013-01-01

Public and animal health controls to limit human exposure to animal prions are focused on bovine spongiform encephalopathy (BSE), but other prion strains in ruminants may also have zoonotic potential. One example is atypical/Nor98 scrapie, which evaded statutory diagnostic methods worldwide until the early 2000s. To investigate whether sheep infected with scrapie prions could be another source of infection, we inoculated transgenic mice that overexpressed human prion protein with brain tissue from sheep with natural field cases of classical and atypical scrapie, sheep with experimental BSE, and cattle with BSE. We found that these mice were susceptible to BSE prions, but disease did not develop after prolonged postinoculation periods when mice were inoculated with classical or atypical scrapie prions. These data are consistent with the conclusion that prion disease is less likely to develop in humans after exposure to naturally occurring prions of sheep than after exposure to epizootic BSE prions of ruminants. PMID:24188521

Genetic Resistance to Scrapie Infection in Experimentally Challenged Goats

PubMed Central

Lacroux, Caroline; Perrin-Chauvineau, Cécile; Corbière, Fabien; Aron, Naima; Aguilar-Calvo, Patricia; Torres, Juan Maria; Costes, Pierrette; Brémaud, Isabelle; Lugan, Séverine; Schelcher, François; Barillet, Francis

2014-01-01

In goats, several field studies have identified coding mutations of the gene encoding the prion protein (I/M142, N/D146, S/D146, R/Q211, and Q/K222) that are associated with a lower risk of developing classical scrapie. However, the data related to the levels of resistance to transmissible spongiform encephalopathies (TSEs) of these different PRNP gene mutations are still considered insufficient for developing large-scale genetic selection against scrapie in this species. In this study, we inoculated wild-type (WT) PRNP (I142R154R211Q222) goats and homozygous and/or heterozygous I/M142, R/H154, R/Q211, and Q/K222 goats with a goat natural scrapie isolate by either the oral or the intracerebral (i.c.) route. Our results indicate that the I/M142 PRNP polymorphism does not provide substantial resistance to scrapie infection following intracerebral or oral inoculation. They also demonstrate that H154, Q211, and K222 PRNP allele carriers are all resistant to scrapie infection following oral exposure. However, in comparison to WT animals, the H154 and Q211 allele carriers displayed only moderate increases in the incubation period following i.c. challenge. After i.c. challenge, heterozygous K222 and a small proportion of homozygous K222 goats also developed the disease, but with incubation periods that were 4 to 5 times longer than those in WT animals. These results support the contention that the K222 goat prion protein variant provides a strong but not absolutely protective effect against classical scrapie. PMID:24284317

Sensitive and specific detection of classical scrapie prions in the brains of goats by real-time quaking-induced conversion.

PubMed

Dassanayake, Rohana P; Orrú, Christina D; Hughson, Andrew G; Caughey, Byron; Graça, Telmo; Zhuang, Dongyue; Madsen-Bouterse, Sally A; Knowles, Donald P; Schneider, David A

2016-03-01

Real-time quaking-induced conversion (RT-QuIC) is a rapid, specific and highly sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect subinfectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully used to detect PrPSc in various tissues from humans and animals, including sheep, tissues from goats infected with classical scrapie have not yet been tested. Therefore, the aims of the present study were to (1) evaluate whether prion seeding activity could be detected in the brain tissues of goats with scrapie using RT-QuIC, (2) optimize reaction conditions to improve scrapie detection in goats, and (3) compare the performance of RT-QuIC for the detection of PrPSc with the more commonly used ELISA and Western blot assays. We further optimized RT-QuIC conditions for sensitive and specific detection of goat scrapie seeding activity in brain tissue from clinical animals. When used with 200  mM sodium chloride, both full-length sheep rPrPSen substrates (PrP genotypes A136R154Q171 and V136R154Q171) provided good discrimination between scrapie-infected and normal goat brain samples at 10(- )3 dilution within 15  h. Our findings indicate that RT-QuIC was at least 10,000-fold more sensitive than ELISA and Western blot assays for the detection of scrapie seeding activity in goat brain samples. In addition to PRNP WT samples, positive RT-QuIC reactions were also observed with three PRNP polymorphic goat brain samples (G/S127, I/M142 and H/R143) tested. Taken together, these findings demonstrate that RT-QuIC sensitively detects prion seeding activity in classical scrapie-infected goat brain samples.

Sensitive and specific detection of classical scrapie prions in the brains of goats by real-time quaking-induced conversion

PubMed Central

Dassanayake, Rohana P.; Orrú, Christina D.; Hughson, Andrew G.; Caughey, Byron; Graça, Telmo; Zhuang, Dongyue; Madsen-Bouterse, Sally A.; Knowles, Donald P.; Schneider, David A.

2016-01-01

Real-time quaking-induced conversion (RT-QuIC) is a rapid, specific and highly sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect subinfectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully used to detect PrPSc in various tissues from humans and animals, including sheep, tissues from goats infected with classical scrapie have not yet been tested. Therefore, the aims of the present study were to (1) evaluate whether prion seeding activity could be detected in the brain tissues of goats with scrapie using RT-QuIC, (2) optimize reaction conditions to improve scrapie detection in goats, and (3) compare the performance of RT-QuIC for the detection of PrPSc with the more commonly used ELISA and Western blot assays. We further optimized RT-QuIC conditions for sensitive and specific detection of goat scrapie seeding activity in brain tissue from clinical animals. When used with 200 mM sodium chloride, both full-length sheep rPrPSen substrates (PrP genotypes A136R154Q171 and V136R154Q171) provided good discrimination between scrapie-infected and normal goat brain samples at 10− 3 dilution within 15 h. Our findings indicate that RT-QuIC was at least 10 000-fold more sensitive than ELISA and Western blot assays for the detection of scrapie seeding activity in goat brain samples. In addition to PRNP WT samples, positive RT-QuIC reactions were also observed with three PRNP polymorphic goat brain samples (G/S127, I/M142 and H/R143) tested. Taken together, these findings demonstrate that RT-QuIC sensitively detects prion seeding activity in classical scrapie-infected goat brain samples. PMID:26653410

Scrapie Agent (Strain 263K) Can Transmit Disease via the Oral Route after Persistence in Soil over Years

PubMed Central

Groschup, Martin H.; Peters, Rainer; Beekes, Michael; Terytze, Konstantin

2007-01-01

The persistence of infectious biomolecules in soil constitutes a substantial challenge. This holds particularly true with respect to prions, the causative agents of transmissible spongiform encephalopathies (TSEs) such as scrapie, bovine spongiform encephalopathy (BSE), or chronic wasting disease (CWD). Various studies have indicated that prions are able to persist in soil for years without losing their pathogenic activity. Dissemination of prions into the environment can occur from several sources, e.g., infectious placenta or amniotic fluid of sheep. Furthermore, environmental contamination by saliva, excrements or non-sterilized agricultural organic fertilizer is conceivable. Natural transmission of scrapie in the field seems to occur via the alimentary tract in the majority of cases, and scrapie-free sheep flocks can become infected on pastures where outbreaks of scrapie had been observed before. These findings point to a sustained contagion in the environment, and notably the soil. By using outdoor lysimeters, we simulated a contamination of standard soil with hamster-adapted 263K scrapie prions, and analyzed the presence and biological activity of the soil-associated PrPSc and infectivity by Western blotting and hamster bioassay, respectively. Our results showed that 263K scrapie agent can persist in soil at least over 29 months. Strikingly, not only the contaminated soil itself retained high levels of infectivity, as evidenced by oral administration to Syrian hamsters, but also feeding of aqueous soil extracts was able to induce disease in the reporter animals. We could also demonstrate that PrPSc in soil, extracted after 21 months, provides a catalytically active seed in the protein misfolding cyclic amplification (PMCA) reaction. PMCA opens therefore a perspective for considerably improving the detectability of prions in soil samples from the field. PMID:17502917

Caprine prion gene polymorphisms are associated with decreased incidence of classical scrapie in goat herds in the United Kingdom

PubMed Central

2011-01-01

The application of genetic breeding programmes to eradicate transmissible spongiform encephalopathies in goats is an important aim for reasons of animal welfare as well as human food safety and food security. Based on the positive impact of Prnp genetics on sheep scrapie in Europe in the past decade, we have established caprine Prnp gene variation in more than 1100 goats from the United Kingdom and studied the association of Prnp alleles with disease phenotypes in 150 scrapie-positive goats. This investigation confirms the association of the Met142 encoding Prnp allele with increased resistance to preclinical and clinical scrapie. It reveals a novel association of the Ser127 encoding allele with a reduced probability to develop clinical signs of scrapie in goats that are already positive for the accumulation of disease-specific prion protein in brain or periphery. A United Kingdom survey of Prnp genotypes in eight common breeds revealed eleven alleles in over thirty genotypes. The Met142 encoding allele had a high overall mean allele frequency of 22.6%, whereas the Ser127 encoding allele frequency was considerably lower with 6.4%. In contrast, a well known resistance associated allele encoding Lys222 was found to be rare (0.9%) in this survey. The analysis of Prnp genotypes in Mexican Criollas goats revealed nine alleles, including a novel Phe to Leu substitution in codon 201, confirming that high genetic variability of Prnp can be found in scrapie-free populations. Our study implies that it should be feasible to lower scrapie prevalence in goat herds in the United Kingdom by genetic selection. PMID:22040234

Different prion disease phenotypes result from inoculation of cattle with two temporally separated sources of sheep scrapie from Great Britain

PubMed Central

Konold, Timm; Lee, Yoon Hee; Stack, Michael J; Horrocks, Claire; Green, Robert B; Chaplin, Melanie; Simmons, Marion M; Hawkins, Steve AC; Lockey, Richard; Spiropoulos, John; Wilesmith, John W; Wells, Gerald AH

2006-01-01

Background Given the theoretical proposal that bovine spongiform encephalopathy (BSE) could have originated from sheep scrapie, this study investigated the pathogenicity for cattle, by intracerebral (i.c.) inoculation, of two pools of scrapie agents sourced in Great Britain before and during the BSE epidemic. Two groups of ten cattle were each inoculated with pools of brain material from sheep scrapie cases collected prior to 1975 and after 1990. Control groups comprised five cattle inoculated with sheep brain free from scrapie, five cattle inoculated with saline, and for comparison with BSE, naturally infected cattle and cattle i.c. inoculated with BSE brainstem homogenate from a parallel study. Phenotypic characterisation of the disease forms transmitted to cattle was conducted by morphological, immunohistochemical, biochemical and biological methods. Results Disease occurred in 16 cattle, nine inoculated with the pre-1975 inoculum and seven inoculated with the post-1990 inoculum, with four cattle still alive at 83 months post challenge (as at June 2006). The different inocula produced predominantly two different disease phenotypes as determined by histopathological, immunohistochemical and Western immunoblotting methods and biological characterisation on transmission to mice, neither of which was identical to BSE. Whilst the disease presentation was uniform in all scrapie-affected cattle of the pre-1975 group, the post-1990 inoculum produced a more variable disease, with two animals sharing immunohistochemical and molecular profile characteristics with animals in the pre-1975 group. Conclusion The study has demonstrated that cattle inoculated with different pooled scrapie sources can develop different prion disease phenotypes, which were not consistent with the phenotype of BSE of cattle and whose isolates did not have the strain typing characteristics of the BSE agent on transmission to mice. PMID:17044917

Differential display detects host nucleic acid motifs altered in scrapie-infected brain.

PubMed

Lathe, Richard; Harris, Alyson

2009-09-25

The transmissible spongiform encephalopathies (TSEs) including scrapie have been attributed to an infectious protein or prion. Infectivity is allied to conversion of the endogenous nucleic-acid-binding protein PrP to an infectious modified form known as PrP(sc). The protein-only theory does not easily explain the enigmatic properties of the agent including strain variation. It was previously suggested that a short nucleic acid, perhaps host-encoded, might contribute to the pathoetiology of the TSEs. No candidate host molecules that might explain transmission of strain differences have yet been put forward. Differential display is a robust technique for detecting nucleic acid differences between two populations. We applied this technique to total nucleic acid preparations from scrapie-infected and control brain. Independent RNA preparations from eight normal and eight scrapie-infected (strain 263K) hamster brains were randomly amplified and visualized in parallel. Though the nucleic acid patterns were generally identical in scrapie-infected versus control brain, some rare bands were differentially displayed. Molecular species consistently overrepresented (or underrepresented) in all eight infected brain samples versus all eight controls were excised from the display, sequenced, and assembled into contigs. Only seven ros contigs (RNAs over- or underrepresented in scrapie) emerged, representing <4 kb from the transcriptome. All contained highly stable regions of secondary structure. The most abundant scrapie-only ros sequence was homologous to a repetitive transposable element (LINE; long interspersed nuclear element). Other ros sequences identified cellular RNA 7SL, clathrin heavy chain, visinin-like protein-1, and three highly specific subregions of ribosomal RNA (ros1-3). The ribosomal ros sequences accurately corresponded to LINE; retrotransposon insertion sites in ribosomal DNA (p<0.01). These differential motifs implicate specific host RNAs in the pathoetiology of the TSEs.

78 FR 25943 - Changes to Scrapie Flock Certification Program

Federal Register 2010, 2011, 2012, 2013, 2014

2013-05-03

.... SUPPLEMENTARY INFORMATION: Scrapie is a degenerative and ultimately fatal disease affecting the central nervous systems of sheep and goats. It belongs to a group of diseases called transmissible spongiform...

9 CFR 71.1 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-01-01

... subchapter, for conducting an active State scrapie program involving the identification of scrapie in sheep... which contains or displays an AIN with an 840 prefix must bear the U.S. shield. The design, size, shape...

9 CFR 71.1 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-01-01

... subchapter, for conducting an active State scrapie program involving the identification of scrapie in sheep... which contains or displays an AIN with an 840 prefix must bear the U.S. shield. The design, size, shape...

Gene expression in scrapie. Cloning of a new scrapie-responsive gene and the identification of increased levels of seven other mRNA transcripts.

PubMed

Dandoy-Dron, F; Guillo, F; Benboudjema, L; Deslys, J P; Lasmézas, C; Dormont, D; Tovey, M G; Dron, M

1998-03-27

To define genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies, we analyzed gene expression in scrapie-infected mouse brain using "mRNA differential display." The RNA transcripts of eight genes were increased 3-8-fold in the brains of scrapie-infected animals. Five of these genes have not previously been reported to exhibit increased expression in this disease: cathepsin S, the C1q B-chain of complement, apolipoprotein D, and two previously unidentified genes denominated scrapie-responsive gene (ScRG)-1 and ScRG-2, which are preferentially expressed in brain tissue. Increased expression of the three remaining genes, beta2 microglobulin, F4/80, and metallothionein II, has previously been reported to occur in experimental scrapie. Kinetic analysis revealed a concomitant increase in the levels of ScRG-1, cathepsin S, the C1q B-chain of complement, and beta2 microglobulin mRNA as well as glial fibrillary acidic protein and F4/80 transcripts, markers of astrocytosis and microglial activation, respectively. In contrast, the level of ScRG-2, apolipoprotein D, and metallothionein II mRNA was only increased at the terminal stage of the disease. ScRG-1 mRNA was found to be preferentially expressed in glial cells and to code for a short protein of 47 amino acids with a strong hydrophobic N-terminal region.

A crucial role for B cells in neuroinvasive scrapie.

PubMed

Brandner, S; Klein, M A; Aguzzi, A

1999-02-01

Although prions are most efficiently propagated via intracerebral inoculation, peripheral administration has caused kuru [Gajdusek et al, 1966], iatrogenic Creutzfeldt-Jakob disease (CJD) [Gibbs et al, 1997], bovine spongiform encephalitis (BSE), and new variant CJD [Hill et al, 1997; Bruce et al, 1997]. Neurological disease after peripheral inoculation depends on prion expansion within cells of the lymphoreticular system (LRS) [Lasmezas et al. 1996; Wilesmith et al, 1992]. In order to identify the nature of the latter cells, we inoculated a panel of immune deficient mice with prions intraperitoneally. While defects affecting only T lymphocytes had no apparent effect, all mutations affecting differentiation and responses of B lymphocytes prevented development of clinical scrapie. Since absence of B cells and of antibodies correlates with severe defects in follicular dendritic cells (FDCs), the lack of any of these three components may prevent clinical scrapie. Yet, mice expressing immunoglobulins exclusively of the M subclass without detectable specificity for PrPc, and mice with differentiated B cells but lacking functional FDCs, developed scrapie after peripheral inoculation: therefore, differentiated B cells appear to play a crucial role in neuroinvasion of scrapie regardless of B-cell receptor specificity.

Scrapie

USDA-ARS?s Scientific Manuscript database

Prion diseases are a heterogeneous group of transmissible spongiform encephalopathies (TSEs) that affect a variety of mammals, causing a slowly progressive and ultimately fatal, degenerative disease of the brain. Classical scrapie, commonly just referred to as ‘scrapie’, is a naturally transmissibl...

Primary transmission of chronic wasting disease versus scrapie prions from small ruminants to transgenic mice expressing ovine or cervid prion protein.

PubMed

Madsen-Bouterse, Sally A; Schneider, David A; Zhuang, Dongyue; Dassanayake, Rohana P; Balachandran, Aru; Mitchell, Gordon B; O'Rourke, Katherine I

2016-09-01

Development of mice expressing either ovine (Tg338) or cervid (TgElk) prion protein (PrP) have aided in characterization of scrapie and chronic wasting disease (CWD), respectively. Experimental inoculation of sheep with CWD prions has demonstrated the potential for interspecies transmission but, infection with CWD versus classical scrapie prions may be difficult to differentiate using validated diagnostic platforms. In this study, mouse bioassay in Tg338 and TgElk was utilized to evaluate transmission of CWD versus scrapie prions from small ruminants. Mice (≥5 per homogenate) were inoculated with brain homogenates from clinically affected sheep or goats with naturally acquired classical scrapie, white-tailed deer with naturally acquired CWD (WTD-CWD) or sheep with experimentally acquired CWD derived from elk (sheep-passaged-CWD). Survival time (time to clinical disease) and attack rates (brain accumulation of protease resistant PrP, PrPres) were determined. Inoculation with classical scrapie prions resulted in clinical disease and 100 % attack rates in Tg338, but no clinical disease at endpoint (>300 days post-inoculation, p.i.) and low attack rates (6.8 %) in TgElk. Inoculation with WTD-CWD prions yielded no clinical disease or brain PrPres accumulation in Tg338 at endpoint (>500 days p.i.), but rapid onset of clinical disease (~121 days p.i.) and 100 % attack rate in TgElk. Sheep-passaged-CWD resulted in transmission to both mouse lines with 100 % attack rates at endpoint in Tg338 and an attack rate of ~73 % in TgElk with some culled due to clinical disease. These primary transmission observations demonstrate the potential of bioassay in Tg338 and TgElk to help differentiate possible infection with CWD versus classical scrapie prions in sheep and goats.

Sheep Feed and Scrapie, France

PubMed Central

Philippe, Sandrine; Ducrot, Christian; Roy, Pascal; Remontet, Laurent; Jarrige, Nathalie

2005-01-01

Scrapie is a small ruminant, transmissible spongiform encephalopathy (TSE). Although in the past scrapie has not been considered a zoonosis, the emergence of bovine spongiform encephalopathy, transmissible to humans and experimentally to sheep, indicates that risk exists for small ruminant TSEs in humans. To identify the risk factors for introducing scrapie into sheep flocks, a case-control study was conducted in France from 1999 to 2000. Ninety-four case and 350 control flocks were matched by location and main breed. Three main hypotheses were tested: direct contact between flocks, indirect environmental contact, and foodborne risk. Statistical analysis was performed by using adjusted generalized linear models with the complementary log-log link function, considering flock size as an offset. A notable effect of using proprietary concentrates and milk replacers was observed. The risk was heterogeneous among feed factories. Contacts between flocks were not shown to be a risk factor. PMID:16102318

Enhanced levels of scrapie responsive gene mRNA in BSE-infected mouse brain.

PubMed

Dandoy-Dron, F; Benboudjema, L; Guillo, F; Jaegly, A; Jasmin, C; Dormont, D; Tovey, M G; Dron, M

2000-03-10

The expression of the mRNA of nine scrapie responsive genes was analyzed in the brains of FVB/N mice infected with bovine spongiform encephalopathy (BSE). The RNA transcripts of eight genes were overexpressed to a comparable extent in both BSE-infected and scrapie-infected mice, indicating a common series of pathogenic events in the two transmissible spongiform encephalopathies (TSEs). In contrast, the serine proteinase inhibitor spi 2, an analogue of the human alpha-1 antichymotrypsin gene, was overexpressed to a greater extent in the brains of scrapie-infected animals than in animals infected with BSE, reflecting either an agent specific or a mouse strain specific response. The levels of spi 2 mRNA were increased during the course of scrapie prior to the onset of clinical signs of the disease and the increase reached 11 to 45 fold relative to uninfected controls in terminally ill mice. Spi 2, in common with four of the other scrapie responsive genes studied, is known to be associated with pro-inflammatory processes. These observations underline the importance of cell reactivity in TSE. In addition, scrg2 mRNA the level of which is enhanced in TSE-infected mouse brain, was identified as a previously unrecognized long transcript of the murine aldolase C gene. However, the level of the principal aldolase C mRNA is unaffected in TSE. The increased representation of the longer transcript in the late stage of the disease may reflect changes in mRNA processing and/or stability in reactive astrocytes or in damaged Purkinje cells.

Experimental transmission of U.S. scrapie agent to neonatal sheep by oral route

USDA-ARS?s Scientific Manuscript database

Scrapie, a transmissible spongiform encephalopathy (TSE), is a naturally occurring fatal neurodegenerative disease of sheep and goats. This study documents incubation periods, pathological findings and distribution of abnormal prion proteins (PrP**Sc) by immunohistochemistry and Western blot in tiss...

White-Tailed Deer Susceptible to Scrapie by Natural Route of Infection

USDA-ARS?s Scientific Manuscript database

Interspecies transmission studies afford the opportunity to better understand the potential host range and origins of prion diseases. Previous experiments demonstrated that white-tailed deer are susceptible to sheep-derived scrapie by intracranial inoculation. The purpose of this study was to determ...

White-Tailed Deer Susceptible to Scrapie by Natural Route of Infection

USDA-ARS?s Scientific Manuscript database

Interspecies transmission studies afford the opportunity to better understand the potential host range and origins of prion diseases. Previous experiments demonstrated that white-tailed deer are susceptible to sheep-derived scrapie by intracranial inoculation. The purpose of an ongoing study is to...

Transmissibility of caprine scrapie in ovine transgenic mice

USDA-ARS?s Scientific Manuscript database

Scrapie is a transmissible spongiform encephalopathy or prion disease of domestic sheep and goats. The current US and Canadian control programs are based on diagnosis by identification of abnormal prion protein in brain or lymphoid tissues with selective culling of genetically susceptible sheep exp...

hTERT-immortalized ovine microglia propagate natural scrapie isolates

USDA-ARS?s Scientific Manuscript database

Scrapie is a transmissible fatal neurodegenerative disease of sheep that is caused by a novel infectious agent called a prion. The study of prions under controlled culture conditions is crucial to advancing knowledge regarding prion processes of replication and accumulation within cells. Unfortunate...

Identification of Two Prion Protein Regions That Modify Scrapie Incubation Time

PubMed Central

Supattapone, Surachai; Muramoto, Tamaki; Legname, Giuseppe; Mehlhorn, Ingrid; Cohen, Fred E.; DeArmond, Stephen J.; Prusiner, Stanley B.; Scott, Michael R.

2001-01-01

A series of prion transmission experiments was performed in transgenic (Tg) mice expressing either wild-type, chimeric, or truncated prion protein (PrP) molecules. Following inoculation with Rocky Mountain Laboratory (RML) murine prions, scrapie incubation times for Tg(MoPrP)4053, Tg(MHM2)294/Prnp0/0, and Tg(MoPrP,Δ23–88)9949/Prnp0/0 mice were ∼50, 120, and 160 days, respectively. Similar scrapie incubation times were obtained after inoculation of these lines of Tg mice with either MHM2(MHM2(RML)) or MoPrP(Δ23–88)(RML) prions, excluding the possibility that sequence-dependent transmission barriers could account for the observed differences. Tg(MHM2)294/Prnp0/0 mice displayed prolonged scrapie incubation times with four different strains of murine prions. These data provide evidence that the N terminus of MoPrP and the chimeric region of MHM2 PrP (residues 108 through 111) both influence the inherent efficiency of prion propagation. PMID:11152514

Comparison of strategies for substantiating freedom from scrapie in a sheep flock.

PubMed

Durand, Benoit; Martinez, Marie-José; Calavas, Didier; Ducrot, Christian

2009-04-30

The public health threat represented by a potential circulation of bovine spongiform encephalopathy agent in sheep population has led European animal health authorities to launch large screening and genetic selection programmes. If demonstrated, such a circulation would have dramatic economic consequences for sheep breeding sector. In this context, it is important to evaluate the feasibility of qualification procedures that would allow sheep breeders demonstrating their flock is free from scrapie. Classical approaches, based on surveys designed to detect disease presence, do not account for scrapie specificities: the genetic variations of susceptibility and the absence of live diagnostic test routinely available. Adapting these approaches leads to a paradoxical situation in which a greater amount of testing is needed to substantiate disease freedom in genetically resistant flocks than in susceptible flocks, whereas probability of disease freedom is a priori higher in the former than in the latter. The goal of this study was to propose, evaluate and compare several qualification strategies for demonstrating a flock is free from scrapie. A probabilistic framework was defined that accounts for scrapie specificities and allows solving the preceding paradox. Six qualification strategies were defined that combine genotyping data, diagnostic tests results and flock pedigree. These were compared in two types of simulated flocks: resistant and susceptible flocks. Two strategies allowed demonstrating disease freedom in several years, for the majority of simulated flocks: a strategy in which all the flock animals are genotyped, and a strategy in which only founders animals are genotyped, the flock pedigree being known. In both cases, diagnostic tests are performed on culled animals. The less costly strategy varied according to the genetic context (resistant or susceptible) and to the relative costs of a genotyping exam and of a diagnostic test. This work demonstrates that combining data sources allows substantiating a flock is free from scrapie within a reasonable time frame. Qualification schemes could thus be a useful tool for voluntary or mandatory scrapie control programmes. However, there is no general strategy that would always minimize the costs and choice of the strategy should be adapted to local genetic conditions.

Ovine Reference Materials and Assays for Prion Genetic Testing

USDA-ARS?s Scientific Manuscript database

Background: Genetic predisposition to scrapie in sheep is associated with variation in the peptide sequence of the ovine prion protein encoded by Prnp. Codon variants implicated in scrapie susceptibility or disease progression include those at amino acid positions 112, 136, 141, 154, and 171. Nin...

Prolonged Incubation Time in Sheep with QK171 Genotype

USDA-ARS?s Scientific Manuscript database

Background: Sheep scrapie susceptibility or resistance is a function of genotype with polymorphisms at codon 171 playing a major role. Glutamine (Q) at 171 contributes to scrapie susceptibility while arginine (R) is associated with resistance. In some breeds, lysine (K) occurs at 171, but its affe...

9 CFR 54.1 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-01-01

... project. A pilot project authorized by the Administrator in writing, designed to test or improve program... CONTROL AND ERADICATION OF LIVESTOCK OR POULTRY DISEASES CONTROL OF SCRAPIE § 54.1 Definitions. Accredited... by the Administrator in accordance with § 54.11 to conduct one or more scrapie tests, or genotype...

9 CFR 54.1 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-01-01

... project. A pilot project authorized by the Administrator in writing, designed to test or improve program... CONTROL AND ERADICATION OF LIVESTOCK OR POULTRY DISEASES CONTROL OF SCRAPIE § 54.1 Definitions. Accredited... by the Administrator in accordance with § 54.11 to conduct one or more scrapie tests, or genotype...

9 CFR 54.1 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-01-01

... project. A pilot project authorized by the Administrator in writing, designed to test or improve program... CONTROL AND ERADICATION OF LIVESTOCK OR POULTRY DISEASES CONTROL OF SCRAPIE § 54.1 Definitions. Accredited... by the Administrator in accordance with § 54.11 to conduct one or more scrapie tests, or genotype...

9 CFR 54.1 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-01-01

... project. A pilot project authorized by the Administrator in writing, designed to test or improve program... CONTROL AND ERADICATION OF LIVESTOCK OR POULTRY DISEASES CONTROL OF SCRAPIE § 54.1 Definitions. Accredited... by the Administrator in accordance with § 54.11 to conduct one or more scrapie tests, or genotype...

9 CFR 54.1 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-01-01

... project. A pilot project authorized by the Administrator in writing, designed to test or improve program... CONTROL AND ERADICATION OF LIVESTOCK OR POULTRY DISEASES CONTROL OF SCRAPIE § 54.1 Definitions. Accredited... by the Administrator in accordance with § 54.11 to conduct one or more scrapie tests, or genotype...

White-tailed Deer are Susceptible to Sheep Scrapie by Intracerebral Inoculation

USDA-ARS?s Scientific Manuscript database

Interspecies transmission studies afford the opportunity to better understand the potential host range and origins of prion diseases. The purpose of this experiment was to determine susceptibility of white-tailed deer to scrapie after intracerebral inoculation and to compare clinical signs and lesi...

Assessment of prion reduction filters in decreasing infectivity of ultracentrifuged 263K scrapie-infected brain homogenates in "spiked" human blood and red blood cells.

PubMed

Cardone, Franco; Sowemimo-Coker, Samuel; Abdel-Haq, Hanin; Sbriccoli, Marco; Graziano, Silvia; Valanzano, Angelina; Berardi, Vito Angelo; Galeno, Roberta; Puopolo, Maria; Pocchiari, Maurizio

2014-04-01

The safety of red blood cells (RBCs) is of concern because of the occurrence of four transfusion-transmitted variant Creutzfeldt-Jakob disease (vCJD) cases in the United Kingdom. The absence of validated screening tests requires the use of procedures to remove prions from blood to minimize the risk of transmission. These procedures must be validated using infectious prions in a form that is as close as possible to one in blood. Units of human whole blood (WB) and RBCs were spiked with high-speed supernatants of 263K scrapie-infected hamster brain homogenates. Spiked samples were leukoreduced and then passed through prion-removing filters (Pall Corporation). In another experiment, RBCs from 263K scrapie-infected hamsters were treated as above, and residual infectivity was measured by bioassay. The overall removal of infectivity by the filters from prion-spiked WB and RBCs was approximately two orders of magnitude. No infectivity was detected in filtered hamster RBCs endogenously infected with scrapie. The use of prion-removing filters may help to reduce the risk of transfusion-transmitted vCJD. To avoid overestimation of prion removal efficiency in validation studies, it may be more appropriate to use supernates from ultracentrifugation of scrapie-infected hamster brain homogenate rather than the current standard brain homogenates. © 2013 American Association of Blood Banks.

Screening of intact yeasts and cell extracts to reduce Scrapie prions during biotransformation of food waste.

PubMed

Huyben, David; Boqvist, Sofia; Passoth, Volkmar; Renström, Lena; Allard Bengtsson, Ulrika; Andréoletti, Olivier; Kiessling, Anders; Lundh, Torbjörn; Vågsholm, Ivar

2018-02-08

Yeasts can be used to convert organic food wastes to protein-rich animal feed in order to recapture nutrients. However, the reuse of animal-derived waste poses a risk for the transmission of infectious prions that can cause neurodegeneration and fatality in humans and animals. The aim of this study was to investigate the ability of yeasts to reduce prion activity during the biotransformation of waste substrates-thereby becoming a biosafety hurdle in such a circular food system. During pre-screening, 30 yeast isolates were spiked with Classical Scrapie prions and incubated for 72 h in casein substrate, as a waste substitute. Based on reduced Scrapie seeding activity, waste biotransformation and protease activities, intact cells and cell extracts of 10 yeasts were further tested. Prion analysis showed that five yeast species reduced Scrapie seeding activity by approximately 1 log10 or 90%. Cryptococcus laurentii showed the most potential to reduce prion activity since both intact and extracted cells reduced Scrapie by 1 log10 and achieved the highest protease activity. These results show that select forms of yeast can act as a prion hurdle during the biotransformation of waste. However, the limited ability of yeasts to reduce prion activity warrants caution as a sole barrier to transmission as higher log reductions are needed before using waste-cultured yeast in circular food systems.

A quantitative risk assessment for the safety of carcase storage systems for scrapie infected farms.

PubMed

Adkin, A; Jones, D L; Eckford, R L; Edwards-Jones, G; Williams, A P

2014-10-01

To determine the risk associated with the use of carcase storage vessels on a scrapie infected farm. A stochastic quantitative risk assessment was developed to determine the rate of accumulation and fate of scrapie in a novel low-input storage system. For an example farm infected with classical scrapie, a mean of 10(3·6) Ovine Oral ID50 s was estimated to accumulate annually. Research indicates that the degradation of any prions present may range from insignificant to a magnitude of one or two logs over several months of storage. For infected farms, the likely partitioning of remaining prion into the sludge phase would necessitate the safe operation and removal of resulting materials from these systems. If complete mixing could be assumed, on average, the concentrations of infectivity are estimated to be slightly lower than that measured in placenta from infected sheep at lambing. This is the first quantitative assessment of the scrapie risk associated with fallen stock on farm and provides guidance to policy makers on the safety of one type of storage system and the relative risk when compared to other materials present on an infected farm. © 2014 Crown Copyright. Journal of Applied Microbiology © 2014 Society for Applied Microbiology This article is published with the permission of the Controller of HMSO and the Queen's Printer for Scotland.

9 CFR 54.22 - State scrapie certification boards.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false State scrapie certification boards. 54.22 Section 54.22 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT... boards are not appointed for the purpose of providing APHIS with consensus advice or policy...

Temporospatial distribution of microglial activation in a murine model of scrapie

USDA-ARS?s Scientific Manuscript database

Mouse models of prion disease offer the advantages of genetic homogeneity and short incubation times while retaining the disease phenotype of natural mammalian hosts. Intracranial (IC) inoculation of C57BL/6 mice with a mouse-adapted scrapie strain (RML) yields uniform incubation periods with a rapi...

Determining the relative susceptibility of four prion protein genotypes to atypical scrapie

USDA-ARS?s Scientific Manuscript database

Atypical scrapie is a sheep prion (PrPSc) disease whose epidemiology is consistent with a sporadic origin and is associated with specific polymorphisms of the normal cellular prion protein (PrPC). We describe a mass spectrometry-based method of detecting and quantifying the polymorphisms of sheep P...

9 CFR 54.21 - Participation.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Participation. 54.21 Section 54.21 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE COOPERATIVE CONTROL AND ERADICATION OF LIVESTOCK OR POULTRY DISEASES CONTROL OF SCRAPIE Scrapie Flock Certification Program § 54.21 Participation. Any...

9 CFR 79.1 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-01-01

... to conduct one or more scrapie tests, or genotype tests, on one or more tissues. Area veterinarian in.... Consistent State. (1) A State that the Administrator has determined conducts an active State scrapie control program that either: (i) Meets the requirements of § 79.6; or (ii) Effectively enforces a State designed...

9 CFR 79.1 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-01-01

... to conduct one or more scrapie tests, or genotype tests, on one or more tissues. Area veterinarian in.... Consistent State. (1) A State that the Administrator has determined conducts an active State scrapie control program that either: (i) Meets the requirements of § 79.6; or (ii) Effectively enforces a State designed...

9 CFR 79.1 - Definitions.

Code of Federal Regulations, 2013 CFR

2013-01-01

... to conduct one or more scrapie tests, or genotype tests, on one or more tissues. Area veterinarian in.... Consistent State. (1) A State that the Administrator has determined conducts an active State scrapie control program that either: (i) Meets the requirements of § 79.6; or (ii) Effectively enforces a State designed...

9 CFR 79.1 - Definitions.

Code of Federal Regulations, 2012 CFR

2012-01-01

... to conduct one or more scrapie tests, or genotype tests, on one or more tissues. Area veterinarian in.... Consistent State. (1) A State that the Administrator has determined conducts an active State scrapie control program that either: (i) Meets the requirements of § 79.6; or (ii) Effectively enforces a State designed...

9 CFR 79.1 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-01-01

... to conduct one or more scrapie tests, or genotype tests, on one or more tissues. Area veterinarian in.... Consistent State. (1) A State that the Administrator has determined conducts an active State scrapie control program that either: (i) Meets the requirements of § 79.6; or (ii) Effectively enforces a State designed...

9 CFR 54.3 - Animals eligible for indemnity payments.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Animals eligible for indemnity payments. 54.3 Section 54.3 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... SCRAPIE Scrapie Indemnification Program § 54.3 Animals eligible for indemnity payments. (a) Indemnity may...

9 CFR 54.3 - Animals eligible for indemnity payments.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Animals eligible for indemnity payments. 54.3 Section 54.3 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... SCRAPIE Scrapie Indemnification Program § 54.3 Animals eligible for indemnity payments. (a) Indemnity may...

9 CFR 54.3 - Animals eligible for indemnity payments.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Animals eligible for indemnity payments. 54.3 Section 54.3 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... SCRAPIE Scrapie Indemnification Program § 54.3 Animals eligible for indemnity payments. (a) Indemnity may...

9 CFR 54.3 - Animals eligible for indemnity payments.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Animals eligible for indemnity payments. 54.3 Section 54.3 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... SCRAPIE Scrapie Indemnification Program § 54.3 Animals eligible for indemnity payments. (a) Indemnity may...

9 CFR 54.3 - Animals eligible for indemnity payments.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Animals eligible for indemnity payments. 54.3 Section 54.3 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... SCRAPIE Scrapie Indemnification Program § 54.3 Animals eligible for indemnity payments. (a) Indemnity may...

Differential immunoreactivity of goat derived scrapie following in vitro misfolding versus mouse bioassay

USDA-ARS?s Scientific Manuscript database

The protein misfolding cyclic amplification (PMCA) assay allows for detection of the disease associated isoform of the prion protein in tissues and fluids of sheep where it was previously undetected by conventional western blot and immunohistochemistry assays. Studies of goats with scrapie have yet ...

Cytokine antibody array analysis in brain and periphery of scrapie-infected Tg338 mice

USDA-ARS?s Scientific Manuscript database

Scrapie is a naturally occurring transmissible spongiform encephalopathy (TSE) that affects sheep and goats. While a change in prion protein conformation has been established as an important aspect of disease, other aspects of TSE pathogenesis are not fully understood. The preset study used protei...

9 CFR 79.2 - Identification of sheep and goats in interstate commerce.

Code of Federal Regulations, 2014 CFR

2014-01-01

... prefix that has been linked in the National Scrapie Database with the assigned premises identification... official identification method or device approved by the Administrator. (3) The owner of the flock of... premises identification if they are linked to the premises in the National Scrapie Database) will be...

9 CFR 79.2 - Identification of sheep and goats in interstate commerce.

Code of Federal Regulations, 2013 CFR

2013-01-01

... prefix that has been linked in the National Scrapie Database with the assigned premises identification... official identification method or device approved by the Administrator. (3) The owner of the flock of... premises identification if they are linked to the premises in the National Scrapie Database) will be...

9 CFR 79.2 - Identification of sheep and goats in interstate commerce.

Code of Federal Regulations, 2012 CFR

2012-01-01

... prefix that has been linked in the National Scrapie Database with the assigned premises identification... official identification method or device approved by the Administrator. (3) The owner of the flock of... premises identification if they are linked to the premises in the National Scrapie Database) will be...

A transfectant RK13 cell line permissive to classical caprine scrapie prion propagation

USDA-ARS?s Scientific Manuscript database

Classical scrapie is a form of transmissible spongiform encephalopathies (TSE) affecting domestic goats and sheep and disease is characterized by the accumulation of abnormal conformational isoform (PrP-Sc) of normal cellular prion protein (PrP-C) in the central nervous system and, in most cases, ly...

Prolonged incubation time in sheep with prion protein containing lysine at position 171

USDA-ARS?s Scientific Manuscript database

Sheep scrapie susceptibility or resistance is a function of genotype with polymorphisms at codon 171 in the sheep prion gene playing a major role. Glutamine (Q) at 171 contributes to scrapie susceptibility while arginine (R) is associated with resistance. In some breeds, lysine (K) occurs at codon 1...

Department of Agriculture Semiannual Regulatory Agenda

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-20

... Import Restrictions on Certain 0579-AC74 Live Fish 189 Scrapie in Sheep and Goats 0579-AC92 190 Plant..., Farming Experience as an Eligibility Requirement; 5201, Eligibility of Equine Farmers and Ranchers for... Phone: 301 734-8695 RIN: 0579-AC74 189. SCRAPIE IN SHEEP AND GOATS Legal Authority: 7 USC 8301 to 8317...

9 CFR 79.7 - Waiver of requirements for scrapie control pilot projects.

Code of Federal Regulations, 2014 CFR

2014-01-01

... contains testing or other procedures that indicate that an animal, despite meeting the definition of high... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Waiver of requirements for scrapie control pilot projects. 79.7 Section 79.7 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION...

9 CFR 54.9 - Waiver of requirements for scrapie control pilot projects.

Code of Federal Regulations, 2012 CFR

2012-01-01

... contains testing or other procedures that indicate that an animal, despite meeting the definition of high... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Waiver of requirements for scrapie control pilot projects. 54.9 Section 54.9 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION...

9 CFR 54.9 - Waiver of requirements for scrapie control pilot projects.

Code of Federal Regulations, 2010 CFR

2010-01-01

... contains testing or other procedures that indicate that an animal, despite meeting the definition of high... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Waiver of requirements for scrapie control pilot projects. 54.9 Section 54.9 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION...

9 CFR 79.7 - Waiver of requirements for scrapie control pilot projects.

Code of Federal Regulations, 2013 CFR

2013-01-01

... contains testing or other procedures that indicate that an animal, despite meeting the definition of high... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Waiver of requirements for scrapie control pilot projects. 79.7 Section 79.7 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION...

9 CFR 79.7 - Waiver of requirements for scrapie control pilot projects.

Code of Federal Regulations, 2011 CFR

2011-01-01

... contains testing or other procedures that indicate that an animal, despite meeting the definition of high... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Waiver of requirements for scrapie control pilot projects. 79.7 Section 79.7 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION...

9 CFR 79.7 - Waiver of requirements for scrapie control pilot projects.

Code of Federal Regulations, 2010 CFR

2010-01-01

... contains testing or other procedures that indicate that an animal, despite meeting the definition of high... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Waiver of requirements for scrapie control pilot projects. 79.7 Section 79.7 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION...

9 CFR 54.9 - Waiver of requirements for scrapie control pilot projects.

Code of Federal Regulations, 2014 CFR

2014-01-01

... contains testing or other procedures that indicate that an animal, despite meeting the definition of high... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Waiver of requirements for scrapie control pilot projects. 54.9 Section 54.9 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION...

9 CFR 79.7 - Waiver of requirements for scrapie control pilot projects.

Code of Federal Regulations, 2012 CFR

2012-01-01

... contains testing or other procedures that indicate that an animal, despite meeting the definition of high... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Waiver of requirements for scrapie control pilot projects. 79.7 Section 79.7 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION...

9 CFR 54.9 - Waiver of requirements for scrapie control pilot projects.

Code of Federal Regulations, 2013 CFR

2013-01-01

... contains testing or other procedures that indicate that an animal, despite meeting the definition of high... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Waiver of requirements for scrapie control pilot projects. 54.9 Section 54.9 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION...

9 CFR 54.9 - Waiver of requirements for scrapie control pilot projects.

Code of Federal Regulations, 2011 CFR

2011-01-01

... contains testing or other procedures that indicate that an animal, despite meeting the definition of high... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Waiver of requirements for scrapie control pilot projects. 54.9 Section 54.9 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION...

Inoculation of Scrapie with the Self-Assembling RADA-Peptide Disrupts Prion Accumulation and Extends Hamster Survival

USDA-ARS?s Scientific Manuscript database

Intercerebral inoculation of 263K Scrapie brain homogenate (PrPsc) with a self-assembling RADA-peptide (RADA) significantly delayed disease onset and increased hamster survival. Time of survival was dependent on the dose of RADA and pre-incubation with PrPsc prior to inoculation. RADA treatment resu...

Different structural stability and toxicity of PrP(ARR) and PrP(ARQ) sheep prion protein variants.

PubMed

Paludi, Domenico; Thellung, Stefano; Chiovitti, Katia; Corsaro, Alessandro; Villa, Valentina; Russo, Claudio; Ianieri, Adriana; Bertsch, Uwe; Kretzschmar, Hans A; Aceto, Antonio; Florio, Tullio

2007-12-01

The polymorphisms at amino acid residues 136, 154, and 171 in ovine prion protein (PrP) have been associated with different susceptibility to scrapie: animals expressing PrP(ARQ) [PrP(Ala136/Arg154/Gln171)] show vulnerability, whereas those that express PrP(ARR) [PrP(Ala136/Arg154/Arg171)] are resistant to scrapie. The aim of this study was to evaluate the in vitro toxic effects of PrP(ARR) and PrP(ARQ) variants in relation with their structural characteristics. We show that both peptides cause cell death inducing apoptosis but, unexpectedly, the scrapie resistant PrP(ARR) form was more toxic than the scrapie susceptible PrP(ARQ) variant. Moreover, the alpha-helical conformation of PrP(ARR) was less stable than that of PrP(ARQ) and the structural determinants responsible of these different conformational stabilities were characterized by spectroscopic analysis. We observed that PrP toxicity was inversely related to protein structural stability, being the unfolded conformation more toxic than the native one. However, the PrP(ARQ) variant displays a higher propensity to form large aggregates than PrP(ARR). Interestingly, in the presence of small amounts of PrP(ARR), PrP(ARQ) aggregability was reduced to levels similar to that of PrP(ARR). Thus, in contrast to PrP(ARR) toxicity, scrapie transmissibility seems to reside in the more stable conformation of PrP(ARQ) that allows the formation of large amyloid fibrils.

Generalized cerebral atrophy seen on MRI in a naturally exposed animal model for creutzfeldt-jakob disease

PubMed Central

2010-01-01

Background Magnetic resonance imaging has been used in the diagnosis of human prion diseases such as sCJD and vCJD, but patients are scanned only when clinical signs appear, often at the late stage of disease. This study attempts to answer the questions "Could MRI detect prion diseases before clinical symptoms appear?, and if so, with what confidence?" Methods Scrapie, the prion disease of sheep, was chosen for the study because sheep can fit into a human sized MRI scanner (and there were no large animal MRI scanners at the time of this study), and because the USDA had, at the time of the study, a sizeable sample of scrapie exposed sheep, which we were able to use for this purpose. 111 genetically susceptible sheep that were naturally exposed to scrapie were used in this study. Results Our MRI findings revealed no clear, consistent hyperintense or hypointense signal changes in the brain on either clinically affected or asymptomatic positive animals on any sequence. However, in all 37 PrPSc positive sheep (28 asymptomatic and 9 symptomatic), there was a greater ventricle to cerebrum area ratio on MRI compared to 74 PrPSc negative sheep from the scrapie exposed flock and 6 control sheep from certified scrapie free flocks as defined by immunohistochemistry (IHC). Conclusions Our findings indicate that MRI imaging can detect diffuse cerebral atrophy in asymptomatic and symptomatic sheep infected with scrapie. Nine of these 37 positive sheep, including 2 one-year old animals, were PrPSc positive only in lymph tissues but PrPSc negative in the brain. This suggests either 1) that the cerebral atrophy/neuronal loss is not directly related to the accumulation of PrPSc within the brain or 2) that the amount of PrPSc in the brain is below the detectable limits of the utilized immunohistochemistry assay. The significance of these findings remains to be confirmed in human subjects with CJD. PMID:21108848

An Economic Evaluation of Preclinical Testing Strategies Compared to the Compulsory Scrapie Flock Scheme in the Control of Classical Scrapie

PubMed Central

Hawkins, Neil; Houston, Fiona; Fryer, Helen; Kao, Rowland

2012-01-01

Cost-benefit is rarely combined with nonlinear dynamic models when evaluating control options for infectious diseases. The current strategy for scrapie in Great Britain requires that all genetically susceptible livestock in affected flocks be culled (Compulsory Scrapie Flock Scheme or CSFS). However, this results in the removal of many healthy sheep, and a recently developed pre-clinical test for scrapie now offers a strategy based on disease detection. We explore the flock level cost-effectiveness of scrapie control using a deterministic transmission model and industry estimates of costs associated with genotype testing, pre-clinical tests and the value of a sheep culled. Benefit was measured in terms of the reduction in the number of infected sheep sold on, compared to a baseline strategy of doing nothing, using Incremental Cost Effectiveness analysis to compare across strategies. As market data was not available for pre-clinical testing, a threshold analysis was used to set a unit-cost giving equal costs for CSFS and multiple pre-clinical testing (MT, one test each year for three consecutive years). Assuming a 40% within-flock proportion of susceptible genotypes and a test sensitivity of 90%, a single test (ST) was cheaper but less effective than either the CSFS or MT strategies (30 infected-sales-averted over the lifetime of the average epidemic). The MT strategy was slightly less effective than the CSFS and would be a dominated strategy unless preclinical testing was cheaper than the threshold price of £6.28, but may be appropriate for flocks with particularly valuable livestock. Though the ST is not currently recommended, the proportion of susceptible genotypes in the national flock is likely to continue to decrease; this may eventually make it a cost-effective alternative to the MT or CSFS. PMID:22412943

Variation in the prion protein sequence in Dutch goat breeds.

PubMed

Windig, J J; Hoving, R A H; Priem, J; Bossers, A; van Keulen, L J M; Langeveld, J P M

2016-10-01

Scrapie is a neurodegenerative disease occurring in goats and sheep. Several haplotypes of the prion protein increase resistance to scrapie infection and may be used in selective breeding to help eradicate scrapie. In this study, frequencies of the allelic variants of the PrP gene are determined for six goat breeds in the Netherlands. Overall frequencies in Dutch goats were determined from 768 brain tissue samples in 2005, 766 in 2008 and 300 in 2012, derived from random sampling for the national scrapie surveillance without knowledge of the breed. Breed specific frequencies were determined in the winter 2013/2014 by sampling 300 breeding animals from the main breeders of the different breeds. Detailed analysis of the scrapie-resistant K222 haplotype was carried out in 2014 for 220 Dutch Toggenburger goats and in 2015 for 942 goats from the Saanen derived White Goat breed. Nine haplotypes were identified in the Dutch breeds. Frequencies for non-wild type haplotypes were generally low. Exception was the K222 haplotype in the Dutch Toggenburger (29%) and the S146 haplotype in the Nubian and Boer breeds (respectively 7 and 31%). The frequency of the K222 haplotype in the Toggenburger was higher than for any other breed reported in literature, while for the White Goat breed it was with 3.1% similar to frequencies of other Saanen or Saanen derived breeds. Further evidence was found for the existence of two M142 haplotypes, M142 /S240 and M142 /P240 . Breeds vary in haplotype frequencies but frequencies of resistant genotypes are generally low and consequently selective breeding for scrapie resistance can only be slow but will benefit from animals identified in this study. The unexpectedly high frequency of the K222 haplotype in the Dutch Toggenburger underlines the need for conservation of rare breeds in order to conserve genetic diversity rare or absent in other breeds. © 2016 Blackwell Verlag GmbH.

Comparison of cattle and sheep colonic permeabilities to horseradish peroxidase and hamster scrapie prion protein in vitro

PubMed Central

McKie, A; Zammit, P; Naftalin, R

1999-01-01

BACKGROUND—Paracellular permeability to solutes across the descending colon is much higher in cattle than sheep. This is a possible route for transmission of infective materials, such as scrapie prion.
AIMS—To compare the permeabilities of labelled scrapie prion protein and other macromolecules in bovine and ovine descending colons in vitro.
METHODS—Using fresh slaughterhouse material, transepithelial fluxes of macromolecules across colonic mucosae mounted in Ussing chambers were measured by monitoring transport of either enzyme activity or radioactivity.
RESULTS—The comparative bovine to ovine permeability ratio of the probes increased with molecular weight: from 3.1 (0.13) for PEG400 to 10.67 (0.20) (p<0.001) for PEG4000; and from 1.64 (0.17) for microperoxidase to 7.03 (0.20) (p<0.001) for horseradish peroxidase (HRP). The permeability of 125I-labelled inactivated Syrian hamster scrapie prion protein (ShaPrPsc) was 7.02 (0.33)-fold higher in bovine than ovine colon (p<0.0025). In each species, the probe permeabilities decreased according to the formula: P = Po.exp(−K.ra). The "ideal" permeabilities, Po are similar, however, K(ovine) = 2.46 (0.20) cm/h/nm exceeds K(bovine) = 0.85 (0.15) cm/h/nm (p<0.001) indicating that bovine colon has a higher proportion of wide pores than ovine. Image analysis confirmed that HRP permeated through the bovine mucosal layer via a pericryptal paracellular route much more rapidly than in sheep.
CONCLUSIONS—These data may imply that scrapie prion is transmitted in vivo more easily across the low resistance bovine colonic barrier than in other species.


Keywords: cattle; sheep; colon; paracellular permeability; horseradish peroxidase; hamster scrapie prion protein PMID:10562587

Primary transmission of chronic wasting disease versus scrapie prions from small ruminants to transgenic mice expressing ovine and cervid prion protein

USDA-ARS?s Scientific Manuscript database

Identifying transmissible spongiform encephalopathy (TSE) reservoirs that could lead to disease re-emergence is imperative to U.S. scrapie eradication efforts. Transgenic mice expressing the cervid (TgElk) or ovine (Tg338) prion protein have aided characterization of chronic wasting disease (CWD) an...

Relationships between PrPSc stability and incubation time for United States scrapie strains in a natural host system

USDA-ARS?s Scientific Manuscript database

Transmissible spongiform encephalopathies (TSEs), including scrapie in sheep (Ovis aries), are fatal neurodegenerative diseases caused by the misfolding of the cellular prion protein (PrP**C) into a beta-rich conformer (PrP**Sc) that accumulates into higher-order structures in the brain and other ti...

Classical scrapie prions in ovine blood are associated with B lymphocytes and platelets-rich plasma

USDA-ARS?s Scientific Manuscript database

Classical scrapie is a naturally occurring fatal brain disease of sheep and goats which is caused by prions, a novel class of infectious agent, and is accompanied by the accumulation of abnormal isoforms of prion protein (PrP-Sc) in certain neural and lymphoid tissues. Although collection of a blood...

Accumulation of PrP-Sc in hemal nodes of naturally and experimentally scrapie-infected sheep

USDA-ARS?s Scientific Manuscript database

Classical scrapie is a naturally occurring fatal disease of sheep and goats which is caused by prions, a novel class of infectious agent. Infection is accompanied by accumulation of abnormal isoforms of the prion protein (PrP-Sc) in certain neural and lymphoid tissues. Hemal nodes, which are unique ...

Transmission of the agent of sheep scrapie to deer results in PrPSc with two distinct molecular profiles

USDA-ARS?s Scientific Manuscript database

The purpose of this work was to determine susceptibility of white-tailed deer (WTD) to the agent of sheep scrapie and to compare the resultant PrPSc to that of the original inoculum and chronic wasting disease (CWD). We inoculated WTD by a natural route of exposure (concurrent oral and intranasal (I...

9 CFR 54.11 - Approval of laboratories to run official scrapie tests and official genotype tests.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Approval of laboratories to run official scrapie tests and official genotype tests. 54.11 Section 54.11 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE COOPERATIVE CONTROL AND ERADICATION OF LIVESTOCK OR POULTRY DISEASES CONTROL OF...

9 CFR 54.11 - Approval of laboratories to run official scrapie tests and official genotype tests.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Approval of laboratories to run official scrapie tests and official genotype tests. 54.11 Section 54.11 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE COOPERATIVE CONTROL AND ERADICATION OF LIVESTOCK OR POULTRY DISEASES CONTROL OF...

9 CFR 54.11 - Approval of laboratories to run official scrapie tests and official genotype tests.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Approval of laboratories to run official scrapie tests and official genotype tests. 54.11 Section 54.11 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE COOPERATIVE CONTROL AND ERADICATION OF LIVESTOCK OR POULTRY DISEASES CONTROL OF...

9 CFR 54.11 - Approval of laboratories to run official scrapie tests and official genotype tests.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Approval of laboratories to run official scrapie tests and official genotype tests. 54.11 Section 54.11 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE COOPERATIVE CONTROL AND ERADICATION OF LIVESTOCK OR POULTRY DISEASES CONTROL OF...

Cell surface expression of PrP-c and the presence of scrapie prions in the blood of goats

USDA-ARS?s Scientific Manuscript database

Classical scrapie is a naturally occurring fatal brain disease of goats and sheep which is caused by prions, a novel class of infectious agent, and is accompanied by the accumulation of abnormal isoforms of prion protein (PrP-Sc) in certain neural and lymphoid tissues. Although collection of a blood...

9 CFR 79.4 - Designation of scrapie-positive animals, high-risk animals, exposed animals, suspect animals...

Code of Federal Regulations, 2013 CFR

2013-01-01

... designated scrapie epidemiologist may conduct testing of animals if he or she determines such testing is... epidemiologist will select animals for testing in a manner that will provide a 95 percent confidence of detecting... lambed in the flock are available for testing, may limit the testing to all exposed and suspect animals...

9 CFR 79.4 - Designation of scrapie-positive animals, high-risk animals, exposed animals, suspect animals...

Code of Federal Regulations, 2014 CFR

2014-01-01

... designated scrapie epidemiologist may conduct testing of animals if he or she determines such testing is... epidemiologist will select animals for testing in a manner that will provide a 95 percent confidence of detecting... lambed in the flock are available for testing, may limit the testing to all exposed and suspect animals...

9 CFR 79.4 - Designation of scrapie-positive animals, high-risk animals, exposed animals, suspect animals...

Code of Federal Regulations, 2012 CFR

2012-01-01

... designated scrapie epidemiologist may conduct testing of animals if he or she determines such testing is... epidemiologist will select animals for testing in a manner that will provide a 95 percent confidence of detecting... lambed in the flock are available for testing, may limit the testing to all exposed and suspect animals...

Scrapie transmits to white-tailed deer by the oral route and has a molecular profile similar to chronic wasting disease

USDA-ARS?s Scientific Manuscript database

The purpose of this work was to determine susceptibility of white-tailed deer (WTD) to the agent of sheep scrapie and to compare the resultant PrPSc to that of the original inoculum and chronic wasting disease (CWD). We inoculated WTD by a natural route of exposure (concurrent oral and intranasal (I...

9 CFR 54.11 - Approval of laboratories to run official scrapie tests and official genotype tests.

Code of Federal Regulations, 2010 CFR

2010-01-01

... request approval to conduct one or more types of scrapie test or genotype test on one or more types of... type of test and for each type of tissue for which they request approval. (c) The Administrator may... the laboratory and shall give the director an opportunity to respond. If there are conflicts as to any...

Surveillance and simulation of bovine spongiform encephalopathy and scrapie in small ruminants in Switzerland

PubMed Central

2010-01-01

Background After bovine spongiform encephalopathy (BSE) emerged in European cattle livestock in 1986 a fundamental question was whether the agent established also in the small ruminants' population. In Switzerland transmissible spongiform encephalopathies (TSEs) in small ruminants have been monitored since 1990. While in the most recent TSE cases a BSE infection could be excluded, for historical cases techniques to discriminate scrapie from BSE had not been available at the time of diagnosis and thus their status remained unclear. We herein applied state-of-the-art techniques to retrospectively classify these animals and to re-analyze the affected flocks for secondary cases. These results were the basis for models, simulating the course of TSEs over a period of 70 years. The aim was to come to a statistically based overall assessment of the TSE situation in the domestic small ruminant population in Switzerland. Results In sum 16 TSE cases were identified in small ruminants in Switzerland since 1981, of which eight were atypical and six were classical scrapie. In two animals retrospective analysis did not allow any further classification due to the lack of appropriate tissue samples. We found no evidence for an infection with the BSE agent in the cases under investigation. In none of the affected flocks, secondary cases were identified. A Bayesian prevalence calculation resulted in most likely estimates of one case of BSE, five cases of classical scrapie and 21 cases of atypical scrapie per 100'000 small ruminants. According to our models none of the TSEs is considered to cause a broader epidemic in Switzerland. In a closed population, they are rather expected to fade out in the next decades or, in case of a sporadic origin, may remain at a very low level. Conclusions In summary, these data indicate that despite a significant epidemic of BSE in cattle, there is no evidence that BSE established in the small ruminant population in Switzerland. Classical and atypical scrapie both occur at a very low level and are not expected to escalate into an epidemic. In this situation the extent of TSE surveillance in small ruminants requires reevaluation based on cost-benefit analysis. PMID:20398417

Spatial reversal learning in preclinical scrapie-inoculated mice.

PubMed

Lysons, A M; Woollard, S J

1996-04-10

Acquisition and reversal of a two-choice spatial discrimination were tested in scrapie-inoculated mice. Both acquisition and reversal were normal in mice tested 138 and 103 days prior to the onset of clinical symptoms. At 65 days before onset of clinical symptoms, scrapie-inoculated mice required more trails to criterion in reversal learning, but this effect was not significant in a second experiment (68 days preclinical) and was transient: no effect was seen 33 days before symptoms. However, the course of reversal learning was abnormal in all three late preclinical groups (68, 65 and 33 days before symptoms). Reversal learning in these three groups was characterized by a rapid extinction of the original discrimination, followed by a period, absent in controls, during which performance showed no further improvement. This effect corresponds in time of onset to the appearance of characteristic neuropathological features.

Relationship of PrPSc molecular properties with incubation time in a natural prion disease host: a characterization of three isolates of U.S. sheep scrapie

USDA-ARS?s Scientific Manuscript database

Determination of aspects of tertiary and quaternary structure of PrPSc associated with differences in disease presentation in the host is a key area of interest in the prion field. Previously, we determined that a U.S. scrapie isolate (136-VDEP) with a short incubation time upon passage in sheep als...

Microsecond Unfolding Kinetics of Sheep Prion Protein Reveals an Intermediate that Correlates with Susceptibility to Classical Scrapie

PubMed Central

Chen, Kai-Chun; Xu, Ming; Wedemeyer, William J.; Roder, Heinrich

2011-01-01

The microsecond folding and unfolding kinetics of ovine prion proteins (ovPrP) were measured under various solution conditions. A fragment comprising residues 94–233 of the full-length ovPrP was studied for four variants with differing susceptibilities to classical scrapie in sheep. The observed biexponential unfolding kinetics of ovPrP provides evidence for an intermediate species. However, in contrast to previous results for human PrP, there is no evidence for an intermediate under refolding conditions. Global analysis of the kinetic data, based on a sequential three-state mechanism, quantitatively accounts for all folding and unfolding data as a function of denaturant concentration. The simulations predict that an intermediate accumulates under both folding and unfolding conditions, but is observable only in unfolding experiments because the intermediate is optically indistinguishable from the native state. The relative population of intermediates in two ovPrP variants, both transiently and under destabilizing equilibrium conditions, correlates with their propensities for classical scrapie. The variant susceptible to classical scrapie has a larger population of the intermediate state than the resistant variant. Thus, the susceptible variant should be favored to undergo the PrPC to PrPSc conversion and oligomerization. PMID:21889460

An overview of animal prion diseases

PubMed Central

2011-01-01

Prion diseases are transmissible neurodegenerative conditions affecting human and a wide range of animal species. The pathogenesis of prion diseases is associated with the accumulation of aggregates of misfolded conformers of host-encoded cellular prion protein (PrPC). Animal prion diseases include scrapie of sheep and goats, bovine spongiform encephalopathy (BSE) or mad cow disease, transmissible mink encephalopathy, feline spongiform encephalopathy, exotic ungulate spongiform encephalopathy, chronic wasting disease of cervids and spongiform encephalopathy of primates. Although some cases of sporadic atypical scrapie and BSE have also been reported, animal prion diseases have basically occurred via the acquisition of infection from contaminated feed or via the exposure to contaminated environment. Scrapie and chronic wasting disease are naturally sustaining epidemics. The transmission of BSE to human has caused more than 200 cases of variant Cruetzfeldt-Jacob disease and has raised serious public health concerns. The present review discusses the epidemiology, clinical neuropathology, transmissibility and genetics of animal prion diseases. PMID:22044871

[From the Scrapie syndrome of sheep and goat to the mad cow disease - the history of the discovery of prion].

PubMed

Liu, Rui; Weng, Yi

2009-05-01

Since the discovery of Scrapie Syndrome in sheep and goats in 1730, there emerged a series of diseases such as Creutzfeldt-Jakob disease, kuru disease and mad cow disease etc. In the research of kuru disease, the American scientist D. Carlteton Gajdusek found a new virus without the characteristic of DNA and RNA, which was awarded the Nobel Prize in physiology in 1976. Since then another American scientist, Stanley B. Prusiner, found a new virus-prion, taking protein as the genetic medium, which was awarded the Nobel prize in physiology and medicine in 1997. The discovery of prion is a great landmark in the research of life science, which laid a theoretical foundation for people to conquer a series of diseases such as Scrapie syndrome in sheep and goats, Creutzfeldt-Jakob disease, kuru disease and mad cow disease etc.

Theoretical Modeling of Molecular Mechanisms, Strains, and Time Scales in Prion Diseases

DTIC Science & Technology

2005-07-01

oversimplification) displays a linear relationship between the change in the free energy difference between cellular and scrapies form of the protein and...and show with molecular dynamics (MD) that the DSTP has more favorable intermonomer hydrogen bonding and proline dihedral strain energy than the BPT...ramping of the overall free energy surface slope in favor of the scrapies form. In Fig. 5 we illustrate two ways in which strains might breed true if

Evidence for more cost-effective surveillance options for bovine spongiform encephalopathy (BSE) and scrapie in Great Britain.

PubMed

Wall, Ben A; Arnold, Mark E; Radia, Devi; Gilbert, Will; Ortiz-Pelaez, Angel; Stärk, Katharina Dc; Van Klink, Ed; Guitian, Javier

2017-08-10

Transmissible spongiform encephalopathies (TSEs) are an important public health concern. Since the emergence of bovine spongiform encephalopathy (BSE) during the 1980s and its link with human Creutzfeldt-Jakob disease, active surveillance has been a key element of the European Union's TSE control strategy. Success of this strategy means that now, very few cases are detected compared with the number of animals tested. Refining surveillance strategies would enable resources to be redirected towards other public health priorities. Cost-effectiveness analysis was performed on several alternative strategies involving reducing the number of animals tested for BSE and scrapie in Great Britain and, for scrapie, varying the ratio of sheep sampled in the abattoir to fallen stock (which died on the farm). The most cost-effective strategy modelled for BSE involved reducing the proportion of fallen stock tested from 100% to 75%, producing a cost saving of ca GBP 700,000 per annum. If 50% of fallen stock were tested, a saving of ca GBP 1.4 million per annum could be achieved. However, these reductions are predicted to increase the period before surveillance can detect an outbreak. For scrapie, reducing the proportion of abattoir samples was the most cost-effective strategy modelled, with limited impact on surveillance effectiveness. This article is copyright of The Authors, 2017.

Phosphorylated human tau associates with mouse prion protein amyloid in scrapie-infected mice but does not increase progression of clinical disease.

PubMed

Race, Brent; Phillips, Katie; Kraus, Allison; Chesebro, Bruce

2016-07-03

Tauopathies are a family of neurodegenerative diseases in which fibrils of human hyperphosphorylated tau (P-tau) are believed to cause neuropathology. In Alzheimer disease, P-tau associates with A-beta amyloid and contributes to disease pathogenesis. In familial human prion diseases and variant CJD, P-tau often co-associates with prion protein amyloid, and might also accelerate disease progression. To test this latter possibility, here we compared progression of amyloid prion disease in vivo after scrapie infection of mice with and without expression of human tau. The mice used expressed both anchorless prion protein (PrP) and membrane-anchored PrP, that generate disease associated amyloid and non-amyloid PrP (PrPSc) after scrapie infection. Human P-tau induced by scrapie infection was only rarely associated with non-amyloid PrPSc, but abundant human P-tau was detected at extracellular, perivascular and axonal deposits associated with amyloid PrPSc. This pathology was quite similar to that seen in familial prion diseases. However, association of human and mouse P-tau with amyloid PrPSc did not diminish survival time following prion infection in these mice. By analogy, human P-tau may not affect prion disease progression in humans. Alternatively, these results might be due to other factors, including rapidity of disease, blocking effects by mouse tau, or low toxicity of human P-tau in this model.

Identification of seven haplotypes of the caprine PrP gene at codons 127, 142, 154, 211, 222 and 240 in French Alpine and Saanen breeds and their association with classical scrapie.

PubMed

Barillet, F; Mariat, D; Amigues, Y; Faugeras, R; Caillat, H; Moazami-Goudarzi, K; Rupp, R; Babilliot, J M; Lacroux, C; Lugan, S; Schelcher, F; Chartier, C; Corbière, F; Andréoletti, O; Perrin-Chauvineau, C

2009-03-01

In sheep, susceptibility to scrapie is mainly influenced by polymorphisms of the PrP gene. In goats, there are to date few data related to scrapie susceptibility association with PrP gene polymorphisms. In this study, we first investigated PrP gene polymorphisms of the French Alpine and Saanen breeds. Based on PrP gene open reading frame sequencing of artificial insemination bucks (n=404), six encoding mutations were identified at codons 127, 142, 154, 211, 222 and 240. However, only seven haplotypes could be detected: four (GIH(154)RQS, GIRQ(211)QS, GIRRK(222)S and GIRRQP(240)) derived from the wild-type allele (G(127)I(142)R(154)R(211)Q(222)S(240)) by a single-codon mutation, and two (S(127)IRRQP(240) and GM(142)RRQP(240)) by a double-codon mutation. A case-control study was then implemented in a highly affected Alpine and Saanen breed herd (90 cases/164 controls). Mutations at codon 142 (I/M), 154 (R/H), 211 (R/Q) and 222 (Q/K) were found to induce a significant degree of protection towards natural scrapie infection. Compared with the baseline homozygote wild-type genotype I(142)R(154)R(211)Q(222)/IRRQ goats, the odds of scrapie cases in IRQ(211)Q/IRRQ and IRRK(222)/IRRQ heterozygous animals were significantly lower [odds ratio (OR)=0.133, P<0.0001; and OR=0.048, P<0.0001, respectively]. The heterozygote M(142)RRQ/IRRQ genotype was only protective (OR=0.243, P=0.0186) in goats also PP(240) homozygous at codon 240. However, mutated allele frequencies in French Alpine and Saanen breeds were low (0.5-18.5 %), which prevent us from assessing the influence of all the possible genotypes in natural exposure conditions.

Partitioning of human and sheep forms of the pathogenic prion protein during the purification of therapeutic proteins from human plasma.

PubMed

Stenland, Christopher J; Lee, Douglas C; Brown, Paul; Petteway, Stephen R; Rubenstein, Richard

2002-11-01

Therapeutic proteins derived from human plasma and other biologic sources have demonstrated an excellent safety record relative to the potential threat of transmissible spongiform encephalopathy (TSE) transmission. Previously, hamster-adapted scrapie was used as a model agent to assess TSE clearance in purification steps leading to the isolation of biopharmaceutical proteins. The current study investigated the validity of hamster scrapie as a model for human TSE clearance studies. The partitioning of the pathogenic forms of the prion protein associated with human variant CJD (PrP(vCJD)), human sporadic CJD (PrP(sCJD)) and Gerstmann-Sträussler-Scheinker (PrP(GSS)) syndrome was compared to the partitioning of hamster scrapie (PrP(Sc)) in three plasma protein purification steps. Sheep scrapie (PrP(Sc)) was similarly evaluated. The starting materials for three plasma protein purification steps, cryoseparation, 3 percent PEG separation, and 11.5 percent PEG separation, were spiked with brain homogenates containing human PrP(vCJD), human PrP(sCJD), human PrP(GSS), sheep PrP(Sc), and hamster 263K PrP(Sc). The partitioning of the pathogenic form of the PrP was analyzed. Clearance of the pathogenic form of the PrP was measured relative to the effluent fraction. Regardless of the source of the pathogenic prion, clearance was similar to hamster PrP(Sc). A nominal amount of clearance (approx., 1 log), an intermediate amount of clearance (approx., 2 log), and a substantial amount of clearance (> or = 3 log) were observed for the cryoseparation, 3 percent PEG separation, and 11.5 percent PEG separation steps, respectively. In the latter step, no PrP was detected in the effluents. These data demonstrate that human prions, including vCJD prions, can be removed during the purification of human therapeutic proteins and indicate that partitioning of human prions is similar to that observed in the hamster scrapie model.

Comparative Susceptibility of Sheep of Different Origins, Breeds and PRNP Genotypes to Challenge with Bovine Spongiform Encephalopathy and Scrapie

PubMed Central

Houston, Fiona; Goldmann, Wilfred; Foster, James; González, Lorenzo; Jeffrey, Martin; Hunter, Nora

2015-01-01

Sheep are natural hosts of the prion disease, scrapie. They are also susceptible to experimental challenge with various scrapie strains and with bovine spongiform encephalopathy (BSE), which affects cattle and has been accidentally transmitted to a range of other species, including man. Incidence and incubation period of clinical disease in sheep following inoculation is controlled by the PRNP gene, which has different alleles defined on the basis of polymorphisms, particularly at codons 136, 154 and 171, although other codons are associated with survival time, and the exact responses of the sheep may be influenced by other breed-related differences. Here we report the results of a long term single study of experimental scrapie and BSE susceptibility of sheep of Cheviot, Poll Dorset and Suffolk breeds, originating from New Zealand and of a wide range of susceptible and resistant PRNP genotypes. Responses were compared with those of sheep from a closed Cheviot flock of UK origin (Roslin Cheviot flock). The unusually long observation period (6–8 years for most, but up to 12 years for others) allows us to draw robust conclusions about rates of survival of animals previously regarded as resistant to infection, particularly PRNP heterozygotes, and is the most comprehensive such study reported to date. BSE inoculation by an intracerebral route produced disease in all genotype groups with differing incubation periods, although M112T and L141F polymorphisms seemed to give some protection. Scrapie isolate SSBP/1, which has the shortest incubation period in sheep with at least one VRQ PRNP allele, also produced disease following sub-cutaneous inoculation in ARQ/ARQ animals of New Zealand origin, but ARQ/ARQ sheep from the Roslin flock survived the challenge. Our results demonstrate that the links between PRNP genotype and clinical prion disease in sheep are much less secure than previously thought, and may break down when, for example, a different breed of sheep is moved into a new flock. PMID:26587837

Changes in Protein Structure and Distribution Observed at Pre-Clinical Stages of Scrapie Pathogenesis

PubMed Central

Kretlow, Ariane; Wang, Qi; Beekes, Michael; Naumann, Dieter; Miller, Lisa M.

2011-01-01

Scrapie is a neurodegenerative disorder that involves the misfolding, aggregation and accumulation of the prion protein (PrP). The normal cellular PrP (PrPC) is rich in α-helical secondary structure, whereas the disease-associated pathogenic form of the protein (PrPSc) has an anomalously high β-sheet content. In this study, protein structural changes were examined in situ in the dorsal root ganglia from perorally 263K scrapie-infected and mock-infected hamsters using synchrotron Fourier Transform InfraRed Microspectroscopy (FTIRM) at four time points over the course of the disease (preclinical, 100 & 130 days post-infection (dpi); first clinical signs (~145 dpi); and terminal (~170 dpi)). Results showed clear changes in the total protein content, structure, and distribution as the disease progressed. At pre-clinical time points, the scrapie-infected animals exhibited a significant increase in protein expression, but the β-sheet protein content was significantly lower than controls. Based on these findings, we suggest that the pre-clinical stages of scrapie are characterized by an overexpression of proteins low in β-sheet content. As the disease progressed, the β-sheet content increased significantly. Immunostaining with a PrP-specific antibody, 3F4, confirmed that this increase was partly – but not solely – due to the formation of PrPSc in the tissue and indicated that other proteins high in β-sheet were produced, either by overexpression or misfolding. Elevated β-sheet was observed near the cell membrane at pre-clinical time points and also in the cytoplasm of infected neurons at later stages of infection. At the terminal stage of the disease, the protein expression declined significantly, likely due to degeneration and death of neurons. These dramatic changes in protein content and structure, especially at pre-clinical time points, emphasize the possibility for identifying other proteins involved in early pathogenesis, which are important for further understanding the disease. PMID:18625306

Analysis of protein levels of 24 cytokines in scrapie agent-infected brain and glial cell cultures from mice differing in prion protein expression levels.

PubMed

Tribouillard-Tanvier, Déborah; Striebel, James F; Peterson, Karin E; Chesebro, Bruce

2009-11-01

Activation of microglia and astroglia is seen in many neurodegenerative diseases including prion diseases. Activated glial cells produce cytokines as a protective response against certain pathogens and as part of the host inflammatory response to brain damage. In addition, cytokines might also exacerbate tissue damage initiated by other processes. In the present work using multiplex assays to analyze protein levels of 24 cytokines in scrapie agent-infected C57BL/10 mouse brains, we observed elevation of CCL2, CCL5, CXCL1, CXCL10, granulocyte-macrophage colony-stimulating factor (GM-CSF), gamma interferon (IFN-gamma), interleukin 1alpha (IL-1alpha), IL-1beta, IL-6, and IL-12p40. Scrapie agent-infected wild-type mice and transgenic mice expressing anchorless prion protein (PrP) had similar cytokine responses in spite of extensive differences in neuropathology. Therefore, these responses may be primarily a reaction to brain damage induced by prion infection rather than specific inducers of a particular type of pathology. To study the roles of astroglia and microglia in these cytokine responses, primary glial cultures were exposed to scrapie agent-infected brain homogenates. Microglia produced only IL-12p40 and CXCL10, whereas astroglia produced these cytokines plus CCL2, CCL3, CCL5, CXCL1, G-CSF, IL-1beta, IL-6, IL-12p70, and IL-13. Glial cytokine responses from wild-type mice and transgenic mice expressing anchorless PrP differed only slightly, but glia from PrP-null mice produced only IL-12p40, indicating that PrP expression was required for scrapie agent induction of other cytokines detected. The difference in cytokine response between microglia and astroglia correlated with 20-fold-higher levels of PrP expression in astroglia versus microglia, suggesting that high-level PrP expression on astroglia might be important for induction of certain cytokines.

Prion protein genotype survey confirms low frequency of scrapie-resistant K222 allele in British goat herds.

PubMed

Goldmann, W; Marier, E; Stewart, P; Konold, T; Street, S; Langeveld, J; Windl, O; Ortiz-Pelaez, A

2016-02-13

Scrapie in goats is a transmissible, fatal prion disease, which is endemic in the British goat population. The recent success in defining caprine PRNP gene variants that provide resistance to experimental and natural classical scrapie has prompted the authors to conduct a survey of PRNP genotypes in 10 goat breeds and 52 herds to find goats with the resistant K222 allele. They report here the frequencies in 1236 tested animals of the resistance-associated K222 and several other alleles by breed and herd. Eight animals were found to be heterozygous QK222 goats (0.64 per cent genotype frequency, 95 per cent CI 0.28 to 1.27 per cent) but no homozygous KK222 goats were detected. The K222 allele was found in Saanen, Toggenburg and Anglo-Nubian goats. The fact that only a few goats with the K222 allele have been identified does not preclude the possibility to design and implement successful breeding programmes at national level. British Veterinary Association.

Perturbation of the Secondary Structure of the Scrapie Prion Protein Under Conditions that Alter Infectivity

NASA Astrophysics Data System (ADS)

Gasset, Maria; Baldwin, Michael A.; Fletterick, Robert J.; Prusiner, Stanley B.

1993-01-01

Limited proteolysis of the scrapie prion protein (PrPSc) generates PrP 27-30, which polymerizes into amyloid. By attenuated total reflection-Fourier transform infrared spectroscopy, PrP 27-30 polymers contained 54% β-sheet, 25% α-helix, 10% turns, and 11% random coil; dispersion into detergent-lipid-protein-complexes preserved infectivity and secondary structure. Almost 60% of the β-sheet was low-frequency infrared-absorbing, reflecting intermolecular aggregation. Decreased low-frequency β-sheet and increased turn content were found after SDS/PAGE, which disassembled the amyloid polymers, denatured PrP 27-30, and diminished scrapie infectivity. Acid-induced transitions were reversible, whereas alkali produced an irreversible transition centered at pH 10 under conditions that diminished infectivity. Whether PrPSc synthesis involves a transition in the secondary structure of one or more domains of the cellular prion protein from α-helical, random coil, or turn into β-sheet remains to be established.

Effects of pH and aggregation in the human prion conversion into scrapie form: a study using molecular dynamics with excited normal modes.

PubMed

Lima, Angelica Nakagawa; de Oliveira, Ronaldo Junio; Braz, Antônio Sérgio Kimus; de Souza Costa, Maurício Garcia; Perahia, David; Scott, Luis Paulo Barbour

2018-03-15

There are two different prion conformations: (1) the cellular natural (PrP C ) and (2) the scrapie (PrP Sc ), an infectious form that tends to aggregate under specific conditions. PrP C and PrP Sc are widely different regarding secondary and tertiary structures. PrP Sc contains more and longer β-strands compared to PrP C . The lack of solved PrP Sc structures precludes a proper understanding of the mechanisms related to the transition between cellular and scrapie forms, as well as the aggregation process. In order to investigate the conformational transition between PrP C and PrP Sc , we applied MDeNM (molecular dynamics with excited normal modes), an enhanced sampling simulation technique that has been recently developed to probe large structural changes. These simulations yielded new structural rearrangements of the cellular prion that would have been difficult to obtain with standard MD simulations. We observed an increase in β-sheet formation under low pH (≤ 4) and upon oligomerization, whose relevance was discussed on the basis of the energy landscape theory for protein folding. The characterization of intermediate structures corresponding to transition states allowed us to propose a conversion model from the cellular to the scrapie prion, which possibly ignites the fibril formation. This model can assist the design of new drugs to prevent neurological disorders related to the prion aggregation mechanism.

Gene expression profiling of mesenteric lymph nodes from sheep with natural scrapie

PubMed Central

2014-01-01

Background Prion diseases are characterized by the accumulation of the pathogenic PrPSc protein, mainly in the brain and the lymphoreticular system. Although prions multiply/accumulate in the lymph nodes without any detectable pathology, transcriptional changes in this tissue may reflect biological processes that contribute to the molecular pathogenesis of prion diseases. Little is known about the molecular processes that occur in the lymphoreticular system in early and late stages of prion disease. We performed a microarray-based study to identify genes that are differentially expressed at different disease stages in the mesenteric lymph node of sheep naturally infected with scrapie. Oligo DNA microarrays were used to identify gene-expression profiles in the early/middle (preclinical) and late (clinical) stages of the disease. Results In the clinical stage of the disease, we detected 105 genes that were differentially expressed (≥2-fold change in expression). Of these, 43 were upregulated and 62 downregulated as compared with age-matched negative controls. Fewer genes (50) were differentially expressed in the preclinical stage of the disease. Gene Ontology enrichment analysis revealed that the differentially expressed genes were largely associated with the following terms: glycoprotein, extracellular region, disulfide bond, cell cycle and extracellular matrix. Moreover, some of the annotated genes could be grouped into 3 specific signaling pathways: focal adhesion, PPAR signaling and ECM-receptor interaction. We discuss the relationship between the observed gene expression profiles and PrPSc deposition and the potential involvement in the pathogenesis of scrapie of 7 specific differentially expressed genes whose expression levels were confirmed by real time-PCR. Conclusions The present findings identify new genes that may be involved in the pathogenesis of natural scrapie infection in the lymphoreticular system, and confirm previous reports describing scrapie-induced alterations in the expression of genes involved in protein misfolding, angiogenesis and the oxidative stress response. Further studies will be necessary to determine the role of these genes in prion replication, dissemination and in the response of the organism to this disease. PMID:24450868

In vitro prion protein conversion suggests risk of bighorn sheep (Ovis canadensis) to transmissible spongiform encephalopathies

PubMed Central

2013-01-01

Background Transmissible spongiform encephalopathies (TSEs) affect both domestic sheep (scrapie) and captive and free-ranging cervids (chronic wasting disease; CWD). The geographical range of bighorn sheep (Ovis canadensis; BHS) overlaps with states or provinces that have contained scrapie-positive sheep or goats and areas with present epizootics of CWD in cervids. No TSEs have been documented in BHS, but the susceptibility of this species to TSEs remains unknown. Results We acquired a library of BHS tissues and found no evidence of preexisting TSEs in these animals. The prion protein gene (Prnp) in all BHS in our library was identical to scrapie-susceptible domestic sheep (A136R154Q171 genotype). Using an in vitro prion protein conversion assay, which has been previously used to assess TSE species barriers and, in our study appears to recollect known species barriers in mice, we assessed the potential transmissibility of TSEs to BHS. As expected based upon Prnp genotype, we observed BHS prion protein conversion by classical scrapie agent and evidence for a species barrier between transmissible mink encephalopathy (TME) and BHS. Interestingly, our data suggest that the species barrier of BHS to white-tailed deer or wapiti CWD agents is likely low. We also used protein misfolding cyclic amplification to confirm that CWD, but not TME, can template prion protein misfolding in A136R154Q171 genotype sheep. Conclusions Our results indicate the in vitro conversion assay used in our study does mimic the species barrier of mice to the TSE agents that we tested. Based on Prnp genotype and results from conversion assays, BHS are likely to be susceptible to infection by classical scrapie. Despite mismatches in amino acids thought to modulate prion protein conversion, our data indicate that A136R154Q171 genotype sheep prion protein is misfolded by CWD agent, suggesting that these animals could be susceptible to CWD. Further investigation of TSE transmissibility to BHS, including animal studies, is warranted. The lack of reported TSEs in BHS may be attributable to other host factors or a lack of TSE surveillance in this species. PMID:23938169

In vitro prion protein conversion suggests risk of bighorn sheep (Ovis canadensis) to transmissible spongiform encephalopathies

USGS Publications Warehouse

Johnson, Christopher J.; Morawski, A.R.; Carlson, C.M.; Chang, H.

2013-01-01

Background: Transmissible spongiform encephalopathies (TSEs) affect both domestic sheep (scrapie) and captive and free-ranging cervids (chronic wasting disease; CWD). The geographical range of bighorn sheep (Ovis canadensis; BHS) overlaps with states or provinces that have contained scrapie-positive sheep or goats and areas with present epizootics of CWD in cervids. No TSEs have been documented in BHS, but the susceptibility of this species to TSEs remains unknown. Results: We acquired a library of BHS tissues and found no evidence of preexisting TSEs in these animals. The prion protein gene (Prnp) in all BHS in our library was identical to scrapie-susceptible domestic sheep (A136R 154Q171). Using an in vitro prion protein conversion assay, which has been previously used to assess TSE species barriers and, in our study appears to recollect known species barriers in mice, we assessed the potential transmissibility of TSEs to BHS. As expected based upon Prnp genotype, we observed BHS prion protein conversion by classical scrapie agent and evidence for a species barrier between transmissible mink encephalopathy (TME) and BHS. Interestingly, our data suggest that the species barrier of BHS to white-tailed deer or wapiti CWD agents is likely low. We also used protein misfolding cyclic amplification to confirm that CWD, but not TME, can template prion protein misfolding in A136R 154Q171genotype sheep. Conclusions: Our results indicate the in vitro conversion assay used in our study does mimic the species barrier of mice to the TSE agents that we tested. Based on Prnp genotype and results from conversion assays, BHS are likely to be susceptible to infection by classical scrapie. Despite mismatches in amino acids thought to modulate prion protein conversion, our data indicate that A136R154Q171 genotype sheep prion protein is misfolded by CWD agent, suggesting that these animals could be susceptible to CWD. Further investigation of TSE transmissibility to BHS, including animal studies, is warranted. The lack of reported TSEs in BHS may be attributable to other host factors or a lack of TSE surveillance in this species.

Scrg1 is induced in TSE and brain injuries, and associated with autophagy.

PubMed

Dron, Michel; Bailly, Yannick; Beringue, Vincent; Haeberlé, Anne-Marie; Griffond, Bernadette; Risold, Pierre-Yves; Tovey, Michael G; Laude, Hubert; Dandoy-Dron, Françoise

2005-07-01

We have previously identified Scrg1, a gene with increased cerebral mRNA levels in transmissible spongiform encephalopathies (TSE) such as scrapie, bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. In this study, Scrg1-immunoreactive cells, essentially neurons, were shown to be widely distributed throughout the brain of scrapie-infected mice, while only rare and weakly immunoreactive cells could be detected in the brain of non-infected normal mice. Induction of the protein was confirmed by Western blot analysis. At the ultrastructural level, Scrg1 protein was associated with dictyosomes of the Golgi apparatus and autophagic vacuoles in the central neurons of the scrapie-infected mice. These results suggested a role for Scrg1 in the pathological changes observed in TSE. We have generated transgenic mice specifically expressing Scrg1 in neurons. No significant differences in the time course of the disease were detected between transgenic and non-transgenic mice infected with scrapie prions. However, tight association of Scrg1 with autophagic vacuoles was again observed in brain neurons of infected transgenic mice. High levels of the protein were also detected in degenerating Purkinje cells of Ngsk Prnp 0/0 mice overexpressing the Prnd gene coding for doppel, a neurotoxic paralogue of the prion protein. Furthermore, induction of Scrg1 protein was observed in the brain of mice injured by canine distemper virus or gold thioglucose treatment. Taken together, our results indicate that Scrg1 is associated with neurodegenerative processes in TSE, but is not directly linked to dysregulation of prion protein.

Prions in Milk from Ewes Incubating Natural Scrapie

PubMed Central

Lacroux, Caroline; Simon, Stéphanie; Benestad, Sylvie L.; Maillet, Séverine; Mathey, Jacinthe; Lugan, Séverine; Corbière, Fabien; Cassard, Hervé; Costes, Pierrette; Bergonier, Dominique; Weisbecker, Jean-Louis; Moldal, Torffin; Simmons, Hugh; Lantier, Frederic; Feraudet-Tarisse, Cécile; Morel, Nathalie; Schelcher, François; Grassi, Jacques; Andréoletti, Olivier

2008-01-01

Since prion infectivity had never been reported in milk, dairy products originating from transmissible spongiform encephalopathy (TSE)-affected ruminant flocks currently enter unrestricted into the animal and human food chain. However, a recently published study brought the first evidence of the presence of prions in mammary secretions from scrapie-affected ewes. Here we report the detection of consistent levels of infectivity in colostrum and milk from sheep incubating natural scrapie, several months prior to clinical onset. Additionally, abnormal PrP was detected, by immunohistochemistry and PET blot, in lacteal ducts and mammary acini. This PrPSc accumulation was detected only in ewes harbouring mammary ectopic lymphoid follicles that developed consequent to Maedi lentivirus infection. However, bioassay revealed that prion infectivity was present in milk and colostrum, not only from ewes with such lympho-proliferative chronic mastitis, but also from those displaying lesion-free mammary glands. In milk and colostrum, infectivity could be recovered in the cellular, cream, and casein-whey fractions. In our samples, using a Tg 338 mouse model, the highest per ml infectious titre measured was found to be equivalent to that contained in 6 µg of a posterior brain stem from a terminally scrapie-affected ewe. These findings indicate that both colostrum and milk from small ruminants incubating TSE could contribute to the animal TSE transmission process, either directly or through the presence of milk-derived material in animal feedstuffs. It also raises some concern with regard to the risk to humans of TSE exposure associated with milk products from ovine and other TSE-susceptible dairy species. PMID:19079578

Genotype-dependent Molecular Evolution of Sheep Bovine Spongiform Encephalopathy (BSE) Prions in Vitro Affects Their Zoonotic Potential*

PubMed Central

Krejciova, Zuzana; Barria, Marcelo A.; Jones, Michael; Ironside, James W.; Jeffrey, Martin; González, Lorenzo; Head, Mark W.

2014-01-01

Prion diseases are rare fatal neurological conditions of humans and animals, one of which (variant Creutzfeldt-Jakob disease) is known to be a zoonotic form of the cattle disease bovine spongiform encephalopathy (BSE). What makes one animal prion disease zoonotic and others not is poorly understood, but it appears to involve compatibility between the prion strain and the host prion protein sequence. Concerns have been raised that the United Kingdom sheep flock may have been exposed to BSE early in the cattle BSE epidemic and that serial BSE transmission in sheep might have resulted in adaptation of the agent, which may have come to phenotypically resemble scrapie while maintaining its pathogenicity for humans. We have modeled this scenario in vitro. Extrapolation from our results suggests that if BSE were to infect sheep in the field it may, with time and in some sheep genotypes, become scrapie-like at the molecular level. However, the results also suggest that if BSE in sheep were to come to resemble scrapie it would lose its ability to affect humans. PMID:25100723

Identification of a novel ovine PrP polymorphism and scrapie-resistant genotypes for St. Croix White and a related composite breed.

PubMed

Seabury, C M; Derr, J N

2003-01-01

Susceptibility to scrapie is primarily controlled by polymorphisms in the ovine prion protein gene (PRNP). Here, we report a novel ovine exon three PRNP polymorphism (SNP G346C; P116), its association with the ovine ARQ allele (P116A136R154Q171), and two new genotypes (PARQ/ARR; PARQ/ARQ) for the St. Croix White (SCW) breed and a related composite (CMP) breed developed for meat production. The (P116) polymorphism occurs between the N-terminal cleavage site and the hydrophobic region of the ovine prion protein, a region which exhibits extreme conservation across mammalian taxa. The relatively high frequency (0.75) of resistant ARR alleles and the absence of ARQ alleles for the SCW ewes used as breeding stock for CMP resulted in significant genic differentiation (P = 0.0123; S.E. = 0.00113). Additionally, the majority of the SCW (66.7%) and CMP (65.4%) sampled possessed genotypes considered resistant or nearly resistant to scrapie and experimental BSE (bovine spongiform encephalopathy. Copyright 2003 S. Karger AG, Basel

Markedly Increased Susceptibility to Natural Sheep Scrapie of Transgenic Mice Expressing Ovine PrP

PubMed Central

Vilotte, Jean-Luc; Soulier, Solange; Essalmani, Rachid; Stinnakre, Marie-George; Vaiman, Daniel; Lepourry, Laurence; Da Silva, Jose Costa; Besnard, Nathalie; Dawson, Mike; Buschmann, Anne; Groschup, Martin; Petit, Stephanie; Madelaine, Marie-Francoise; Rakatobe, Sabine; Le Dur, Annick; Vilette, Didier; Laude, Hubert

2001-01-01

The susceptibility of sheep to scrapie is known to involve, as a major determinant, the nature of the prion protein (PrP) allele, with the VRQ allele conferring the highest susceptibility to the disease. Transgenic mice expressing in their brains three different ovine PrPVRQ-encoding transgenes under an endogenous PrP-deficient genetic background were established. Nine transgenic (tgOv) lines were selected and challenged with two scrapie field isolates derived from VRQ-homozygous affected sheep. All inoculated mice developed neurological signs associated with a transmissible spongiform encephalopathy (TSE) disease and accumulated a protease-resistant form of PrP (PrPres) in their brains. The incubation duration appeared to be inversely related to the PrP steady-state level in the brain, irrespective of the transgene construct. The survival time for animals from the line expressing the highest level of PrP was reduced by at least 1 year compared to those of two groups of conventional mice. With one isolate, the duration of incubation was as short as 2 months, which is comparable to that observed for the rodent TSE models with the briefest survival times. No survival time reduction was observed upon subpassaging of either isolate, suggesting no need for adaptation of the agent to its new host. Overexpression of the transgene was found not to be required for transmission to be accelerated compared to that observed with wild-type mice. Conversely, transgenic mice overexpressing murine PrP were found to be less susceptible than tgOv lines expressing ovine PrP at physiological levels. These data argue that ovine PrPVRQ provided a better substrate for sheep prion replication than did mouse PrP. Altogether, these tgOv mice could be an improved model for experimental studies on natural sheep scrapie. PMID:11390599

Neurodegenerative Disease Transmission and Transgenesis in Mice.

PubMed

Dugger, Brittany N; Perl, Daniel P; Carlson, George A

2017-11-01

Although the discovery of the prion protein (PrP) resulted from its co-purification with scrapie infectivity in Syrian hamsters, work with genetically defined and genetically modified mice proved crucial for understanding the fundamental processes involved not only in prion diseases caused by PrP misfolding, aggregation, and spread but also in other, much more common, neurodegenerative brain diseases. In this review, we focus on methodological and conceptual approaches used to study scrapie and related PrP misfolding diseases in mice and how these approaches have advanced our understanding of related disorders including Alzheimer's and Parkinson's disease. Copyright © 2017 Cold Spring Harbor Laboratory Press; all rights reserved.

Application of temperature-gradient gel electrophoresis for detection of prion protein gene polymorphisms in Polish Swiniarka sheep.

PubMed

Jasik, Agnieszka; Reichert, Michal

2006-05-01

This study presents preliminary data on the polymorphism in the prion protein gene of Swiniarka sheep using temperature gradient gel electrophoresis (TGGE). Available data indicate that sensitivity to scrapie is associated with polymorphisms in three codons of prion protein gene: 136,154, and 171. The TGGE method was used to detect point mutations in these codons responsible for sensitivity or resistance to scrapie. This study revealed presence of an allele encoding valine (V) in codon 136, which is associated with high sensitivity to scrapie and occurred in the form of heterozygous allele together with alanine (AV). The highest variability was observed in codon 171, with presence of arginine (R) and glutamine (Q) in the homozygous (RR or QQ) as well as the heterozygous form (RQ). The results of examination of fifty sheep DNA samples with mutations in codons 136, 154, and 171 demonstrated that TGGE can be used as a simple and rapid method to detect mutations in the PrP gene of sheep. Several samples can be run at the same time, making TGGE ideal for the screening of large numbers of samples.

SCRG1, a potential marker of autophagy in transmissible spongiform encephalopathies.

PubMed

Dron, Michel; Bailly, Yannick; Beringue, Vincent; Haeberlé, Anne-Marie; Griffond, Bernadette; Risold, Pierre-Yves; Tovey, Michael G; Laude, Hubert; Dandoy-Dron, Françoise

2006-01-01

The Scrg1 gene was initially discovered as one of the genes upregulated in transmissible spongiform encephalopathies (TSE). Scrg1 encodes a highly conserved, cysteine-rich protein expressed principally in the central nervous system. The protein is targeted to the Golgi apparatus and large dense-core vesicles/secretory granules in neurons. We have recently shown that the Scrg1 protein is widely induced in neurons of scrapie-infected mice, suggesting that Scrg1 is involved in the host response to stress and/or the death of neurons. At the ultrastructural level, Scrg1 is associated with dictyosomes of the Golgi apparatus and autophagic vacuoles of degenerative neurons. It is well known that apoptosis plays a major role in the events leading to neuronal cell death in TSE. However, autophagy was identified in experimentally induced scrapie a long time ago and was recently reevaluated as a possible cell death program in prion diseases. The consistent association of Scrg1 with autophagic structures typical of scrapie is in agreement with the recruitment of Golgi-specific proteins in this degradation process and we suggest that Scrg1 might be used as a specific probe to identify neuronal autophagy in TSE.

Use of Murine Bioassay to Resolve Ovine Transmissible Spongiform Encephalopathy Cases Showing a Bovine Spongiform Encephalopathy Molecular Profile

PubMed Central

Beck, Katy E; Sallis, Rosemary E; Lockey, Richard; Vickery, Christopher M; Béringue, Vincent; Laude, Hubert; Holder, Thomas M; Thorne, Leigh; Terry, Linda A; Tout, Anna C; Jayasena, Dhanushka; Griffiths, Peter C; Cawthraw, Saira; Ellis, Richard; Balkema-Buschmann, Anne; Groschup, Martin H; Simmons, Marion M; Spiropoulos, John

2012-01-01

Two cases of unusual transmissible spongiform encephalopathy (TSE) were diagnosed on the same farm in ARQ/ARQ PrP sheep showing attributes of both bovine spongiform encephalopathy (BSE) and scrapie. These cases, UK-1 and UK-2, were investigated further by transmissions to wild-type and ovine transgenic mice. Lesion profiles (LP) on primary isolation and subpassage, incubation period (IP) of disease, PrPSc immunohistochemical (IHC) deposition pattern and Western blot profiles were used to characterize the prions causing disease in these sheep. Results showed that both cases were compatible with scrapie. The presence of BSE was contraindicated by the following: LP on primary isolation in RIII and/or MR (modified RIII) mice; IP and LP after serial passage in wild-type mice; PrPSc deposition pattern in wild-type mice; and IP and Western blot data in transgenic mice. Furthermore, immunohistochemistry (IHC) revealed that each case generated two distinct PrPSc deposition patterns in both wild-type and transgenic mice, suggesting that two scrapie strains coexisted in the ovine hosts. Critically, these data confirmed the original differential IHC categorization that these UK-1 and UK-2 cases were not compatible with BSE. PMID:21919992

Effect of Polymorphisms at Codon 146 of the Goat PRNP Gene on Susceptibility to Challenge with Classical Scrapie by Different Routes.

PubMed

Papasavva-Stylianou, Penelope; Simmons, Marion Mathieson; Ortiz-Pelaez, Angel; Windl, Otto; Spiropoulos, John; Georgiadou, Soteria

2017-11-15

This report presents the results of experimental challenges of goats with scrapie by both the intracerebral (i.c.) and oral routes, exploring the effects of polymorphisms at codon 146 of the goat PRNP gene on resistance to disease. The results of these studies illustrate that while goats of all genotypes can be infected by i.c. challenge, the survival distribution of the animals homozygous for asparagine at codon 146 was significantly shorter than those of animals of all other genotypes (chi-square value, 10.8; P = 0.001). In contrast, only those animals homozygous for asparagine at codon 146 (NN animals) succumbed to oral challenge. The results also indicate that any cases of infection in non-NN animals can be detected by the current confirmatory test (immunohistochemistry), although successful detection with the rapid enzyme-linked immunosorbent assay (ELISA) was more variable and dependent on the polymorphism. Together with data from previous studies of goats exposed to infection in the field, these data support the previously reported observations that polymorphisms at this codon have a profound effect on susceptibility to disease. It is concluded that only animals homozygous for asparagine at codon 146 succumb to scrapie under natural conditions. IMPORTANCE In goats, like in sheep, there are PRNP polymorphisms that are associated with susceptibility or resistance to scrapie. However, in contrast to the polymorphisms in sheep, they are more numerous in goats and may be restricted to certain breeds or geographical regions. Therefore, eradication programs must be specifically designed depending on the identification of suitable polymorphisms. An initial analysis of surveillance data suggested that such a polymorphism in Cypriot goats may lie in codon 146. In this study, we demonstrate experimentally that NN animals are highly susceptible after i.c. inoculation. The presence of a D or S residue prolonged incubation periods significantly, and prions were detected in peripheral tissues only in NN animals. In oral challenges, prions were detected only in NN animals, and the presence of a D or S residue at this position conferred resistance to the disease. This study provides an experimental transmission model for assessing the genetic susceptibility of goats to scrapie. © Crown copyright 2017.

Methodological approach for substantiating disease freedom in a heterogeneous small population. Application to ovine scrapie, a disease with a strong genetic susceptibility.

PubMed

Martinez, Marie-José; Durand, Benoit; Calavas, Didier; Ducrot, Christian

2010-06-01

Demonstrating disease freedom is becoming important in different fields including animal disease control. Most methods consider sampling only from a homogeneous population in which each animal has the same probability of becoming infected. In this paper, we propose a new methodology to calculate the probability of detecting the disease if it is present in a heterogeneous population of small size with potentially different risk groups, differences in risk being defined using relative risks. To calculate this probability, for each possible arrangement of the infected animals in the different groups, the probability that all the animals tested are test-negative given this arrangement is multiplied by the probability that this arrangement occurs. The probability formula is developed using the assumption of a perfect test and hypergeometric sampling for finite small size populations. The methodology is applied to scrapie, a disease affecting small ruminants and characterized in sheep by a strong genetic susceptibility defining different risk groups. It illustrates that the genotypes of the tested animals influence heavily the confidence level of detecting scrapie. The results present the statistical power for substantiating disease freedom in a small heterogeneous population as a function of the design prevalence, the structure of the sample tested, the structure of the herd and the associated relative risks. (c) 2010 Elsevier B.V. All rights reserved.

Structure and polymorphism of the mouse prion protein gene.

PubMed Central

Westaway, D; Cooper, C; Turner, S; Da Costa, M; Carlson, G A; Prusiner, S B

1994-01-01

Missense mutations in the prion protein (PrP) gene, overexpression of the cellular isoform of PrP (PrPC), and infection with prions containing the scrapie isoform of PrP (PrPSc) all cause neurodegenerative disease. To understand better the physiology and expression of PrPC, we retrieved mouse PrP gene (Prn-p) yeast artificial chromosome (YAC), cosmid, phage, and cDNA clones. Physical mapping positions Prn-p approximately 300 kb from ecotropic virus integration site number 4 (Evi-4), compatible with failure to detect recombination between Prn-p and Evi-4 in genetic crosses. The Prn-pa allele encompasses three exons, with exons 1 and 2 encoding the mRNA 5' untranslated region. Exon 2 has no equivalent in the Syrian hamster and human PrP genes. The Prn-pb gene shares this intron/exon structure but harbors an approximately 6-kb deletion within intron 2. While the Prn-pb open reading frame encodes two amino acid substitutions linked to prolonged scrapie incubation periods, a deletion of intron 2 sequences also characterizes inbred strains such as RIII/S and MOLF/Ei with shorter incubation periods, making a relationship between intron 2 size and scrapie pathogenesis unlikely. The promoter regions of a and b Prn-p alleles include consensus Sp1 and AP-1 sites, as well as other conserved motifs which may represent binding sites for as yet unidentified transcription factors. Images PMID:7912827

Ovine progressive pneumonia provirus levels are unaffected by the prion 171R allele in an Idaho sheep flock.

PubMed

Harrington, Robert D; Herrmann-Hoesing, Lynn M; White, Stephen N; O'Rourke, Katherine I; Knowles, Donald P

2009-01-22

Selective breeding of sheep for arginine (R) at prion gene (PRNP) codon 171 confers resistance to classical scrapie. However, other effects of 171R selection are uncertain. Ovine progressive pneumonia/Maedi-Visna virus (OPPV) may infect up to 66% of a flock thus any affect of 171R selection on OPPV susceptibility or disease progression could have major impact on the sheep industry. Hypotheses that the PRNP 171R allele is 1) associated with the presence of OPPV provirus and 2) associated with higher provirus levels were tested in an Idaho ewe flock. OPPV provirus was found in 226 of 358 ewes by quantitative PCR. The frequency of ewes with detectable provirus did not differ significantly among the 171QQ, 171QR, and 171RR genotypes (p > 0.05). Also, OPPV provirus levels in infected ewes were not significantly different among codon 171 genotypes (p > 0.05). These results show that, in the flock examined, the presence of OPPV provirus and provirus levels are not related to the PRNP 171R allele. Therefore, a genetic approach to scrapie control is not expected to increase or decrease the number of OPPV infected sheep or the progression of disease. This study provides further support to the adoption of PRNP 171R selection as a scrapie control measure.

The kuru infectious agent is a unique geographic isolate distinct from Creutzfeldt–Jakob disease and scrapie agents

PubMed Central

Manuelidis, Laura; Chakrabarty, Trisha; Miyazawa, Kohtaro; Nduom, Nana-Aba; Emmerling, Kaitlin

2009-01-01

Human sporadic Creutzfeldt–Jakob disease (sCJD), endemic sheep scrapie, and epidemic bovine spongiform encephalopathy (BSE) are caused by a related group of infectious agents. The new U.K. BSE agent spread to many species, including humans, and clarifying the origin, specificity, virulence, and diversity of these agents is critical, particularly because infected humans do not develop disease for many years. As with viruses, transmissible spongiform encephalopathy (TSE) agents can adapt to new species and become more virulent yet maintain fundamentally unique and stable identities. To make agent differences manifest, one must keep the host genotype constant. Many TSE agents have revealed their independent identities in normal mice. We transmitted primate kuru, a TSE once epidemic in New Guinea, to mice expressing normal and ≈8-fold higher levels of murine prion protein (PrP). High levels of murine PrP did not prevent infection but instead shortened incubation time, as would be expected for a viral receptor. Sporadic CJD and BSE agents and representative scrapie agents were clearly different from kuru in incubation time, brain neuropathology, and lymphoreticular involvement. Many TSE agents can infect monotypic cultured GT1 cells, and unlike sporadic CJD isolates, kuru rapidly and stably infected these cells. The geographic independence of the kuru agent provides additional reasons to explore causal environmental pathogens in these infectious neurodegenerative diseases. PMID:19633190

Enzymatic Formulation Capable of Degrading Scrapie Prion under Mild Digestion Conditions

PubMed Central

Okoroma, Emeka A.; Purchase, Diane; Garelick, Hemda; Morris, Roger; Neale, Michael H.; Windl, Otto; Abiola, Oduola O.

2013-01-01

The prion agent is notoriously resistant to common proteases and conventional sterilisation procedures. The current methods known to destroy prion infectivity such as incineration, alkaline and thermal hydrolysis are harsh, destructive, environmentally polluting and potentially hazardous, thus limit their applications for decontamination of delicate medical and laboratory devices, remediation of prion contaminated environment and for processing animal by-products including specified risk materials and carcases. Therefore, an environmentally friendly, non-destructive enzymatic degradation approach is highly desirable. A feather-degrading Bacillus licheniformis N22 keratinase has been isolated which degraded scrapie prion to undetectable level of PrPSc signals as determined by Western Blot analysis. Prion infectivity was verified by ex vivo cell-based assay. An enzymatic formulation combining N22 keratinase and biosurfactant derived from Pseudomonas aeruginosa degraded PrPSc at 65°C in 10 min to undetectable level -. A time-course degradation analysis carried out at 50°C over 2 h revealed the progressive attenuation of PrPSc intensity. Test of residual infectivity by standard cell culture assay confirmed that the enzymatic formulation reduced PrPSc infectivity to undetectable levels as compared to cells challenged with untreated standard scrapie sheep prion (SSBP/1) (p-value = 0.008 at 95% confidence interval). This novel enzymatic formulation has significant potential application for prion decontamination in various environmentally friendly systems under mild treatment conditions. PMID:23874511

Transmissible Spongiform Encephalopathies (Prion Diseases)

MedlinePlus

... have resulted from human consumption of beef from cattle with a TSE disease called bovine spongiform encephalopathy (BSE), also known as "mad cow disease." Other TSEs found in animals include scrapie, ...

Characteristics of 263K Scrapie Agent in Multiple Hamster Species

PubMed Central

Barbian, Kent D.; Race, Brent; Favara, Cynthia; Gardner, Don; Taubner, Lara; Porcella, Stephen; Race, Richard

2009-01-01

Transmissible spongiform encephalopathy (TSE) diseases are known to cross species barriers, but the pathologic and biochemical changes that occur during transmission are not well understood. To better understand these changes, we infected 6 hamster species with 263K hamster scrapie strain and, after each of 3 successive passages in the new species, analyzed abnormal proteinase K (PK)–resistant prion protein (PrPres) glycoform ratios, PrPres PK sensitivity, incubation periods, and lesion profiles. Unique 263K molecular and biochemical profiles evolved in each of the infected hamster species. Characteristics of 263K in the new hamster species seemed to correlate best with host factors rather than agent strain. Furthermore, 2 polymorphic regions of the prion protein amino acid sequence correlated with profile differences in these TSE-infected hamster species. PMID:19193264

Creutzfeldt-Jakob disease

MedlinePlus

... be the same one that causes vCJD in humans. Variant CJD causes less than 1% of all ... Scrapie (found in sheep) Other very rare inherited human diseases, such as Gerstmann-Straussler-Scheinker disease and ...

Genetic profile of scrapie codons 146, 211 and 222 in the PRNP gene locus in three breeds of dairy goats.

PubMed

Vouraki, Sotiria; Gelasakis, Athanasios I; Alexandri, Panoraia; Boukouvala, Evridiki; Ekateriniadou, Loukia V; Banos, Georgios; Arsenos, Georgios

2018-01-01

Polymorphisms at PRNP gene locus have been associated with resistance against classical scrapie in goats. Genetic selection on this gene within appropriate breeding programs may contribute to the control of the disease. The present study characterized the genetic profile of codons 146, 211 and 222 in three dairy goat breeds in Greece. A total of 766 dairy goats from seven farms were used. Animals belonged to two indigenous Greek, Eghoria (n = 264) and Skopelos (n = 287) and a foreign breed, Damascus (n = 215). Genomic DNA was extracted from blood samples from individual animals. Polymorphisms were detected in these codons using Real-Time PCR analysis and four different Custom TaqMan® SNP Genotyping Assays. Genotypic, allelic and haplotypic frequencies were calculated based on individual animal genotypes. Chi-square tests were used to examine Hardy-Weinberg equilibrium state and compare genotypic distribution across breeds. Genetic distances among the three breeds, and between these and 30 breeds reared in other countries were estimated based on haplotypic frequencies using fixation index FST with Arlequin v3.1 software; a Neighbor-Joining tree was created using PHYLIP package v3.695. Level of statistical significance was set at P = 0.01. All scrapie resistance-associated alleles (146S, 146D, 211Q and 222K) were detected in the studied population. Significant frequency differences were observed between the indigenous Greek and Damascus breeds. Alleles 222K and 146S had the highest frequency in the two indigenous and the Damascus breed, respectively (ca. 6.0%). The studied breeds shared similar haplotypic frequencies with most South Italian and Turkish breeds but differed significantly from North-Western European, Far East and some USA goat breeds. Results suggest there is adequate variation in the PRNP gene locus to support breeding programs for enhanced scrapie resistance in goats reared in Greece. Genetic comparisons among goat breeds indicate that separate breeding programs should apply to the two indigenous and the imported Damascus breeds.

A New Method for the Characterization of Strain-Specific Conformational Stability of Protease-Sensitive and Protease-Resistant PrPSc

PubMed Central

Pirisinu, Laura; Di Bari, Michele; Marcon, Stefano; Vaccari, Gabriele; D'Agostino, Claudia; Fazzi, Paola; Esposito, Elena; Galeno, Roberta; Langeveld, Jan; Agrimi, Umberto; Nonno, Romolo

2010-01-01

Although proteinacious in nature, prions exist as strains with specific self-perpetuating biological properties. Prion strains are thought to be associated with different conformers of PrPSc, a disease-associated isoform of the host-encoded cellular protein (PrPC). Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrPSc. However, PrPSc is composed not only of protease-resistant but also of protease-sensitive isoforms. The aim of this work was to develop a protocol for the molecular characterization of both, protease-resistant and protease-sensitive PrPSc aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrPC and PrPSc by means of differential centrifugation. The conformational solubility and stability assay (CSSA) was then developed by measuring PrPSc solubility as a function of increased exposure to GdnHCl. Brain homogenates from voles infected with human and sheep prion isolates were analysed by CSSA and showed strain-specific conformational stabilities, with mean [GdnHCl]1/2 values ranging from 1.6 M for MM2 sCJD to 2.1 for scrapie and to 2.8 M for MM1/MV1 sCJD and E200K gCJD. Interestingly, the rank order of [GdnHCl]1/2 values observed in the human and sheep isolates used as inocula closely matched those found following transmission in voles, being MM1 sCJD the most resistant (3.3 M), followed by sheep scrapie (2.2 M) and by MM2 sCJD (1.6 M). In order to test the ability of CSSA to characterise protease-sensitive PrPSc, we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrPSc aggregates were mainly protease-sensitive and showed a conformational stability much lower than in classical scrapie. Our results show that CSSA is able to reveal strain-specified PrPSc conformational stabilities of protease-resistant and protease-sensitive PrPSc and that it is a valuable tool for strain typing in natural hosts, such as humans and sheep. PMID:20856860

Are humans getting 'mad-cow disease' from eating beef, or something else?

PubMed

Concepcion, G P; Padlan, E A

2003-05-01

Bovine spongiform encephalopathy (BSE) or 'mad-cow disease' is believed to have been caused by the consumption of scrapie-infected sheep matter that had been added to cattle feed. BSE is then believed to have been transmitted to humans by the consumption of infected beef. We have compared the sequences of human and various animal prion proteins with regards to the fragments that could result from gastric digestion. We noted the close similarity of the sequences of human and rodent prion proteins in a peptic fragment that corresponds very closely to one that had been shown by others to be protease resistant and infective. Since rats and mice are known to be susceptible to prion disease, we propose that ingestion of infected rodent parts, possibly droppings, may be a possible mode of transmission of scrapie or BSE to humans.

Molecular Barriers to Zoonotic Transmission of Prions

PubMed Central

Barria, Marcelo A.; Balachandran, Aru; Morita, Masanori; Kitamoto, Tetsuyuki; Barron, Rona; Manson, Jean; Knight, Richard; Ironside, James W.

2014-01-01

The risks posed to human health by individual animal prion diseases cannot be determined a priori and are difficult to address empirically. The fundamental event in prion disease pathogenesis is thought to be the seeded conversion of normal prion protein to its pathologic isoform. We used a rapid molecular conversion assay (protein misfolding cyclic amplification) to test whether brain homogenates from specimens of classical bovine spongiform encephalopathy (BSE), atypical BSE (H-type BSE and L-type BSE), classical scrapie, atypical scrapie, and chronic wasting disease can convert normal human prion protein to the abnormal disease-associated form. None of the tested prion isolates from diseased animals were as efficient as classical BSE in converting human prion protein. However, in the case of chronic wasting disease, there was no absolute barrier to conversion of the human prion protein. PMID:24377702

Prion protein genotypes of sheep as determined from 3343 samples submitted from Ontario and other provinces of Canada from 2005 to 2012

PubMed Central

Cameron, Colin; Bell-Rogers, Patricia; McDowall, Rebeccah; Rebelo, Ana R.; Cai, Hugh Y.

2014-01-01

This study analyzed sheep prion protein (PrP) genotypes of samples submitted from Ontario and other provinces of Canada to the Animal Health Laboratory at the University of Guelph, Guelph, Ontario, between 2005 and 2012. In Ontario, the proportion of scrapie-resistant sheep increased from 2005 to 2012 as evidenced by an increase in the ARR haplotype. When Canadian provinces (Alberta, Ontario, Quebec, and Nova Scotia) were compared from 2008 to 2012, a high proportion of scrapie-resistant sheep was found in all the provinces. The proportions of resistant sheep were lower in Alberta and Quebec than in Ontario and Nova Scotia. Alberta had higher proportions of susceptible sheep and a higher frequency of VRQ alleles, and Quebec had a higher frequency of the ARQ allele. PMID:25355994

The prion diseases of animals

USDA-ARS?s Scientific Manuscript database

Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative diseases that affect several species of animals and include bovine spongiform encephalopathy (BSE), scrapie in sheep and goats, chronic wasting disease (CWD) in cervids, and transmissible mink encephalopat...

Prion Protein Devoid of the Octapeptide Repeat Region Delays Bovine Spongiform Encephalopathy Pathogenesis in Mice.

PubMed

Hara, Hideyuki; Miyata, Hironori; Das, Nandita Rani; Chida, Junji; Yoshimochi, Tatenobu; Uchiyama, Keiji; Watanabe, Hitomi; Kondoh, Gen; Yokoyama, Takashi; Sakaguchi, Suehiro

2018-01-01

Conformational conversion of the cellular isoform of prion protein, PrP C , into the abnormally folded, amyloidogenic isoform, PrP Sc , is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy (BSE) in animals. We previously reported that the octapeptide repeat (OR) region could be dispensable for converting PrP C into PrP Sc after infection with RML prions. We demonstrated that mice transgenically expressing mouse PrP with deletion of the OR region on the PrP knockout background, designated Tg(PrPΔOR)/ Prnp 0 / 0 mice, did not show reduced susceptibility to RML scrapie prions, with abundant accumulation of PrP Sc ΔOR in their brains. We show here that Tg(PrPΔOR)/ Prnp 0 / 0 mice were highly resistant to BSE prions, developing the disease with markedly elongated incubation times after infection with BSE prions. The conversion of PrPΔOR into PrP Sc ΔOR was markedly delayed in their brains. These results suggest that the OR region may have a crucial role in the conversion of PrP C into PrP Sc after infection with BSE prions. However, Tg(PrPΔOR)/ Prnp 0 / 0 mice remained susceptible to RML and 22L scrapie prions, developing the disease without elongated incubation times after infection with RML and 22L prions. PrP Sc ΔOR accumulated only slightly less in the brains of RML- or 22L-infected Tg(PrPΔOR)/ Prnp 0 / 0 mice than PrP Sc in control wild-type mice. Taken together, these results indicate that the OR region of PrP C could play a differential role in the pathogenesis of BSE prions and RML or 22L scrapie prions. IMPORTANCE Structure-function relationship studies of PrP C conformational conversion into PrP Sc are worthwhile to understand the mechanism of the conversion of PrP C into PrP Sc We show here that, by inoculating Tg(PrPΔOR)/ Prnp 0 / 0 mice with the three different strains of RML, 22L, and BSE prions, the OR region could play a differential role in the conversion of PrP C into PrP Sc after infection with RML or 22L scrapie prions and BSE prions. PrPΔOR was efficiently converted into PrP Sc ΔOR after infection with RML and 22L prions. However, the conversion of PrPΔOR into PrP Sc ΔOR was markedly delayed after infection with BSE prions. Further investigation into the role of the OR region in the conversion of PrP C into PrP Sc after infection with BSE prions might be helpful for understanding the pathogenesis of BSE prions. Copyright © 2017 American Society for Microbiology.

The transmissible spongiform encephalopathies of livestock

USDA-ARS?s Scientific Manuscript database

Prion diseases or transmissible spongiform encephalopathies (TSEs) are fatal protein misfolding neurodegenerative diseases. TSEs have been described in several species including bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep and goats, chronic wasting disease (CWD) in cervids, tr...

Immunoreactivity of specific epitopes of PrPSc is enhanced by pretreatment in a hydrated autoclave.

PubMed Central

Yokoyama, T; Momotani, E; Kimura, K; Yuasa, N

1996-01-01

An abnormal protein (PrPSc) accumulates in animals affected with scrapie. Immunoblotting procedures have been used widely to detect PrPSc. Blotted membranes were subjected to pretreatment in a hydrated autoclave, and the subsequent immunoreactivity of PrPSc was examined. The immunoreactivity of PrPSc to antisera against the synthetic peptides of the mouse PrP amino acid sequences 199 to 208 and 213 to 226 was enhanced by the pretreatment. However, the reactivity to antisera of peptide sequences 100 to 115 and 165 to 174 was not affected. The antibody-binding ability of the specific epitopes which are located close to the C-terminal end of PrP27-30 the proteinase-resistant portion of PrPSc, was enhanced by pretreatment in a hydrated autoclave. This pretreatment increased the sensitivity of PrPSc, and it would be useful for diagnosis of scrapie. PMID:8807215

Fate of Prions in Soil: A Review

PubMed Central

Smith, Christen B.; Booth, Clarissa J.; Pedersen, Joel A.

2011-01-01

Prions are the etiological agents of transmissible spongiform encephalopathies (TSEs), a class of fatal neurodegenerative diseases affecting humans and other mammals. The pathogenic prion protein is a misfolded form of the host-encoded prion protein and represents the predominant, if not sole, component of the infectious agent. Environmental routes of TSE transmission are implicated in epizootics of sheep scrapie and chronic wasting disease (CWD) of deer, elk, and moose. Soil represents a plausible environmental reservoir of scrapie and CWD agents, which can persist in the environment for years. Attachment to soil particles likely influences the persistence and infectivity of prions in the environment. Effective methods to inactivate TSE agents in soil are currently lacking, and the effects of natural degradation mechanisms on TSE infectivity are largely unknown. An improved understanding of the processes affecting the mobility, persistence, and bioavailability of prions in soil is needed for the management of TSE-contaminated environments. PMID:21520752

[Disease concept of the slow virus infection].

PubMed

Takasu, Toshiaki

2007-08-01

This article gives a brief history of the terminology of slow virus infection, the conceptual change that occurred in it, the features common to slow infection and the current concept of slow virus infection. Björn Sigurdsson from the field of veterinary medicine proposed slow virus infection as unique mode of infection in 1954. Its initial concept was remodeled along with the general acceptance of prion theory of sheep scrapie that was proposed in 1982. The features common to slow infection include very long latency, unanimous poor prognosis, central nervous system involvement, etc. Currently the slow infection comprises those caused by slow conventional viruses that is the slow virus infection (for example subacute sclerosing panencephalitis and progressive multifocal encephalopathy in human and visna-maedi in sheep) and prion diseases (for example kuru, Creutzfeldt-Jakob disease, Gerstmann-Sträussler-Scheinker syndrome in human, scrapie and bovine spongiform encephalopathy).

Pathological findings in retina and visual pathways associated to natural Scrapie in sheep.

PubMed

Hortells, Paloma; Monzón, Marta; Monleón, Eva; Acín, Cristina; Vargas, Antonia; Bolea, Rosa; Luján, Lluís; Badiola, Juan José

2006-09-07

This work represents a comprehensive pathological description of the retina and visual pathways in naturally affected Scrapie sheep. Twenty naturally affected Scrapie sheep and 6 matched controls were used. Eyes, optic nerves and brain from each animal were fixed and histologically processed using hematoxylin-eosin, followed by immunohistochemical staining for prion protein (PrPsc) and glial fibrillar acidic protein (GFAP). Retinal histopathological changes were observed in only 7 clinically affected animals and mainly consisted of loss of outer limitant layer definition, outer plexiform layer atrophy, disorganization and loss of nuclei in both nuclear layers, and Müller glia hypertrophy. PrPsc was detected in the retina of 19 of the 20 sheep and characterized by a disseminated granular deposit across layers and intraneuronally in ganglion cells. The inner plexiform and the ganglion cell layers were the structures most severely affected by PrPsc deposits. PrPsc exhibited a tendency to spread from these two layers to the others. A marked increase in the number and intensity of GFAP-expressing Müller cells was observed in the clinical stage, especially at the terminal stage of the disease. Spongiosis and PrPsc were detected within the visual pathways at the preclinical stage, their values increasing during the course of the disease but varying between the areas examined. PrPsc was detected in only 3 optic nerves. The results suggest that the presence of PrPsc in the retina correlates with disease progression during the preclinical and clinical stages, perhaps using the inner plexiform layer as a first entry site and diffusing from the brain using a centrifugal model.

Quantitative Risk Assessment of Bovine Spongiform Encephalopathy

NASA Astrophysics Data System (ADS)

Tsutsui, Toshiyuki; Kasuga, Fumiko

Bovine spongiform encephalopathy (BSE) is a progressive neurological disease of cattle affecting the central nervous system and was first diagnosed in the United Kingdom (UK) in 1986 (Wells et al., 1987). This disease is one of the transmissible spongiform encephalopathy (TSE) which includes Creutzfeldt-Jakob disease (CJD) in humans and scrapie in sheep. The causative agent of TSE is considered to be an abnormal form of prion protein. However, the details of its pathogenic mechanism have not been fully identified. Scrapie, which causes neurological symptoms in sheep and goats, has existed in the UK for 200 years (Hoinville, 1996) and spread across the rest of the world in the 1900s (Detwiler & Baylis, 2003). There has been no report so far that scrapie can be transmitted to humans. Initially, BSE was also considered as a disease affecting only animals. However, a variant type of Creutzfeldt-Jakob disease (vCJD) was first reported in the UK, and exposure to a BSE agent was suspected (Collinge, Sidle, Meads, Ironside, & Hill, 1996). vCJD is clinically and pathologically different from the sporadic type of CJD, and age at clinical onset of vCJD is younger than sporadic type (Will et al., 1996). Since the UK government announced the possible association between BSE and vCJD in 1996, BSE has become a huge public health concern all over the world. Of particular concern about vCJD, the fatal disease in younger age, distorted consumer confidence in beef safety, and as a result reduced beef consumption has been seen in many BSE-affected countries.

Subcellular colocalization of the cellular and scrapie prion proteins in caveolae-like membranous domains

PubMed Central

Vey, Martin; Pilkuhn, Susanne; Wille, Holger; Nixon, Randal; DeArmond, Stephen J.; Smart, Eric J.; Anderson, Richard G. W.; Taraboulos, Albert; Prusiner, Stanley B.

1996-01-01

Results of transgenetic studies argue that the scrapie isoform of the prion protein (PrPSc) interacts with the substrate cellular PrP (PrPC) during conversion into nascent PrPSc. While PrPSc appears to accumulate primarily in lysosomes, caveolae-like domains (CLDs) have been suggested to be the site where PrPC is converted into PrPSc. We report herein that CLDs isolated from scrapie-infected neuroblastoma (ScN2a) cells contain PrPC and PrPSc. After lysis of ScN2a cells in ice-cold Triton X-100, both PrP isoforms and an N-terminally truncated form of PrPC (PrPC-II) were found concentrated in detergent-insoluble complexes resembling CLDs that were isolated by flotation in sucrose gradients. Similar results were obtained when CLDs were purified from plasma membranes by sonication and gradient centrifugation; with this procedure no detergents are used, which minimizes artifacts that might arise from redistribution of proteins among subcellular fractions. The caveolar markers ganglioside GM1 and H-ras were found concentrated in the CLD fractions. When plasma membrane proteins were labeled with the impermeant reagent sulfo-N-hydroxysuccinimide-biotin, both PrPC and PrPSc were found biotinylated in CLD fractions. Similar results on the colocalization of PrPC and PrPSc were obtained when CLDs were isolated from Syrian hamster brains. Our findings demonstrate that both PrPC and PrPSc are present in CLDs and, thus, support the hypothesis that the PrPSc formation occurs within this subcellular compartment. PMID:8962161

Bovine Spongiform Encephalopathy: Atypical Pros and Cons

USDA-ARS?s Scientific Manuscript database

Transmissible spongiform encephalopathies (TSEs) are fatal neurologic diseases that affect several mammalian species including human beings. Four animal TSE agents have been reported: scrapie of sheep and goats; chronic wasting disease (CWD) of deer, elk, and moose; transmissible mink encephalopath...

Detecting and quantifying prions: Mass spectrometry-based approaches

USDA-ARS?s Scientific Manuscript database

Prions are novel pathogens that cause a set of rare fatal neurological diseases know as transmissible spongiform encephalopathies. Examples of these diseases include Creutzfeldt-Jakob disease, scrapie and chronic wasting disease. Prions are able to recruit a normal cellular prion protein and convert...

Explaining the heterogeneous scrapie surveillance figures across Europe: a meta-regression approach.

PubMed

Del Rio Vilas, Victor J; Hopp, Petter; Nunes, Telmo; Ru, Giuseppe; Sivam, Kumar; Ortiz-Pelaez, Angel

2007-06-28

Two annual surveys, the abattoir and the fallen stock, monitor the presence of scrapie across Europe. A simple comparison between the prevalence estimates in different countries reveals that, in 2003, the abattoir survey appears to detect more scrapie in some countries. This is contrary to evidence suggesting the greater ability of the fallen stock survey to detect the disease. We applied meta-analysis techniques to study this apparent heterogeneity in the behaviour of the surveys across Europe. Furthermore, we conducted a meta-regression analysis to assess the effect of country-specific characteristics on the variability. We have chosen the odds ratios between the two surveys to inform the underlying relationship between them and to allow comparisons between the countries under the meta-regression framework. Baseline risks, those of the slaughtered populations across Europe, and country-specific covariates, available from the European Commission Report, were inputted in the model to explain the heterogeneity. Our results show the presence of significant heterogeneity in the odds ratios between countries and no reduction in the variability after adjustment for the different risks in the baseline populations. Three countries contributed the most to the overall heterogeneity: Germany, Ireland and The Netherlands. The inclusion of country-specific covariates did not, in general, reduce the variability except for one variable: the proportion of the total adult sheep population sampled as fallen stock by each country. A large residual heterogeneity remained in the model indicating the presence of substantial effect variability between countries. The meta-analysis approach was useful to assess the level of heterogeneity in the implementation of the surveys and to explore the reasons for the variation between countries.

A Bayesian zero-truncated approach for analysing capture-recapture count data from classical scrapie surveillance in France.

PubMed

Vergne, Timothée; Calavas, Didier; Cazeau, Géraldine; Durand, Benoît; Dufour, Barbara; Grosbois, Vladimir

2012-06-01

Capture-recapture (CR) methods are used to study populations that are monitored with imperfect observation processes. They have recently been applied to the monitoring of animal diseases to evaluate the number of infected units that remain undetected by the surveillance system. This paper proposes three bayesian models to estimate the total number of scrapie-infected holdings in France from CR count data obtained from the French classical scrapie surveillance programme. We fitted two zero-truncated Poisson (ZTP) models (with and without holding size as a covariate) and a zero-truncated negative binomial (ZTNB) model to the 2006 national surveillance count dataset. We detected a large amount of heterogeneity in the count data, making the use of the simple ZTP model inappropriate. However, including holding size as a covariate did not bring any significant improvement over the simple ZTP model. The ZTNB model proved to be the best model, giving an estimation of 535 (CI(95%) 401-796) infected and detectable sheep holdings in 2006, although only 141 were effectively detected, resulting in a holding-level prevalence of 4.4‰ (CI(95%) 3.2-6.3) and a sensitivity of holding-level surveillance of 26% (CI(95%) 18-35). The main limitation of the present study was the small amount of data collected during the surveillance programme. It was therefore not possible to build complex models that would allow depicting more accurately the epidemiological and detection processes that generate the surveillance data. We discuss the perspectives of capture-recapture count models in the context of animal disease surveillance. Copyright © 2012 Elsevier B.V. All rights reserved.

Different 2-Aminothiazole Therapeutics Produce Distinct Patterns of Scrapie Prion Neuropathology in Mouse Brains.

PubMed

Giles, Kurt; Berry, David B; Condello, Carlo; Hawley, Ronald C; Gallardo-Godoy, Alejandra; Bryant, Clifford; Oehler, Abby; Elepano, Manuel; Bhardwaj, Sumita; Patel, Smita; Silber, B Michael; Guan, Shenheng; DeArmond, Stephen J; Renslo, Adam R; Prusiner, Stanley B

2015-10-01

Because no drug exists that halts or even slows any neurodegenerative disease, developing effective therapeutics for any prion disorder is urgent. We recently reported two compounds (IND24 and IND81) with the 2-aminothiazole (2-AMT) chemical scaffold that almost doubled the incubation times in scrapie prion-infected, wild-type (wt) FVB mice when given in a liquid diet. Remarkably, oral prophylactic treatment with IND24 beginning 14 days prior to intracerebral prion inoculation extended survival from ∼120 days to over 450 days. In addition to IND24, we evaluated the pharmacokinetics and efficacy of five additional 2-AMTs; one was not followed further because its brain penetration was poor. Of the remaining four new 2-AMTs, IND114338 doubled and IND125 tripled the incubation times of RML-inoculated wt and Tg4053 mice overexpressing wt mouse prion protein (PrP), respectively. Neuropathological examination of the brains from untreated controls showed a widespread deposition of self-propagating, β-sheet-rich "scrapie" isoform (PrP(Sc)) prions accompanied by a profound astrocytic gliosis. In contrast, mice treated with 2-AMTs had lower levels of PrP(Sc) and associated astrocytic gliosis, with each compound resulting in a distinct pattern of deposition. Notably, IND125 prevented both PrP(Sc) accumulation and astrocytic gliosis in the cerebrum. Progressive central nervous system dysfunction in the IND125-treated mice was presumably due to the PrP(Sc) that accumulated in their brainstems. Disappointingly, none of the four new 2-AMTs prolonged the lives of mice expressing a chimeric human/mouse PrP transgene inoculated with Creutzfeldt-Jakob disease prions. Copyright © 2015 by The American Society for Pharmacology and Experimental Therapeutics.

Protecting effect of PrP codons M142 and K222 in goats orally challenged with bovine spongiform encephalopathy prions.

PubMed

Fast, C; Goldmann, W; Berthon, P; Tauscher, K; Andréoletti, O; Lantier, I; Rossignol, C; Bossers, A; Jacobs, J G; Hunter, N; Groschup, M H; Lantier, F; Langeveld, J P M

2017-09-19

Breeding towards genetic resistance to prion disease is effective in eliminating scrapie. In sheep, classical forms of scrapie have been eradicated almost completely in several countries by breeding programs using a prion protein (PrP) gene (PRNP) amino acid polymorphism. For goats, field and experimental studies have provided evidence for several amino acid polymorphisms that are associated with resistance to scrapie, but only limited data are available concerning the susceptibility of caprine PRNP genotypes to BSE. In this study, goat kids representing five PRNP genotypes based on three polymorphisms (M142, Q211 and K222 and the wild type I142, R211 and Q222) were orally challenged with bovine or goat BSE. Wild type goats were killed with clinical signs between 24-28 months post inoculation (mpi) to both challenges, and goats with genotype R/Q211 succumbed between 29-36 mpi. I/M142 goats developed clinical signs at 44-45 mpi and M/M142 goats remained healthy until euthanasia at 48 mpi. None of the Q/K222 goats showed definite clinical signs. Taken together the highest attack ratios were seen in wild type and R/Q211 goats, and the lowest in I/M142, M/M142 and Q/K222. In all genotype groups, one or more goats remained healthy within the incubation period in both challenges and without detectable PrP deposition in the tissues. Our data show that both the K222 and M142 polymorphisms lengthen the incubation period significantly compared to wild type animals, but only K222 was associated with a significant increase in resistance to BSE infection after oral exposure to both BSE sources.

Prominent pancreatic endocrinopathy and altered control of food intake disrupt energy homeostasis in prion diseases

USGS Publications Warehouse

Bailey, J. D.; Berardinelli, J.G.; Rocke, T.E.; Bessen, R.A.

2008-01-01

Prion diseases are fatal neurodegenerative diseases that can induce endocrinopathies. The basis of altered endocrine function in prion diseases is not well understood, and the purpose of this study was to investigate the spatiotemporal relationship between energy homeostasis and prion infection in hamsters inoculated with either the 139H strain of scrapie agent, which induces preclinical weight gain, or the HY strain of transmissible mink encephalopathy (TME), which induces clinical weight loss. Temporal changes in body weight, feed, and water intake were measured as well as both non-fasted and fasted concentrations of serum glucose, insulin, glucagon, ??-ketones, and leptin. In 139H scrapie-infected hamsters, polydipsia, hyperphagia, non-fasted hyperinsulinemia with hyperglycemia, and fasted hyperleptinemia were found at preclinical stages and are consistent with an anabolic syndrome that has similarities to type II diabetes mellitus and/or metabolic syndrome X. In HY TME-infected hamsters, hypodipsia, hypersecretion of glucagon (in both non-fasted and fasted states), increased fasted ??-ketones, fasted hypoglycemia, and suppressed non-fasted leptin concentrations were found while feed intake was normal. These findings suggest a severe catabolic syndrome in HY TME infection mediated by chronic increases in glucagon secretion. In both models, alterations of pancreatic endocrine function were not associated with PrPSc deposition in the pancreas. The results indicate that prominent endocrinopathy underlies alterations in body weight, pancreatic endocrine function, and intake of food. The prion-induced alterations of energy homeostasis in 139H scrapie- or HY TME-infected hamsters could occur within areas of the hypothalamus that control food satiety and/or within autonomic centers that provide neural outflow to the pancreas. ?? 2008 Society for Endocrinology.

Ovine Reference Materials and Assays for Prion Genetic Testing

USDA-ARS?s Scientific Manuscript database

Codon variants implicated in scrapie susceptibility or disease progression include those at amino acid positions 112, 136, 141, 154, and 171. Nine single nucleotide polymorphisms (SNPs) determine which residues are encoded by the five implicated codons and accurately scoring these SNPs is essential...

Stability properties of PrPSc from cattle with experimental transmissible spongiform encephalopathies

USDA-ARS?s Scientific Manuscript database

Transmissible Spongiform Encephalopathies (TSEs), including scrapie in sheep, chronic wasting disease (CWD) in cervids, and bovine spongiform encephalopathy (BSE), are fatal diseases of the nervous system associated with accumulation of misfolded prion protein (PrPSc). Different strains of BSE exist...

76 FR 77767 - Submission for OMB Review; Comment Request

Federal Register 2010, 2011, 2012, 2013, 2014

2011-12-14

...; post-exposure management and monitoring plans; scrapie test records; application for indemnity payments... authority to promulgate regulations designed to prevent the importation, preparation, sale, or shipment of... applications, product and test report forms and field study summaries. Need and Use of the Information: APHIS...

9 CFR 71.1 - Definitions.

Code of Federal Regulations, 2014 CFR

2014-01-01

... § 79.6 of this subchapter, for conducting an active State scrapie program involving the identification... shield. Beginning March 11, 2015, all official eartags applied to animals must bear an official eartag shield. The design, size, shape, color, and other characteristics of the official eartag will depend on...

9 CFR 54.20 - Administration.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Administration. 54.20 Section 54.20 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE... Certification Program § 54.20 Administration. The Scrapie Flock Certification Program is a cooperative effort...

9 CFR 54.20 - Administration.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Administration. 54.20 Section 54.20 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE... Certification Program § 54.20 Administration. The Scrapie Flock Certification Program is a cooperative effort...

9 CFR 54.20 - Administration.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Administration. 54.20 Section 54.20 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE, DEPARTMENT OF AGRICULTURE... Certification Program § 54.20 Administration. The Scrapie Flock Certification Program is a cooperative effort...

PRNP variants in goats reduce sensitivity of detection of PrPSc by immunoassay

USDA-ARS?s Scientific Manuscript database

Immunoassays are extensively utilized in disease diagnostics with monoclonal antibodies serving as critical tools within the assay. Detection of scrapie in sheep and goats relies heavily on immunoassays including immunohistochemistry, western blotting, and ELISA. In the United States, regulatory tes...

Rapid Typing of Transmissible Spongiform Encephalopathy Strains with Differential ELISA

PubMed Central

Simon, Stéphanie; Nugier, Jérôme; Morel, Nathalie; Boutal, Hervé; Créminon, Christophe; Benestad, Sylvie L.; Andréoletti, Olivier; Lantier, Frédéric; Bilheude, Jean-Marc; Feyssaguet, Muriel; Biacabe, Anne-Gaëlle; Baron, Thierry

2008-01-01

The bovine spongiform encephalopathy (BSE) agent has been transmitted to humans, leading to variant Creutzfeldt-Jakob disease. Sheep and goats can be experimentally infected by BSE and have been potentially exposed to natural BSE; however, whether BSE can be transmitted to small ruminants is not known. Based on the particular biochemical properties of the abnormal prion protein (PrPsc) associated with BSE, and particularly the increased degradation induced by proteinase K in the N terminal part of PrPsc, we have developed a rapid ELISA designed to distinguish BSE from other scrapie strains. This assay clearly discriminates experimental ovine BSE from other scrapie strains and was used to screen 260 transmissible spongiform encephalopathy (TSE)–infected small ruminant samples identified by the French active surveillance network (2002/2003). In this context, this test has helped to identify the first case of natural BSE in a goat and can be used to classify TSE isolates based on the proteinase K sensitivity of PrPsc. PMID:18394279

Physical, chemical and kinetic factors affecting prion infectivity

PubMed Central

Properzi, Francesca; Badhan, Anjna; Klier, Steffi; Schmidt, Christian; Klöhn, Peter C.; Wadsworth, Jonathan D. F.; Clarke, Anthony R.; Jackson, Graham S.; Collinge, John

2016-01-01

ABSTRACT The mouse-adapted scrapie prion strain RML is one of the most widely used in prion research. The introduction of a cell culture-based assay of RML prions, the scrapie cell assay (SCA) allows more rapid and precise prion titration. A semi-automated version of this assay (ASCA) was applied to explore a range of conditions that might influence the infectivity and properties of RML prions. These include resistance to freeze-thaw procedures; stability to endogenous proteases in brain homogenate despite prolonged exposure to varying temperatures; distribution of infective material between pellet and supernatant after centrifugation, the effect of reducing agents and the influence of detergent additives on the efficiency of infection. Apparent infectivity is increased significantly by interaction with cationic detergents. Importantly, we have also elucidated the relationship between the duration of exposure of cells to RML prions and the transmission of infection. We established that the infection process following contact of cells with RML prions is rapid and followed an exponential time course, implying a single rate-limiting process. PMID:27282252

Physical, chemical and kinetic factors affecting prion infectivity.

PubMed

Properzi, Francesca; Badhan, Anjna; Klier, Steffi; Schmidt, Christian; Klöhn, Peter C; Wadsworth, Jonathan D F; Clarke, Anthony R; Jackson, Graham S; Collinge, John

2016-05-03

The mouse-adapted scrapie prion strain RML is one of the most widely used in prion research. The introduction of a cell culture-based assay of RML prions, the scrapie cell assay (SCA) allows more rapid and precise prion titration. A semi-automated version of this assay (ASCA) was applied to explore a range of conditions that might influence the infectivity and properties of RML prions. These include resistance to freeze-thaw procedures; stability to endogenous proteases in brain homogenate despite prolonged exposure to varying temperatures; distribution of infective material between pellet and supernatant after centrifugation, the effect of reducing agents and the influence of detergent additives on the efficiency of infection. Apparent infectivity is increased significantly by interaction with cationic detergents. Importantly, we have also elucidated the relationship between the duration of exposure of cells to RML prions and the transmission of infection. We established that the infection process following contact of cells with RML prions is rapid and followed an exponential time course, implying a single rate-limiting process.

Divergent prion strain evolution driven by PrPC expression level in transgenic mice

PubMed Central

Le Dur, Annick; Laï, Thanh Lan; Stinnakre, Marie-George; Laisné, Aude; Chenais, Nathalie; Rakotobe, Sabine; Passet, Bruno; Reine, Fabienne; Soulier, Solange; Herzog, Laetitia; Tilly, Gaëlle; Rézaei, Human; Béringue, Vincent; Vilotte, Jean-Luc; Laude, Hubert

2017-01-01

Prions induce a fatal neurodegenerative disease in infected host brain based on the refolding and aggregation of the host-encoded prion protein PrPC into PrPSc. Structurally distinct PrPSc conformers can give rise to multiple prion strains. Constrained interactions between PrPC and different PrPSc strains can in turn lead to certain PrPSc (sub)populations being selected for cross-species transmission, or even produce mutation-like events. By contrast, prion strains are generally conserved when transmitted within the same species, or to transgenic mice expressing homologous PrPC. Here, we compare the strain properties of a representative sheep scrapie isolate transmitted to a panel of transgenic mouse lines expressing varying levels of homologous PrPC. While breeding true in mice expressing PrPC at near physiological levels, scrapie prions evolve consistently towards different strain components in mice beyond a certain threshold of PrPC overexpression. Our results support the view that PrPC gene dosage can influence prion evolution on homotypic transmission. PMID:28112164

Scrapie in swine: a diagnostic challenge

USDA-ARS?s Scientific Manuscript database

A naturally occurring prion disease has not been recognized in swine, but the agent of bovine spongiform encephalopathy does transmit to swine by experimental routes. Swine are thought to have a robust species barrier when exposed to the naturally occurring prion diseases of other species, but the s...

9 CFR 71.1 - Definitions.

Code of Federal Regulations, 2011 CFR

2011-01-01

... the Animal and Plant Health Inspection Service in the State concerned. Breeding sheep and goats. Any sexually intact sheep or goat that is not moving either directly to slaughter or through one or more... and goats for the purpose of controlling the spread of scrapie. Department. The United States...

9 CFR 71.1 - Definitions.

Code of Federal Regulations, 2010 CFR

2010-01-01

... the Animal and Plant Health Inspection Service in the State concerned. Breeding sheep and goats. Any sexually intact sheep or goat that is not moving either directly to slaughter or through one or more... and goats for the purpose of controlling the spread of scrapie. Department. The United States...

Prion peripheralization is a host-driven trait of prion infection, independent of strain

USDA-ARS?s Scientific Manuscript database

Chronic wasting disease (CWD), like scrapie of sheep, is a horizontally transmissible spongiform encephalopathy. Proposed natural routes of transmission for both agents include saliva, urine, and feces, and are likely related to an accumulation of misfolded prion proteins in peripheral excretory tis...

Transmission of scrapie prions to primate after an extended silent incubation period

USDA-ARS?s Scientific Manuscript database

Classical bovine spongiform encephalopathy (c-BSE) is an animal prion disease that also causes variant Creutzfeldt-Jakob disease in humans. Over the past decades, c-BSE's zoonotic potential has been the driving force in establishing extensive protective measures for animal and human health. In compl...

Development and characterization of an ex-vivo brain slice culture model of chronic wasting disease

USDA-ARS?s Scientific Manuscript database

Prion diseases have long incubation times in vivo, therefore, modeling the diseases ex-vivo will advance the development of rationale-based therapeutic strategies. An organotypic slice culture assay (POSCA) was recently developed for scrapie prions by inoculating mouse cerebellar brain slices with R...

Comparison of Prion Allele Frequency found in Suffolk and Targhee Sheep

USDA-ARS?s Scientific Manuscript database

Scrapie is a class of Transmissible Spongiform Encephalopathy that affects sheep and goats. The objective of this study was to compare genotypic and allelic frequencies among USSES Targhee and Suffolk sheep. A total of 122 sheep were genotyped for codon 171 with allele specific primers in 2 separate...

Department of Agriculture Semiannual Regulatory Agenda

Federal Register 2010, 2011, 2012, 2013, 2014

2010-04-26

... Goats 0579-AC92 17 Plant Pest Regulations; Update of General Provisions 0579-AC98 18 Bovine Spongiform... than birds bred for use in research. Timetable: Action Date FR Cite NPRM 08/00/10 NPRM Comment Period... AND GOATS Legal Authority: 7 USC 8301 to 8317 Abstract: This rulemaking would amend the scrapie...

Correlation of cellular factors and differential scrapie prion permissiveness in ovine microglia

USDA-ARS?s Scientific Manuscript database

Prion diseases are fatal neurodegenerative disorders by which the native cellular prion protein (PrP-C) is misfolded into an accumulating, disease-associated isoform (PrP-D). To improve the understanding of prion pathogenesis and develop effective treatments, it is essential to elucidate factors con...

Experimental Interspecies Transmission Studies of the Transmissible Spongiform Encephalopathies to Cattle: Comparison to Bovine Spongiform Encephalopathy in Cattle

USDA-ARS?s Scientific Manuscript database

Prion diseases or transmissible spongiform encephalopathies (TSEs) of animals include scrapie of sheep and goats; transmissible mink encephalopathy (TME); chronic wasting disease (CWD) of deer, elk and moose; and bovine spongiform encephalopathy (BSE) of cattle. Since the emergence of BSE and its pr...

Molecular approaches to detecting and discriminating among prions, a class of pathogenic molecules(Abstract)

USDA-ARS?s Scientific Manuscript database

Prions (PrPSc)are the pathogens that cause a set of fatal neurological diseases that include scrapie and chronic wasting disease (CWD). They are composed solely of protein and unlike viral, bacterial, or fungal pathogens, the information necessary to convert the normal cellular prion protein (PrPC) ...

Time-course study of retinal pathology in C57BL/6 mice infected with RML scrapie

USDA-ARS?s Scientific Manuscript database

Prions are proteinaceous pathogens that cause transmissible spongiform encephalopathies (TSEs). These diseases develop slowly as the misfolded and protease-resistant prion protein, PrP**Sc, interacts with the normal cellular form, PrP**C, a cell-surface protein found throughout the nervous system. T...

Does methionine oxidation influence the progression of classical or atypical scrapie

USDA-ARS?s Scientific Manuscript database

Introduction Prions are pathological proteins that propagate by converting a normal cellular prion protein (PrPC) into a prion (PrPSc). PrPC and PrPSc possess identical covalent structures and only differ in their conformations. The conversion of the PrPC conformation to the PrPSc one is template ...

Transmission of chronic wasting disease of white-tailed deer to Suffolk sheep following intracranial inoculation

USDA-ARS?s Scientific Manuscript database

Background: Interspecies transmission studies are an opportunity to better understand the potential host ranges of prion diseases. Chronic wasting disease (CWD) of cervids and scrapie of sheep and goats have a similar tissue distribution of abnormal prion protein (PrPSc) and prion disease exposure a...

75 FR 79709 - Semiannual Regulatory Agenda, Fall 2010

Federal Register 2010, 2011, 2012, 2013, 2014

2010-12-20

... in Sheep and Goats 0579-AC92 190 Plant Pest Regulations; Update of General Provisions (Reg Plan Seq... Payment Loan Program; 5101, Farming Experience as an Eligibility Requirement; 5201, Eligibility of Equine... Phone: 301 734-8695 RIN: 0579-AC74 189. SCRAPIE IN SHEEP AND GOATS Legal Authority: 7 USC 8301 to 8317...

9 CFR 54.2 - Cooperative agreements and memoranda of understanding with States.

Code of Federal Regulations, 2010 CFR

2010-01-01

... of understanding with States. 54.2 Section 54.2 Animals and Animal Products ANIMAL AND PLANT HEALTH... DISEASES CONTROL OF SCRAPIE § 54.2 Cooperative agreements and memoranda of understanding with States. APHIS will execute cooperative agreements and/or memoranda of understanding with the animal health agency of...

76 FR 43649 - Notice of Request for Extension of Approval of an Information Collection; Scrapie in Sheep and...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-07-21

...; Interstate Movement Restrictions and Indemnity Program AGENCY: Animal and Plant Health Inspection Service... with regulations for the interstate movement of sheep and goats and an indemnity program to control the... Movement Restrictions and Indemnity Program. OMB Number: 0579-0101. Type of Request: Extension of approval...

9 CFR 98.35 - Declaration, health certificate, and other documents for animal semen.

Code of Federal Regulations, 2012 CFR

2012-01-01

... donor animal as a condition for importing the semen; (7) The seal number on the shipping container; (8... compulsorily notifiable disease; and (ii) An effective surveillance and monitoring system for scrapie is in... shipping containers carrying animal semen for importation into the United States must be sealed with an...

9 CFR 98.35 - Declaration, health certificate, and other documents for animal semen.

Code of Federal Regulations, 2014 CFR

2014-01-01

... donor animal as a condition for importing the semen; (7) The seal number on the shipping container; (8... compulsorily notifiable disease; and (ii) An effective surveillance and monitoring system for scrapie is in... shipping containers carrying animal semen for importation into the United States must be sealed with an...

9 CFR 98.35 - Declaration, health certificate, and other documents for animal semen.

Code of Federal Regulations, 2013 CFR

2013-01-01

... donor animal as a condition for importing the semen; (7) The seal number on the shipping container; (8... compulsorily notifiable disease; and (ii) An effective surveillance and monitoring system for scrapie is in... shipping containers carrying animal semen for importation into the United States must be sealed with an...

Pathology in practice: pituitary adenoma in a sheep

USDA-ARS?s Scientific Manuscript database

A 5-year-old Suffolk ewe maintained as breeding stock in the scrapie-free flock at the USDA National Animal Disease Center was euthanized and presented for necropsy because of poor reproductive performance. The ewe produced 5 lambs in 3 prior lambings, but failed to carry lambs to term in her 4th se...

Characterization of the human analogue of a Scrapie-responsive gene.

PubMed

Dron, M; Dandoy-Dron, F; Guillo, F; Benboudjema, L; Hauw, J J; Lebon, P; Dormont, D; Tovey, M G

1998-07-17

We have recently described a novel mRNA denominated ScRG-1, the level of which is increased in the brains of Scrapie-infected mice (Dandoy-Dron, F., Guillo, F., Benboudjema, L., Deslys, J.-P., Lasmézas, C., Dormont, D., Tovey, M. G., and Dron, M. (1998) J. Biol. Chem. 273, 7691-7697). The increase in ScRG-1 mRNA in the brain follows the accumulation of PrPSc, the proteinase K-resistant form of the prion protein (PrP), and precedes the widespread neuronal death that occurs in late stage disease. In the present study, we have isolated a cDNA encoding the human counterpart of ScRG-1. Comparison of the human and mouse transcripts firmly established that both sequences encode a highly conserved protein of 98 amino acids that contains a signal peptide, suggesting that the protein may be secreted. Examination of the distribution of human ScRG-1 mRNA in adult and fetal tissues revealed that the gene was expressed primarily in the central nervous system as a 0.7-kilobase message and was under strict developmental control.

Recombinant human prion protein inhibits prion propagation in vitro.

PubMed

Yuan, Jue; Zhan, Yi-An; Abskharon, Romany; Xiao, Xiangzhu; Martinez, Manuel Camacho; Zhou, Xiaochen; Kneale, Geoff; Mikol, Jacqueline; Lehmann, Sylvain; Surewicz, Witold K; Castilla, Joaquín; Steyaert, Jan; Zhang, Shulin; Kong, Qingzhong; Petersen, Robert B; Wohlkonig, Alexandre; Zou, Wen-Quan

2013-10-09

Prion diseases are associated with the conformational conversion of the cellular prion protein (PrP(C)) into the pathological scrapie isoform (PrP(Sc)) in the brain. Both the in vivo and in vitro conversion of PrP(C) into PrP(Sc) is significantly inhibited by differences in amino acid sequence between the two molecules. Using protein misfolding cyclic amplification (PMCA), we now report that the recombinant full-length human PrP (rHuPrP23-231) (that is unglycosylated and lacks the glycophosphatidylinositol anchor) is a strong inhibitor of human prion propagation. Furthermore, rHuPrP23-231 also inhibits mouse prion propagation in a scrapie-infected mouse cell line. Notably, it binds to PrP(Sc), but not PrP(C), suggesting that the inhibitory effect of recombinant PrP results from blocking the interaction of brain PrP(C) with PrP(Sc). Our findings suggest a new avenue for treating prion diseases, in which a patient's own unglycosylated and anchorless PrP is used to inhibit PrP(Sc) propagation without inducing immune response side effects.

Protein conformation determines the sensibility to high pressure treatment of infectious scrapie prions.

PubMed

Heindl, Philipp; García, Avelina Fernández; Butz, Peter; Pfaff, Eberhard; Tauscher, Bernhard

2006-03-01

Application of high pressure can be used for gentle pasteurizing of food, minimizing undesirable alterations such as vitamin losses and changes in taste and color. In addition, pressure has become a useful tool for investigating structural changes in proteins. Treatments of proteins with high pressure can reveal conformations that are not obtainable by other physical variables like temperature, since pressure favors structural transitions accompanied with smaller volumes. Here, we discuss both the potential use of high pressure to inactivate infectious TSE material and the application of this thermodynamic parameter for the investigation of prion folding. This review summarizes our findings on the effects of pressure on the structure of native infectious scrapie prions in hamster brain homogenates and on the structure of infectious prion rods isolated from diseased hamsters brains. Native prions were found to be pressure sensitive, whereas isolated prions revealed an extreme pressure-resistant structure. The discussion will be focused on the different pressure behavior of these prion isoforms, which points out differences in the protein structure that have not been taken into consideration before.

Substitutions of PrP N-terminal histidine residues modulate scrapie disease pathogenesis and incubation time in transgenic mice

PubMed Central

Eigenbrod, Sabina; Frick, Petra; Bertsch, Uwe; Mitteregger-Kretzschmar, Gerda; Mielke, Janina; Maringer, Marko; Piening, Niklas; Hepp, Alexander; Daude, Nathalie; Windl, Otto; Levin, Johannes; Giese, Armin; Sakthivelu, Vignesh; Tatzelt, Jörg

2017-01-01

Prion diseases have been linked to impaired copper homeostasis and copper induced-oxidative damage to the brain. Divalent metal ions, such as Cu2+ and Zn2+, bind to cellular prion protein (PrPC) at octapeptide repeat (OR) and non-OR sites within the N-terminal half of the protein but information on the impact of such binding on conversion to the misfolded isoform often derives from studies using either OR and non-OR peptides or bacterially-expressed recombinant PrP. Here we created new transgenic mouse lines expressing PrP with disrupted copper binding sites within all four histidine-containing OR's (sites 1–4, H60G, H68G, H76G, H84G, "TetraH>G" allele) or at site 5 (composed of residues His-95 and His-110; "H95G" allele) and monitored the formation of misfolded PrP in vivo. Novel transgenic mice expressing PrP(TetraH>G) at levels comparable to wild-type (wt) controls were susceptible to mouse-adapted scrapie strain RML but showed significantly prolonged incubation times. In contrast, amino acid replacement at residue 95 accelerated disease progression in corresponding PrP(H95G) mice. Neuropathological lesions in terminally ill transgenic mice were similar to scrapie-infected wt controls, but less severe. The pattern of PrPSc deposition, however, was not synaptic as seen in wt animals, but instead dense globular plaque-like accumulations of PrPSc in TgPrP(TetraH>G) mice and diffuse PrPSc deposition in (TgPrP(H95G) mice), were observed throughout all brain sections. We conclude that OR and site 5 histidine substitutions have divergent phenotypic impacts and that cis interactions between the OR region and the site 5 region modulate pathogenic outcomes by affecting the PrP globular domain. PMID:29220360

A "shotgun" method for tracing the birth locations of sheep from flock tags, applied to scrapie surveillance in Great Britain.

PubMed

Birch, Colin P D; Del Rio Vilas, Victor J; Chikukwa, Ambrose C

2010-09-01

Movement records are often used to identify animal sample provenance by retracing the movements of individuals. Here we present an alternative method, which uses the same identity tags and movement records as are used to retrace movements, but ignores individual movement paths. The first step uses a simple query to identify the most likely birth holding for every identity tag included in a database recording departures from agricultural holdings. The second step rejects a proportion of the birth holding locations to leave a list of birth holding locations that are relatively reliable. The method was used to trace the birth locations of sheep sampled for scrapie in abattoirs, or on farm as fallen stock. Over 82% of the sheep sampled in the fallen stock survey died at the holding of birth. This lack of movement may be an important constraint on scrapie transmission. These static sheep provided relatively reliable birth locations, which were used to define criteria for selecting reliable traces. The criteria rejected 16.8% of fallen stock traces and 11.9% of abattoir survey traces. Two tests provided estimates that selection reduced error in fallen stock traces from 11.3% to 3.2%, and in abattoir survey traces from 8.1% to 1.8%. This method generated 14,591 accepted traces of fallen stock from samples taken during 2002-2005 and 83,136 accepted traces from abattoir samples. The absence or ambiguity of flock tag records at the time of slaughter prevented the tracing of 16-24% of abattoir samples during 2002-2004, although flock tag records improved in 2005. The use of internal scoring to generate and evaluate results from the database query, and the confirmation of results by comparison with other database fields, are analogous to methods used in web search engines. Such methods may have wide application in tracing samples and in adding value to biological datasets. Crown Copyright 2010. Published by Elsevier B.V. All rights reserved.

Inactivation of Prions and Amyloid Seeds with Hypochlorous Acid

PubMed Central

Kraus, Allison; Phillips, Katie; Contreras, Luis; Zanusso, Gianluigi; Caughey, Byron

2016-01-01

Hypochlorous acid (HOCl) is produced naturally by neutrophils and other cells to kill conventional microbes in vivo. Synthetic preparations containing HOCl can also be effective as microbial disinfectants. Here we have tested whether HOCl can also inactivate prions and other self-propagating protein amyloid seeds. Prions are deadly pathogens that are notoriously difficult to inactivate, and standard microbial disinfection protocols are often inadequate. Recommended treatments for prion decontamination include strongly basic (pH ≥~12) sodium hypochlorite bleach, ≥1 N sodium hydroxide, and/or prolonged autoclaving. These treatments are damaging and/or unsuitable for many clinical, agricultural and environmental applications. We have tested the anti-prion activity of a weakly acidic aqueous formulation of HOCl (BrioHOCl) that poses no apparent hazard to either users or many surfaces. For example, BrioHOCl can be applied directly to skin and mucous membranes and has been aerosolized to treat entire rooms without apparent deleterious effects. Here, we demonstrate that immersion in BrioHOCl can inactivate not only a range of target microbes, including spores of Bacillus subtilis, but also prions in tissue suspensions and on stainless steel. Real-time quaking-induced conversion (RT-QuIC) assays showed that BrioHOCl treatments eliminated all detectable prion seeding activity of human Creutzfeldt-Jakob disease, bovine spongiform encephalopathy, cervine chronic wasting disease, sheep scrapie and hamster scrapie; these findings indicated reductions of ≥103- to 106-fold. Transgenic mouse bioassays showed that all detectable hamster-adapted scrapie infectivity in brain homogenates or on steel wires was eliminated, representing reductions of ≥~105.75-fold and >104-fold, respectively. Inactivation of RT-QuIC seeding activity correlated with free chlorine concentration and higher order aggregation or destruction of proteins generally, including prion protein. BrioHOCl treatments had similar effects on amyloids composed of human α-synuclein and a fragment of human tau. These results indicate that HOCl can block the self-propagating activity of prions and other amyloids. PMID:27685252

Dysfunction of mitochondrial dynamics in the brains of scrapie-infected mice

DOE Office of Scientific and Technical Information (OSTI.GOV)

Choi, Hong-Seok; Ilsong Institute of Life Science, Hallym University, 1605-4 Gwanyang-dong, Dongan-gu, Anyang, Gyeonggi-do 431-060; Choi, Yeong-Gon

Highlights: • Mfn1 and Fis1 are significantly increased in the hippocampal region of the ME7 prion-infected brain, whereas Dlp1 is significantly decreased in the infected brain. • Dlp1 is significantly decreased in the cytosolic fraction of the hippocampus in the infected brain. • Neuronal mitochondria in the prion-infected brains are enlarged and swollen compared to those of control brains. • There are significantly fewer mitochondria in the ME7-infected brain compared to the number in control brain. - Abstract: Mitochondrial dysfunction is a common and prominent feature of many neurodegenerative diseases, including prion diseases; it is induced by oxidative stress inmore » scrapie-infected animal models. In previous studies, we found swelling and dysfunction of mitochondria in the brains of scrapie-infected mice compared to brains of controls, but the mechanisms underlying mitochondrial dysfunction remain unclear. To examine whether the dysregulation of mitochondrial proteins is related to the mitochondrial dysfunction associated with prion disease, we investigated the expression patterns of mitochondrial fusion and fission proteins in the brains of ME7 prion-infected mice. Immunoblot analysis revealed that Mfn1 was up-regulated in both whole brain and specific brain regions, including the cerebral cortex and hippocampus, of ME7-infected mice compared to controls. Additionally, expression levels of Fis1 and Mfn2 were elevated in the hippocampus and the striatum, respectively, of the ME7-infected brain. In contrast, Dlp1 expression was significantly reduced in the hippocampus in the ME7-infected brain, particularly in the cytosolic fraction. Finally, we observed abnormal mitochondrial enlargement and histopathological change in the hippocampus of the ME7-infected brain. These observations suggest that the mitochondrial dysfunction, which is presumably caused by the dysregulation of mitochondrial fusion and fission proteins, may contribute to the neuropathological changes associated with prion disease.« less

9 CFR 98.34 - Import permits for poultry semen and animal semen.

Code of Federal Regulations, 2014 CFR

2014-01-01

... associated antigen (VIAA) in serum. (Animals having responses to the AGID test or reacting to the VN test at...). (C) Swine vesicular disease: Virus neutralization test at 1:40 dilution (serums to be tested at FADDL... section have been met. (d) Sheep and goat semen from regions where scrapie exists. Importation of semen of...

9 CFR 98.34 - Import permits for poultry semen and animal semen.

Code of Federal Regulations, 2013 CFR

2013-01-01

... associated antigen (VIAA) in serum. (Animals having responses to the AGID test or reacting to the VN test at...). (C) Swine vesicular disease: Virus neutralization test at 1:40 dilution (serums to be tested at FADDL... section have been met. (d) Sheep and goat semen from regions where scrapie exists. Importation of semen of...

9 CFR 98.34 - Import permits for poultry semen and animal semen.

Code of Federal Regulations, 2012 CFR

2012-01-01

... associated antigen (VIAA) in serum. (Animals having responses to the AGID test or reacting to the VN test at...). (C) Swine vesicular disease: Virus neutralization test at 1:40 dilution (serums to be tested at FADDL... section have been met. (d) Sheep and goat semen from regions where scrapie exists. Importation of semen of...

Differential effects of divalent cations on elk prion protein fibril formation and stability

USDA-ARS?s Scientific Manuscript database

Misfolding of the normally folded prion protein of mammals (PrPC) into infectious fibrils causes a variety of different diseases, from scrapie in sheep to bovine spongiform encephalopathy in cattle to chronic wasting disease (CWD) in deer and elk. The misfolded form of PrPC, termed PrPSc, or in this...

Detecting and discriminating among pathogenic protein conformers(prions), using mass spectrometry-based and antibody-based approaches(Abstract)

USDA-ARS?s Scientific Manuscript database

A set of fatal neurological diseases that includes scrapie and chronic wasting disease (CWD) are caused by a pathological protein referred to as a prion (PrPSc). A prion propagates an infection by converting a normal cellular protein (PrPC) into a prion. Unlike viral, bacterial, or fungal pathogens,...

Raman spectroscopy reveals spectroscopic changes in histologically normal retinas in a mouse model of alpha-synucleinopathy

USDA-ARS?s Scientific Manuscript database

The retina is an extension of the nervous system and is accessible for in vivo assessments. We have previously demonstrated changes in retinal function and pathology associated with scrapie, TME and BSE. The purpose of this work was to determine the utility of the retina to identify early CNS change...

76 FR 34033 - Notice of Request for Extension of Approval of an Information Collection; Interstate Movement of...

Federal Register 2010, 2011, 2012, 2013, 2014

2011-06-10

...] Notice of Request for Extension of Approval of an Information Collection; Interstate Movement of Sheep... movement of sheep and goats to control the spread of scrapie. DATES: We will consider all comments that we... . FOR FURTHER INFORMATION CONTACT: For information on regulations for the interstate movement of sheep...

Ovine leukocyte profiles do not associate with variation in the prion gene, but are breed-dependent

USDA-ARS?s Scientific Manuscript database

Prion genotype in sheep confer resistance to scrapie. In cattle, lymphocyte profile has been found to be associated with prion genotype. Therefore, the aim of this study was to determine if variations in the sheep prion gene were associated with leukocyte populations as measured by complete blood ce...

Chronic wasting disease and atypical forms of BSE and scrapie are not transmissible to mice expressing wild-type levels of human PrP

USDA-ARS?s Scientific Manuscript database

The association between bovine spongiform encephalopathy (BSE) and variant Creutzfeldt-Jakob disease (vCJD) has demonstrated that cattle TSEs can pose a risk to human health and raises the possibility that other ruminant TSEs may be transmissible to humans. In recent years, several new TSEs in shee...

Novel epitopes identified by Anti-PrP monoclonal antibodies produced following immunization of Prnp0/0 Balb/cJ mice with purified scrapie prions

USDA-ARS?s Scientific Manuscript database

Prions, or infectious proteins, cause a class of uniformly fatal neurodegenerative diseases. Prions are composed solely of an aberrantly folded isoform(PrPSc)of a normal cellular protein (PrPC). Shared sequence identity of PrPSc with PrPC has limited the detection sensitivity of immunochemical assay...

9 CFR 79.2 - Identification of sheep and goats in interstate commerce.

Code of Federal Regulations, 2010 CFR

2010-01-01

... goats; the breed or cross if known; the name, street address, including city and State, or the township... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Identification of sheep and goats in... SCRAPIE IN SHEEP AND GOATS § 79.2 Identification of sheep and goats in interstate commerce. (a) No sheep...

9 CFR 79.2 - Identification of sheep and goats in interstate commerce.

Code of Federal Regulations, 2011 CFR

2011-01-01

... goats; the breed or cross if known; the name, street address, including city and State, or the township... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Identification of sheep and goats in... SCRAPIE IN SHEEP AND GOATS § 79.2 Identification of sheep and goats in interstate commerce. (a) No sheep...

Examining relationships between production and gastrointestinal nematode (GIN) measures in organic and conventionally reared Katahdin lambs

USDA-ARS?s Scientific Manuscript database

Katahdin sheep numbers are growing in the Southeastern US. It is a breed committed to the sheep industry’s goal of eradicating Scrapie by selecting for sheep with RR or QR at codon 171. Gastrointestinal nematodes remain a threat to the health and well-being of sheep due to widespread anthelmintic r...

Sensitive and specific detection of classical scrapie prions in the brain of goats by real-time quaking-induced conversion

USDA-ARS?s Scientific Manuscript database

The real-time quaking-induced conversion (RT-QuIC) is a rapid, specific, and sensitive prion seeding activity detection assay that uses recombinant prion protein (rPrPSen) to detect sub-infectious levels of the abnormal isoforms of the prion protein (PrPSc). Although RT-QuIC has been successfully us...

Prions: Beyond a Single Protein

PubMed Central

Das, Alvin S.

2016-01-01

SUMMARY Since the term protein was first coined in 1838 and protein was discovered to be the essential component of fibrin and albumin, all cellular proteins were presumed to play beneficial roles in plants and mammals. However, in 1967, Griffith proposed that proteins could be infectious pathogens and postulated their involvement in scrapie, a universally fatal transmissible spongiform encephalopathy in goats and sheep. Nevertheless, this novel hypothesis had not been evidenced until 1982, when Prusiner and coworkers purified infectious particles from scrapie-infected hamster brains and demonstrated that they consisted of a specific protein that he called a “prion.” Unprecedentedly, the infectious prion pathogen is actually derived from its endogenous cellular form in the central nervous system. Unlike other infectious agents, such as bacteria, viruses, and fungi, prions do not contain genetic materials such as DNA or RNA. The unique traits and genetic information of prions are believed to be encoded within the conformational structure and posttranslational modifications of the proteins. Remarkably, prion-like behavior has been recently observed in other cellular proteins—not only in pathogenic roles but also serving physiological functions. The significance of these fascinating developments in prion biology is far beyond the scope of a single cellular protein and its related disease. PMID:27226089

Disease associated prion protein may deposit in the peripheral nervous system in human transmissible spongiform encephalopathies.

PubMed

Hainfellner, J A; Budka, H

1999-11-01

There is increasing evidence indicating involvement of the peripheral nervous system (PNS) in the pathogenesis of transmissible spongiform encephalopathies (TSEs). Immunocytochemically detectable deposits of TSE-specific abnormal prion protein (PrP(sc)) are considered as a surrogate marker for infectivity. We used anti-PrP immunocytochemistry to trace PrP(sc) deposition in spinal and enteric ganglia, and peripheral nerve in Creutzfeldt-Jakob disease (CJD), Gerstmann-Sträussler-Scheinker disease (GSS), and fatal familial insomnia. Discrete PrP(sc) deposits were detectable only in a few posterior root nerve fibers in an adaxonal location in one of nine CJD and the one GSS patients examined. Follicular dendritic cells of the gut and enteric nervous system were not labeled. Thus, PrP(sc) may spread to the PNS in different forms of human prion disease. In contrast to our observations in experimental scrapie (Groschup et al., Acta Neuropathol, this issue), the deposits were scant. Possible explanations for this discrepancy comprise strain difference, or centripetal (experimental scrapie) versus centrifugal (sporadic and genetic human prion diseases) spread of PrP(sc), resulting in different patterns and amounts of PrP(sc) accumulation in the PNS.

Efficient and Rapid Development of Transgenic Hamster Models of TSEs Using a Radical New Technology

DTIC Science & Technology

2007-09-01

clinical signs but did not have the typical brain lesions that had formerly been required for a positive diagno- sis.19 Indeed, bovine spongiform...scrapie as shown by PCR and DNA sequence analyses. J Neuropathol Exp Neurol 2001;60:613–620. 25. Ebringer A, Rashid T, Wilson C. Bovine spongiform...Transmissible spongiform encephalopathy BSE Bovine spongiform encephalopathy CWD

Temporal resolution of misfolded prion protein transport, accumulation, glial activation, and neuronal death in the retinas of mice inoculated with scrapie

USDA-ARS?s Scientific Manuscript database

Currently, there is a lack of pathologic landmarks to describe the progression of prion disease in vivo. The goal of this work was to determine the temporal relationship between the transport of misfolded prion protein from the brain to the retina, the accumulation of PrPSc in the retina, the respon...

Infection by ME7 prion is not modified in transgenic mice expressing the yeast chaperone Hsp104 in neurons.

PubMed

Dandoy-Dron, Françoise; Bogdanova, Anna; Beringue, Vincent; Bailly, Yannick; Tovey, Michael G; Laude, Hubert; Dron, Michel

2006-09-25

The Hsp104 chaperone induces thermo-tolerance in yeast and rescues proteins trapped in aggregates. In this study, we showed that xenogenic expression of Hsp104 dramatically increased the viability of the neuronal mouse CAD cell line after exposure to heat shock. These results indicate that the Hsp104 protein confers thermo-resistance to mammalian neuronal cells, the canonical property of Hsp104 in yeast. Hsp104 also determines the prion state of prion-like proteins in yeast and to investigate whether Hsp104 expression may modify mammalian prion infection in vivo, transgenic mice with specific expression of Hsp104 in neurons were generated. Mice develop and reproduce normally, they show no detectable physical defect and may constitute valuable model for the study of aggregation-prone neuropathological disorders. Hsp104 transgenic and control littermates were infected intracerebrally with the ME7 strain of scrapie. No differences in the incubation time of the disease or in PrP(Sc) accumulation were observed between transgenic and control mice. These results suggest that the heat-shock protein Hsp104 is not efficient to modulate the multiplication of mammalian prions and/or to counteract neurodegeneration in the brain of scrapie-infected mice.

Pressure-assisted dissociation and degradation of "proteinase K-resistant" fibrils prepared by seeding with scrapie-infected hamster prion protein.

PubMed

Akasaka, Kazuyuki; Maeno, Akihiro; Murayama, Taichi; Tachibana, Hideki; Fujita, Yuzo; Yamanaka, Hitoki; Nishida, Noriyuki; Atarashi, Ryuichiro

2014-01-01

The crucial step for the fatal neurodegenerative prion diseases involves the conversion of a normal cellular protein, PrP(C), into a fibrous pathogenic form, PrP(Sc), which has an unusual stability against heat and resistance against proteinase K digestion. A successful challenge to reverse the reaction from PrP(Sc) into PrP(C) is considered valuable, as it would give a key to dissolving the complex molecular events into thermodynamic and kinetic analyses and may also provide a means to prevent the formation of PrP(Sc) from PrP(C) eventually in vivo. Here we show that, by applying pressures at kbar range, the "proteinase K-resistant" fibrils (rHaPrP(res)) prepared from hamster prion protein (rHaPrP [23-231]) by seeding with brain homogenate of scrapie-infected hamster, becomes easily digestible. The result is consistent with the notion that rHaPrP(res) fibrils are dissociated into rHaPrP monomers under pressure and that the formation of PrP(Sc) from PrP(C) is thermodynamically controlled. Moreover, the efficient degradation of prion fibrils under pressure provides a novel means of eliminating infectious PrP(Sc) from various systems of pathogenic concern.

Propagation of prion strains through specific conformers of the prion protein.

PubMed Central

Scott, M R; Groth, D; Tatzelt, J; Torchia, M; Tremblay, P; DeArmond, S J; Prusiner, S B

1997-01-01

Two prion strains with identical incubation periods in mice exhibited distinct incubation periods and different neuropathological profiles upon serial transmission to transgenic mice expressing chimeric Syrian hamster/mouse (MH2M) prion protein (PrP) genes [Tg(MH2M) mice] and subsequent transmission to Syrian hamsters. After transmission to Syrian hamsters, the Me7 strain was indistinguishable from the previously established Syrian hamster strain Sc237, despite having been derived from an independent ancestral source. This apparent convergence suggests that prion diversity may be limited. The Me7 mouse strain could also be transmitted directly to Syrian hamsters, but when derived in this way, its properties were distinct from those of Me7 passaged through Tg(MH2M) mice. The Me7 strain did not appear permanently altered in either case, since the original incubation period could be restored by effectively reversing the series of passages. Prion diversity enciphered in the conformation of the scrapie isoform of PrP (PrP(Sc)) (G. C. Telling et al., Science 274:2079-2082, 1996) seems to be limited by the sequence of the PrP substrates serially converted into PrP(Sc), while prions are propagated through interactions between the cellular and scrapie isoforms of PrP. PMID:9371560

Pruritus is a common feature in sheep infected with the BSE agent

PubMed Central

Konold, Timm; Bone, Gemma; Vidal-Diez, Alberto; Tortosa, Raul; Davis, Andrew; Dexter, Glenda; Hill, Peter; Jeffrey, Martin; Simmons, Marion M; Chaplin, Melanie J; Bellworthy, Susan J; Berthelin-Baker, Christine

2008-01-01

Background The variability in the clinical or pathological presentation of transmissible spongiform encephalopathies (TSEs) in sheep, such as scrapie and bovine spongiform encephalopathy (BSE), has been attributed to prion protein genotype, strain, breed, clinical duration, dose, route and type of inoculum and the age at infection. The study aimed to describe the clinical signs in sheep infected with the BSE agent throughout its clinical course to determine whether the clinical signs were as variable as described for classical scrapie in sheep. The clinical signs were compared to BSE-negative sheep to assess if disease-specific clinical markers exist. Results Forty-seven (34%) of 139 sheep, which comprised 123 challenged sheep and 16 undosed controls, were positive for BSE. Affected sheep belonged to five different breeds and three different genotypes (ARQ/ARQ, VRQ/VRQ and AHQ/AHQ). None of the controls or BSE exposed sheep with ARR alleles were positive. Pruritus was present in 41 (87%) BSE positive sheep; the remaining six were judged to be pre-clinically infected. Testing of the response to scratching along the dorsum of a sheep proved to be a good indicator of clinical disease with a test sensitivity of 85% and specificity of 98% and usually coincided with weight loss. Clinical signs that were displayed significantly earlier in BSE positive cases compared to negative cases were behavioural changes, pruritic behaviour, a positive scratch test, alopecia, skin lesions, teeth grinding, tremor, ataxia, loss of weight and loss of body condition. The frequency and severity of each specific clinical sign usually increased with the progression of disease over a period of 16–20 weeks. Conclusion Our results suggest that BSE in sheep presents with relatively uniform clinical signs, with pruritus of increased severity and abnormalities in behaviour or movement as the disease progressed. Based on the studied sheep, these clinical features appear to be independent of breed, affected genotype, dose, route of inoculation and whether BSE was passed into sheep from cattle or from other sheep, suggesting that the clinical phenotype of BSE is influenced by the TSE strain more than by other factors. The clinical phenotype of BSE in the genotypes and breed studied was indistinguishable from that described for classical scrapie cases. PMID:18445253

Temporal resolution of PrPSc transport, PrPSc accumulation, activation of glia and neuronal death in retinas from C57Bl/6 mice inoculated with RML scrapie: Relevance to biomarkers of prion disease progression

USDA-ARS?s Scientific Manuscript database

Currently, there is a lack of pathologic landmarks to objectively evaluate the progression of prion disease in vivo. The goal of this work was to determine the temporal relationship between transport of misfolded prion protein to the retina from the brain, accumulation of PrPSc in the retina, the re...

Polymorphism analysis of prion protein gene in 11 Pakistani goat breeds

PubMed Central

Hassan, Mohammad Farooque; Khan, Sher Hayat; Babar, Masroor Ellahi; Yang, Lifeng; Ali, Tariq; Khan, Jamal Muhammad; Shah, Syed Zahid Ali; Zhou, Xiangmei; Hussain, Tanveer; Zhu, Ting; Hussain, Tariq; Zhao, Deming

2016-01-01

ABSTRACT The association between caprine PrP gene polymorphisms and its susceptibility to scrapie has been investigated in current years. As the ORF of the PrP gene is extremely erratic in different breeds of goats, we studied the PrP gene polymorphisms in 80 goats which belong to 11 Pakistani indigenous goat breeds from all provinces of Pakistan. A total of 6 distinct polymorphic sites (one novel) with amino acid substitutions were identified in the PrP gene which includes 126 (A -> G), 304 (G -> T), 379 (A -> G), 414 (C -> T), 428 (A -> G) and 718 (C -> T). The locus c.428 was found highly polymorphic in all breeds as compare to other loci. On the basis of these PrP variants NJ phylogenetic tree was constructed through MEGA6.1 which showed that all goat breeds along with domestic sheep and Mauflon sheep appeared as in one clade and sharing its most recent common ancestors (MRCA) with deer species while Protein analysis has shown that these polymorphisms can lead to varied primary, secondary and tertiary structure of protein. Based on these polymorphic variants, genetic distance, multidimensional scaling plot and principal component analyses revealed the clear picture regarding greater number of substitutions in cattle PrP regions as compared to the small ruminant species. In particular these findings may pinpoint the fundamental control over the scrapie in Capra hircus on genetic basis. PMID:27388702

Production of Prnp-/- goats by gene targeting in adult fibroblasts.

PubMed

Zhu, Caihong; Li, Bei; Yu, Guohua; Chen, Jianquan; Yu, Huiqing; Chen, Juan; Xu, Xujun; Wu, Youbing; Zhang, Aimin; Cheng, Guoxiang

2009-04-01

Homozygous mice devoid of functional Prnp are resistant to scrapie and prion propagation, but heterozygous mice for Prnp disruption still suffer from prion disease and prion deposition. We have previously generated heterozygous cloned goats with one allele of Prnp functional disruption. To obtain goats with both alleles of Prnp be disrupted which would be resistant to scrapie completely, a second-round gene targeting was applied to disrupt the wild type allele of Prnp in the heterozygous goats. By second-round gene targeting, we successfully disrupted the wild type allele of Prnp in primary Prnp (+/-) goat skin fibroblasts and obtained a Prnp (-/-) cell line without Prnp expression. This is the first report on successful targeting modification in primary adult somatic cells of animals. These cells were used as nuclear donors for somatic cell cloning to produce Prnp (-/-) goats. A total of 57 morulae or blastocytes developed from the reconstructed embryos were transferred to 31 recipients, which produced 7 pregnancies at day 35. At 73 days of gestation, we obtained one cloned fetus with Prnp (-/-) genotype. Our research not only indicated that multiple genetic modifications could be accomplished by multi-round gene targeting in primary somatic cells, but also provided strong evidence that gene targeting in adult cells other than fetal cells could be applied to introduce precise genetic modifications in animals without destroying the embryos.

Origin and implications of bovine spongiform encephalopathy.

PubMed

Narang, H

1996-04-01

All spongiform encephalopathies in animals, including humans, are slow developing infectious diseases. The current working theory links the origin of bovine spongiform encephalopathy (BSE) to the feeding of cattle with meat and bone meal prepared from scrapie-infected sheep remains. Recycling of cattle meat and bones (MBM) essentially resulted in the selection of a single strain from the "wild type", a mixture of 20 strains. The BSE agent is easily transmitted through ingestion, with some evidence of vertical transmission. Paradoxically, cattle have selected a major new strain which appears to be more virulent than an unselected strain found in scrapie sheep. The same strain of BSE agent is implicated in the occurrence of spongiform encephalopathy in domestic cats, tiger, and some exotic species of ruminants in zoos. The properties of BSE and its spread into cattle are still disputed. Since our understanding of the disease and its transmissibility in humans must await observations that will be made over some years to come, it is important to keep a reasonable perspective and ensure that any speculative comment is consistent with fact. In risk assessment in such circumstances, it is tempting give too much credence to persuasive parallels when direct relevant information is not available. On the other hand, it would also not be wise to assume that the disease will die by itself and will have no effect on humans.

EU-approved rapid tests might underestimate bovine spongiform encephalopathy infection in goats.

PubMed

Meloni, Daniela; Bozzetta, Elena; Langeveld, Jan P M; Groschup, Martin H; Goldmann, Wilfred; Andrèoletti, Olivier; Lantier, Isabelle; Van Keulen, Lucien; Bossers, Alex; Pitardi, Danilo; Nonno, Romolo; Sklaviadis, Theodoros; Ingravalle, Francesco; Peletto, Simone; Colussi, Silvia; Acutis, Pier Luigi

2017-03-01

We report the diagnostic sensitivity of 3 EU-approved rapid tests (ELISAs; 1 from IDEXX and 2 from Bio-Rad) for the detection of transmissible spongiform encephalopathy diseases in goats. Ninety-eight goat brainstem samples were tested. All the rapid tests had 100% specificity and ≥80% sensitivity, with the IDEXX test significantly more sensitive than the 2 Bio-Rad tests. All tests detected 100% of samples from goats with clinical scrapie, but missed 8% (IDEXX) to 33% (Bio-Rad SG) of samples from preclinical goats. Importantly, only IDEXX picked up all samples from clinical bovine spongiform encephalopathy (BSE)-infected goats, whereas the other 2 rapid tests missed 15% (Bio-Rad SG) to 25% (Bio-Rad SAP). These results show that a fraction of preclinical scrapie infections are likely missed by EU surveillance, with sensitivity of detection strongly dependent on the choice of the rapid test. Moreover, a significant proportion of clinical BSE infections are underestimated by using either Bio-Rad test. Assuming that the same sensitivity on preclinical goats would also occur in BSE-infected goats, our data suggest that IDEXX is likely the most sensitive test for detecting preclinical field cases of BSE infection in goats, although with an 8% failure rate. These results raise some concerns about the reliability of current EU surveillance figures on BSE infection in goats.

BSE: a consequence of cattle feeding with glycated molecules host-unknown?

PubMed

Boratyński, J; Górski, A

2002-04-01

Although there is much evidence supporting a prion contribution in the pathogenesis of transmissible spongiform encephalopathies, a novel point of view as to the induction of the diseases can be proposed. It is hypothesized that neurodegenerative diseases, such as scrapie in sheep and goats and bovine spongiform encephalopathy in cattle (BSE), originate from the consumption of glycated proteins contained in their feed. These components are obtained during a high-temperature glycation process. Copyright 2002 Elsevier Science Ltd. All rights reserved.

Cleaning, disinfection and sterilization of surface prion contamination.

PubMed

McDonnell, G; Dehen, C; Perrin, A; Thomas, V; Igel-Egalon, A; Burke, P A; Deslys, J P; Comoy, E

2013-12-01

Prion contamination is a risk during device reprocessing, being difficult to remove and inactivate. Little is known of the combined effects of cleaning, disinfection and sterilization during a typical reprocessing cycle in clinical practice. To investigate the combination of cleaning, disinfection and/or sterilization on reducing the risk of surface prion contamination. In vivo test methods were used to study the impact of cleaning alone and cleaning combined with thermal disinfection and high- or low-temperature sterilization processes. A standardized test method, based on contamination of stainless steel wires with high titres of scrapie-infected brain homogenates, was used to determine infectivity reduction. Traditional chemical methods of surface decontamination against prions were confirmed to be effective, but extended steam sterilization was more variable. Steam sterilization alone reduced the risk of prion contamination under normal or extended exposure conditions, but did show significant variation. Thermal disinfection had no impact in these studies. Cleaning with certain defined formulations in combination with steam sterilization can be an effective prion decontamination process, in particular with alkaline formulations. Low-temperature, gaseous hydrogen peroxide sterilization was also confirmed to reduce infectivity in the presence and absence of cleaning. Prion decontamination is affected by the full reprocessing cycle used on contaminated surfaces. The correct use of defined cleaning, disinfection and sterilization methods as tested in this report in the scrapie infectivity assay can provide a standard precaution against prion contamination. Copyright © 2013 The Healthcare Infection Society. Published by Elsevier Ltd. All rights reserved.

A brief history of prions.

PubMed

Zabel, Mark D; Reid, Crystal

2015-12-01

Proteins were described as distinct biological molecules and their significance in cellular processes was recognized as early as the 18th century. At the same time, Spanish shepherds observed a disease that compelled their Merino sheep to pathologically scrape against fences, a defining clinical sign that led to the disease being named scrapie. In the late 19th century, Robert Koch published his postulates for defining causative agents of disease. In the early 20th century, pathologists Creutzfeldt and Jakob described a neurodegenerative disease that would later be included with scrapie into a group of diseases known as transmissible spongiform encephalopathies (TSEs). Later that century, mounting evidence compelled a handful of scientists to betray the prevailing biological dogma governing pathogen replication that Watson and Crick so convincingly explained by cracking the genetic code just two decades earlier. Because TSEs seemed to defy these new rules, J.S. Griffith theorized mechanisms by which a pathogenic protein could encipher its own replication blueprint without a genetic code. Stanley Prusiner called this proteinaceous infectious pathogen a prion. Here we offer a concise account of the discovery of prions, the causative agent of TSEs, in the wider context of protein biochemistry and infectious disease. We highlight the discovery of prions in yeast and discuss the implication of prions as epigenomic carriers of biological and pathological information. We also consider expanding the prion hypothesis to include other proteins whose alternate isoforms confer new biological or pathological properties. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A brief history of prions

PubMed Central

Zabel, Mark D.; Reid, Crystal

2015-01-01

Proteins were described as distinct biological molecules and their significance in cellular processes was recognized as early as the 18th century. At the same time, Spanish shepherds observed a disease that compelled their Merino sheep to pathologically scrape against fences, a defining clinical sign that led to the disease being named scrapie. In the late 19th century, Robert Koch published his postulates for defining causative agents of disease. In the early 20th century, pathologists Creutzfeldt and Jakob described a neurodegenerative disease that would later be included with scrapie into a group of diseases known as transmissible spongiform encephalopathies (TSEs). Later that century, mounting evidence compelled a handful of scientists to betray the prevailing biological dogma governing pathogen replication that Watson and Crick so convincingly explained by cracking the genetic code just two decades earlier. Because TSEs seemed to defy these new rules, J.S. Griffith theorized mechanisms by which a pathogenic protein could encipher its own replication blueprint without a genetic code. Stanley Prusiner called this proteinaceous infectious pathogen a prion. Here we offer a concise account of the discovery of prions, the causative agent of TSEs, in the wider context of protein biochemistry and infectious disease. We highlight the discovery of prions in yeast and discuss the implication of prions as epigenomic carriers of biological and pathological information. We also consider expanding the prion hypothesis to include other proteins whose alternate isoforms confer new biological or pathological properties. PMID:26449713

Physical and chemical properties of the transmissible mink encephalopathy agent.

PubMed

Marsh, R F; Hanson, R P

1969-02-01

The size of the transmissible mink encephalopathy (TME) agent is estimated to be less than 50 nm on the basis of its passage through membrane filters. The agent is sensitive to ether, relatively resistant to 10% Formalin, resistant to ultraviolet irradiation, and susceptible to proteolytic digestion with Pronase. Attempts to extract an infectious nucleic acid fraction with hot phenol were unsuccessful. The results of these studies indicate that the TME agent has biochemical properties which are similar to those described for the transmissible agent of scrapie.

Clinical, pathological, and molecular features of classical and L-type atypical-BSE in goats

PubMed Central

D’Angelo, Antonio; Mazza, Maria; Meloni, Daniela; Baioni, Elisa; Maurella, Cristiana; Colussi, Silvia; Martinelli, Nicola; Lo Faro, Monica; Favole, Alessandra; Grifoni, Silvia; Gallo, Marina; Lombardi, Guerino; Iulini, Barbara; Casalone, Cristina; Corona, Cristiano

2018-01-01

Monitoring of small ruminants for transmissible spongiform encephalopathies (TSEs) has recently become more relevant after two natural scrapie suspected cases of goats were found to be positive for classical BSE (C-BSE). C-BSE probably established itself in this species unrecognized, undermining disease control measures. This opens the possibility that TSEs in goats may remain an animal source for human prion diseases. Currently, there are no data regarding the natural presence of the atypical BSE in caprines. Here we report that C-BSE and L-type atypical BSE (L-BSE) isolates from bovine species are intracerebrally transmissible to goats, with a 100% attack rate and a significantly shorter incubation period and survival time after C-BSE than after L-BSE experimental infection, suggesting a lower species barrier for classical agentin goat. All animals showed nearly the same clinical features of disease characterized by skin lesions, including broken hair and alopecia, and abnormal mental status. Histology and immunohistochemistry showed several differences between C-BSE and L-BSE infection, allowing discrimination between the two different strains. The lymphoreticular involvement we observed in the C-BSE positive goats argues in favour of a peripheral distribution of PrPSc similar to classical scrapie. Western blot and other currently approved screening tests detected both strains in the goats and were able to classify negative control animals. These data demonstrate that active surveillance of small ruminants, as applied to fallen stock and/or healthy slaughter populations in European countries, is able to correctly identify and classify classical and L-BSE and ultimately protect public health. PMID:29795663

Prion propagation and toxicity occur in vitro with two-phase kinetics specific to strain and neuronal type.

PubMed

Hannaoui, Samia; Maatouk, Layal; Privat, Nicolas; Levavasseur, Etienne; Faucheux, Baptiste A; Haïk, Stéphane

2013-03-01

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders that occur in humans and animals. The neuropathological hallmarks of TSEs are spongiosis, glial proliferation, and neuronal loss. The only known specific molecular marker of TSEs is the abnormal isoform (PrP(Sc)) of the host-encoded prion protein (PrP(C)), which accumulates in the brain of infected subjects and forms infectious prion particles. Although this transmissible agent lacks a specific nucleic acid component, several prion strains have been isolated. Prion strains are characterized by differences in disease outcome, PrP(Sc) distribution patterns, and brain lesion profiles at the terminal stage of the disease. The molecular factors and cellular mechanisms involved in strain-specific neuronal tropism and toxicity remain largely unknown. Currently, no cellular model exists to facilitate in vitro studies of these processes. A few cultured cell lines that maintain persistent scrapie infections have been developed, but only two of them have shown the cytotoxic effects associated with prion propagation. In this study, we have developed primary neuronal cultures to assess in vitro neuronal tropism and toxicity of different prion strains (scrapie strains 139A, ME7, and 22L). We have tested primary neuronal cultures enriched in cerebellar granular, striatal, or cortical neurons. Our results showed that (i) a strain-specific neuronal tropism operated in vitro; (ii) the cytotoxic effect varied among strains and neuronal cell types; (iii) prion propagation and toxicity occurred in two kinetic phases, a replicative phase followed by a toxic phase; and (iv) neurotoxicity peaked when abnormal PrP accumulation reached a plateau.

Prion Propagation and Toxicity Occur In Vitro with Two-Phase Kinetics Specific to Strain and Neuronal Type

PubMed Central

Hannaoui, Samia; Maatouk, Layal; Privat, Nicolas; Levavasseur, Etienne; Faucheux, Baptiste A.

2013-01-01

Prion diseases, or transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative disorders that occur in humans and animals. The neuropathological hallmarks of TSEs are spongiosis, glial proliferation, and neuronal loss. The only known specific molecular marker of TSEs is the abnormal isoform (PrPSc) of the host-encoded prion protein (PrPC), which accumulates in the brain of infected subjects and forms infectious prion particles. Although this transmissible agent lacks a specific nucleic acid component, several prion strains have been isolated. Prion strains are characterized by differences in disease outcome, PrPSc distribution patterns, and brain lesion profiles at the terminal stage of the disease. The molecular factors and cellular mechanisms involved in strain-specific neuronal tropism and toxicity remain largely unknown. Currently, no cellular model exists to facilitate in vitro studies of these processes. A few cultured cell lines that maintain persistent scrapie infections have been developed, but only two of them have shown the cytotoxic effects associated with prion propagation. In this study, we have developed primary neuronal cultures to assess in vitro neuronal tropism and toxicity of different prion strains (scrapie strains 139A, ME7, and 22L). We have tested primary neuronal cultures enriched in cerebellar granular, striatal, or cortical neurons. Our results showed that (i) a strain-specific neuronal tropism operated in vitro; (ii) the cytotoxic effect varied among strains and neuronal cell types; (iii) prion propagation and toxicity occurred in two kinetic phases, a replicative phase followed by a toxic phase; and (iv) neurotoxicity peaked when abnormal PrP accumulation reached a plateau. PMID:23255799

Estimating chronic wasting disease susceptibility in cervids using real-time quaking-induced conversion

PubMed Central

Haley, Nicholas J.; Rielinger, Rachel; Davenport, Kristen A.; O'Rourke, Katherine; Mitchell, Gordon; Richt, Jürgen A.

2017-01-01

In mammals, susceptibility to prion infection is primarily modulated by the host’s cellular prion protein (PrPC) sequence. In the sheep scrapie model, a graded scale of susceptibility has been established both in vivo and in vitro based on PrPC amino acids 136, 154 and 171, leading to global breeding programmes to reduce the prevalence of scrapie in sheep. Chronic wasting disease (CWD) resistance in cervids is often characterized as decreased prevalence and/or protracted disease progression in individuals with specific alleles; at present, no PrPC allele conferring absolute resistance in cervids has been identified. To model the susceptibility of various naturally occurring and hypothetical cervid PrPC alleles in vitro, we compared the amplification rates and amyloid extension efficiencies of eight distinct CWD isolates in recombinant cervid PrPC substrates using real-time quaking-induced conversion. We hypothesized that the in vitro conversion characteristics of these isolates in cervid substrates would correlate to in vivo susceptibility – permitting susceptibility prediction for the rare alleles found in nature. We also predicted that hypothetical alleles with multiple resistance-associated codons would be more resistant to in vitro conversion than natural alleles with a single resistant codon. Our studies demonstrate that in vitro conversion metrics align with in vivo susceptibility, and that alleles with multiple amino acid substitutions, each influencing resistance independently, do not necessarily contribute additively to conversion resistance. Importantly, we found that the naturally occurring whitetail deer QGAK substrate exhibited the slowest amplification rate among those evaluated, suggesting that further investigation of this allele and its resistance in vivo is warranted. PMID:29058651

Evaluation of a combinatorial approach to prion inactivation using an oxidizing agent, SDS, and proteinase K.

PubMed

Smith, Jodi D; Nicholson, Eric M; Greenlee, Justin J

2013-07-25

Prions demonstrate an unusual resistance to methods effective at inactivating conventional microorganisms. This has resulted in a very tangible and difficult infection control challenge to the medical and veterinary communities, as well as animal agriculture and related industries. Currently accepted practices of harsh chemical treatments such as prolonged exposure to sodium hydroxide or sodium hypochlorite, or autoclaving are not suitable in many situations. Less caustic and more readily applicable treatments to contaminated environments are therefore desirable. We recently demonstrated that exposure of the RML scrapie agent to a commercial product containing sodium percarbonate (SPC-P) with or without sodium dodecyl sulfate (SDS) rendered PrP(Sc) sensitive to proteinase K (PK), but did not eliminate infectivity. The current study was designed to evaluate the efficacy of a combinatorial approach to inactivating prions by exposing RML-positive brain homogenate to SPC-P and SDS followed by PK. Treated samples were evaluated for PrP(Sc)-immunoreactivity by western blot, and residual infectivity by mouse bioassay. Treatment of infected brain homogenate with SPC-P and SDS followed by PK exposure resulted in a 4-5 log10 reduction in infectivity when bioassayed in tga20 mice. This study demonstrates that exposure of the RML scrapie agent to SPC-P and SDS followed by PK markedly reduces, but does not eliminate infectivity. The results of this study encourage further investigation into whether consecutive or concomitant exposure to sodium percarbonate, SDS, and a protease may serve as a viable and non-caustic option for prion inactivation.

Replication and spread of CJD, kuru and scrapie agents in vivo and in cell culture

PubMed Central

Miyazawa, Kohtaro; Emmerling, Kaitlin

2011-01-01

Transmissible Spongiform Encephalopathy (TSE) agents are defined by their virulence for particular species, their spread in the population, their incubation time to cause disease, and their neuropathological sequelae. Murine adapted human agents, including sporadic CJD (sCJD), New Guinea kuru, and Japanese CJD agents, display particularly distinct incubation times and maximal infectious brain titers. They also induce agent-specific patterns of neurodegeneration. When these TSE agents are transmitted to cultured hypothalamic GT1 cells they maintain their unique identities. Nevertheless, the human kuru (kCJD) and Japanese FU-CJD agents, as well as the sheep 22L and 263K scrapie agents display doubling times that are 8× to 33× faster in cells than in brain, indicating release from complex innate immune responses. These data are most consistent with a foreign viral structure, rather than an infectious form of host prion protein (PrP-res). Profound agent-specific inhibitory effects are also apparent in GT1 cells, and maximal titer plateau in kCJD and FU-CJD differed by 1,000-fold in a cell-based assay. Remarkably, the lower titer kCJD agent rapidly induced de novo PrP-res in GT1 cells, whereas the high titer FU-CJD agent replicated silently for multiple passages. Although PrP-res is often considered to be toxic, PrP-res instead may be part of a primal defense and/or clearance mechanism against TSE environmental agents. Limited spread of particular TSE agents through nanotubes and cell-to-cell contacts probably underlies the long peripheral phase of human CJD. PMID:21527829

Conversion of truncated and elongated prion proteins into the scrapie isoform in cultured cells.

PubMed Central

Rogers, M; Yehiely, F; Scott, M; Prusiner, S B

1993-01-01

The only known component of the infectious prion is a posttranslationally modified protein known as the scrapie isoform of the prion protein, PrPSc. Upon limited proteolysis, a protease-resistant fragment designated PrP 27-30 is formed. Using in vitro mutagenesis, we examined the role of the N and C termini in the formation of PrPSc in persistently infected, mouse neuroblastoma (ScN2a) cells. Neither deletion of amino acids 23-88, which are also removed by proteinase K in the formation of PrP 27-30, nor deletion of the five octapeptide repeats within this region altered synthesis of PrPSc. Elongation of PrP with one, two, four, or six octapeptide repeats in addition to the five found in wild-type PrP did not alter the synthesis of PrPSc. Truncation of the C terminus was accomplished by substituting a translation stop codon for the predicted glycosylinositol phospholipid (GPI) anchor-attachment signal corresponding to amino acids 231-254. Expression of this C-terminal PrP mutant in ScN2a cells produced PrPSc that appeared to lack a GPI anchor. We conclude that neither the GPI anchor nor the N-terminal 66 amino acids are required for the synthesis of PrPSc as measured by the acquisition of limited resistance to proteinase K digestion. Whether these truncated or elongated PrP molecules are competent to participate in the formation of infectious prions remains to be established. Images Fig. 1 Fig. 2 Fig. 3 Fig. 4 Fig. 5 PMID:8475059

The risk of bovine spongiform encephalopathy ('mad cow disease') to human health.

PubMed

Brown, P

1997-09-24

Some human cases of the transmissible neurodegenerative disorder Creutzfeldt-Jakob disease recently seen in Great Britain are thought to have resulted from eating beef infected with the agent of bovine spongiform encephalopathy. Reasons for and against this presumption are explained, and the question of a similar situation occurring in countries other than Britain-in particular, the United States-is discussed in terms of the existence of scrapie (in sheep) or unrecognized bovine spongiform encephalopathy (in cattle), the practice of recycling nonedible sheep and cattle tissue for animal nutrition, and precautionary measures already taken or under consideration by government agencies

Overexpression of PLK3 Mediates the Degradation of Abnormal Prion Proteins Dependent on Chaperone-Mediated Autophagy.

PubMed

Wang, Hui; Tian, Chan; Sun, Jing; Chen, Li-Na; Lv, Yan; Yang, Xiao-Dong; Xiao, Kang; Wang, Jing; Chen, Cao; Shi, Qi; Shao, Qi-Xiang; Dong, Xiao-Ping

2017-08-01

Polo-like kinase 3 (PLK3) is the main cause of cell cycle reentry-related neuronal apoptosis which has been implicated in the pathogenesis of prion diseases. Previous work also showed the regulatory activity of exogenous PLK3 on the degradation of PrP (prion protein) mutants and pathogenic PrP Sc ; however, the precise mechanisms remain unknown. In this study, we identified that the overexpression of PLK3-mediated degradation of PrP mutant and PrP Sc was repressed by lysosome rather than by proteasomal and macroautophagy inhibitors. Core components of chaperone-mediated autophagy (CMA) effectors, lysosome-associated membrane protein type 2A (LAMP2a), and heat shock cognate protein 70 (Hsc70) are markedly decreased in the HEK293T cells expressing PrP mutant and scrapie-infected cell line SMB-S15. Meanwhile, PrP mutant showed ability to interact with LAMP2a and Hsc70. Overexpression of PLK3 sufficiently increased the cellular levels of LAMP2a and Hsc70, accompanying with declining the accumulations of PrP mutant and PrP Sc . The kinase domain (KD) of PLK3 was responsible for elevating LAMP2a and Hsc70. Knockdown of endogenous PLK3 enhanced the activity of macroautophagy in the cultured cells. Moreover, time-dependent reductions of LAMP2a and Hsc70 were also observed in the brain tissues of hamster-adapted scrapie agent 263K-infected hamsters, indicating an impairment of CMA during prion infection. Those data indicate that the overexpression of PLK3-mediated degradation of abnormal PrP is largely dependent on CMA pathway.

Mechanism of Scrapie Prion Precipitation with Phosphotungstate Anions

PubMed Central

2015-01-01

The phosphotungstate anion (PTA) is widely used to facilitate the precipitation of disease-causing prion protein (PrPSc) from infected tissue for applications in structural studies and diagnostic approaches. However, the mechanism of this precipitation is not understood. In order to elucidate the nature of the PTA interaction with PrPSc under physiological conditions, solutions of PTA were characterized by NMR spectroscopy at varying pH. At neutral pH, the parent [PW12O40]3– ion decomposes to give a lacunary [PW11O39]7– (PW11) complex and a single orthotungstate anion [WO4]2– (WO4). To measure the efficacy of each component of PTA, increasing concentrations of PW11, WO4, and mixtures thereof were used to precipitate PrPSc from brain homogenates of scrapie prion-infected mice. The amount of PrPSc isolated, quantified by ELISA and immunoblotting, revealed that both PW11 and WO4 contribute to PrPSc precipitation. Incubation with sarkosyl, PTA, or individual components of PTA resulted in separation of higher-density PrP aggregates from the neuronal lipid monosialotetrahexosylganglioside (GM1), as observed by sucrose gradient centrifugation. These experiments revealed that yield and purity of PrPSc were greater with polyoxometalates (POMs), which substantially supported the separation of lipids from PrPSc in the samples. Interaction of POMs and sarkosyl with brain homogenates promoted the formation of fibrillar PrPSc aggregates prior to centrifugation, likely through the separation of lipids like GM1 from PrPSc. We propose that this separation of lipids from PrP is a major factor governing the facile precipitation of PrPSc by PTA from tissue and might be optimized further for the detection of prions. PMID:25695325

Estimating chronic wasting disease susceptibility in cervids using real-time quaking-induced conversion.

PubMed

Haley, Nicholas J; Rielinger, Rachel; Davenport, Kristen A; O'Rourke, Katherine; Mitchell, Gordon; Richt, Jürgen A

2017-11-01

In mammals, susceptibility to prion infection is primarily modulated by the host's cellular prion protein (PrP C ) sequence. In the sheep scrapie model, a graded scale of susceptibility has been established both in vivo and in vitro based on PrP C amino acids 136, 154 and 171, leading to global breeding programmes to reduce the prevalence of scrapie in sheep. Chronic wasting disease (CWD) resistance in cervids is often characterized as decreased prevalence and/or protracted disease progression in individuals with specific alleles; at present, no PrP C allele conferring absolute resistance in cervids has been identified. To model the susceptibility of various naturally occurring and hypothetical cervid PrP C alleles in vitro, we compared the amplification rates and amyloid extension efficiencies of eight distinct CWD isolates in recombinant cervid PrP C substrates using real-time quaking-induced conversion. We hypothesized that the in vitro conversion characteristics of these isolates in cervid substrates would correlate to in vivo susceptibility - permitting susceptibility prediction for the rare alleles found in nature. We also predicted that hypothetical alleles with multiple resistance-associated codons would be more resistant to in vitro conversion than natural alleles with a single resistant codon. Our studies demonstrate that in vitro conversion metrics align with in vivo susceptibility, and that alleles with multiple amino acid substitutions, each influencing resistance independently, do not necessarily contribute additively to conversion resistance. Importantly, we found that the naturally occurring whitetail deer QGAK substrate exhibited the slowest amplification rate among those evaluated, suggesting that further investigation of this allele and its resistance in vivo is warranted.

Prion Strain Differences in Accumulation of PrPSc on Neurons and Glia Are Associated with Similar Expression Profiles of Neuroinflammatory Genes: Comparison of Three Prion Strains

PubMed Central

Carroll, James A.; Striebel, James F.; Rangel, Alejandra; Woods, Tyson; Phillips, Katie; Peterson, Karin E.; Race, Brent; Chesebro, Bruce

2016-01-01

Misfolding and aggregation of host proteins are important features of the pathogenesis of neurodegenerative diseases including Alzheimer’s disease, Parkinson’s disease, frontotemporal dementia and prion diseases. In all these diseases, the misfolded protein increases in amount by a mechanism involving seeded polymerization. In prion diseases, host prion protein is misfolded to form a pathogenic protease-resistant form, PrPSc, which accumulates in neurons, astroglia and microglia in the CNS. Here using dual-staining immunohistochemistry, we compared the cell specificity of PrPSc accumulation at early preclinical times post-infection using three mouse scrapie strains that differ in brain regional pathology. PrPSc from each strain had a different pattern of cell specificity. Strain 22L was mainly associated with astroglia, whereas strain ME7 was mainly associated with neurons and neuropil. In thalamus and cortex, strain RML was similar to 22L, but in substantia nigra, RML was similar to ME7. Expression of 90 genes involved in neuroinflammation was studied quantitatively using mRNA from thalamus at preclinical times. Surprisingly, despite the cellular differences in PrPSc accumulation, the pattern of upregulated genes was similar for all three strains, and the small differences observed correlated with variations in the early disease tempo. Gene upregulation correlated with activation of both astroglia and microglia detected in early disease prior to vacuolar pathology or clinical signs. Interestingly, the profile of upregulated genes in scrapie differed markedly from that seen in two acute viral CNS diseases (LaCrosse virus and BE polytropic Friend retrovirus) that had reactive gliosis at levels similar to our prion-infected mice. PMID:27046083

Prion Infected Meat-and-Bone Meal Is Still Infectious after Biodiesel Production

PubMed Central

Bruederle, Cathrin E.; Hnasko, Robert M.; Kraemer, Thomas; Garcia, Rafael A.; Haas, Michael J.; Marmer, William N.; Carter, John Mark

2008-01-01

The epidemic of bovine spongiform encephalopathy (BSE) has led to a world-wide drop in the market for beef by-products, such as Meat-and-Bone Meal (MBM), a fat-containing but mainly proteinaceaous product traditionally used as an animal feed supplement. While normal rendering is insufficient, the production of biodiesel from MBM has been suggested to destroy infectivity from transmissible spongiform encephalopathies (TSEs). In addition to producing fuel, this method simultaneously generates a nutritious solid residue. In our study we produced biodiesel from MBM under defined conditions using a modified form of alkaline methanolysis. We evaluated the presence of prion in the three resulting phases of the biodiesel reaction (Biodiesel, Glycerol and Solid Residue) in vitro and in vivo. Analysis of the reaction products from 263K scrapie infected MBM led to no detectable immunoreactivity by Western Blot. Importantly, and in contrast to the biochemical results the solid MBM residue from the reaction retained infectivity when tested in an animal bioassay. Histochemical analysis of hamster brains inoculated with the solid residue showed typical spongiform degeneration and vacuolation. Re-inoculation of these brains into a new cohort of hamsters led to onset of clinical scrapie symptoms within 75 days, suggesting that the specific infectivity of the prion protein was not changed during the biodiesel process. The biodiesel reaction cannot be considered a viable prion decontamination method for MBM, although we observed increased survival time of hamsters and reduced infectivity greater than 6 log orders in the solid MBM residue. Furthermore, results from our study compare for the first time prion detection by Western Blot versus an infectivity bioassay for analysis of biodiesel reaction products. We could show that biochemical analysis alone is insufficient for detection of prion infectivity after a biodiesel process. PMID:18698417

Infectious Prion Protein Alters Manganese Transport and Neurotoxicity in a Cell Culture Model of Prion Disease

PubMed Central

Martin, Dustin P.; Anantharam, Vellareddy; Jin, Huajun; Witte, Travis; Houk, Robert; Kanthasamy, Arthi; Kanthasamy, Anumantha G.

2011-01-01

Protein misfolding and aggregation are considered key features of many neurodegenerative diseases, but biochemical mechanisms underlying protein misfolding and the propagation of protein aggregates are not well understood. Prion disease is a classical neurodegenerative disorder resulting from the misfolding of endogenously expressed normal cellular prion protein (PrPC). Although the exact function of PrPC has not been fully elucidated, studies have suggested that it can function as a metal binding protein. Interestingly, increased brain manganese (Mn) levels have been reported in various prion diseases indicating divalent metals also may play a role in the disease process. Recently, we reported that PrPC protects against Mn-induced cytotoxicity in a neural cell culture model. To further understand the role of Mn in prion diseases, we examined Mn neurotoxicity in an infectious cell culture model of prion disease. Our results show CAD5 scrapie-infected cells were more resistant to Mn neurotoxicity as compared to uninfected cells (EC50 = 428.8 μM for CAD5 infected cells vs. 211.6 μM for uninfected cells). Additionally, treatment with 300 μM Mn in persistently infected CAD5 cells showed a reduction in mitochondrial impairment, caspase-3 activation, and DNA fragmentation when compared to uninfected cells. Scrapie-infected cells also showed significantly reduced Mn uptake as measured by inductively coupled plasma-mass spectrometry (ICP-MS), and altered expression of metal transporting proteins DMT1 and transferrin. Together, our data indicate that conversion of PrP to the pathogenic isoform enhances its ability to regulate Mn homeostasis, and suggest that understanding the interaction of metals with disease-specific proteins may provide further insight to protein aggregation in neurodegenerative diseases. PMID:21871919

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

PubMed Central

Chaplin, M. J.; Vickery, C. M.; Simon, S.; Davis, L.; Denyer, M.; Lockey, R.; Stack, M. J.; O'Connor, M. J.; Bishop, K.; Gough, K. C.; Maddison, B. C.; Thorne, L.; Spiropoulos, J.

2015-01-01

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 new in vitro discriminatory 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. PMID:26041899

Amyloid-beta-sheet formation at the air-water interface.

PubMed Central

Schladitz, C; Vieira, E P; Hermel, H; Möhwald, H

1999-01-01

An amyloid(1-40) solution rich in coil, turn, and alpha-helix, but poor in beta-sheet, develops monolayers with a high beta-sheet content when spread at the air-water interface. These monolayers are resistant to repeated compression-dilatation cycles and interaction with trifluoroethanol. The secondary structure motifs were detected by circular dichroism (CD) in solution and with infrared reflection-absorption spectroscopy (IRRAS) at the interface. Hydrophobic influences are discussed for the structure conversion in an effort to understand the completely unknown reason for the natural change of the normal prion protein cellular (PrP(C)) into the abnormal prion protein scrapie (PrP(Sc)). PMID:10585952

A novel copper-hydrogen peroxide formulation for prion decontamination.

PubMed

Solassol, Jerome; Pastore, Manuela; Crozet, Carole; Perrier, Veronique; Lehmann, Sylvain

2006-09-15

With the appearance of variant Creutzfeldt-Jakob disease (CJD) and the detection of infectious prions in the peripheral organs of persons with sporadic CJD, the development of decontamination methods that are compatible with medical equipment has become a major issue. Here, we show that a formulation of copper metal ions in combination with hydrogen peroxide dramatically reduces the level of prion protein (PrP)(Sc) (the scrapie isoform of PrP) present in homogenates of samples from prion-infected brains, including brain samples from humans with CJD. An animal bioassay confirmed the reduction in prion infectivity, indicating that this novel Cu(2+)-H(2)O(2) formulation has great potential for prion decontamination.

High-Throughput Screening of Compounds for Anti-Transmissible Spongiform Encephalopathy Activity Using Cell-Culture and Cell-Free Models and Infected Animals

DTIC Science & Technology

2006-07-01

cattle humans (33). One important experimental model that has been lacking in CWD research is a CWD-infected cell line. Several scrapie- infected...Table of Contents Introduction…………………………………………………………….…………....4 Body…………………………………………………………………………………….5 Key Research ...INTRODUCTION This research is aimed at finding therapeutics against the Transmissible Spongiform Encephalopathies (TSEs) or prion diseases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Marín-Moreno, Alba; Espinosa, Juan-Carlos; Fernánd

The environment plays a key role in horizontal transmission of prion diseases, since prions are extremely resistant to classical inactivation procedures. In prior work, we observed the high stability of bovine spongiform encephalopathy (BSE) infectivity when these prions were incubated in aqueous media such as phosphate-buffered saline (PBS) or wastewater for nearly nine months. As a continuation of this experiment, the same samples were maintained in PBS or wastewater for five additional years and residual BSE infectivity was assessed in bovine PrP{sup C} transgenic mice. Over this long time period (more than six years), BSE infectivity was reduced by threemore » and one orders of magnitude in wastewater and PBS respectively. To rule out a possible agent specific effect, sheep scrapie prions were subjected to the same experimental protocol, using eight years as the experimental end-point. No significant reduction in scrapie infectivity was observed over the first nine months of wastewater incubation while PBS incubation for eight years only produced a two logarithmic unit reduction in infectivity. By contrast, the dynamics of PrP{sup Res} persistence was different, disappearing progressively over the first year. The long persistence of prion infectivity observed in this study for two different agents provides supporting evidence of the assumed high stability of these agents in aquatic environments and that environmental processes or conventional wastewater treatments with low retention times would have little impact on prion infectivity. These results could have great repercussions in terms of risk assessment and safety for animals and human populations. - Highlights: • Prion infectivity resists long term incubations in aquatic environments. • Infectivity persistence in wastewater is reduced when compared to PBS. • In this study PrPRes fails as a marker for prion detection. • Mice bioassay is the most powerful tool for assessing prion presence. • Wastewater conventional treatment would not eliminate prion infectivity.« less

High hydrophobic amino acid exposure is responsible of the neurotoxic effects induced by E200K or D202N disease-related mutations of the human prion protein.

PubMed

Corsaro, Alessandro; Thellung, Stefano; Bucciarelli, Tonino; Scotti, Luca; Chiovitti, Katia; Villa, Valentina; D'Arrigo, Cristina; Aceto, Antonio; Florio, Tullio

2011-03-01

Mutations in prion protein are thought to be causative of inherited prion diseases favoring the spontaneous conversion of the normal prion protein into the scrapie-like pathological prion protein. We previously reported that, by controlled thermal denaturation, human prion protein fragment 90-231 acquires neurotoxic properties when transformed in a β-rich conformation, resembling the scrapie-like conformation. In this study we generated prion protein fragment 90-231 bearing mutations identified in familial prion diseases (D202N and E200K), to analyze their role in the induction of a neurotoxic conformation. Prion protein fragment 90-231(wild type) and the D202N mutant were not toxic in native conformation but induced cell death only after thermal denaturation. Conversely, prion protein fragment 90-231(E200K) was highly toxic in its native structure, suggesting that E200K mutation per se favors the acquisition of a peptide neurotoxic conformation. To identify the structural determinants of prion protein fragment 90-231 toxicity, we show that while the wild type peptide is structured in α-helix, hPrP90-231 E200K is spontaneously refolded in a β-structured conformer characterized by increased proteinase K resistance and propensity to generate fibrils. However, the most significant difference induced by E200K mutation in prion protein fragment 90-231 structure in native conformation we observed, was an increase in the exposure of hydrophobic amino-acids on protein surface that was detected in wild type and D202N proteins only after thermal denaturation. In conclusion, we propose that increased hydrophobicity is one of the main determinants of toxicity induced by different mutations in prion protein-derived peptides. Copyright © 2010 Elsevier Ltd. All rights reserved.

Invited review: Current state of genetic improvement in dairy sheep.

PubMed

Carta, A; Casu, Sara; Salaris, S

2009-12-01

Dairy sheep have been farmed traditionally in the Mediterranean basin in southern Europe, central Europe, eastern Europe, and in Near East countries. Currently, dairy sheep farming systems vary from extensive to intensive according to the economic relevance of the production chain and the specific environment and breed. Modern breeding programs were conceived in the 1960s. The most efficient selection scheme for local dairy sheep breeds is based on pyramidal management of the population with the breeders of nucleus flocks at the top, where pedigree and official milk recording, artificial insemination, controlled natural mating, and breeding value estimation are carried out to generate genetic progress. The genetic progress is then transferred to the commercial flocks through artificial insemination or natural-mating rams. Increasing milk yield is still the most profitable breeding objective for several breeds. Almost all milk is used for cheese production and, consequently, milk content traits are very important. Moreover, other traits are gaining interest for selection: machine milking ability and udder morphology, resistance to diseases (mastitis, internal parasites, scrapie), and traits related to the nutritional value of milk (fatty acid composition). Current breeding programs based on the traditional quantitative approach have achieved appreciable genetic gains for milk yield. In many cases, further selection goals such as milk composition, udder morphology, somatic cell count, and scrapie resistance have been implemented. However, the possibility of including other traits of selective interest is limited by high recording costs. Also, the organizational effort needed to apply the traditional quantitative approach limits the diffusion of current selection programs outside the European Mediterranean area. In this context, the application of selection schemes assisted by molecular information, to improve either traditional dairy traits or traits costly to record, seems to be attractive in dairy sheep. At the moment, the most effective strategy seems to be the strengthening of research projects aimed at finding causal mutations along the genes affecting traits of economic importance. However, genome-wide selection seems to be unfeasible in most dairy sheep breeds.

Correlation between Infectivity and Disease Associated Prion Protein in the Nervous System and Selected Edible Tissues of Naturally Affected Scrapie Sheep

PubMed Central

Chianini, Francesca; Cosseddu, Gian Mario; Steele, Philip; Hamilton, Scott; Hawthorn, Jeremy; Síso, Sílvia; Pang, Yvonne; Finlayson, Jeanie; Eaton, Samantha L.; Reid, Hugh W.; Dagleish, Mark P.; Di Bari, Michele Angelo; D’Agostino, Claudia; Agrimi, Umberto; Terry, Linda; Nonno, Romolo

2015-01-01

The transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of fatal neurodegenerative disorders characterised by the accumulation of a pathological form of a host protein known as prion protein (PrP). The validation of abnormal PrP detection techniques is fundamental to allow the use of high-throughput laboratory based tests, avoiding the limitations of bioassays. We used scrapie, a prototype TSE, to examine the relationship between infectivity and laboratory based diagnostic tools. The data may help to optimise strategies to prevent exposure of humans to small ruminant TSE material via the food chain. Abnormal PrP distribution/accumulation was assessed by immunohistochemistry (IHC), Western blot (WB) and ELISA in samples from four animals. In addition, infectivity was detected using a sensitive bank vole bioassay with selected samples from two of the four sheep and protein misfolding cyclic amplification using bank vole brain as substrate (vPMCA) was also carried out in selected samples from one animal. Lymph nodes, oculomotor muscles, sciatic nerve and kidney were positive by IHC, WB and ELISA, although at levels 100–1000 fold lower than the brain, and contained detectable infectivity by bioassay. Tissues not infectious by bioassay were also negative by all laboratory tests including PMCA. Although discrepancies were observed in tissues with very low levels of abnormal PrP, there was an overall good correlation between IHC, WB, ELISA and bioassay results. Most importantly, there was a good correlation between the detection of abnormal PrP in tissues using laboratory tests and the levels of infectivity even when the titre was low. These findings provide useful information for risk modellers and represent a first step toward the validation of laboratory tests used to quantify prion infectivity, which would greatly aid TSE risk assessment policies. PMID:25807559

Copper and the Prion Protein: Methods, Structures, Function, and Disease

NASA Astrophysics Data System (ADS)

Millhauser, Glenn L.

2007-05-01

The transmissible spongiform encephalopathies (TSEs) arise from conversion of the membrane-bound prion protein from PrPC to PrPSc. Examples of the TSEs include mad cow disease, chronic wasting disease in deer and elk, scrapie in goats and sheep, and kuru and Creutzfeldt-Jakob disease in humans. Although the precise function of PrPC in healthy tissues is not known, recent research demonstrates that it binds Cu(II) in an unusual and highly conserved region of the protein termed the octarepeat domain. This review describes recent connections between copper and PrPC, with an emphasis on the electron paramagnetic resonance elucidation of the specific copper-binding sites, insights into PrPC function, and emerging connections between copper and prion disease.

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

PubMed

Workman, Rob G; Maddison, Ben C; Gough, Kevin C

2017-07-04

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.

An Enzymatic Treatment of Soil-Bound Prions Effectively Inhibits Replication ▿

PubMed Central

Saunders, Samuel E.; Bartz, Jason C.; Vercauteren, Kurt C.; Bartelt-Hunt, Shannon L.

2011-01-01

Chronic wasting disease (CWD) and scrapie can be transmitted through indirect environmental routes, possibly via soil, and a practical decontamination strategy for prion-contaminated soil is currently unavailable. In the laboratory, an enzymatic treatment under environmentally relevant conditions (22°C, pH 7.4) can degrade soil-bound PrPSc below the limits of Western blot detection. We developed and used a quantitative serial protein misfolding cyclic amplification (PMCA) protocol to characterize the amplification efficiency of treated soil samples relative to controls of known infectious titer. Our results suggest large (104- to >106-fold) decreases in soil-bound prion infectivity following enzyme treatment, demonstrating that a mild enzymatic treatment could effectively reduce the risk of prion disease transmission via soil or other environmental surfaces. PMID:21571886

Applications of fluorescence spectroscopy to problems of food safety: detection of fecal contamination and of the presence of central nervous system tissue and diagnosis of neurological disease

NASA Astrophysics Data System (ADS)

Adhikary, Ramkrishna; Bose, Sayantan; Casey, Thomas A.; Gapsch, Al; Rasmussen, Mark A.; Petrich, Jacob W.

2010-02-01

Applications of fluorescence spectroscopy that enable the real-time or rapid detection of fecal contamination on beef carcasses and the presence of central nervous system tissue in meat products are discussed. The former is achieved by employing spectroscopic signatures of chlorophyll metabolites; the latter, by exploiting the characteristic structure and intensity of lipofuscin in central nervous system tissue. The success of these techniques has led us to investigate the possibility of diagnosing scrapie in sheep by obtaining fluorescence spectra of the retina. Crucial to this diagnosis is the ability to obtain baseline correlations of lipofuscin fluorescence with age. A murine model was employed as a proof of principle of this correlation.

The myth of maternal transmission of spongiform encephalopathy.

PubMed Central

Ridley, R. M.; Baker, H. F.

1995-01-01

It has long been accepted that the pattern of occurrence of scrapie--the form of spongiform encephalopathy associated with sheep--is determined mainly by maternal transmission, and this view has had a profound influence on policy decisions in the control of bovine spongiform encephalopathy and on public concern over the risk to human health form this disease. The occurrence of maternal transmission is, however, not predicted by modern knowledge of the aetiology of spongiform encephalopathy, and even though claims of maternal transmission have been reiterated frequently in the literature, re-examination of the source data reveals that these data are extremely scanty, unreplicated, and probably subject to ascertainment bias. The probability of maternal transmission of spongiform encephalopathy in any species should be viewed with the greatest scepticism. Images p1073-a PMID:7580668

NEUROPATHOLOGIC FINDINGS IN CETACEANS STRANDED IN ITALY (2002-14).

PubMed

Pintore, Maria Domenica; Mignone, Walter; Di Guardo, Giovanni; Mazzariol, Sandro; Ballardini, Marco; Florio, Caterina Lucia; Goria, Maria; Romano, Angelo; Caracappa, Santo; Giorda, Federica; Serracca, Laura; Pautasso, Alessandra; Tittarelli, Cristiana; Petrella, Antonio; Lucifora, Giuseppe; Di Nocera, Fabio; Uberti, Barbara Degli; Corona, Cristiano; Casalone, Cristina; Iulini, Barbara

2018-04-01

  We summarized the neuropathologic findings in 60 cetaceans stranded along the Italian coastline from 2002 to 2014. The following neuropathologic changes were detected in 45% (27/60) of animals: nonsuppurative meningo-encephalitides (30%, 18/60), nonspecific lesions (12%, 7/60), suppurative encephalitis (2%, 1/60), and neoplasm (2%, 1/60). No histologic lesions were found in 47% (28/60) of the specimens. Five (8%, 5/60) samples were unsuitable for analysis. Analysis with PCR detected Brucella spp., morbillivirus, and Toxoplasma gondii infection in one, six, and seven individuals, respectively. Immunohistochemical analysis confirmed positivity for morbillivirus and for T. gondii infection in three cases each. No evidence of the scrapie-associated prion protein PrPSc was detected. Our findings underscore the importance of an adequate surveillance system for monitoring aquatic mammal pathologies and for protecting both animal and human health.

Control and eradication of animal diseases in New Zealand.

PubMed

Davidson, R M

2002-01-01

New Zealand is free from all the major epidemic (Office International des Epizooties List A) diseases of animals and other important diseases, such as rabies and the transmissible spongiform encephalopathies. The once endemic conditions of sheep scab (Psoroptes ovis), bovine brucellosis (Brucella abortus), hydatids (Echinococcus granulosus) and Aujeszky's disease have been eradicated. Anthrax (Bacillus anthracis) is no longer considered endemic and Pullorum disease (Salmonella Pullorum) has effectively been eradicated from commercial poultry flocks. There are current control programmes for bovine tuberculosis (Mycobacterium bovis), enzootic bovine leucosis in dairy cattle, infectious bursal disease, ovine epididymitis (Brucella ovis), and caprine arthritis encephalitis. Historically, incursions by three important non-endemic diseases, contagious bovine pleuropneumonia, classical swine fever and scrapie, have been successfully eliminated. Any new occurrence of a serious exotic disease would be dealt with swiftly using powerful legislative authorities available for the purpose.

Bovine spongiform encephalopathy: is it time to relax BSE-related measures in the context of international trade?

PubMed

Matthews, D; Adkin, A

2011-04-01

Bovine spongiform encephalopathy (BSE) has presented serious challenges to both the World Organisation for Animal Health and national governments, in defining and implementing appropriate national control measures, and in agreeing trade rules that permit safe trade in cattle and bovine products. Precautionary trade rules were initially necessary, based upon the science of sheep scrapie, but research into BSE later enabled BSE-specific trade rules to be developed. As a result, current rules on trade are underpinned by a sound body of knowledge on BSE. Declining epidemics in most affected countries confirm the appropriateness of current precautions. Nevertheless, risk is primarily dependent on the prevalence of infection with BSE. In the face of low prevalence scenarios, certain precautionary measures in the Terrestrial Animal Health Code may now be considered excessive. A thorough review is therefore deemed appropriate.

PrPC Undergoes Basal to Apical Transcytosis in Polarized Epithelial MDCK Cells

PubMed Central

Arkhipenko, Alexander; Syan, Sylvie; Victoria, Guiliana Soraya

2016-01-01

The Prion Protein (PrP) is an ubiquitously expressed glycosylated membrane protein attached to the external leaflet of the plasma membrane via a glycosylphosphatidylinositol anchor (GPI). While the misfolded PrPSc scrapie isoform is the infectious agent of prion disease, the cellular isoform (PrPC) is an enigmatic protein with unclear function. Of interest, PrP localization in polarized MDCK cells is controversial and its mechanism of trafficking is not clear. Here we investigated PrP traffic in MDCK cells polarized on filters and in three-dimensional MDCK cysts, a more physiological model of polarized epithelia. We found that, unlike other GPI-anchored proteins (GPI-APs), PrP undergoes basolateral-to-apical transcytosis in fully polarized MDCK cells. Following this event full-length PrP and its cleavage fragments are segregated in different domains of the plasma membrane in polarized cells in both 2D and 3D cultures. PMID:27389581

Cyclic Tetrapyrrole Sulfonation, Metals, and Oligomerization in Antiprion Activity▿

PubMed Central

Caughey, Winslow S.; Priola, Suzette A.; Kocisko, David A.; Raymond, Lynne D.; Ward, Anne; Caughey, Byron

2007-01-01

Cyclic tetrapyrroles are among the most potent compounds with activity against transmissible spongiform encephalopathies (TSEs; or prion diseases). Here the effects of differential sulfonation and metal binding to cyclic tetrapyrroles were investigated. Their potencies in inhibiting disease-associated protease-resistant prion protein were compared in several types of TSE-infected cell cultures. In addition, prophylactic antiscrapie activities were determined in scrapie-infected mice. The activity of phthalocyanine was relatively insensitive to the number of peripheral sulfonate groups but varied with the type of metal bound at the center of the molecule. The tendency of the various phthalocyanine sulfonates to oligomerize (i.e., stack) correlated with anti-TSE activity. Notably, aluminum(III) phthalocyanine tetrasulfonate was both the poorest anti-TSE compound and the least prone to oligomerization in aqueous media. Similar comparisons of iron- and manganese-bound porphyrin sulfonates confirmed that stacking ability correlates with anti-TSE activity among cyclic tetrapyrroles. PMID:17709470

Close Vicinity of PrP Expressing Cells (FDC) with Noradrenergic Fibers in Healthy Sheep Spleen

PubMed Central

Lezmi, S.; Hunsmann, G.; Baron, T.

2001-01-01

In naturally and experimentally occurring scrapie in sheep, prions invade the immune system and replicate in lymphoid organs. Here we analysed immunohistochemically, in seven spleens of 6-month-old healthy sheep, the nature of the cells expressing prion protein (PrP) potentially supporting prion replication, as well as their relationship with autonomic innervation. PrP was identified using either RB1 rabbit antiserum or 4F2 monoclonal antibody directed against AA 108–123 portion of the bovine and AA 79–92 of human prion protein respectively. Using double labelling analysis, we demonstrated that PrPc is expressed by follicular dendritic cells using a specific monoclonal antibody (CNA42). We also showed the close vicinity of these PrP expressing cells with noradrenergic fibers, using a polyclonal tyrosine hydroxylase antibody. Our results may help the study of the cellular requirements for the possible neuroinvasion from the spleen. PMID:11785673

The Priority position paper: Protecting Europe's food chain from prions.

PubMed

Requena, Jesús R; Kristensson, Krister; Korth, Carsten; Zurzolo, Chiara; Simmons, Marion; Aguilar-Calvo, Patricia; Aguzzi, Adriano; Andreoletti, Olivier; Benestad, Sylvie L; Böhm, Reinhard; Brown, Karen; Calgua, Byron; Del Río, José Antonio; Espinosa, Juan Carlos; Girones, Rosina; Godsave, Sue; Hoelzle, Ludwig E; Knittler, Michael R; Kuhn, Franziska; Legname, Giuseppe; Laeven, Paul; Mabbott, Neil; Mitrova, Eva; Müller-Schiffmann, Andreas; Nuvolone, Mario; Peters, Peter J; Raeber, Alex; Roth, Klaus; Schmitz, Matthias; Schroeder, Björn; Sonati, Tiziana; Stitz, Lothar; Taraboulos, Albert; Torres, Juan María; Yan, Zheng-Xin; Zerr, Inga

2016-05-03

Bovine spongiform encephalopathy (BSE) created a global European crisis in the 1980s and 90s, with very serious health and economic implications. Classical BSE now appears to be under control, to a great extent as a result of a global research effort that identified the sources of prions in meat and bone meal (MBM) and developed new animal-testing tools that guided policy. Priority ( www.prionpriority.eu ) was a European Union (EU) Framework Program 7 (FP7)-funded project through which 21 European research institutions and small and medium enterprises (SMEs) joined efforts between 2009 and 2014, to conduct coordinated basic and applied research on prions and prion diseases. At the end of the project, the Priority consortium drafted a position paper ( www.prionpriority.eu/Priority position paper) with its main conclusions. In the present opinion paper, we summarize these conclusions. With respect to the issue of re-introducing ruminant protein into the feed-chain, our opinion is that sustaining an absolute ban on feeding ruminant protein to ruminants is essential. In particular, the spread and impact of non-classical forms of scrapie and BSE in ruminants is not fully understood and the risks cannot be estimated. Atypical prion agents will probably continue to represent the dominant form of prion diseases in the near future in Europe. Atypical L-type BSE has clear zoonotic potential, as demonstrated in experimental models. Similarly, there are now data indicating that the atypical scrapie agent can cross various species barriers. More epidemiological data from large cohorts are necessary to reach any conclusion on the impact of its transmissibility on public health. Re-evaluations of safety precautions may become necessary depending on the outcome of these studies. Intensified searching for molecular determinants of the species barrier is recommended, since this barrier is key for important policy areas and risk assessment. Understanding the structural basis for strains and the basis for adaptation of a strain to a new host will require continued fundamental research, also needed to understand mechanisms of prion transmission, replication and how they cause nervous system dysfunction and death. Early detection of prion infection, ideally at a preclinical stage, also remains crucial for development of effective treatment strategies.

The Priority position paper: Protecting Europe's food chain from prions

PubMed Central

Kristensson, Krister; Korth, Carsten; Zurzolo, Chiara; Simmons, Marion; Aguilar-Calvo, Patricia; Aguzzi, Adriano; Andreoletti, Olivier; Benestad, Sylvie L.; Böhm, Reinhard; Brown, Karen; Calgua, Byron; del Río, José Antonio; Espinosa, Juan Carlos; Girones, Rosina; Godsave, Sue; Hoelzle, Ludwig E.; Knittler, Michael R.; Kuhn, Franziska; Legname, Giuseppe; Laeven, Paul; Mitrova, Eva; Müller-Schiffmann, Andreas; Nuvolone, Mario; Peters, Peter J.; Raeber, Alex; Roth, Klaus; Schmitz, Matthias; Schroeder, Björn; Sonati, Tiziana; Stitz, Lothar; Taraboulos, Albert; Torres, Juan María; Yan, Zheng-Xin; Zerr, Inga

2016-01-01

ABSTRACT Bovine spongiform encephalopathy (BSE) created a global European crisis in the 1980s and 90s, with very serious health and economic implications. Classical BSE now appears to be under control, to a great extent as a result of a global research effort that identified the sources of prions in meat and bone meal (MBM) and developed new animal-testing tools that guided policy. Priority (www.prionpriority.eu) was a European Union (EU) Framework Program 7 (FP7)-funded project through which 21 European research institutions and small and medium enterprises (SMEs) joined efforts between 2009 and 2014, to conduct coordinated basic and applied research on prions and prion diseases. At the end of the project, the Priority consortium drafted a position paper (www.prionpriority.eu/Priority position paper) with its main conclusions. In the present opinion paper, we summarize these conclusions. With respect to the issue of re-introducing ruminant protein into the feed-chain, our opinion is that sustaining an absolute ban on feeding ruminant protein to ruminants is essential. In particular, the spread and impact of non-classical forms of scrapie and BSE in ruminants is not fully understood and the risks cannot be estimated. Atypical prion agents will probably continue to represent the dominant form of prion diseases in the near future in Europe. Atypical L-type BSE has clear zoonotic potential, as demonstrated in experimental models. Similarly, there are now data indicating that the atypical scrapie agent can cross various species barriers. More epidemiological data from large cohorts are necessary to reach any conclusion on the impact of its transmissibility on public health. Re-evaluations of safety precautions may become necessary depending on the outcome of these studies. Intensified searching for molecular determinants of the species barrier is recommended, since this barrier is key for important policy areas and risk assessment. Understanding the structural basis for strains and the basis for adaptation of a strain to a new host will require continued fundamental research, also needed to understand mechanisms of prion transmission, replication and how they cause nervous system dysfunction and death. Early detection of prion infection, ideally at a preclinical stage, also remains crucial for development of effective treatment strategies. PMID:27220820

PrPc Does Not Mediate Internalization of PrPSc but Is Required at an Early Stage for De Novo Prion Infection of Rov Cells▿

PubMed Central

Paquet, Sophie; Daude, Nathalie; Courageot, Marie-Pierre; Chapuis, Jérôme; Laude, Hubert; Vilette, Didier

2007-01-01

We have studied the interactions of exogenous prions with an epithelial cell line inducibly expressing PrPc protein and permissive to infection by a sheep scrapie agent. We demonstrate that abnormal PrP (PrPSc) and prion infectivity are efficiently internalized in Rov cells, whether or not PrPc is expressed. At odds with earlier studies implicating cellular heparan sulfates in PrPSc internalization, we failed to find any involvement of such molecules in Rov cells, indicating that prions can enter target cells by several routes. We further show that PrPSc taken up in the absence of PrPc was unable to promote efficient prion multiplication once PrPc expression was restored in the cells. This observation argues that interaction of PrPSc with PrPc has to occur early, in a specific subcellular compartment(s), and is consistent with the view that the first prion multiplication events may occur at the cell surface. PMID:17626095

A closer look at prion strains

PubMed Central

Solforosi, Laura; Milani, Michela; Mancini, Nicasio; Clementi, Massimo; Burioni, Roberto

2013-01-01

Prions are infectious proteins that are responsible for transmissible spongiform encephalopathies (TSEs) and consist primarily of scrapie prion protein (PrPSc), a pathogenic isoform of the host-encoded cellular prion protein (PrPC). The absence of nucleic acids as essential components of the infectious prions is the most striking feature associated to these diseases. Additionally, different prion strains have been isolated from animal diseases despite the lack of DNA or RNA molecules. Mounting evidence suggests that prion-strain-specific features segregate with different PrPSc conformational and aggregation states. Strains are of practical relevance in prion diseases as they can drastically differ in many aspects, such as incubation period, PrPSc biochemical profile (e.g., electrophoretic mobility and glycoform ratio) and distribution of brain lesions. Importantly, such different features are maintained after inoculation of a prion strain into genetically identical hosts and are relatively stable across serial passages. This review focuses on the characterization of prion strains and on the wide range of important implications that the study of prion strains involves. PMID:23357828

Potential approaches for heterologous prion protein treatment of prion diseases

PubMed Central

Seelig, Davis M.; Goodman, Patricia A.; Skinner, Pamela J.

2016-01-01

ABSTRACT Prion diseases, or transmissible spongiform encephalopathies (TSEs) are progressive, fatal neurodegenerative diseases with no effective treatment. The pathology of these diseases involves the conversion of a protease sensitive form of the cellular prion protein (PrPC) into a protease resistant infectious form (PrPres). The efficiency of this conversion is predicated upon a number of factors, most notably a strong homology between cellular PrPC and PrPres. In our recently published study, we infected mice with the RML-Chandler strain of scrapie and treated them with heterologous hamster prion proteins. This treatment was seen to reduce clinical signs of prion disease, to delay the onset of clinical symptoms and to prolong survival. In this current article we discuss potential mechanisms of action of treatment with heterologous prion proteins. We also discuss potential extensions of these studies using a heterologous rabbit PrP-based treatment strategy or a peptide based strategy, and improvement of treatment delivery including a lentiviral-based system. PMID:26636482

Molecular modelling indicates that the pathological conformations of prion proteins might be beta-helical.

PubMed Central

Downing, D T; Lazo, N D

1999-01-01

Creutzfeldt-Jakob disease, kuru, scrapie and bovine spongiform encephalopathy are diseases of the mammalian central nervous system that involve the conversion of a cellular protein into an insoluble extracellular isoform. Spectroscopic studies have shown that the precursor protein contains mainly alpha-helical and random-coil conformations, whereas the prion isoform is largely in the beta conformation. The pathogenic prion is resistant to denaturation and protease digestion and can promote the conversion of the precursor protein to the pathogenic form. These properties have yet to be explained in terms of the structural conformations of the proteins. In the present study, molecular modelling showed that prion proteins could adopt the beta-helical conformation, which has been established for a number of fibrous proteins and has been suggested previously as the basis of amyloid fibrils. The beta-helical conformation provides explanations for the biophysical and biochemical stability of prions, their ability to form templates for the transmission of pathological conformation, and the existence of phenotypical strains of the prion diseases. PMID:10510313

Food Safety: Bovine Spongiform Encephalopathy (Mad Cow Disease).

PubMed

Acheson, David W. K.

2002-01-01

Bovine spongiform encephalopathy is just one of a group of diseases known as transmissible spongiform encephalopathies. Only recently has it become recognized that transmissible spongiform encephalopathies are likely due to proteins known as prions. Although it has been recognized that transmissible spongiform encephalopathies may readily spread within species, the recent observations that bovine spongiform encephalopathy in cattle may have originated from another transmissible spongiform encephalopathy in sheep, known as scrapie, is cause for concern. Further, bovine spongiform encephalopathy has now been strongly linked with a universally fatal human neurologic disease known as new variant Creutzfeldt-Jakob disease. Currently the only approach to preventing bovine spongiform encephalopathy, and subsequent new variant Creutzfeldt-Jakob disease in humans, from ingestion of bovine spongiform encephalopathy-infected material is to avoid consumption of contaminated food. Little can be done to treat food that will destroy prions and leave a palatable product. At this stage we are continuing to learn about transmissible spongiform encephalopathies and their implications on human health. This is an ever-changing situation and has an unpredictable element in terms of the extent of the current outbreaks in England and other parts of Europe.

Feasibility of infectious prion digestion using mild conditions and commercial subtilisin.

PubMed

Pilon, John L; Nash, Paul B; Arver, Terry; Hoglund, Don; Vercauteren, Kurt C

2009-10-01

Two serine protease enzymes, subtilisin 309 and subtilisin 309-v, were used to digest brain homogenates containing high levels of prion infectivity using mildly alkaline conditions to investigate prion decontamination methods. To establish that PrP(res) infectivity was eliminated, we utilized the Rocky Mountain Laboratory (RML) mouse-adapted scrapie model system for bioassay. Only one digestion condition (subtilisin 309 at 138mAU/ml, 55 degrees C and 14h digestion time pH 7.9) was considered to be highly relevant statistically (P<0.001) compared to control, with 52% of challenged mice surviving until the end of the study period. In contrast, treatment of PrP(res) by autoclaving at 134 degrees C or treatment with hypochlorite at a concentration of 20,000 ppm completely protected mice from prionosis. Further, in vitro assays suggest that potential proteolytic based PrP(res) decontamination methods must use high enzyme concentration, pH values >9.0, and elevated temperatures to be a safely efficacious, thereby limiting applicability on delicate surgical instruments and use in the environment.

Resistance of Soil-Bound Prions to Rumen Digestion

PubMed Central

Saunders, Samuel E.; Bartelt-Hunt, Shannon L.; Bartz, Jason C.

2012-01-01

Before prion uptake and infection can occur in the lower gastrointestinal system, ingested prions are subjected to anaerobic digestion in the rumen of cervids and bovids. The susceptibility of soil-bound prions to rumen digestion has not been evaluated previously. In this study, prions from infectious brain homogenates as well as prions bound to a range of soils and soil minerals were subjected to in vitro rumen digestion, and changes in PrP levels were measured via western blot. Binding to clay appeared to protect noninfectious hamster PrPc from complete digestion, while both unbound and soil-bound infectious PrPSc proved highly resistant to rumen digestion. In addition, no change in intracerebral incubation period was observed following active rumen digestion of unbound hamster HY TME prions and HY TME prions bound to a silty clay loam soil. These results demonstrate that both unbound and soil-bound prions readily survive rumen digestion without a reduction in infectivity, further supporting the potential for soil-mediated transmission of chronic wasting disease (CWD) and scrapie in the environment. PMID:22937149

A closer look at prion strains: characterization and important implications.

PubMed

Solforosi, Laura; Milani, Michela; Mancini, Nicasio; Clementi, Massimo; Burioni, Roberto

2013-01-01

Prions are infectious proteins that are responsible for transmissible spongiform encephalopathies (TSEs) and consist primarily of scrapie prion protein (PrP (Sc) ), a pathogenic isoform of the host-encoded cellular prion protein (PrP (C) ). The absence of nucleic acids as essential components of the infectious prions is the most striking feature associated to these diseases. Additionally, different prion strains have been isolated from animal diseases despite the lack of DNA or RNA molecules. Mounting evidence suggests that prion-strain-specific features segregate with different PrP (Sc) conformational and aggregation states. Strains are of practical relevance in prion diseases as they can drastically differ in many aspects, such as incubation period, PrP (Sc) biochemical profile (e.g., electrophoretic mobility and glycoform ratio) and distribution of brain lesions. Importantly, such different features are maintained after inoculation of a prion strain into genetically identical hosts and are relatively stable across serial passages. This review focuses on the characterization of prion strains and on the wide range of important implications that the study of prion strains involves.

Biochemical Characterization of Prion Strains in Bank Voles

PubMed Central

Pirisinu, Laura; Marcon, Stefano; Di Bari, Michele Angelo; D’Agostino, Claudia; Agrimi, Umberto; Nonno, Romolo

2013-01-01

Prions exist as different strains exhibiting distinct disease phenotypes. Currently, the identification of prion strains is still based on biological strain typing in rodents. However, it has been shown that prion strains may be associated with distinct PrPSc biochemical types. Taking advantage of the availability of several prion strains adapted to a novel rodent model, the bank vole, we investigated if any prion strain was actually associated with distinctive PrPSc biochemical characteristics and if it was possible to univocally identify strains through PrPSc biochemical phenotypes. We selected six different vole-adapted strains (three human-derived and three animal-derived) and analyzed PrPSc from individual voles by epitope mapping of protease resistant core of PrPSc (PrPres) and by conformational stability and solubility assay. Overall, we discriminated five out of six prion strains, while two different scrapie strains showed identical PrPSc types. Our results suggest that the biochemical strain typing approach here proposed was highly discriminative, although by itself it did not allow us to identify all prion strains analyzed. PMID:25437201

Mitigation of Prion Infectivity and Conversion Capacity by a Simulated Natural Process—Repeated Cycles of Drying and Wetting

PubMed Central

Yuan, Qi; Eckland, Thomas; Telling, Glenn; Bartz, Jason; Bartelt-Hunt, Shannon

2015-01-01

Prions enter the environment from infected hosts, bind to a wide range of soil and soil minerals, and remain highly infectious. Environmental sources of prions almost certainly contribute to the transmission of chronic wasting disease in cervids and scrapie in sheep and goats. While much is known about the introduction of prions into the environment and their interaction with soil, relatively little is known about prion degradation and inactivation by natural environmental processes. In this study, we examined the effect of repeated cycles of drying and wetting on prion fitness and determined that 10 cycles of repeated drying and wetting could reduce PrPSc abundance, PMCA amplification efficiency and extend the incubation period of disease. Importantly, prions bound to soil were more susceptible to inactivation by repeated cycles of drying and wetting compared to unbound prions, a result which may be due to conformational changes in soil-bound PrPSc or consolidation of the bonding between PrPSc and soil. This novel finding demonstrates that naturally-occurring environmental process can degrade prions. PMID:25665187

Mother to Offspring Transmission of Chronic Wasting Disease in Reeves’ Muntjac Deer

PubMed Central

Nalls, Amy V.; McNulty, Erin; Powers, Jenny; Seelig, Davis M.; Hoover, Clare; Haley, Nicholas J.; Hayes-Klug, Jeanette; Anderson, Kelly; Stewart, Paula; Goldmann, Wilfred; Hoover, Edward A.; Mathiason, Candace K.

2013-01-01

The horizontal transmission of prion diseases has been well characterized in bovine spongiform encephalopathy (BSE), chronic wasting disease (CWD) of deer and elk and scrapie of sheep, and has been regarded as the primary mode of transmission. Few studies have monitored the possibility of vertical transmission occurring within an infected mother during pregnancy. To study the potential for and pathway of vertical transmission of CWD in the native cervid species, we used a small cervid model–the polyestrous breeding, indoor maintainable, Reeves’ muntjac deer–and determined that the susceptibility and pathogenesis of CWD in these deer reproduce that in native mule and white-tailed deer. Moreover, we demonstrate here that CWD prions are transmitted from doe to fawn. Maternal CWD infection also appears to result in lower percentage of live birth offspring. In addition, evolving evidence from protein misfolding cyclic amplification (PMCA) assays on fetal tissues suggest that covert prion infection occurs in utero. Overall, our findings demonstrate that transmission of prions from mother to offspring can occur, and may be underestimated for all prion diseases. PMID:23977159

Piperazine derivatives inhibit PrP/PrP(res) propagation in vitro and in vivo.

PubMed

Leidel, Fabienne; Eiden, Martin; Geissen, Markus; Hirschberger, Thomas; Tavan, Paul; Giese, Armin; Kretzschmar, Hans A; Schätzl, Hermann; Groschup, Martin H

2014-02-28

Prion diseases are fatal neurodegenerative disorders, which are not curable and no effective treatment exists so far. The major neuropathological change in diseased brains is the conversion of the normal cellular form of the prion protein PrPc(C) into a disease-associated isoform PrP(Sc). PrP(Sc) accumulates into multimeres and fibrillar aggregates, which leads to the formation of amyloid plaques. Increasing evidence indicates a fundamental role of PrP(Sc) species and its aggregation in the pathogenesis of prion diseases, which initiates the pathological cascade and leads to neurodegeneration accompanied by spongiform changes. In search of compounds that have the potential to interfere with PrP(Sc) formation and propagation, we used a cell based assay for the screening of potential aggregation inhibitors. The assay deals with a permanently prion infected cell line that was adapted for a high-throughput screening of a compound library composed of 10,000 compounds (DIVERset 2, ChemBridge). We could detect six different classes of highly potent inhibitors of PrP(Sc) propagation in vitro and identified piperazine derivatives as a new inhibitory lead structure, which increased incubation time of scrapie infected mice. Copyright © 2014 Elsevier Inc. All rights reserved.

Sex effects in mouse prion disease incubation time.

PubMed

Akhtar, Shaheen; Wenborn, Adam; Brandner, Sebastian; Collinge, John; Lloyd, Sarah E

2011-01-01

Prion disease incubation time in mice is determined by many factors including PrP expression level, Prnp alleles, genetic background, prion strain and route of inoculation. Sex differences have been described in age of onset for vCJD and in disease duration for both vCJD and sporadic CJD and have also been shown in experimental models. The sex effects reported for mouse incubation times are often contradictory and detail only one strain of mice or prions, resulting in broad generalisations and a confusing picture. To clarify the effect of sex on prion disease incubation time in mice we have compared male and female transmission data from twelve different inbred lines of mice inoculated with at least two prion strains, representing both mouse-adapted scrapie and BSE. Our data show that sex can have a highly significant difference on incubation time. However, this is limited to particular mouse and prion strain combinations. No sex differences were seen in endogenous PrP(C) levels nor in the neuropathological markers of prion disease: PrP(Sc) distribution, spongiosis, neuronal loss and gliosis. These data suggest that when comparing incubation times between experimental groups, such as testing the effects of modifier genes or therapeutics, single sex groups should be used.

Infectious particles, stress, and induced prion amyloids

PubMed Central

2013-01-01

Transmissible encephalopathies (TSEs) are believed by many to arise by spontaneous conversion of host prion protein (PrP) into an infectious amyloid (PrP-res, PrPSc) without nucleic acid. Many TSE agents reside in the environment, with infection controlled by public health measures. These include the disappearance of kuru with the cessation of ritual cannibalism, the dramatic reduction of epidemic bovine encephalopathy (BSE) by removal of contaminated feed, and the lack of endemic scrapie in geographically isolated Australian sheep with susceptible PrP genotypes. While prion protein modeling has engendered an intense focus on common types of protein misfolding and amyloid formation in diverse organisms and diseases, the biological characteristics of infectious TSE agents, and their recognition by the host as foreign entities, raises several fundamental new directions for fruitful investigation such as: (1) unrecognized microbial agents in the environmental metagenome that may cause latent neurodegenerative disease, (2) the evolutionary social and protective functions of different amyloid proteins in diverse organisms from bacteria to mammals, and (3) amyloid formation as a beneficial innate immune response to stress (infectious and non-infectious). This innate process however, once initiated, can become unstoppable in accelerated neuronal aging. PMID:23633671

RML prions act through Mahogunin and Attractin-independent pathways.

PubMed

Gunn, Teresa M; Carlson, George A

2013-01-01

While the conversion of the normal form of prion protein to a conformationally distinct pathogenic form is recognized to be the primary cause of prion disease, it is not clear how this leads to spongiform change, neuronal dysfunction and death. Mahogunin ring finger-1 (Mgrn1) and Attractin (Atrn) null mutant mice accumulate vacuoles throughout the brain that appear very similar to those associated with prion disease, but they do not accumulate the protease-resistant scrapie form of the prion protein or become sick. A study demonstrating an interaction between cytosolically-exposed prion protein and MGRN1 suggested that disruption of MGRN1 function may contribute to prion disease pathogenesis, but we recently showed that neither loss of MGRN1 nor MGRN1 overexpression influences the onset or progression of prion disease following intracerebral inoculation with Rocky Mountain Laboratory prions. Here, we show that loss of ATRN also has no effect on prion disease onset or progression and discuss possible mechanisms that could cause vacuolation of the central nervous system in Mgrn1 and Atrn null mutant mice and whether the same pathways might contribute to this intriguing phenotype in prion disease.

Amyloid Structure and Assembly: Insights from Scanning Transmission Electron Microscopy

PubMed Central

Goldsbury, Claire; Baxa, Ulrich; Simon, Martha N.; Steven, Alasdair C.; Engel, Andreas; Wall, Joseph S.; Aebi, Ueli; Müller, Shirley A.

2010-01-01

Amyloid fibrils are filamentous protein aggregates implicated in several common diseases like Alzheimer’s disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies like Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR). PMID:20868754

Virus inactivation studies using ion beams, electron and gamma irradiation

NASA Astrophysics Data System (ADS)

Smolko, Eduardo E.; Lombardo, Jorge H.

2005-07-01

Known methods of virus inactivation are based on the chemical action of some substances such as acetylethylenimine, betapropiolactone, glycidalaldehyde, formaldehyde, etc. In such a process, the viral suspension should be kept at room or higher temperatures for 24-48 h. Under these conditions, physical and chemical agents act to degrade the virus antigenic proteins. On the contrary with ionizing radiations at low temperatures, the treatment does not cause such degradation allowing the study of different viral functions. In this work, particle (α, d and ß) and γ irradiations were used for partial and total inactivation of Foot and Mouth Disease Virus (FMDV), Rauscher Leukemia Virus (RLV) and Herpes Simplex Virus (HSV). Obtention of the D37 dose from survival curves and the application of the target theory, permitted the determination of molecular weight of the nucleic acid genomes, EBR values and useful information for vaccine preparation. For RLV virus, a two target model of the RNA genome was deduced in accordance with biological information while from data from the literature and our own work on the structure of the scrapie prion, considering the molecular weight obtained by application of the theory, a new model for prion replication is presented, based on a trimer molecule.

A Sequence-Dependent DNA Condensation Induced by Prion Protein

PubMed Central

2018-01-01

Different studies indicated that the prion protein induces hybridization of complementary DNA strands. Cell culture studies showed that the scrapie isoform of prion protein remained bound with the chromosome. In present work, we used an oxazole dye, YOYO, as a reporter to quantitative characterization of the DNA condensation by prion protein. We observe that the prion protein induces greater fluorescence quenching of YOYO intercalated in DNA containing only GC bases compared to the DNA containing four bases whereas the effect of dye bound to DNA containing only AT bases is marginal. DNA-condensing biological polyamines are less effective than prion protein in quenching of DNA-bound YOYO fluorescence. The prion protein induces marginal quenching of fluorescence of the dye bound to oligonucleotides, which are resistant to condensation. The ultrastructural studies with electron microscope also validate the biophysical data. The GC bases of the target DNA are probably responsible for increased condensation in the presence of prion protein. To our knowledge, this is the first report of a human cellular protein inducing a sequence-dependent DNA condensation. The increased condensation of GC-rich DNA by prion protein may suggest a biological function of the prion protein and a role in its pathogenesis. PMID:29657864

A Sequence-Dependent DNA Condensation Induced by Prion Protein.

PubMed

Bera, Alakesh; Biring, Sajal

2018-01-01

Different studies indicated that the prion protein induces hybridization of complementary DNA strands. Cell culture studies showed that the scrapie isoform of prion protein remained bound with the chromosome. In present work, we used an oxazole dye, YOYO, as a reporter to quantitative characterization of the DNA condensation by prion protein. We observe that the prion protein induces greater fluorescence quenching of YOYO intercalated in DNA containing only GC bases compared to the DNA containing four bases whereas the effect of dye bound to DNA containing only AT bases is marginal. DNA-condensing biological polyamines are less effective than prion protein in quenching of DNA-bound YOYO fluorescence. The prion protein induces marginal quenching of fluorescence of the dye bound to oligonucleotides, which are resistant to condensation. The ultrastructural studies with electron microscope also validate the biophysical data. The GC bases of the target DNA are probably responsible for increased condensation in the presence of prion protein. To our knowledge, this is the first report of a human cellular protein inducing a sequence-dependent DNA condensation. The increased condensation of GC-rich DNA by prion protein may suggest a biological function of the prion protein and a role in its pathogenesis.

Polythiophenes Inhibit Prion Propagation by Stabilizing Prion Protein (PrP) Aggregates*

PubMed Central

Margalith, Ilan; Suter, Carlo; Ballmer, Boris; Schwarz, Petra; Tiberi, Cinzia; Sonati, Tiziana; Falsig, Jeppe; Nyström, Sofie; Hammarström, Per; Åslund, Andreas; Nilsson, K. Peter R.; Yam, Alice; Whitters, Eric; Hornemann, Simone; Aguzzi, Adriano

2012-01-01

Luminescent conjugated polymers (LCPs) interact with ordered protein aggregates and sensitively detect amyloids of many different proteins, suggesting that they may possess antiprion properties. Here, we show that a variety of anionic, cationic, and zwitterionic LCPs reduced the infectivity of prion-containing brain homogenates and of prion-infected cerebellar organotypic cultured slices and decreased the amount of scrapie isoform of PrPC (PrPSc) oligomers that could be captured in an avidity assay. Paradoxically, treatment enhanced the resistance of PrPSc to proteolysis, triggered the compaction, and enhanced the resistance to proteolysis of recombinant mouse PrP(23–231) fibers. These results suggest that LCPs act as antiprion agents by transitioning PrP aggregates into structures with reduced frangibility. Moreover, ELISA on cerebellar organotypic cultured slices and in vitro conversion assays with mouse PrP(23–231) indicated that poly(thiophene-3-acetic acid) may additionally interfere with the generation of PrPSc by stabilizing the conformation of PrPC or of a transition intermediate. Therefore, LCPs represent a novel class of antiprion agents whose mode of action appears to rely on hyperstabilization, rather than destabilization, of PrPSc deposits. PMID:22493452

Newer knowledge in comparative virology--its contribution to human health research.

PubMed

Cabasso, J J

1975-06-28

Like other comparative sciences, and despite its recent beginning comparative virology has already contributed useful applications and observations to human health research. Teachings derived from the study of Marek's disease found application in that of Burkitt's lymphoma, and may lead to a possible vaccine against the human disease. Equally useful information came from the study of canine distemper in the development of a chorio-allantoic membrane attenuated measles vaccine, and in our knowledge of subacute sclerosing panencephalitis (SSPE) of humans; from the study of reovirus-like agents of infant mice and neonatal calves in that of an acute nonbacterial gastro-enteritis of infants and young children; and from that of the cancer-producing viruses of chickens, cats, and dogs to a better understanding of some human neoplasias. Finally, Aleutian mink disease may be an excellent natural model for the study of the collagen diseases of man, and scrapie of sheep one for that of a human chronic degenerative disease of the central nervous system of humans such as Kuru. Comparative virology has proved quite productive in a relatively short period, and is unlikely to be neglected in the future.

Prion Diseases as Transmissible Zoonotic Diseases

PubMed Central

Lee, Jeongmin; Kim, Su Yeon; Hwang, Kyu Jam; Ju, Young Ran; Woo, Hee-Jong

2013-01-01

Prion diseases, also called transmissible spongiform encephalopathies (TSEs), lead to neurological dysfunction in animals and are fatal. Infectious prion proteins are causative agents of many mammalian TSEs, including scrapie (in sheep), chronic wasting disease (in deer and elk), bovine spongiform encephalopathy (BSE; in cattle), and Creutzfeldt–Jakob disease (CJD; in humans). BSE, better known as mad cow disease, is among the many recently discovered zoonotic diseases. BSE cases were first reported in the United Kingdom in 1986. Variant CJD (vCJD) is a disease that was first detected in 1996, which affects humans and is linked to the BSE epidemic in cattle. vCJD is presumed to be caused by consumption of contaminated meat and other food products derived from affected cattle. The BSE epidemic peaked in 1992 and decreased thereafter; this decline is continuing sharply owing to intensive surveillance and screening programs in the Western world. However, there are still new outbreaks and/or progression of prion diseases, including atypical BSE, and iatrogenic CJD and vCJD via organ transplantation and blood transfusion. This paper summarizes studies on prions, particularly on prion molecular mechanisms, BSE, vCJD, and diagnostic procedures. Risk perception and communication policies of the European Union for the prevention of prion diseases are also addressed to provide recommendations for appropriate government policies in Korea. PMID:24159531

The prion-ZIP connection: From cousins to partners in iron uptake

PubMed Central

Singh, Neena; Asthana, Abhishek; Baksi, Shounak; Desai, Vilok; Haldar, Swati; Hari, Sahi; Tripathi, Ajai K

2015-01-01

ABSTRACT Converging observations from disparate lines of inquiry are beginning to clarify the cause of brain iron dyshomeostasis in sporadic Creutzfeldt-Jakob disease (sCJD), a neurodegenerative condition associated with the conversion of prion protein (PrPC), a plasma membrane glycoprotein, from α-helical to a β-sheet rich PrP-scrapie (PrPSc) isoform. Biochemical evidence indicates that PrPC facilitates cellular iron uptake by functioning as a membrane-bound ferrireductase (FR), an activity necessary for the transport of iron across biological membranes through metal transporters. An entirely different experimental approach reveals an evolutionary link between PrPC and the Zrt, Irt-like protein (ZIP) family, a group of proteins involved in the transport of zinc, iron, and manganese across the plasma membrane. Close physical proximity of PrPC with certain members of the ZIP family on the plasma membrane and increased uptake of extracellular iron by cells that co-express PrPC and ZIP14 suggest that PrPC functions as a FR partner for certain members of this family. The connection between PrPC and ZIP proteins therefore extends beyond common ancestry to that of functional cooperation. Here, we summarize evidence supporting the facilitative role of PrPC in cellular iron uptake, and implications of this activity on iron metabolism in sCJD brains. PMID:26689487

Kinetics of Ozone Inactivation of Infectious Prion Protein

PubMed Central

Ding, Ning; Price, Luke M.; Braithwaite, Shannon L.; Balachandran, Aru; Mitchell, Gordon; Belosevic, Miodrag

2013-01-01

The kinetics of ozone inactivation of infectious prion protein (PrPSc, scrapie 263K) was investigated in ozone-demand-free phosphate-buffered saline (PBS). Diluted infectious brain homogenates (IBH) (0.01%) were exposed to a predetermined ozone dose (10.8 ± 2.0 mg/liter) at three pHs (pH 4.4, 6.0, and 8.0) and two temperatures (4°C and 20°C). The inactivation of PrPSc was quantified by determining the in vitro destruction of PrPSc templating properties using the protein misfolding cyclic amplification (PMCA) assay and bioassay, which were shown to correlate well. The inactivation kinetics were characterized by both Chick-Watson (CW) and efficiency factor Hom (EFH) models. It was found that the EFH model fit the experimental data more appropriately. The efficacy of ozone inactivation of PrPSc was both pH and temperature dependent. Based on the EFH model, CT (disinfectant concentration multiplied by contact time) values were determined for 2-log10, 3-log10, and 4-log10 inactivation at the conditions under which they were achieved. Our results indicated that ozone is effective for prion inactivation in ozone-demand-free water and may be applied for the inactivation of infectious prion in prion-contaminated water and wastewater. PMID:23416994

The evolution of internet-based map server applications in the United States Department of Agriculture, Veterinary Services.

PubMed

Maroney, Susan A; McCool, Mary Jane; Geter, Kenneth D; James, Angela M

2007-01-01

The internet is used increasingly as an effective means of disseminating information. For the past five years, the United States Department of Agriculture (USDA) Veterinary Services (VS) has published animal health information in internet-based map server applications, each oriented to a specific surveillance or outbreak response need. Using internet-based technology allows users to create dynamic, customised maps and perform basic spatial analysis without the need to buy or learn desktop geographic information systems (GIS) software. At the same time, access can be restricted to authorised users. The VS internet mapping applications to date are as follows: Equine Infectious Anemia Testing 1972-2005, National Tick Survey tick distribution maps, the Emergency Management Response System-Mapping Module for disease investigations and emergency outbreaks, and the Scrapie mapping module to assist with the control and eradication of this disease. These services were created using Environmental Systems Research Institute (ESRI)'s internet map server technology (ArcIMS). Other leading technologies for spatial data dissemination are ArcGIS Server, ArcEngine, and ArcWeb Services. VS is prototyping applications using these technologies, including the VS Atlas of Animal Health Information using ArcGIS Server technology and the Map Kiosk using ArcEngine for automating standard map production in the case of an emergency.

Mechanisms of triggering H1 helix in prion proteins unfolding revealed by molecular dynamic simulation

NASA Astrophysics Data System (ADS)

Tseng, Chih-Yuan; Lee, H. C.

2006-03-01

In template-assistance model, normal Prion protein (PrP^C), the pathogen to cause several prion diseases such as Creutzfeldt-Jakob (CJD) in human, Bovine Spongiform Encephalopathy (BSE) in cow, and scrapie in sheep, converts to infectious prion (PrP^Sc) through a transient interaction with PrP^Sc. Furthermore, conventional studies showed S1-H1-S2 region in PrP^C to be the template of S1-S2 β-sheet in PrP^Sc, and Prion protein's conformational conversion may involve an unfolding of H1 and refolding into β-sheet. Here we prepare several mouse prion peptides that contain S1-H1-S2 region with specific different structures, which are corresponding to specific interactions, to investigate possible mechanisms to trigger H1 α-helix unfolding process via molecular dynamic simulation. Three properties, conformational transition, salt-bridge in H1, and hydrophobic solvent accessible surface (SAS) are analyzed. From these studies, we found the interaction that triggers H1 unfolding to be the one that causes dihedral angle at residue Asn^143 changes. Whereas interactions that cause S1 segment's conformational changes play a minor in this process. These studies offers an additional evidence for template-assistance model.

Inhibition of protease-resistant prion protein formation by porphyrins and phthalocyanines

PubMed Central

Caughey, Winslow S.; Raymond, Lynne D.; Horiuchi, Motohiro; Caughey, Byron

1998-01-01

A central aspect of pathogenesis in the transmissible spongiform encephalopathies or prion diseases is the conversion of normal protease-sensitive prion protein (PrP-sen) to the abnormal protease-resistant form, PrP-res. Here we identify porphyrins and phthalocyanines as inhibitors of PrP-res accumulation. The most potent of these tetrapyrroles had IC50 values of 0.5–1 μM in scrapie-infected mouse neuroblastoma (ScNB) cell cultures. Inhibition was observed without effects on protein biosynthesis in general or PrP-sen biosynthesis in particular. Tetrapyrroles also inhibited PrP-res formation in a cell-free reaction composed predominantly of hamster PrP-res and PrP-sen. Inhibitors were found among phthalocyanines, deuteroporphyrins IX, and meso-substituted porphines; examples included compounds containing anionic, neutral protic, and cationic peripheral substituents and various metals. We conclude that certain tetrapyrroles specifically inhibit the conversion of PrP-sen to PrP-res without apparent cytotoxic effects. The inhibition observed in the cell-free conversion reaction suggests that the mechanism involved direct interactions of the tetrapyrrole with PrP-res and/or PrP-sen. These findings introduce a new class of inhibitors of PrP-res formation that represents a potential source of therapeutic agents for transmissible spongiform encephalopathies. PMID:9770449

Prion disease tempo determined by host-dependent substrate reduction

PubMed Central

Mays, Charles E.; Kim, Chae; Haldiman, Tracy; van der Merwe, Jacques; Lau, Agnes; Yang, Jing; Grams, Jennifer; Di Bari, Michele A.; Nonno, Romolo; Telling, Glenn C.; Kong, Qingzhong; Langeveld, Jan; McKenzie, Debbie; Westaway, David; Safar, Jiri G.

2014-01-01

The symptoms of prion infection can take years or decades to manifest following the initial exposure. Molecular markers of prion disease include accumulation of the misfolded prion protein (PrPSc), which is derived from its cellular precursor (PrPC), as well as downregulation of the PrP-like Shadoo (Sho) glycoprotein. Given the overlapping cellular environments for PrPC and Sho, we inferred that PrPC levels might also be altered as part of a host response during prion infection. Using rodent models, we found that, in addition to changes in PrPC glycosylation and proteolytic processing, net reductions in PrPC occur in a wide range of prion diseases, including sheep scrapie, human Creutzfeldt-Jakob disease, and cervid chronic wasting disease. The reduction in PrPC results in decreased prion replication, as measured by the protein misfolding cyclic amplification technique for generating PrPSc in vitro. While PrPC downregulation is not discernible in animals with unusually short incubation periods and high PrPC expression, slowly evolving prion infections exhibit downregulation of the PrPC substrate required for new PrPSc synthesis and as a receptor for pathogenic signaling. Our data reveal PrPC downregulation as a previously unappreciated element of disease pathogenesis that defines the extensive, presymptomatic period for many prion strains. PMID:24430187

Prions and prion diseases: fundamentals and mechanistic details.

PubMed

Ryou, Chongsuk

2007-07-01

Prion diseases, often called transmissible spongiform encephalopathies (TSEs), are infectious diseases that accompany neurological dysfunctions in many mammalian hosts. Prion diseases include Creutzfeldt-Jakob disease (CJD) in humans, bovine spongiform encephalopathy (BSE, "mad cow disease") in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and elks. The cause of these fatal diseases is a proteinaceous pathogen termed prion that lacks functional nucleic acids. As demonstrated in the BSE outbreak and its transmission to humans, the onset of disease is not limited to a certain species but can be transmissible from one host species to another. Such a striking nature ofprions has generated huge concerns in public health and attracted serious attention in the scientific communities. To date, the potential transmission ofprions to humans via foodbome infectiorn and iatrogenic routes has not been alleviated. Rather, the possible transmission of human to human or cervids to human aggravates the terrifying situation across the globe. In this review, basic features about prion diseases including clinical and pathological characteristics, etiology, and transmission of diseases are described. Based on recently accumulated evidences, the molecular and biochemical aspects of prions, with an emphasis on the molecular interactions involved in prion conversion that is critical during prion replication and pathogenesis, are also addressed.

Parkin Overexpression Ameliorates PrP106-126-Induced Neurotoxicity via Enhanced Autophagy in N2a Cells.

PubMed

Khan, Sher Hayat; Zhao, Deming; Shah, Syed Zahid Ali; Hassan, Mohammad Farooque; Zhu, Ting; Song, Zhiqi; Zhou, Xiangmei; Yang, Lifeng

2017-05-01

Transmissible spongiform encephalopathies (TSEs) are caused by the accumulation of the abnormal prion protein scrapie (PrP Sc ). Prion protein aggregation, misfolding, and cytotoxicity in the brain are the major causes of neuronal dysfunction and ultimate neurodegeneration in all TSEs. Parkin, an E3 ubiquitin ligase, has been studied extensively in all major protein misfolding aggregating diseases, especially Parkinson's disease and Alzheimer's disease, but the role of parkin in TSEs remains unknown. Here we investigated the role of parkin in a prion disease cell model in which neuroblastoma2a (N2a) cells were treated with prion peptide PrP106-126. We observed a gradual decrease in the soluble parkin level upon treatment with PrP106-126 in a time-dependent manner. Furthermore, endogenous parkin colocalized with FITC-tagged prion fragment106-126. Overexpression of parkin in N2a cells via transfection repressed apoptosis by enhancing autophagy. Parkin-overexpressing cells also showed reductions in apoptotic BAX translocation to the mitochondria and cytochrome c release to the cytosol, which ultimately inhibited activation of proapoptotic caspases. Taken together, our findings reveal a parkin-mediated cytoprotective mechanism against PrP106-126 toxicity, which is a novel potential therapeutic target for treating prion diseases.

Progress and problems in the biology, diagnostics, and therapeutics of prion diseases

PubMed Central

Aguzzi, Adriano; Heikenwalder, Mathias; Miele, Gino

2004-01-01

The term “prion” was introduced by Stanley Prusiner in 1982 to describe the atypical infectious agent that causes transmissible spongiform encephalopathies, a group of infectious neurodegenerative diseases that include scrapie in sheep, Creutzfeldt-Jakob disease in humans, chronic wasting disease in cervids, and bovine spongiform encephalopathy in cattle. Over the past twenty years, the word “prion” has been taken to signify various subtly different concepts. In this article, we refer to the prion as the transmissible principle underlying prion diseases, without necessarily implying any specific biochemical or structural identity. When Prusiner started his seminal work, the study of transmissible spongiform encephalopathies was undertaken by only a handful of scientists. Since that time, the “mad cow” crisis has put prion diseases on the agenda of both politicians and the media. Significant progress has been made in prion disease research, and many aspects of prion pathogenesis are now understood. And yet the diagnostic procedures available for prion diseases are not nearly as sensitive as they ought to be, and no therapeutic intervention has been shown to reliably affect the course of the diseases. This article reviews recent progress in the areas of pathogenesis of, diagnostics of, and therapy for prion diseases and highlights some conspicuous problems that remain to be addressed in each of these fields. PMID:15254579

Analysis of nucleic acid chaperoning by the prion protein and its inhibition by oligonucleotides.

PubMed

Guichard, Cécile; Ivanyi-Nagy, Roland; Sharma, Kamal Kant; Gabus, Caroline; Marc, Daniel; Mély, Yves; Darlix, Jean-Luc

2011-10-01

Prion diseases are unique neurodegenerative illnesses associated with the conversion of the cellular prion protein (PrP(C)) into the aggregated misfolded scrapie isoform, named PrP(Sc). Recent studies on the physiological role of PrP(C) revealed that this protein has probably multiple functions, notably in cell-cell adhesion and signal transduction, and in assisting nucleic acid folding. In fact, in vitro findings indicated that the human PrP (huPrP) possesses nucleic acid binding and annealing activities, similarly to nucleic acid chaperone proteins that play essential roles in cellular DNA and RNA metabolism. Here, we show that a peptide, representing the N-terminal domain of huPrP, facilitates nucleic acid annealing by two parallel pathways nucleated through the stem termini. We also show that PrP of human or ovine origin facilitates DNA strand exchange, ribozyme-directed cleavage of an RNA template and RNA trans-splicing in a manner similar to the nucleocapsid protein of HIV-1. In an attempt to characterize inhibitors of PrP-chaperoning in vitro we discovered that the thioaptamer 5'-GACACAAGCCGA-3' was extensively inhibiting the PrP chaperoning activities. At the same time a recently characterized methylated oligoribonucleotide inhibiting the chaperoning activity of the HIV-1 nucleocapsid protein was poorly impairing the PrP chaperoning activities.

Direct Detection of Soil-Bound Prions

PubMed Central

Genovesi, Sacha; Leita, Liviana; Sequi, Paolo; Andrighetto, Igino; Sorgato, M. Catia; Bertoli, Alessandro

2007-01-01

Scrapie and chronic wasting disease are contagious prion diseases affecting sheep and cervids, respectively. Studies have indicated that horizontal transmission is important in sustaining these epidemics, and that environmental contamination plays an important role in this. In the perspective of detecting prions in soil samples from the field by more direct methods than animal-based bioassays, we have developed a novel immuno-based approach that visualises in situ the major component (PrPSc) of prions sorbed onto agricultural soil particles. Importantly, the protocol needs no extraction of the protein from soil. Using a cell-based assay of infectivity, we also report that samples of agricultural soil, or quartz sand, acquire prion infectivity after exposure to whole brain homogenates from prion-infected mice. Our data provide further support to the notion that prion-exposed soils retain infectivity, as recently determined in Syrian hamsters intracerebrally or orally challanged with contaminated soils. The cell approach of the potential infectivity of contaminated soil is faster and cheaper than classical animal-based bioassays. Although it suffers from limitations, e.g. it can currently test only a few mouse prion strains, the cell model can nevertheless be applied in its present form to understand how soil composition influences infectivity, and to test prion-inactivating procedures. PMID:17957252

Alkaloidal glycosidase inhibitors (AGIs) as the cause of sporadic scrapie, and the potential treatment of both transmissible spongiform encephalopathies (TSEs) and human immunodeficiency virus (HIV) infection.

PubMed

Dealler, S

1994-02-01

AGIs are produced by plants and microorgansims in the environment. They are absorbed from the gut, distributed throughout the body and are concentrated inside cells. AGIs alter the glycan chains of cellular glycoproteins (CGP) during their formation so that the same CGP produced by different clones of cells (and hence with different glycan chains) becomes structurally the same. Prion protein (PrP), a CGP, is rendered indestructable to cellular mechanisms (as PrPi) by the TSE infective process; it is suggested that AGIs could both cause and prevent this by altering the primary structure of PrP. HIV envelope protein, gp120, carries glycan chains that are decided by the clone of the cells by which it is produced. Each cellular clone would be expected to add a specific group of glycan chains, making the gp120 antigenically separate. As HIV infection progresses, infected clone numbers rise, the antigenic diversity of gp120 may rise as would antibody production, trying to keep pace. Antigenically stimulated CD4+ cells carrying HIV genes, increase HIV production with gp120 antigenically different from its stimulant. AGIs prevent the glycan diversity and may prevent the extension of HIV infection.

Prion protein degradation by lichens of the genus Cladonia

USGS Publications Warehouse

Bennett, James P.; Rodriguez, Cynthia M.; Johnson, Christopher J.

2012-01-01

It has recently been discovered that lichens contain a serine protease capable of degrading the pathogenic prion protein, the etiological agent of prion diseases such as sheep scrapie and cervid chronic wasting disease. Limited methods are available to degrade or inactivate prion disease agents, especially in the environment, and lichens or their serine protease could prove important for management of these diseases. Scant information is available regarding the presence or absence of the protease responsible for degrading prion protein (PrP) in lichen species and, in this study, we tested the hypothesis that PrP degradation activity in lichens is phylogenetically-based by testing 44 species of Cladonia lichens, a genus for which a significant portion of the phylogeny is well established. We categorized PrP degradation activity among the 44 species (high, moderate, low or none) and found that activity in Cladonia species did not correspond with phylogenetic position of the species. Degradation of PrP did correspond, however, with three classical taxonomic characters within the genus: species with brown apothecia, no usnic acid, and the presence of a cortex. Of the 44 species studied, 18 (41%) had either high or moderate PrP degradation activity, suggesting the protease may be frequent in this genus of lichens.

Impact of strong selection for the PrP major gene on genetic variability of four French sheep breeds (Open Access publication)

PubMed Central

Palhiere, Isabelle; Brochard, Mickaël; Moazami-Goudarzi, Katayoun; Laloë, Denis; Amigues, Yves; Bed'hom, Bertrand; Neuts, Étienne; Leymarie, Cyril; Pantano, Thais; Cribiu, Edmond Paul; Bibé, Bernard; Verrier, Étienne

2008-01-01

Effective selection on the PrP gene has been implemented since October 2001 in all French sheep breeds. After four years, the ARR "resistant" allele frequency increased by about 35% in young males. The aim of this study was to evaluate the impact of this strong selection on genetic variability. It is focussed on four French sheep breeds and based on the comparison of two groups of 94 animals within each breed: the first group of animals was born before the selection began, and the second, 3–4 years later. Genetic variability was assessed using genealogical and molecular data (29 microsatellite markers). The expected loss of genetic variability on the PrP gene was confirmed. Moreover, among the five markers located in the PrP region, only the three closest ones were affected. The evolution of the number of alleles, heterozygote deficiency within population, expected heterozygosity and the Reynolds distances agreed with the criteria from pedigree and pointed out that neutral genetic variability was not much affected. This trend depended on breed, i.e. on their initial states (population size, PrP frequencies) and on the selection strategies for improving scrapie resistance while carrying out selection for production traits. PMID:18990357

Lichens: Unexpected anti-prion agents?

USGS Publications Warehouse

Rodriguez, Cynthia M.; Bennett, James P.; Johnson, Christopher J.

2012-01-01

The prion diseases sheep scrapie and cervid chronic wasting disease are transmitted, in part, via an environmental reservoir of infectivity; prions released from infected animals persist in the environment and can cause disease years later. Central to controlling disease transmission is the identification of methods capable of inactivating these agents on the landscape. We have found that certain lichens, common, ubiquitous, symbiotic organisms, possess a serine protease capable of degrading prion protein (PrP) from prion-infected animals. The protease functions against a range of prion strains from various hosts and reduces levels of abnormal PrP by at least two logs. We have now tested more than 20 lichen species from several geographical locations and from various taxa and found that approximately half of these species degrade PrP. Critical next steps include examining the effect of lichens on prion infectivity and cloning the protease responsible for PrP degradation. The impact of lichens on prions in the environment remains unknown. We speculate that lichens could have the potential to degrade prions when they are shed from infected animals onto lichens or into environments where lichens are abundant. In addition, lichens are frequently consumed by cervids and many other animals and the effect of dietary lichens on prion disease transmission should also be considered.

Lions and prions and deer demise.

PubMed

Miller, Michael W; Swanson, Heather M; Wolfe, Lisa L; Quartarone, Fred G; Huwer, Sherri L; Southwick, Charles H; Lukacs, Paul M

2008-01-01

Contagious prion diseases--scrapie of sheep and chronic wasting disease of several species in the deer family--give rise to epidemics that seem capable of compromising host population viability. Despite this prospect, the ecological consequences of prion disease epidemics in natural populations have received little consideration. Using a cohort study design, we found that prion infection dramatically lowered survival of free-ranging adult (>2-year-old) mule deer (Odocoileus hemionus): estimated average life expectancy was 5.2 additional years for uninfected deer but only 1.6 additional years for infected deer. Prion infection also increased nearly fourfold the rate of mountain lions (Puma concolor) preying on deer, suggesting that epidemics may alter predator-prey dynamics by facilitating hunting success. Despite selective predation, about one fourth of the adult deer we sampled were infected. High prevalence and low survival of infected deer provided a plausible explanation for the marked decline in this deer population since the 1980s. Remarkably high infection rates sustained in the face of intense predation show that even seemingly complete ecosystems may offer little resistance to the spread and persistence of contagious prion diseases. Moreover, the depression of infected populations may lead to local imbalances in food webs and nutrient cycling in ecosystems in which deer are important herbivores.

Sod1 deficiency reduces incubation time in mouse models of prion disease.

PubMed

Akhtar, Shaheen; Grizenkova, Julia; Wenborn, Adam; Hummerich, Holger; Fernandez de Marco, Mar; Brandner, Sebastian; Collinge, John; Lloyd, Sarah E

2013-01-01

Prion infections, causing neurodegenerative conditions such as Creutzfeldt-Jakob disease and kuru in humans, scrapie in sheep and BSE in cattle are characterised by prolonged and variable incubation periods that are faithfully reproduced in mouse models. Incubation time is partly determined by genetic factors including polymorphisms in the prion protein gene. Quantitative trait loci studies in mice and human genome-wide association studies have confirmed that multiple genes are involved. Candidate gene approaches have also been used and identified App, Il1-r1 and Sod1 as affecting incubation times. In this study we looked for an association between App, Il1-r1 and Sod1 representative SNPs and prion disease incubation time in the Northport heterogeneous stock of mice inoculated with the Chandler/RML prion strain. No association was seen with App, however, significant associations were seen with Il1-r1 (P = 0.02) and Sod1 (P<0.0001) suggesting that polymorphisms at these loci contribute to the natural variation observed in incubation time. Furthermore, following challenge with Chandler/RML, ME7 and MRC2 prion strains, Sod1 deficient mice showed highly significant reductions in incubation time of 20, 13 and 24%, respectively. No differences were detected in Sod1 expression or activity. Our data confirm the protective role of endogenous Sod1 in prion disease.

Enzymatic Digestion of Chronic Wasting Disease Prions Bound to Soil

PubMed Central

SAUNDERS, SAMUEL E.; BARTZ, JASON C.; VERCAUTEREN, KURT C.; BARTELT-HUNT, SHANNON L.

2010-01-01

Chronic wasting disease (CWD) and sheep scrapie can be transmitted via indirect environmental routes, and it is known that soil can serve as a reservoir of prion infectivity. Given the strong interaction between the prion protein (PrP) and soil, we hypothesized that binding to soil enhances prion resistance to enzymatic digestion, thereby facilitating prion longevity in the environment and providing protection from host degradation. We characterized the performance of a commercially available subtilisin enzyme, the Prionzyme, to degrade soil-bound and unbound CWD and HY TME PrP as a function of pH, temperature, and treatment time. The subtilisin enzyme effectively degraded PrP adsorbed to a wide range of soils and soil minerals below the limits of detection. Signal loss occurred rapidly at high pH (12.5) and within 7 d under conditions representative of the natural environment (pH 7.4, 22°C). We observed no apparent difference in enzyme effectiveness between bound and unbound CWD PrP. Our results show that although adsorbed prions do retain relative resistance to enzymatic digestion compared with other brain homogenate proteins, they can be effectively degraded when bound to soil. Our results also suggest a topical application of a subtilisin enzyme solution may be an effective decontamination method to limit disease transmission via environmental ‘hot spots’ of prion infectivity. PMID:20450190

Sulforaphane-induced autophagy flux prevents prion protein-mediated neurotoxicity through AMPK pathway.

PubMed

Lee, J-H; Jeong, J-K; Park, S-Y

2014-10-10

Prion diseases are neurodegenerative and infectious disorders that involve accumulation of misfolded scrapie prion protein, and which are characterized by spongiform degeneration. Autophagy, a major homeostatic process responsible for the degradation of cytoplasmic components, has garnered attention as the potential target for neurodegenerative diseases such as prion disease. We focused on protective effects of sulforaphane found in cruciferous vegetables on prion-mediated neurotoxicity and the mechanism of sulforaphane related to autophagy. In human neuroblastoma cells, sulforaphane protected prion protein (PrP) (106-126)-mediated neurotoxicity and increased autophagy flux marker microtubule-associated protein 1 light chain 3-II protein levels, following a decrease of p62 protein level. Pharmacological and genetical inhibition of autophagy by 3MA, wortmannin and knockdown of autophagy-related 5 (ATG5) led to block the effect of sulforaphane against PrP (106-126)-induced neurotoxicity. Furthermore we demonstrated that both sulforaphane-induced autophagy and protective effect of sulforaphane against PrP (106-126)-induced neurotoxicity are dependent on the AMP-activated protein kinase (AMPK) signaling. The present results indicated that sulforaphane of cruciferous vegetables enhanced autophagy flux led to the protection effects against prion-mediated neurotoxicity, which was regulated by AMPK signaling pathways in human neuron cells. Our data also suggest that sulforaphane has a potential value as a therapeutic tool in neurodegenerative disease including prion diseases. Copyright © 2014 IBRO. Published by Elsevier Ltd. All rights reserved.

Semi-purification procedures of prions from a prion-infected brain using sucrose has no influence on the nonenzymatic glycation of the disease-associated prion isoform.

PubMed

Choi, Yeong-Gon; Kim, Jae-Il; Choi, Eun-Kyoung; Carp, Richard I; Kim, Yong-Sun

2016-01-01

Previous studies have shown that the Nε-carboxymethyl group is linked to not only one or more N-terminal Lys residues but also to one or more Lys residues of the protease-resistant core region of the pathogenic prion isoform (PrPSc) in prion-infected brains. Using an anti-advanced glycation end product (AGE) antibody, we detected nonenzymatically glycated PrPSc (AGE-PrPSc) in prion-infected brains following concentration by a series of ultracentrifugation steps with a sucrose cushion. In the present study, the levels of in vitro nonenzymatic glycation of PrPSc using sucrose were investigated to determine whether sucrose cushion can artificially and nonenzymatically induce in vitro glycation during ultracentrifugation. The first insoluble pellet fraction following the first ultracentrifugation (PU1st) collected from 263K scrapie-infected brains was incubated with sucrose, glucose or colloidal silica coated with polyvinylpyrrolidone (percoll). None of the compounds in vitro resulted in AGE-PrPSc. Nonetheless, glucose and percoll produced AGEs in vitro from other proteins within PU1st of the infected brains. This reaction could lead to the AGE-modified polymer(s) of nonenzymatic glycation-prone protein(s). This study showed that PrPSc is not nonenzymatically glycated in vitro with sucrose, glucose or percoll and that AGE-modified PrPSc can be isolated and enriched from prion-infected brains.

Bank Vole Prion Protein As an Apparently Universal Substrate for RT-QuIC-Based Detection and Discrimination of Prion Strains.

PubMed

Orrú, Christina D; Groveman, Bradley R; Raymond, Lynne D; Hughson, Andrew G; Nonno, Romolo; Zou, Wenquan; Ghetti, Bernardino; Gambetti, Pierluigi; Caughey, Byron

2015-06-01

Prions propagate as multiple strains in a wide variety of mammalian species. The detection of all such strains by a single ultrasensitive assay such as Real Time Quaking-induced Conversion (RT-QuIC) would facilitate prion disease diagnosis, surveillance and research. Previous studies have shown that bank voles, and transgenic mice expressing bank vole prion protein, are susceptible to most, if not all, types of prions. Here we show that bacterially expressed recombinant bank vole prion protein (residues 23-230) is an effective substrate for the sensitive RT-QuIC detection of all of the different prion types that we have tested so far--a total of 28 from humans, cattle, sheep, cervids and rodents, including several that have previously been undetectable by RT-QuIC or Protein Misfolding Cyclic Amplification. Furthermore, comparison of the relative abilities of different prions to seed positive RT-QuIC reactions with bank vole and not other recombinant prion proteins allowed discrimination of prion strains such as classical and atypical L-type bovine spongiform encephalopathy, classical and atypical Nor98 scrapie in sheep, and sporadic and variant Creutzfeldt-Jakob disease in humans. Comparison of protease-resistant RT-QuIC conversion products also aided strain discrimination and suggested the existence of several distinct classes of prion templates among the many strains tested.

Prion protein β2–α2 loop conformational landscape

PubMed Central

Caldarulo, Enrico; Wüthrich, Kurt; Parrinello, Michele

2017-01-01

In transmissible spongiform encephalopathies (TSEs), which are lethal neurodegenerative diseases that affect humans and a wide range of other mammalian species, the normal “cellular” prion protein (PrPC) is transformed into amyloid aggregates representing the “scrapie form” of the protein (PrPSc). Continued research on this system is of keen interest, since new information on the physiological function of PrPC in healthy organisms is emerging, as well as new data on the mechanism of the transformation of PrPC to PrPSc. In this paper we used two different approaches: a combination of the well-tempered ensemble (WTE) and parallel tempering (PT) schemes and metadynamics (MetaD) to characterize the conformational free-energy surface of PrPC. The focus of the data analysis was on an 11-residue polypeptide segment in mouse PrPC(121–231) that includes the β2–α2 loop of residues 167–170, for which a correlation between structure and susceptibility to prion disease has previously been described. This study includes wild-type mouse PrPC and a variant with the single-residue replacement Y169A. The resulting detailed conformational landscapes complement in an integrative manner the available experimental data on PrPC, providing quantitative insights into the nature of the structural transition-related function of the β2–α2 loop. PMID:28827331

Prion protein β2-α2 loop conformational landscape.

PubMed

Caldarulo, Enrico; Barducci, Alessandro; Wüthrich, Kurt; Parrinello, Michele

2017-09-05

In transmissible spongiform encephalopathies (TSEs), which are lethal neurodegenerative diseases that affect humans and a wide range of other mammalian species, the normal "cellular" prion protein ([Formula: see text]) is transformed into amyloid aggregates representing the "scrapie form" of the protein ([Formula: see text]). Continued research on this system is of keen interest, since new information on the physiological function of [Formula: see text] in healthy organisms is emerging, as well as new data on the mechanism of the transformation of [Formula: see text] to [Formula: see text] In this paper we used two different approaches: a combination of the well-tempered ensemble (WTE) and parallel tempering (PT) schemes and metadynamics (MetaD) to characterize the conformational free-energy surface of [Formula: see text] The focus of the data analysis was on an 11-residue polypeptide segment in mouse [Formula: see text](121-231) that includes the [Formula: see text]2-[Formula: see text]2 loop of residues 167-170, for which a correlation between structure and susceptibility to prion disease has previously been described. This study includes wild-type mouse [Formula: see text] and a variant with the single-residue replacement Y169A. The resulting detailed conformational landscapes complement in an integrative manner the available experimental data on [Formula: see text], providing quantitative insights into the nature of the structural transition-related function of the [Formula: see text]2-[Formula: see text]2 loop.

Pathogenesis of bovine spongiform encephalopathy in sheep.

PubMed

van Keulen, L J M; Vromans, M E W; Dolstra, C H; Bossers, A; van Zijderveld, F G

2008-01-01

The pathogenesis of bovine spongiform encephalopathy (BSE) in sheep was studied by immunohistochemical detection of scrapie-associated prion protein (PrP(Sc)) in the gastrointestinal, lymphoid and neural tissues following oral inoculation with BSE brain homogenate. First accumulation of PrP(Sc) was detected after 6 months in the tonsil and the ileal Peyer's patches. At 9 months postinfection, PrP(Sc) accumulation involved all gut-associated lymphoid tissues and lymph nodes as well as the spleen. At this time point, PrP(Sc) accumulation in the peripheral neural tissues was first seen in the enteric nervous system of the caudal jejunum and ileum and in the coeliac-mesenteric ganglion. In the central nervous system, PrP(Sc) was first detected in the dorsal motor nucleus of the nervus Vagus in the medulla oblongata and in the intermediolateral column in the spinal cord segments T7-L1. At subsequent time points, PrP(Sc) was seen to spread within the lymphoid system to also involve all non-gut-associated lymphoid tissues. In the enteric nervous system, further spread of PrP(Sc) involved the neural plexi along the entire gastrointestinal tract and in the CNS the complete neuraxis. These findings indicate a spread of the BSE agent in sheep from the enteric nervous system through parasympathetic and sympathetic nerves to the medulla oblongata and the spinal cord.

Pathogenesis of bovine spongiform encephalopathy in sheep

PubMed Central

Vromans, M. E. W.; Dolstra, C. H.; Bossers, A.; van Zijderveld, F. G.

2007-01-01

The pathogenesis of bovine spongiform encephalopathy (BSE) in sheep was studied by immunohistochemical detection of scrapie-associated prion protein (PrPSc) in the gastrointestinal, lymphoid and neural tissues following oral inoculation with BSE brain homogenate. First accumulation of PrPSc was detected after 6 months in the tonsil and the ileal Peyer’s patches. At 9 months postinfection, PrPSc accumulation involved all gut-associated lymphoid tissues and lymph nodes as well as the spleen. At this time point, PrPSc accumulation in the peripheral neural tissues was first seen in the enteric nervous system of the caudal jejunum and ileum and in the coeliac-mesenteric ganglion. In the central nervous system, PrPSc was first detected in the dorsal motor nucleus of the nervus Vagus in the medulla oblongata and in the intermediolateral column in the spinal cord segments T7–L1. At subsequent time points, PrPSc was seen to spread within the lymphoid system to also involve all non-gut-associated lymphoid tissues. In the enteric nervous system, further spread of PrPSc involved the neural plexi along the entire gastrointestinal tract and in the CNS the complete neuraxis. These findings indicate a spread of the BSE agent in sheep from the enteric nervous system through parasympathetic and sympathetic nerves to the medulla oblongata and the spinal cord. PMID:18092124

Decision Support Tools for Clinical Diagnosis of Disease in Cows with Suspected Bovine Spongiform Encephalopathy

PubMed Central

Saegerman, C.; Speybroeck, N.; Roels, S.; Vanopdenbosch, E.; Thiry, E.; Berkvens, D.

2004-01-01

Reporting of clinically suspected cattle is currently the most common method for detecting cases of bovine spongiform encephalopathy (BSE). Improvement of clinical diagnosis and decision-making remains crucial. A comparison of clinical patterns, consisting of 25 signs, was made between all 30 BSE cases, confirmed in Belgium before October 2002, and 272 suspected cases that were subsequently determined to be histologically, immunohistochemically, and scrapie-associated-fiber negative. Seasonality in reporting suspected cases was observed, with more cases being reported during wintertime when animals were kept indoors. The median duration of illness was 30 days. The 10 most relevant signs of BSE were kicking in the milking parlor, hypersensitivity to touch and/or sound, head shyness, panic-stricken response, reluctance to enter in the milking parlor, abnormal ear movement or carriage, increased alertness behavior, reduced milk yield, teeth grinding, and temperament change. Ataxia did not appear to be a specific sign of BSE. A classification and regression tree was constructed by using the following four features: age of the animal, year of birth, number of relevant BSE signs noted, and number of clinical signs, typical for listeriosis, noted. The model had a sensitivity of 100% and a specificity of 85%. This approach allows the use of an interactive decision-support tool, based entirely on odds ratios, a statistic independent of disease prevalence. PMID:14715749

Analysis of nucleic acid chaperoning by the prion protein and its inhibition by oligonucleotides

PubMed Central

Guichard, Cécile; Ivanyi-Nagy, Roland; Sharma, Kamal Kant; Gabus, Caroline; Marc, Daniel; Mély, Yves; Darlix, Jean-Luc

2011-01-01

Prion diseases are unique neurodegenerative illnesses associated with the conversion of the cellular prion protein (PrPC) into the aggregated misfolded scrapie isoform, named PrPSc. Recent studies on the physiological role of PrPC revealed that this protein has probably multiple functions, notably in cell–cell adhesion and signal transduction, and in assisting nucleic acid folding. In fact, in vitro findings indicated that the human PrP (huPrP) possesses nucleic acid binding and annealing activities, similarly to nucleic acid chaperone proteins that play essential roles in cellular DNA and RNA metabolism. Here, we show that a peptide, representing the N-terminal domain of huPrP, facilitates nucleic acid annealing by two parallel pathways nucleated through the stem termini. We also show that PrP of human or ovine origin facilitates DNA strand exchange, ribozyme-directed cleavage of an RNA template and RNA trans-splicing in a manner similar to the nucleocapsid protein of HIV-1. In an attempt to characterize inhibitors of PrP-chaperoning in vitro we discovered that the thioaptamer 5′-GACACAAGCCGA-3′ was extensively inhibiting the PrP chaperoning activities. At the same time a recently characterized methylated oligoribonucleotide inhibiting the chaperoning activity of the HIV-1 nucleocapsid protein was poorly impairing the PrP chaperoning activities. PMID:21737432

Animal breeding and disease

PubMed Central

Nicholas, Frank W

2005-01-01

Single-locus disorders in domesticated animals were among the first Mendelian traits to be documented after the rediscovery of Mendelism, and to be included in early linkage maps. The use of linkage maps and (increasingly) comparative genomics has been central to the identification of the causative gene for single-locus disorders of considerable practical importance. The ‘score-card’ in domestic animals is now more than 100 disorders for which the molecular lesion has been identified and hence for which a DNA test is available. Because of the limited lifespan of any such test, a cost-effective and hence popular means of protecting the intellectual property inherent in a DNA test is not to publish the discovery. While understandable, this practice creates a disconcerting precedent. For multifactorial disorders that are scored on an all-or-none basis or into many classes, the effectiveness of control schemes could be greatly enhanced by selection on estimated breeding values for liability. Genetic variation for resistance to pathogens and parasites is ubiquitous. Selection for resistance can therefore be successful. Because of the technical and welfare challenges inherent in the requirement to expose animals to pathogens or parasites in order to be able to select for resistance, there is a very active search for DNA markers for resistance. The first practical fruits of this research were seen in 2002, with the launch of a national scrapie control programme in the UK. PMID:16048793

Serial transmission in rodents of neurodegeneration from transgenic mice expressing mutant prion protein.

PubMed Central

Hsiao, K K; Groth, D; Scott, M; Yang, S L; Serban, H; Rapp, D; Foster, D; Torchia, M; Dearmond, S J; Prusiner, S B

1994-01-01

Two lines of transgenic (Tg) mice expressing high (H) levels of the mutant P101L prion protein (PrP) developed a neurologic illness and central nervous system pathology indistinguishable from experimental murine scrapie; these mice were designated Tg(MoPrP-P101L)H. Brain homogenates from Tg(MoPrP-P101L)H mice were inoculated intracerebrally into CD-1 Swiss mice, Syrian hamsters, and Tg196 mice, Tg mice expressing the MoPrP-P101L transgene at low levels. None of the CD-1 mice developed central nervous system dysfunction, whereas approximately 10% of hamsters and approximately 40% of the Tg196 mice manifested neurologic signs between 117 and 639 days after inoculation. Serial transmission of neurodegeneration in Tg196 mice and Syrian hamsters was initiated with brain extracts, producing incubation times of approximately 400 and approximately 75 days, respectively. Although the Tg(MoPrP-P101L)H mice appear to accumulate only low levels of infections prions in their brains, the serial transmission of disease to inoculated recipients argues that prion formation occurs de novo in the brains of these uninoculated animals. These Tg mouse studies, taken together with similar findings in humans dying of inherited prion diseases, provide additional evidence that prions lack a foreign nucleic acid. Images PMID:7916462

[Still a small problem with the mad cow disease? Creutzfeldt-Jakob disease and other prion diseases: current status].

PubMed

Lundberg, P O

2001-01-10

This review is based on recent published research on the BSE/CJD/vCJD problem mainly from UK, Germany and France. The situation in Sweden seems to be fortunate for several reasons. The use of meat and bonemeal as animal fodder was forbidden in this country 13 years ago. Sweden has not had any sheep with scrapie for many years. No animals with BSE have so far been found in our country. The incidence of sporadic CJD in this country followed retrospectively from 1985 to 1996 and prospectively from 1997 to 1999 has been around 1.2 per million per year with no significant increase. Only few cases of familial CJD are known. No patient with iatrogenic CJD has ever been found. The use of growth hormone derived from human pituitary glands was abandoned in 1985 when recombinant human growth hormone became available. So far there is no indication that any of the CJD cases diagnosed in Sweden has been of the vCJD type, the one linked to BSE. However, as the incubation period for prion diseases is very long and the Swedes are frequent travellers there is a risk that people from our country could have contracted vCJD through consuming meat products in countries with BSE. As a precaution the consumption of brain, spinal cord, lymphatic tissue, lungs, and gastrointestinal tract should be avoided. Human pituitary derived growth hormone is still available in some countries and might be illegally imported into Sweden.

Mouse scrapie responsive gene 1 (Scrg1): genomic organization, physical linkage to sap30, genetic mapping on chromosome 8, and expression in neuronal primary cell cultures.

PubMed

Dron, M; Tartare, X; Guillo, F; Haik, S; Barbin, G; Maury, C; Tovey, M; Dandoy-Dron, F

2000-11-15

We have previously reported a transcript of a novel mouse gene (Scrg1) with increased expression in transmissible spongiform encephalopathies and the cloning of the human mRNA analogue. In this paper, we present the genomic organization of the mouse and human SCRG1 loci, which exhibit a high degree of conservation. The genes are composed of three exons; the two downstream exons contain the protein coding region. The mouse gene is expressed in brain tissue essentially as a 0.7-kb message but also as a minor 2.6-kb mRNA. We have sequenced 20 kb of DNA at the mouse Scrg1 locus and found that the longer transcript is the prolongation of the 0.7-kb mRNA to a polyadenylation site located about 2 kb further downstream. Sequencing revealed that the mouse Scrg1 gene is physically linked to Sap30, a gene that encodes a protein of the histone deacetylase complex, and genetic linkage mapping assigned the localization of Scrg1 to chromosome 8 between Ant1 and Hmg2. Northern blot analysis showed that Scrg1 is under strict developmental control in mouse embryo and is expressed by cells of neuronal origin in vitro. Comparison of the rat, mouse, and human SCRG1 proteins identified a box of 35 identical contiguous amino acids and a characteristic cysteine distribution pattern defining a new protein signature. Copyright 2000 Academic Press.

Inactivation of Template-Directed Misfolding of Infectious Prion Protein by Ozone

PubMed Central

Ding, Ning; Price, Luke M.; Braithwaite, Shannon L.; Balachandran, Aru; Belosevic, Miodrag

2012-01-01

Misfolded prions (PrPSc) are well known for their resistance to conventional decontamination processes. The potential risk of contamination of the water environment, as a result of disposal of specified risk materials (SRM), has raised public concerns. Ozone is commonly utilized in the water industry for inactivation of microbial contaminants and was tested in this study for its ability to inactivate prions (263K hamster scrapie = PrPSc). Treatment variables included initial ozone dose (7.6 to 25.7 mg/liter), contact time (5 s and 5 min), temperature (4°C and 20°C), and pH (pH 4.4, 6.0, and 8.0). Exposure of dilute suspensions of the infected 263K hamster brain homogenates (IBH) (0.01%) to ozone resulted in the in vitro destruction of the templating properties of PrPSc, as measured by the protein misfolding cyclic amplification (PMCA) assay. The highest levels of prion inactivation (≥4 log10) were observed with ozone doses of 13.0 mg/liter, at pH 4.4 and 20°C, resulting in a CT (the product of residual ozone concentration and contact time) value as low as 0.59 mg · liter−1 min. A comparison of ozone CT requirements among various pathogens suggests that prions are more susceptible to ozone degradation than some model bacteria and protozoa and that ozone treatment may be an effective solution for inactivating prions in water and wastewater. PMID:22138993

Plasminogen stimulates propagation of protease-resistant prion protein in vitro.

PubMed

Mays, Charles E; Ryou, Chongsuk

2010-12-01

To clarify the role of plasminogen as a cofactor for prion propagation, we conducted functional assays using a cell-free prion protein (PrP) conversion assay termed protein misfolding cyclic amplification (PMCA) and prion-infected cell lines. Here, we report that plasminogen stimulates propagation of the protease-resistant scrapie PrP (PrP(Sc)). Compared to control PMCA conducted without plasminogen, addition of plasminogen in PMCA using wild-type brain material significantly increased PrP conversion, with an EC(50) = ∼56 nM. PrP conversion in PMCA was substantially less efficient with plasminogen-deficient brain material than with wild-type material. The activity stimulating PrP conversion was specific for plasminogen and conserved in its kringle domains. Such activity was abrogated by modification of plasminogen structure and interference of PrP-plasminogen interaction. Kinetic analysis of PrP(Sc) generation demonstrated that the presence of plasminogen in PMCA enhanced the PrP(Sc) production rate to ∼0.97 U/μl/h and reduced turnover time to ∼1 h compared to those (∼0.4 U/μl/h and ∼2.5 h) obtained without supplementation. Furthermore, as observed in PMCA, plasminogen and kringles promoted PrP(Sc) propagation in ScN2a and Elk 21(+) cells. Our results demonstrate that plasminogen functions in stimulating conversion processes and represents the first cellular protein cofactor that enhances the hypothetical mechanism of prion propagation.

Overexpression of the Hspa13 (Stch) gene reduces prion disease incubation time in mice.

PubMed

Grizenkova, Julia; Akhtar, Shaheen; Hummerich, Holger; Tomlinson, Andrew; Asante, Emmanuel A; Wenborn, Adam; Fizet, Jérémie; Poulter, Mark; Wiseman, Frances K; Fisher, Elizabeth M C; Tybulewicz, Victor L J; Brandner, Sebastian; Collinge, John; Lloyd, Sarah E

2012-08-21

Prion diseases are fatal neurodegenerative disorders that include bovine spongiform encephalopathy (BSE) and scrapie in animals and Creutzfeldt-Jakob disease (CJD) in humans. They are characterized by long incubation periods, variation in which is determined by many factors including genetic background. In some cases it is possible that incubation time may be directly correlated to the level of gene expression. To test this hypothesis, we combined incubation time data from five different inbred lines of mice with quantitative gene expression profiling in normal brains and identified five genes with expression levels that correlate with incubation time. One of these genes, Hspa13 (Stch), is a member of the Hsp70 family of ATPase heat shock proteins, which have been previously implicated in prion propagation. To test whether Hspa13 plays a causal role in determining the incubation period, we tested two overexpressing mouse models. The Tc1 human chromosome 21 (Hsa21) transchromosomic mouse model of Down syndrome is trisomic for many Hsa21 genes including Hspa13 and following Chandler/Rocky Mountain Laboratory (RML) prion inoculation, shows a 4% reduction in incubation time. Furthermore, a transgenic model with eightfold overexpression of mouse Hspa13 exhibited highly significant reductions in incubation time of 16, 15, and 7% following infection with Chandler/RML, ME7, and MRC2 prion strains, respectively. These data further implicate Hsp70-like molecular chaperones in protein misfolding disorders such as prion disease.

Biological and biochemical characterization of mice expressing prion protein devoid of the octapeptide repeat region after infection with prions.

PubMed

Yamaguchi, Yoshitaka; Miyata, Hironori; Uchiyama, Keiji; Ootsuyama, Akira; Inubushi, Sachiko; Mori, Tsuyoshi; Muramatsu, Naomi; Katamine, Shigeru; Sakaguchi, Suehiro

2012-01-01

Accumulating lines of evidence indicate that the N-terminal domain of prion protein (PrP) is involved in prion susceptibility in mice. In this study, to investigate the role of the octapeptide repeat (OR) region alone in the N-terminal domain for the susceptibility and pathogenesis of prion disease, we intracerebrally inoculated RML scrapie prions into tg(PrPΔOR)/Prnp(0/0) mice, which express mouse PrP missing only the OR region on the PrP-null background. Incubation times of these mice were not extended. Protease-resistant PrPΔOR, or PrP(Sc)ΔOR, was easily detectable but lower in the brains of these mice, compared to that in control wild-type mice. Consistently, prion titers were slightly lower and astrogliosis was milder in their brains. However, in their spinal cords, PrP(Sc)ΔOR and prion titers were abundant and astrogliosis was as strong as in control wild-type mice. These results indicate that the role of the OR region in prion susceptibility and pathogenesis of the disease is limited. We also found that the PrP(Sc)ΔOR, including the pre-OR residues 23-50, was unusually protease-resistant, indicating that deletion of the OR region could cause structural changes to the pre-OR region upon prion infection, leading to formation of a protease-resistant structure for the pre-OR region.

Nerve Growth Factor Increases mRNA Levels for the Prion Protein and the β -amyloid Protein Precursor in Developing Hamster Brain

NASA Astrophysics Data System (ADS)

Mobley, William C.; Neve, Rachael L.; Prusiner, Stanley B.; McKinley, Michael P.

1988-12-01

Deposition of amyloid filaments serves as a pathologic hallmark for some neurodegenerative disorders. The prion protein (PrP) is found in amyloid of animals with scrapie and humans with Creutzfeldt-Jakob disease; the β protein is present in amyloid deposits in Alzheimer disease and Down syndrome patients. These two proteins are derived from precursors that in the brain are expressed primarily in neurons and are membrane bound. We found that gene expression for PrP and the β -protein precursor (β -PP) is regulated in developing hamster brain. Specific brain regions showed distinct patterns of ontogenesis for PrP and β -PP mRNAs. The increases in PrP and β -PP mRNAs in developing basal forebrain coincided with an increase in choline acetyltransferase activity, raising the possibility that these markers might be coordinately controlled in cholinergic neurons and regulated by nerve growth factor (NGF). Injections of NGF into the brains of neonatal hamsters increased both PrP and β -PP mRNA levels. Increased PrP and β -PP mRNA levels induced by NGF were confined to regions that contain NGF-responsive cholinergic neurons and were accompanied by elevations in choline acetyltransferase. It remains to be established whether or not exogenous NGF acts to increase PrP and β -PP gene expression selectively in forebrain cholinergic neurons in the developing hamster and endogenous NGF regulates expression of these genes.

Instability of buried hydration sites increases protein subdomains fluctuations in the human prion protein by the pathogenic mutation T188R

NASA Astrophysics Data System (ADS)

Tomobe, Katsufumi; Yamamoto, Eiji; Akimoto, Takuma; Yasui, Masato; Yasuoka, Kenji

2016-05-01

The conformational change from the cellular prion protein (PrPc) to scrapie prion protein (PrPsc) is a key process in prion diseases. The prion protein has buried water molecules which significantly contribute to the stability of the protein; however, there has been no report investigating the influence on the buried hydration sites by a pathogenic mutation not adjacent to the buried hydration sites. Here, we perform molecular dynamics simulations of wild type (WT) PrPc and pathogenic point mutant T188R to investigate conformational changes and the buried hydration sites. In WT-PrPc, four buried hydration sites are identified by residence time and rotational relaxation analysis. However, there are no stable buried hydration sites in one of T188R simulations, which indicates that T188R sometimes makes the buried hydration sites fragile. We also find that fluctuations of subdomains S1-H1-S2 and H1-H2 increase in T188R when the buried hydration sites become unstable. Since the side chain of arginine which is replaced from threonine in T188R is larger than of threonine, the side chain cannot be embedded in the protein, which is one of the causes of the instability of subdomains. These results show correlations between the buried hydration sites and the mutation which is far from them, and provide a possible explanation for the instability by mutation.

The prion protein has RNA binding and chaperoning properties characteristic of nucleocapsid protein NCP7 of HIV-1.

PubMed

Gabus, C; Derrington, E; Leblanc, P; Chnaiderman, J; Dormont, D; Swietnicki, W; Morillas, M; Surewicz, W K; Marc, D; Nandi, P; Darlix, J L

2001-06-01

Transmissible spongiform encephalopathies are fatal neurodegenerative diseases associated with the accumulation of a protease-resistant form of the prion protein (PrP). Although PrP is conserved in vertebrates, its function remains to be identified. In vitro PrP binds large nucleic acids causing the formation of nucleoprotein complexes resembling human immunodeficiency virus type 1 (HIV-1) nucleocapsid-RNA complexes and in vivo MuLV replication accelerates the scrapie infectious process, suggesting possible interactions between retroviruses and PrP. Retroviruses, including HIV-1 encode a major nucleic acid binding protein (NC protein) found within the virus where 2000 NC protein molecules coat the dimeric genome. NC is required in virus assembly and infection to chaperone RNA dimerization and packaging and in proviral DNA synthesis by reverse transcriptase (RT). In HIV-1, 5'-leader RNA/NC interactions appear to control these viral processes. This prompted us to compare and contrast the interactions of human and ovine PrP and HIV-1 NCp7 with HIV-1 5'-leader RNA. Results show that PrP has properties characteristic of NCp7 with respect to viral RNA dimerization and proviral DNA synthesis by RT. The NC-like properties of huPrP map to the N-terminal region of huPrP. Interestingly, PrP localizes in the membrane and cytoplasm of PrP-expressing cells. These findings suggest that PrP is a multifunctional protein possibly participating in nucleic acid metabolism.

PrP(C) signalling in neurons: from basics to clinical challenges.

PubMed

Hirsch, Théo Z; Hernandez-Rapp, Julia; Martin-Lannerée, Séverine; Launay, Jean-Marie; Mouillet-Richard, Sophie

2014-09-01

The cellular prion protein PrP(C) was identified over twenty-five years ago as the normal counterpart of the scrapie prion protein PrP(Sc), itself the main if not the sole component of the infectious agent at the root of Transmissible Spongiform Encephalopathies (TSEs). PrP(C) is a ubiquitous cell surface protein, abundantly expressed in neurons, which constitute the targets of PrP(Sc)-mediated toxicity. Converging evidence have highlighted that neuronal, GPI-anchored PrP(C) is absolutely required for prion-induced neuropathogenesis, which warrants investigating into the normal function exerted by PrP(C) in a neuronal context. It is now well-established that PrP(C) can serve as a cell signalling molecule, able to mobilize transduction cascades in response to interactions with partners. This function endows PrP(C) with the capacity to participate in multiple neuronal processes, ranging from survival to synaptic plasticity. A diverse array of data have allowed to shed light on how this function is corrupted by PrP(Sc). Recently, amyloid Aβ oligomers, whose accumulation is associated with Alzheimer's disease (AD), were shown to similarly instigate toxic events by deviating PrP(C)-mediated signalling. Here, we provide an overview of the various signal transduction cascades ascribed to PrP(C) in neurons, summarize how their subversion by PrP(Sc) or Aβ oligomers contributes to TSE or AD neuropathogenesis and discuss the ensuing clinical implications. Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Sex and PRNP genotype determination in preimplantation caprine embryos.

PubMed

Guignot, F; Perreau, C; Cavarroc, C; Touzé, J-L; Pougnard, J-L; Dupont, F; Beckers, J-F; Rémy, B; Babilliot, J-M; Bed'Hom, B; Lamorinière, J M; Mermillod, P; Baril, G

2011-08-01

The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer. Whole genome amplification (WGA) was performed using Multiple Displacement Amplification (MDA). Sex and prion protein (PRNP) genotypes were determined. Sex diagnosis was carried out by PCR amplification of ZFX/ZFY and Y chromosome-specific sequences. Prion protein genotype determination was performed on codons 142, 154, 211, 222 and 240. Embryos were collected at day 7 after oestrus and biopsied either immediately after collection (blastocysts and expanded blastocysts) or after 24 h of in vitro culture (compacted morulae). Biopsied embryos were frozen by vitrification. Vitrified whole embryos were kept as control. DNA of biopsies was extracted and amplified using MDA. Sex diagnosis was efficient for 97.4% of biopsies and PRNP genotyping was determined in 78.7% of biopsies. After embryo transfer, no significant difference was observed in kidding rate between biopsied and vitrified control embryos, whereas embryo survival rate was different between biopsied and whole vitrified embryos (p = 0.032). At birth, 100% of diagnosed sex and 98.2% of predetermined codons were correct. Offspring PRNP profiles were in agreement with parental genotype. Whole genome amplification with MDA kit coupled with sex diagnosis and PRNP genotype predetermination are very accurate techniques to genotype goat embryos before transfer. These novel results allow us to plan selection of scrapie-resistant genotypes and kid sex before transfer of cryopreserved embryo. © 2010 Blackwell Verlag GmbH.

Effect of Q211 and K222 PRNP Polymorphic Variants in the Susceptibility of Goats to Oral Infection With Goat Bovine Spongiform Encephalopathy.

PubMed

Aguilar-Calvo, Patricia; Fast, Christine; Tauscher, Kerstin; Espinosa, Juan-Carlos; Groschup, Martin H; Nadeem, Muhammad; Goldmann, Wilfred; Langeveld, Jan; Bossers, Alex; Andreoletti, Olivier; Torres, Juan-María

2015-08-15

The prion protein-encoding gene (PRNP) is one of the major determinants for scrapie occurrence in sheep and goats. However, its effect on bovine spongiform encephalopathy (BSE) transmission to goats is not clear. Goats harboring wild-type, R/Q211 or Q/K222 PRNP genotypes were orally inoculated with a goat-BSE isolate to assess their relative susceptibility to BSE infection. Goats were killed at different time points during the incubation period and after the onset of clinical signs, and their brains as well as several peripheral tissues were analyzed for the accumulation of pathological prion protein (PrP(Sc)) and prion infectivity by mouse bioassay. R/Q211 goats displayed delayed clinical signs compared with wild-type goats. Deposits of PrP(Sc) were detected only in brain, whereas infectivity was present in peripheral tissues too. In contrast, none of the Q/K222 goats showed any evidence of clinical prion disease. No PrP(Sc) accumulation was observed in their brains or peripheral tissues, but very low infectivity was detected in some tissues very long after inoculation (44-45 months). These results demonstrate that transmission of goat BSE is genotype dependent, and they highlight the pivotal protective effect of the K222 PRNP variant in the oral susceptibility of goats to BSE. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Orally administered indomethacin acutely reduces cellular prion protein in the small intestine and modestly increases survival of mice exposed to infectious prions.

PubMed

Martin, Gary R; Sharkey, Keith A; Jirik, Frank R

2015-05-01

The oral uptake of infectious prions represents a common way to acquire a prion disease; thus, host factors, such as gut inflammation and intestinal "leakiness", have the potential to influence infectivity. For example, the ingestion of nonsteroidal anti-inflammatory drugs (NSAIDs) is known to induce intestinal inflammation and increase intestinal permeability. Previously, we reported that normal cellular prion protein (PrP(C)) expression was increased in experimental colitis, and since the level of PrP(C) expressed is a determinant of prion disease propagation, we hypothesized that NSAID administration prior to the oral inoculation of mice with infectious prions would increase intestinal PrP(C) expression and accelerate the onset of neurological disease. In the long-term experiments, one group of mice was gavaged with indomethacin, followed by a second gavage with brain homogenate containing mouse-adapted scrapie (ME7). Control mice received ME7 brain homogenate alone. Brain and splenic tissues were harvested at several time points for immunoblotting, including at the onset of clinical signs of disease. In a second series of experiments, mice were gavaged with indomethacin to assess the acute effects of this treatment on intestinal PrP(C) expression. Acutely, NSAID treatment reduced intestinal PrP(C) expression, and chronically, there was a modest delay in the onset of neurological disease. In contrast to our hypothesis, brief exposure to an NSAID decreased intestinal PrP(C) expression and led to a modest survival advantage following oral ingestion of infectious prions.

Prion Replication Elicits Cytopathic Changes in Differentiated Neurosphere Cultures

PubMed Central

Iwamaru, Yoshifumi; Takenouchi, Takato; Imamura, Morikazu; Shimizu, Yoshihisa; Miyazawa, Kohtaro; Mohri, Shirou; Yokoyama, Takashi

2013-01-01

The molecular mechanisms of prion-induced cytotoxicity remain largely obscure. Currently, only a few cell culture models have exhibited the cytopathic changes associated with prion infection. In this study, we introduced a cell culture model based on differentiated neurosphere cultures isolated from the brains of neonatal prion protein (PrP)-null mice and transgenic mice expressing murine PrP (dNP0 and dNP20 cultures). Upon exposure to mouse Chandler prions, dNP20 cultures supported the de novo formation of abnormal PrP and the resulting infectivity, as assessed by bioassays. Furthermore, this culture was susceptible to various prion strains, including mouse-adapted scrapie, bovine spongiform encephalopathy, and Gerstmann-Sträussler-Scheinker syndrome prions. Importantly, a subset of the cells in the infected culture that was mainly composed of astrocyte lineage cells consistently displayed late-occurring, progressive signs of cytotoxicity as evidenced by morphological alterations, decreased cell viability, and increased lactate dehydrogenase release. These signs of cytotoxicity were not observed in infected dNP0 cultures, suggesting the requirement of endogenous PrP expression for prion-induced cytotoxicity. Degenerated cells positive for glial fibrillary acidic protein accumulated abnormal PrP and exhibited features of apoptotic death as assessed by active caspase-3 and terminal deoxynucleotidyltransferase nick-end staining. Furthermore, caspase inhibition provided partial protection from prion-mediated cell death. These results suggest that differentiated neurosphere cultures can provide an in vitro bioassay for mouse prions and permit the study of the molecular basis for prion-induced cytotoxicity at the cellular level. PMID:23740992

Rapid and Highly Sensitive Detection of Variant Creutzfeldt - Jakob Disease Abnormal Prion Protein on Steel Surfaces by Protein Misfolding Cyclic Amplification: Application to Prion Decontamination Studies

PubMed Central

Belondrade, Maxime; Nicot, Simon; Béringue, Vincent; Coste, Joliette; Lehmann, Sylvain; Bougard, Daisy

2016-01-01

The prevalence of variant Creutzfeldt-Jakob disease (vCJD) in the population remains uncertain, although it has been estimated that 1 in 2000 people in the United Kingdom are positive for abnormal prion protein (PrPTSE) by a recent survey of archived appendix tissues. The prominent lymphotropism of vCJD prions raises the possibility that some surgical procedures may be at risk of iatrogenic vCJD transmission in healthcare facilities. It is therefore vital that decontamination procedures applied to medical devices before their reprocessing are thoroughly validated. A current limitation is the lack of a rapid model permissive to human prions. Here, we developed a prion detection assay based on protein misfolding cyclic amplification (PMCA) technology combined with stainless-steel wire surfaces as carriers of prions (Surf-PMCA). This assay allowed the specific detection of minute quantities (10−8 brain dilution) of either human vCJD or ovine scrapie PrPTSE adsorbed onto a single steel wire, within a two week timeframe. Using Surf-PMCA we evaluated the performance of several reference and commercially available prion-specific decontamination procedures. Surprisingly, we found the efficiency of several marketed reagents to remove human vCJD PrPTSE was lower than expected. Overall, our results demonstrate that Surf-PMCA can be used as a rapid and ultrasensitive assay for the detection of human vCJD PrPTSE adsorbed onto a metallic surface, therefore facilitating the development and validation of decontamination procedures against human prions. PMID:26800081

Similar folds with different stabilization mechanisms: the cases of prion and doppel proteins

PubMed Central

Colacino, Stefano; Tiana, Guido; Colombo, Giorgio

2006-01-01

Background Protein misfolding is the main cause of a group of fatal neurodegenerative diseases in humans and animals. In particular, in Prion-related diseases the normal cellular form of the Prion Protein PrP (PrPC) is converted into the infectious PrPSc through a conformational process during which it acquires a high β-sheet content. Doppel is a protein that shares a similar native fold, but lacks the scrapie isoform. Understanding the molecular determinants of these different behaviours is important both for biomedical and biophysical research. Results In this paper, the dynamical and energetic properties of the two proteins in solution is comparatively analyzed by means of long time scale explicit solvent, all-atom molecular dynamics in different temperature conditions. The trajectories are analyzed by means of a recently introduced energy decomposition approach (Tiana et al, Prot. Sci. 2004) aimed at identifying the key residues for the stabilization and folding of the protein. Our analysis shows that Prion and Doppel have two different cores stabilizing the native state and that the relative contribution of the nucleus to the global stability of the protein for Doppel is sensitively higher than for PrP. Moreover, under misfolding conditions the Doppel core is conserved, while the energy stabilization network of PrP is disrupted. Conclusion These observations suggest that different sequences can share similar native topology with different stabilizing interactions and that the sequences of the Prion and Doppel proteins may have diverged under different evolutionary constraints resulting in different folding and stabilization mechanisms. PMID:16857062

Development of a bifunctional filter for prion protein and leukoreduction of red blood cell components.

PubMed

Yokomizo, Tomo; Kai, Takako; Miura, Morikazu; Ohto, Hitoshi

2015-02-01

Leukofiltration of blood components is currently implemented worldwide as a precautionary measure against white blood cell-associated adverse effects and the potential transmission of variant Creutzfeldt-Jakob disease (vCJD). A newly developed bifunctional filter (Sepacell Prima, Asahi Kasei Medical) was assessed for prion removal, leukoreduction (LR), and whether the filter significantly affected red blood cells (RBCs). Sepacell Prima's postfiltration effects on RBCs, including hemolysis, complement activation, and RBC chemistry, were compared with those of a conventional LR filter (Sepacell Pure RC). Prion removal was measured by Western blot after spiking RBCs with microsomal fractions derived from scrapie-infected hamster brain homogenate. Serially diluted exogenous prion solutions (0.05 mL), with or without filtration, were injected intracerebrally into Golden Syrian hamsters. LR efficiency of 4.44 log with the Sepacell Prima was comparable to 4.11 log with the conventional LR filter. There were no significant differences between the two filters in hemoglobin loss, hemolysis, complement activation, and RBC biomarkers. In vitro reduction of exogenously spiked prions by the filter exceeded 3 log. The titer, 6.63 (log ID50 /mL), of prefiltration infectivity of healthy hamsters was reduced to 2.52 (log ID50 /mL) after filtration. The reduction factor was calculated as 4.20 (log ID50 ). With confirmed removal efficacy for exogenous prion protein, this new bifunctional prion and LR filter should reduce the residual risk of vCJD transmission through blood transfusion without adding complexity to component processing. © 2014 AABB.

Region-specific protein misfolding cyclic amplification reproduces brain tropism of prion strains.

PubMed

Privat, Nicolas; Levavasseur, Etienne; Yildirim, Serfildan; Hannaoui, Samia; Brandel, Jean-Philippe; Laplanche, Jean-Louis; Béringue, Vincent; Seilhean, Danielle; Haïk, Stéphane

2017-10-06

Human prion diseases such as Creutzfeldt-Jakob disease are transmissible brain proteinopathies, characterized by the accumulation of a misfolded isoform of the host cellular prion protein (PrP) in the brain. According to the prion model, prions are defined as proteinaceous infectious particles composed solely of this abnormal isoform of PrP (PrP Sc ). Even in the absence of genetic material, various prion strains can be propagated in experimental models. They can be distinguished by the pattern of disease they produce and especially by the localization of PrP Sc deposits within the brain and the spongiform lesions they induce. The mechanisms involved in this strain-specific targeting of distinct brain regions still are a fundamental, unresolved question in prion research. To address this question, we exploited a prion conversion in vitro assay, protein misfolding cyclic amplification (PMCA), by using experimental scrapie and human prion strains as seeds and specific brain regions from mice and humans as substrates. We show here that region-specific PMCA in part reproduces the specific brain targeting observed in experimental, acquired, and sporadic Creutzfeldt-Jakob diseases. Furthermore, we provide evidence that, in addition to cellular prion protein, other region- and species-specific molecular factors influence the strain-dependent prion conversion process. This important step toward understanding prion strain propagation in the human brain may impact research on the molecular factors involved in protein misfolding and the development of ultrasensitive methods for diagnosing prion disease. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The prion protein has DNA strand transfer properties similar to retroviral nucleocapsid protein.

PubMed

Gabus, C; Auxilien, S; Péchoux, C; Dormont, D; Swietnicki, W; Morillas, M; Surewicz, W; Nandi, P; Darlix, J L

2001-04-06

The transmissible spongiform encephalopathies are fatal neurodegenerative diseases that are associated with the accumulation of a protease-resistant form of the cellular prion protein (PrP). Although PrP is highly conserved and widely expressed in vertebrates, its function remains a matter of speculation. Indeed PrP null mice develop normally and are healthy. Recent results show that PrP binds to nucleic acids in vitro and is found associated with retroviral particles. Furthermore, in mice the scrapie infectious process appears to be accelerated by MuLV replication. These observations prompted us to further investigate the interaction between PrP and nucleic acids, and compare it with that of the retroviral nucleocapsid protein (NC). As the major nucleic acid-binding protein of the retroviral particle, NC protein is tightly associated with the genomic RNA in the virion nucleocapsid, where it chaperones proviral DNA synthesis by reverse transcriptase. Our results show that the human prion protein (huPrP) functionally resembles NCp7 of HIV-1. Both proteins form large nucleoprotein complexes upon binding to DNA. They accelerate the hybridization of complementary DNA strands and chaperone viral DNA synthesis during the minus and plus DNA strand transfers necessary to generate the long terminal repeats. The DNA-binding and strand transfer properties of huPrP appear to map to the N-terminal fragment comprising residues 23 to 144, whereas the C-terminal domain is inactive. These findings suggest that PrP could be involved in nucleic acid metabolism in vivo. Copyright 2001 Academic Press.

Comparative prion disease gene expression profiling using the prion disease mimetic, cuprizone

PubMed Central

Moody, Laura R; Herbst, Allen J; Yoo, Han Sang; Vanderloo, Joshua P

2009-01-01

Identification of genes expressed in response to prion infection may elucidate biomarkers for disease, identify factors involved in agent replication, mechanisms of neuropathology and therapeutic targets. Although several groups have sought to identify gene expression changes specific to prion disease, expression profiles rife with cell population changes have consistently been identified. Cuprizone, a neurotoxicant, qualitatively mimics the cell population changes observed in prion disease, resulting in both spongiform change and astrocytosis. The use of cuprizone-treated animals as an experimental control during comparative expression profiling allows for the identification of transcripts whose expression increases during prion disease and remains unchanged during cuprizone-triggered neuropathology. In this study, expression profiles from the brains of mice preclinically and clinically infected with Rocky Mountain Laboratory (RML) mouse-adapted scrapie agent and age-matched controls were profiled using Affymetrix gene arrays. In total, 164 genes were differentially regulated during prion infection. Eighty-three of these transcripts have been previously undescribed as differentially regulated during prion disease. A 0.4% cuprizone diet was utilized as a control for comparative expression profiling. Cuprizone treatment induced spongiosis and astrocyte proliferation as indicated by glial fibrillary acidic protein (Gfap) transcriptional activation and immunohistochemistry. Gene expression profiles from brain tissue obtained from cuprizone-treated mice identified 307 differentially regulated transcript changes. After comparative analysis, 17 transcripts unaffected by cuprizone treatment but increasing in expression from preclinical to clinical prion infection were identified. Here we describe the novel use of the prion disease mimetic, cuprizone, to control for cell population changes in the brain during prion infection. PMID:19535908

Prion Disease Induces Alzheimer Disease-Like Neuropathologic Changes

PubMed Central

Tousseyn, Thomas; Bajsarowicz, Krystyna; Sánchez, Henry; Gheyara, Ania; Oehler, Abby; Geschwind, Michael; DeArmond, Bernadette; DeArmond, Stephen J.

2016-01-01

We examined the brains of 266 patients with prion diseases (PrionD) and found that 46 (17%) had Alzheimer disease (AD)-like changes. To explore potential mechanistic links between PrionD and AD, we exposed human brain aggregates (Hu BrnAggs) to brain homogenate from a patient with sporadic Creutzfeldt-Jakob disease (CJD) and found that the neurons in the Hu BrnAggs produced many β-amyloid (β42) inclusions, whereas uninfected, control-exposed Hu BrnAggs did not. Western blots of 20-pooled CJD-infected BrnAggs verified higher Aβ42 levels than controls. We next examined the CA1 region of the hippocampus from 14 patients with PrionD and found that 5 patients had low levels of scrapie-associated prion protein (PrPSc), many Aβ42 intraneuronal inclusions, low APOE-4, and no significant nerve cell loss. Seven patients had high levels of PrPSc, low Aβ42, high APOE-4 and 40% nerve cell loss, suggesting that APOE-4 and PrPSc together cause neuron loss in PrionD. There were also increased levels of hyperphosphorylated tau protein (Hτ) and Hτ-positive neuropil threads and neuron bodies in both PrionD and AD groups. The brains of 6 age-matched control patients without dementia did not contain Aβ42 deposits; however, there were rare Hτ-positive threads in 5 controls and 2 controls had a few Hτ-positive nerve cell bodies. We conclude that PrionD may trigger biochemical changes similar to AD and suggest that PrionD are diseases of PrPSc, Aβ42, APOE-4 and abnormal tau. PMID:26226132

The mechanisms of humic substances self-assembly with biological molecules: The case study of the prion protein

PubMed Central

Nepravishta, Ridvan; Mandaliti, Walter; Melino, Sonia; Margon, Alja; Scaini, Denis; Mazzei, Pierluigi; Piccolo, Alessandro; Legname, Giuseppe; Paci, Maurizio; Leita, Liviana

2017-01-01

Humic substances (HS) are the largest constituent of soil organic matter and are considered as a key component of the terrestrial ecosystem. HS may facilitate the transport of organic and inorganic molecules, as well as the sorption interactions with environmentally relevant proteins such as prions. Prions enter the environment through shedding from live hosts, facilitating a sustained incidence of animal prion diseases such as Chronic Wasting Disease and scrapie in cervid and ovine populations, respectively. Changes in prion structure upon environmental exposure may be significant as they can affect prion infectivity and disease pathology. Despite its relevance, the mechanisms of prion interaction with HS are still not completely understood. The goal of this work is to advance a structural-level picture of the encapsulation of recombinant, non-infectious, prion protein (PrP) into different natural HS. We observed that PrP precipitation upon addition of HS is mainly driven by a mechanism of “salting-out” whereby PrP molecules are rapidly removed from the solution and aggregate in insoluble adducts with humic molecules. Importantly, this process does not alter the protein folding since insoluble PrP retains its α-helical content when in complex with HS. The observed ability of HS to promote PrP insolubilization without altering its secondary structure may have potential relevance in the context of “prion ecology”. These results suggest that soil organic matter interacts with prions possibly without altering the protein structures. This may facilitate prions preservation from biotic and abiotic degradation leading to their accumulation in the environment. PMID:29161325

Dominant-Negative Inhibition of Prion Formation Diminished by Deletion Mutagenesis of the Prion Protein

PubMed Central

Zulianello, Laurence; Kaneko, Kiyotoshi; Scott, Michael; Erpel, Susanne; Han, Dong; Cohen, Fred E.; Prusiner, Stanley B.

2000-01-01

Polymorphic basic residues near the C terminus of the prion protein (PrP) in humans and sheep appear to protect against prion disease. In heterozygotes, inhibition of prion formation appears to be dominant negative and has been simulated in cultured cells persistently infected with scrapie prions. The results of nuclear magnetic resonance and mutagenesis studies indicate that specific substitutions at the C-terminal residues 167, 171, 214, and 218 of PrPC act as dominant-negative, inhibitors of PrPSc formation (K. Kaneko et al., Proc. Natl. Acad. Sci. USA 94:10069–10074, 1997). Trafficking of substituted PrPC to caveaola-like domains or rafts by the glycolipid anchor was required for the dominant-negative phenotype; interestingly, amino acid replacements at multiple sites were less effective than single-residue substitutions. To elucidate which domains of PrPC are responsible for dominant-negative inhibition of PrPSc formation, we analyzed whether N-terminally truncated PrP(Q218K) molecules exhibited dominant-negative effects in the conversion of full-length PrPC to PrPSc. We found that the C-terminal domain of PrP is not sufficient to impede the conversion of the full-length PrPC molecule and that N-terminally truncated molecules (with residues 23 to 88 and 23 to 120 deleted) have reduced dominant-negative activity. Whether the N-terminal region of PrP acts by stabilizing the C-terminal domain of the molecule or by modulating the binding of PrPC to an auxiliary molecule that participates in PrPSc formation remains to be established. PMID:10756050

Prion Protein Promotes Kidney Iron Uptake via Its Ferrireductase Activity*

PubMed Central

Haldar, Swati; Tripathi, Ajai; Qian, Juan; Beserra, Amber; Suda, Srinivas; McElwee, Matthew; Turner, Jerrold; Hopfer, Ulrich; Singh, Neena

2015-01-01

Brain iron-dyshomeostasis is an important cause of neurotoxicity in prion disorders, a group of neurodegenerative conditions associated with the conversion of prion protein (PrPC) from its normal conformation to an aggregated, PrP-scrapie (PrPSc) isoform. Alteration of iron homeostasis is believed to result from impaired function of PrPC in neuronal iron uptake via its ferrireductase activity. However, unequivocal evidence supporting the ferrireductase activity of PrPC is lacking. Kidney provides a relevant model for this evaluation because PrPC is expressed in the kidney, and ∼370 μg of iron are reabsorbed daily from the glomerular filtrate by kidney proximal tubule cells (PT), requiring ferrireductase activity. Here, we report that PrPC promotes the uptake of transferrin (Tf) and non-Tf-bound iron (NTBI) by the kidney in vivo and mainly NTBI by PT cells in vitro. Thus, uptake of 59Fe administered by gastric gavage, intravenously, or intraperitoneally was significantly lower in PrP-knock-out (PrP−/−) mouse kidney relative to PrP+/+ controls. Selective in vivo radiolabeling of plasma NTBI with 59Fe revealed similar results. Expression of exogenous PrPC in immortalized PT cells showed localization on the plasma membrane and intracellular vesicles and increased transepithelial transport of 59Fe-NTBI and to a smaller extent 59Fe-Tf from the apical to the basolateral domain. Notably, the ferrireductase-deficient mutant of PrP (PrPΔ51–89) lacked this activity. Furthermore, excess NTBI and hemin caused aggregation of PrPC to a detergent-insoluble form, limiting iron uptake. Together, these observations suggest that PrPC promotes retrieval of iron from the glomerular filtrate via its ferrireductase activity and modulates kidney iron metabolism. PMID:25572394

Scrg1, a novel protein of the CNS is targeted to the large dense-core vesicles in neuronal cells.

PubMed

Dandoy-Dron, Françoise; Griffond, Bernadette; Mishal, Zohar; Tovey, Michael G; Dron, Michel

2003-11-01

Scrapie responsive gene one (Scrg1) is a novel transcript discovered through identification of the genes associated with or responsible for the neurodegenerative changes observed in transmissible spongiform encephalopathies. Scrg1 mRNA is distributed principally in the central nervous system and the cDNA sequence predicts a small cysteine-rich protein 98 amino acids in length, with a N-terminal signal peptide. In this study, we have generated antibodies against the predicted protein and revealed expression of a predominant immunoreactive protein of 10 kDa in mouse brain by Western blot analysis. We have established CAD neuronal cell lines stably expressing Scrg1 to determine its subcellular localization. Several lines of evidence show that the protein is targeted to dense-core vesicles in these cells. (i) Scrg1 is detected by immunocytochemistry as very punctate signals especially in the Golgi apparatus and tips of neurites, suggesting a vesicular localization for the protein. Moreover, Scrg1 exhibits a high degree of colocalization with secretogranin II, a dense-core vesicle marker and a very limited colocalization with markers for small synaptic vesicles. (ii) Scrg1 immunoreactivity is associated with large secretory granules/dense-core vesicles, as indicated by immuno-electron microscopy. (iii) Scrg1 is enriched in fractions of sucrose density gradient where synaptotagmin V, a dense-core vesicle-associated protein, is also enriched. The characteristic punctate immunostaining of Scrg1 is observed in N2A cells transfected with Scrg1 and for the endogenous protein in cultured primary neurons, attesting to the generality of the observations. Our findings strongly suggest that Scrg1 is associated with the secretory pathway of neuronal cells.

Highly Efficient Protein Misfolding Cyclic Amplification

PubMed Central

Ostapchenko, Valeriy G.; Savtchenk, Regina; Alexeeva, Irina; Rohwer, Robert G.; Baskakov, Ilia V.

2011-01-01

Protein misfolding cyclic amplification (PMCA) provides faithful replication of mammalian prions in vitro and has numerous applications in prion research. However, the low efficiency of conversion of PrPC into PrPSc in PMCA limits the applicability of PMCA for many uses including structural studies of infectious prions. It also implies that only a small sub-fraction of PrPC may be available for conversion. Here we show that the yield, rate, and robustness of prion conversion and the sensitivity of prion detection are significantly improved by a simple modification of the PMCA format. Conducting PMCA reactions in the presence of Teflon beads (PMCAb) increased the conversion of PrPC into PrPSc from ∼10% to up to 100%. In PMCAb, a single 24-hour round consistently amplified PrPSc by 600-700-fold. Furthermore, the sensitivity of prion detection in one round (24 hours) increased by 2-3 orders of magnitude. Using serial PMCAb, a 1012-fold dilution of scrapie brain material could be amplified to the level detectible by Western blotting in 3 rounds (72 hours). The improvements in amplification efficiency were observed for the commonly used hamster 263K strain and for the synthetic strain SSLOW that otherwise amplifies poorly in PMCA. The increase in the amplification efficiency did not come at the expense of prion replication specificity. The current study demonstrates that poor conversion efficiencies observed previously have not been due to the scarcity of a sub-fraction of PrPC susceptible to conversion nor due to limited concentrations of essential cellular cofactors required for conversion. The new PMCAb format offers immediate practical benefits and opens new avenues for developing fast ultrasensitive assays and for producing abundant quantities of PrPSc in vitro. PMID:21347353

Estimating Prion Adsorption Capacity of Soil by BioAssay of Subtracted Infectivity from Complex Solutions (BASICS)

PubMed Central

Wyckoff, A. Christy; Lockwood, Krista L.; Meyerett-Reid, Crystal; Michel, Brady A.; Bender, Heather; VerCauteren, Kurt C.; Zabel, Mark D.

2013-01-01

Prions, the infectious agent of scrapie, chronic wasting disease and other transmissible spongiform encephalopathies, are misfolded proteins that are highly stable and resistant to degradation. Prions are known to associate with clay and other soil components, enhancing their persistence and surprisingly, transmissibility. Currently, few detection and quantification methods exist for prions in soil, hindering an understanding of prion persistence and infectivity in the environment. Variability in apparent infectious titers of prions when bound to soil has complicated attempts to quantify the binding capacity of soil for prion infectivity. Here, we quantify the prion adsorption capacity of whole, sandy loam soil (SLS) typically found in CWD endemic areas in Colorado; and purified montmorillonite clay (Mte), previously shown to bind prions, by BioAssay of Subtracted Infectivity in Complex Solutions (BASICS). We incubated prion positive 10% brain homogenate from terminally sick mice infected with the Rocky Mountain Lab strain of mouse-adapted prions (RML) with 10% SLS or Mte. After 24 hours samples were centrifuged five minutes at 200×g and soil-free supernatant was intracerebrally inoculated into prion susceptible indicator mice. We used the number of days post inoculation to clinical disease to calculate the infectious titer remaining in the supernatant, which we subtracted from the starting titer to determine the infectious prion binding capacity of SLS and Mte. BASICS indicated SLS bound and removed ≥ 95% of infectivity. Mte bound and removed lethal doses (99.98%) of prions from inocula, effectively preventing disease in the mice. Our data reveal significant prion-binding capacity of soil and the utility of BASICS to estimate prion loads and investigate persistence and decomposition in the environment. Additionally, since Mte successfully rescued the mice from prion disease, Mte might be used for remediation and decontamination protocols. PMID:23484043

Epithelial and endothelial expression of the green fluorescent protein reporter gene under the control of bovine prion protein (PrP) gene regulatory sequences in transgenic mice

NASA Astrophysics Data System (ADS)

Lemaire-Vieille, Catherine; Schulze, Tobias; Podevin-Dimster, Valérie; Follet, Jérome; Bailly, Yannick; Blanquet-Grossard, Françoise; Decavel, Jean-Pierre; Heinen, Ernst; Cesbron, Jean-Yves

2000-05-01

The expression of the cellular form of the prion protein (PrPc) gene is required for prion replication and neuroinvasion in transmissible spongiform encephalopathies. The identification of the cell types expressing PrPc is necessary to understanding how the agent replicates and spreads from peripheral sites to the central nervous system. To determine the nature of the cell types expressing PrPc, a green fluorescent protein reporter gene was expressed in transgenic mice under the control of 6.9 kb of the bovine PrP gene regulatory sequences. It was shown that the bovine PrP gene is expressed as two populations of mRNA differing by alternative splicing of one 115-bp 5' untranslated exon in 17 different bovine tissues. The analysis of transgenic mice showed reporter gene expression in some cells that have been identified as expressing PrP, such as cerebellar Purkinje cells, lymphocytes, and keratinocytes. In addition, expression of green fluorescent protein was observed in the plexus of the enteric nervous system and in a restricted subset of cells not yet clearly identified as expressing PrP: the epithelial cells of the thymic medullary and the endothelial cells of both the mucosal capillaries of the intestine and the renal capillaries. These data provide valuable information on the distribution of PrPc at the cellular level and argue for roles of the epithelial and endothelial cells in the spread of infection from the periphery to the brain. Moreover, the transgenic mice described in this paper provide a model that will allow for the study of the transcriptional activity of the PrP gene promoter in response to scrapie infection.

The Physical Relationship between Infectivity and Prion Protein Aggregates Is Strain-Dependent

PubMed Central

Tixador, Philippe; Herzog, Laëtitia; Reine, Fabienne; Jaumain, Emilie; Chapuis, Jérôme; Le Dur, Annick; Laude, Hubert; Béringue, Vincent

2010-01-01

Prions are unconventional infectious agents thought to be primarily composed of PrPSc, a multimeric misfolded conformer of the ubiquitously expressed host-encoded prion protein (PrPC). They cause fatal neurodegenerative diseases in both animals and humans. The disease phenotype is not uniform within species, and stable, self-propagating variations in PrPSc conformation could encode this ‘strain’ diversity. However, much remains to be learned about the physical relationship between the infectious agent and PrPSc aggregation state, and how this varies according to the strain. We applied a sedimentation velocity technique to a panel of natural, biologically cloned strains obtained by propagation of classical and atypical sheep scrapie and BSE infectious sources in transgenic mice expressing ovine PrP. Detergent-solubilized, infected brain homogenates were used as starting material. Solubilization conditions were optimized to separate PrPSc aggregates from PrPC. The distribution of PrPSc and infectivity in the gradient was determined by immunoblotting and mouse bioassay, respectively. As a general feature, a major proteinase K-resistant PrPSc peak was observed in the middle part of the gradient. This population approximately corresponds to multimers of 12–30 PrP molecules, if constituted of PrP only. For two strains, infectivity peaked in a markedly different region of the gradient. This most infectious component sedimented very slowly, suggesting small size oligomers and/or low density PrPSc aggregates. Extending this study to hamster prions passaged in hamster PrP transgenic mice revealed that the highly infectious, slowly sedimenting particles could be a feature of strains able to induce a rapidly lethal disease. Our findings suggest that prion infectious particles are subjected to marked strain-dependent variations, which in turn could influence the strain biological phenotype, in particular the replication dynamics. PMID:20419156

Fate of prions in soil: trapped conformation of full-length ovine prion protein induced by adsorption on clays.

PubMed

Revault, M; Quiquampoix, H; Baron, M H; Noinville, S

2005-08-05

Studying the mechanism of retention of ovine prion protein in soils will tackle the environmental aspect of potential dissemination of scrapie infectious agent. We consider the surface-induced conformational changes that the recombinant ovine prion protein (ovPrP) may undergo under different pH conditions when interacting with soil minerals of highly adsorptive capacities such as montmorillonite. The conformational states of the full-length ovine prion protein adsorbed on the electronegative clay surface are compared to its solvated state in deuterated buffer in the pD range 3.5-9, using FTIR spectroscopy. The in vitro pH-induced conversion of the alpha-helical monomer of ovPrP into oligomers of beta-like structure prone to self-aggregation does not occur when the protein is adsorbed on the clay surface. The conformation of the trapped ovPrP molecules on montmorillonite is pH-independent and looks like that of the ovPrP solvated state at pD higher than 7, suggesting the major role of Arg and Lys residues in the electrostatic origin of adsorption. The uneven distribution of positively and negatively charged residues of the ovPrP protein would promote a favored orientation of the protein towards the clay, so that not only the basic residues embedded in the N-terminal flexible part but also external basic residues in the globular part of the protein might participate to the attractive interaction. From these results, it appears unlikely that the interaction of normal prions (PrP(C)) with soil clay surfaces could induce a change of conformation leading to the pathogenic form of prions (PrP(Sc)).

Isolation of Novel Synthetic Prion Strains by Amplification in Transgenic Mice Coexpressing Wild-Type and Anchorless Prion Proteins

PubMed Central

Raymond, Gregory J.; Race, Brent; Hollister, Jason R.; Offerdahl, Danielle K.; Moore, Roger A.; Kodali, Ravindra; Raymond, Lynne D.; Hughson, Andrew G.; Rosenke, Rebecca; Long, Dan; Dorward, David W.

2012-01-01

Mammalian prions are thought to consist of misfolded aggregates (protease-resistant isoform of the prion protein [PrPres]) of the cellular prion protein (PrPC). Transmissible spongiform encephalopathy (TSE) can be induced in animals inoculated with recombinant PrP (rPrP) amyloid fibrils lacking mammalian posttranslational modifications, but this induction is inefficient in hamsters or transgenic mice overexpressing glycosylphosphatidylinositol (GPI)-anchored PrPC. Here we show that TSE can be initiated by inoculation of misfolded rPrP into mice that express wild-type (wt) levels of PrPC and that synthetic prion strain propagation and selection can be affected by GPI anchoring of the host's PrPC. To create prions de novo, we fibrillized mouse rPrP in the absence of molecular cofactors, generating fibrils with a PrPres-like protease-resistant banding profile. These fibrils induced the formation of PrPres deposits in transgenic mice coexpressing wt and GPI-anchorless PrPC (wt/GPI−) at a combined level comparable to that of PrPC expression in wt mice. Secondary passage into mice expressing wt, GPI−, or wt plus GPI− PrPC induced TSE disease with novel clinical, histopathological, and biochemical phenotypes. Contrary to laboratory-adapted mouse scrapie strains, the synthetic prion agents exhibited a preference for conversion of GPI− PrPC and, in one case, caused disease only in GPI− mice. Our data show that novel TSE agents can be generated de novo solely from purified mouse rPrP after amplification in mice coexpressing normal levels of wt and anchorless PrPC. These observations provide insight into the minimal elements required to create prions in vitro and suggest that the PrPC GPI anchor can modulate the propagation of synthetic TSE strains. PMID:22915801

Mechanism of Unfolding of Human Prion Protein.

PubMed

Singh, Reman K; Chamachi, Neharika G; Chakrabarty, Suman; Mukherjee, Arnab

2017-01-26

Misfolding and aggregation of prion proteins are associated with several neurodegenerative diseases. Therefore, understanding the mechanism of the misfolding process is of enormous interest in the scientific community. It has been speculated and widely discussed that the native cellular prion protein (PrP C ) form needs to undergo substantial unfolding to a more stable PrP C* state, which may further oligomerize into the toxic scrapie (PrP Sc ) form. Here, we have studied the mechanism of the unfolding of the human prion protein (huPrP) using a set of extensive well-tempered metadynamics simulations. Through multiple microsecond-long metadynamics simulations, we find several possible unfolding pathways. We show that each pathway leads to an unfolded state of lower free energy than the native state. Thus, our study may point to the signature of a PrP C* form that corresponds to a global minimum on the conformational free-energy landscape. Moreover, we find that these global minima states do not involve an increased β-sheet content, as was assumed to be a signature of PrP Sc formation in previous simulation studies. We have further analyzed the origin of metastability of the PrP C form through free-energy surfaces of the chopped helical segments to show that the helices, particularly H2 and H3 of the prion protein, have the tendency to form either a random coil or a β-structure. Therefore, the secondary structural elements of the prion protein are only weakly stabilized by tertiary contacts and solvation forces so that relatively weak perturbations induced by temperature, pressure, pH, and so forth can lead to substantial unfolding with characteristics of intrinsically disordered proteins.

Population-Level Retrospective Study of Neurologically Expressed Disorders in Ruminants before the Onset of Bovine Spongiform Encephalopathy (BSE) in Belgium, a BSE Risk III Country

PubMed Central

Saegerman, C.; Berkvens, D.; Claes, L.; Dewaele, A.; Coignoul, F.; Ducatelle, R.; Cassart, D.; Brochier, B.; Costy, F.; Roels, S.; Deluyker, H.; Vanopdenbosch, E.; Thiry, E.

2005-01-01

A retrospective epidemiological study (n = 7,875) of neurologically expressed disorders (NED) in ruminants before the onset of the bovine spongiform encephalopathy epidemic (years studied, 1980 to 1997) was carried out in Belgium. The archives of all veterinary laboratories and rabies and transmissible spongiform encephalopathy (TSE) epidemiosurveillance networks were consulted. For all species, a significantly higher number of NED with virological causes (rabies) was reported south of the Sambre-Meuse Valley. During the period 1992 to 1997, for which the data were complete, (i) the predicted annual incidence of NED varied significantly as a function of species and area (higher numbers in areas where rabies was present) but was always above 100 cases per million, and (ii) the mean incidence of suspected TSE cases and, among them, those investigated by histopathological examination varied significantly as a function of species and area. The positive predictive value of a presumptive clinical diagnosis of NED ranged from 0.13 (game) to 0.63 (sheep). Knowledge of the positive predictive value permits the definition of a reference point before certain actions (e.g., awareness and training campaigns) are undertaken. It also shows the usefulness of a systematic necropsy or complementary laboratory tests to establish an etiological diagnosis. TSE analysis of a small, targeted historical sampling (n = 48) permitted the confirmation of one case and uncovered another case of scrapie. The results of the present study help to develop and maintain the quality of the worldwide clinical epidemiological networks for TSE, especially in countries that in the past imported live animals, animal products, and feedstuffs from countries with TSE cases. PMID:15695693

Population-level retrospective study of neurologically expressed disorders in ruminants before the onset of bovine spongiform encephalopathy (BSE) in Belgium, a BSE risk III country.

PubMed

Saegerman, C; Berkvens, D; Claes, L; Dewaele, A; Coignoul, F; Ducatelle, R; Cassart, D; Brochier, B; Costy, F; Roels, S; Deluyker, H; Vanopdenbosch, E; Thiry, E

2005-02-01

A retrospective epidemiological study (n = 7,875) of neurologically expressed disorders (NED) in ruminants before the onset of the bovine spongiform encephalopathy epidemic (years studied, 1980 to 1997) was carried out in Belgium. The archives of all veterinary laboratories and rabies and transmissible spongiform encephalopathy (TSE) epidemiosurveillance networks were consulted. For all species, a significantly higher number of NED with virological causes (rabies) was reported south of the Sambre-Meuse Valley. During the period 1992 to 1997, for which the data were complete, (i) the predicted annual incidence of NED varied significantly as a function of species and area (higher numbers in areas where rabies was present) but was always above 100 cases per million, and (ii) the mean incidence of suspected TSE cases and, among them, those investigated by histopathological examination varied significantly as a function of species and area. The positive predictive value of a presumptive clinical diagnosis of NED ranged from 0.13 (game) to 0.63 (sheep). Knowledge of the positive predictive value permits the definition of a reference point before certain actions (e.g., awareness and training campaigns) are undertaken. It also shows the usefulness of a systematic necropsy or complementary laboratory tests to establish an etiological diagnosis. TSE analysis of a small, targeted historical sampling (n = 48) permitted the confirmation of one case and uncovered another case of scrapie. The results of the present study help to develop and maintain the quality of the worldwide clinical epidemiological networks for TSE, especially in countries that in the past imported live animals, animal products, and feedstuffs from countries with TSE cases.

Estimating prion adsorption capacity of soil by BioAssay of Subtracted Infectivity from Complex Solutions (BASICS).

PubMed

Wyckoff, A Christy; Lockwood, Krista L; Meyerett-Reid, Crystal; Michel, Brady A; Bender, Heather; VerCauteren, Kurt C; Zabel, Mark D

2013-01-01

Prions, the infectious agent of scrapie, chronic wasting disease and other transmissible spongiform encephalopathies, are misfolded proteins that are highly stable and resistant to degradation. Prions are known to associate with clay and other soil components, enhancing their persistence and surprisingly, transmissibility. Currently, few detection and quantification methods exist for prions in soil, hindering an understanding of prion persistence and infectivity in the environment. Variability in apparent infectious titers of prions when bound to soil has complicated attempts to quantify the binding capacity of soil for prion infectivity. Here, we quantify the prion adsorption capacity of whole, sandy loam soil (SLS) typically found in CWD endemic areas in Colorado; and purified montmorillonite clay (Mte), previously shown to bind prions, by BioAssay of Subtracted Infectivity in Complex Solutions (BASICS). We incubated prion positive 10% brain homogenate from terminally sick mice infected with the Rocky Mountain Lab strain of mouse-adapted prions (RML) with 10% SLS or Mte. After 24 hours samples were centrifuged five minutes at 200 × g and soil-free supernatant was intracerebrally inoculated into prion susceptible indicator mice. We used the number of days post inoculation to clinical disease to calculate the infectious titer remaining in the supernatant, which we subtracted from the starting titer to determine the infectious prion binding capacity of SLS and Mte. BASICS indicated SLS bound and removed ≥ 95% of infectivity. Mte bound and removed lethal doses (99.98%) of prions from inocula, effectively preventing disease in the mice. Our data reveal significant prion-binding capacity of soil and the utility of BASICS to estimate prion loads and investigate persistence and decomposition in the environment. Additionally, since Mte successfully rescued the mice from prion disease, Mte might be used for remediation and decontamination protocols.

Removal process of prion and parvovirus from human platelet lysates used as clinical-grade supplement for ex vivo cell expansion.

PubMed

Kao, Yu-Chun; Bailey, Andy; Samminger, Bernhard; Tanimoto, Junji; Burnouf, Thierry

2016-07-01

Pooled human platelet lysate (HPL) is becoming the new gold standard as supplement for ex vivo cell culture for clinical protocols. However, the risk of pathogen contamination of HPL increases with the platelet pool size. We hypothesized that hollow fiber anion exchange membrane chromatography using QyuSpeed D (QSD) could remove resistant and untested bloodborne pathogens, such as parvoviruses and prions, from HPL-supplemented growth media without substantially affecting their capacity to support ex vivo cell expansion. Frozen or thawed platelet concentrates were serum-converted and centrifuged for obtaining HPL that was added to various growth media (ca. 100 mL), filtered through a 0.6-mL QSD membrane and characterized for proteins, growth factors and chemical composition. Capacity to expand Chinese hamster ovary, periodontal ligament, gingival fibroblast cells and Wharton's jelly mesenchymal stromal cells was studied. Removal of porcine parvovirus (PPV) and of the 263K prion strain of hamster-adapted scrapie was studied by spiking experiments following international guidelines. QSD had minimal impact on HPL-supplemented medium composition in proteins, growth factors and chemical content, nor capacity to expand and differentiate cells. In addition, QSD could remove ≥5.58 log10 [TCID50/mL] and ≥3.72 log10 of PPV and the 263K prion, respectively. QSD hollow fiber chromatography can be used to improve the virus and prion safety of HPL-supplemented media to safely expand cells for clinical protocols. These data bring new perspectives for increasingly safer use of pooled HPL in cell therapy and regenerative medicine applications. Copyright © 2016 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.

Insect Cell-Derived Cofactors Become Fully Functional after Proteinase K and Heat Treatment for High-Fidelity Amplification of Glycosylphosphatidylinositol-Anchored Recombinant Scrapie and BSE Prion Proteins

PubMed Central

Imamura, Morikazu; Kato, Nobuko; Okada, Hiroyuki; Yoshioka, Miyako; Iwamaru, Yoshifumi; Shimizu, Yoshihisa; Mohri, Shirou; Yokoyama, Takashi; Murayama, Yuichi

2013-01-01

The central event in prion infection is the conformational conversion of host-encoded cellular prion protein (PrPC) into the pathogenic isoform (PrPSc). Diverse mammalian species possess the cofactors required for in vitro replication of PrPSc by protein-misfolding cyclic amplification (PMCA), but lower organisms, such as bacteria, yeasts, and insects, reportedly lack the essential cofactors. Various cellular components, such as RNA, lipids, and other identified cofactor molecules, are commonly distributed in both eukaryotes and prokaryotes, but the reasons for the absence of cofactor activity in lower organisms remain to be elucidated. Previously, we reported that brain-derived factors were necessary for the in vitro replication of glycosylphosphatidylinositol-anchored baculovirus-derived recombinant PrP (Bac-PrP). Here, we demonstrate that following protease digestion and heat treatment, insect cell lysates had the functional cofactor activity required for Bac-PrP replication by PMCA. Mammalian PrPSc seeds and Bac-PrPSc generated by PMCA using Bac-PrP and insect cell-derived cofactors showed similar pathogenicity and produced very similar lesions in the brains of inoculated mice. These results suggested that the essential cofactors required for the high-fidelity replication of mammalian PrPSc were present in the insect cells but that the cofactor activity was masked or inhibited in the native state. We suggest that not only RNA, but also DNA, are the key components of PMCA, although other cellular factors were necessary for the expression of the cofactor activity of nucleic acids. PMCA using only insect cell-derived substances (iPMCA) was highly useful for the ultrasensitive detection of PrPSc of some prion strains. PMID:24367521

Transmissible spongiform encephalopathies in non-domestic animals: origin, transmission and risk factors.

PubMed

Williams, E S; Miller, M W

2003-04-01

The transmissible spongiform encephalopathies (TSEs), scrapie and bovine spongiform encephalopathy (BSE), are serious diseases of domestic animals. Although not as significant in terms of numbers of animals affected or geographical distribution, TSEs also affect non-domestic animals. Transmissible mink encephalopathy (TME) was the first TSE to be identified in non-domestic animals. This disease of captive mink (Mustela vison) is very rare and is associated with exposure through feed contaminated by a TSE agent. The second TSE to be identified in non-domestic animals was chronic wasting disease (CWD) of deer and elk. This disease is not known to be associated with feedstuffs contaminated with the agent of CWD, but the natural route of exposure appears to be oral, possibly through direct interaction between animals or through environmental contamination. Over the last five years, the known distribution of CWD across North America has expanded, increasing concerns over the impact of this disease on populations of free-ranging cervids and the viability of game farming industries. Concurrent with the epidemic of BSE, a variety of non-domestic ruminants and felid species were also affected in the United Kingdom, presumably through exposure to the agent in contaminated feed. These examples illustrate that when non-domestic animals are held in captivity, they depend upon feeds supplied by their caretakers and may show degrees of susceptibility to infectious agents in feeds which vary from those of domestic species. Although humans have less influence over exposure of free-ranging species to infectious agents, monitoring these populations for diseases may be important for managing the health of these animals. It is important to institute or continue surveillance for an entire range of infectious diseases, including TSEs, in free-ranging and captive non-domestic species. Study of diseases in these species may provide important information about infectious agents of concern for domestic animals and humans.

Risk of transmission of bovine spongiform encephalopathy to humans in the United States: report of the Council on Scientific Affairs. American Medical Association.

PubMed

Tan, L; Williams, M A; Khan, M K; Champion, H C; Nielsen, N H

The risk of possible transmission of bovine spongiform encephalopathy (BSE) in the United States is a substantial public health concern. To systematically review the current scientific literature and discuss legislation and regulations that have been implemented to prevent the disease. Literature review using the MEDLINE, EMBASE, and Lexis/Nexis databases for 1975 through 1997 on the terms bovine spongiform encephalopathy, prion diseases, prions, and Creutzfeldt-Jakob syndrome. The Internet was used to identify regulatory actions and health surveillance. MEDLINE, EMBASE, and Lexis/Nexis databases were searched from 1975 through 1997 for English-language articles that provided information on assessment of transmission risk. Unique circumstances in the United Kingdom caused the emergence and propagation of BSE in cattle, including widespread use of meat and bonemeal cattle feed derived from scrapie-infected sheep and the adoption of a new type of processing that did not reduce the amount of infectious prions prior to feeding. Many of these circumstances do not exist in the United States. In the United Kingdom, new variant Creutzfeldt-Jakob disease probably resulted from the ingestion of BSE-contaminated processed beef. The United Kingdom and the European Union now have strong regulations in place to stop the spread of BSE. While BSE has not been observed in the United States, the US government has surveillance and response plans in effect. Current risk of transmission of BSE in the United States is minimal because (1) BSE has not been shown to exist in this country; (2) adequate regulations exist to prevent entry of foreign sources of BSE into the United States; (3) adequate regulations exist to prevent undetected cases of BSE from uncontrolled amplification within the US cattle population; and (4) adequate preventive guidelines exist to prevent high-risk bovine materials from contaminating products intended for human consumption.

The first report of prion-related protein gene (PRNT) polymorphisms in goat.

PubMed

Kim, Yong-Chan; Jeong, Byung-Hoon

2017-06-01

Prion protein is encoded by the prion protein gene (PRNP). Polymorphisms of several members of the prion gene family have shown association with prion diseases in several species. Recent studies on a novel member of the prion gene family in rams have shown that prion-related protein gene (PRNT) has a linkage with codon 26 of prion-like protein (PRND). In a previous study, codon 26 polymorphism of PRND has shown connection with PRNP haplotype which is strongly associated with scrapie vulnerability. In addition, the genotype of a single nucleotide polymorphism (SNP) at codon 26 of PRND is related to fertilisation capacity. These findings necessitate studies on the SNP of PRNT gene which is connected with PRND. In goat, several polymorphism studies have been performed for PRNP, PRND, and shadow of prion protein gene (SPRN). However, polymorphism on PRNT has not been reported. Hence, the objective of this study was to determine the genotype and allelic distribution of SNPs of PRNT in 238 Korean native goats and compare PRNT DNA sequences between Korean native goats and several ruminant species. A total of five SNPs, including PRNT c.-114G > T, PRNT c.-58A > G in the upstream of PRNT gene, PRNT c.71C > T (p.Ala24Val) and PRNT c.102G > A in the open reading frame (ORF) and c.321C > T in the downstream of PRNT gene, were found in this study. All five SNPs of caprine PRNT gene in Korean native goat are in complete linkage disequilibrium (LD) with a D' value of 1.0. Interestingly, comparative sequence analysis of the PRNT gene revealed five mismatches between DNA sequences of Korean native goats and those of goats deposited in the GenBank. Korean native black goats also showed 5 mismatches in PRNT ORF with cattle. To the best of our knowledge, this is the first genetic research of the PRNT gene in goat.

The roles of the conserved tyrosine in the β2-α2 loop of the prion protein.

PubMed

Huang, Danzhi; Caflisch, Amedeo

2015-01-01

Prions cause neurodegenerative diseases for which no cure exists. Despite decades of research activities the function of the prion protein (PrP) in mammalians is not known. Moreover, little is known on the molecular mechanisms of the self-assembly of the PrP from its monomeric state (cellular PrP, PrP(C)) to the multimeric state. The latter state includes the toxic species (scrapie PrP, PrP(Sc)) knowledge of which would facilitate the development of drugs against prion diseases. Here we analyze the role of a tyrosine residue (Y169) which is strictly conserved in mammalian PrPs. Nuclear magnetic resonance (NMR) spectroscopy studies of many mammalian PrP(C) proteins have provided evidence of a conformational equilibrium between a 3(10)-helical turn and a type I β turn conformation in the β2-α2 loop (residues 165-175). In vitro cell-free experiments of the seeded conversion of PrP(C) indicate that non-aromatic residues at position 169 reduce the formation of proteinase K-resistant PrP. Recent molecular dynamics (MD) simulations of monomeric PrP and several single-point mutants show that Y169 stabilizes the 3(10)-helical turn conformation more than single-point mutants at position 169 or residues in contact with it. In the 3(10)-helical turn conformation the hydrophobic and aggregation-prone segment 169-YSNQNNF-175 is buried and thus not-available for self-assembly. From the combined analysis of simulation and experimental results it emerges that Y169 is an aggregation gatekeeper with a twofold role. Mutations related to 3 human prion diseases are interpreted on the basis of the gatekeeper role in the monomeric state. Another potential role of the Y169 side chain is the stabilization of the ordered aggregates, i.e., reduction of frangibility of filamentous protofibrils and fibrils, which is likely to reduce the generation of toxic species.

Risk management of transmissible spongiform encephalopathies in Asia.

PubMed

Ozawa, Y

2003-04-01

A questionnaire-based survey was distributed to the Office International des Epizooties (OIE: World organisation for animal health) Member Countries in Asia to assess the use of risk management for transmissible spongiform encephalopathies. The author presents a summary of 16 responses received in July 2002. The survey revealed that import risk analysis on bovine spongiform encephalopathy (BSE) is not routinely carried out in ten countries, indicating an urgent need for further training courses. Although the number of ruminants imported from Europe is relatively small, significant quantities of feedstuffs of ruminant origin have been imported into Asia, which may mean that the BSE agent could have reached domestic cattle in most countries. The external challenge has been considerably reduced in recent years as most countries in Asia banned the importation of feedstuffs from countries with BSE, but a few weak spots which enable imports of risk materials still persist. Recycling of BSE through rendering plants is unlikely but cannot be totally excluded in some countries such as the People's Republic of China, India, Japan, Pakistan and Taipei China. Therefore, much more stringent management at slaughterhouses and rendering plants, as well as extensive surveillance programmes, are required in those countries. Bovine spongiform encephalopathy is not notifiable in six countries, indicating a total absence of risk management of BSE in those countries. Immediate actions by these governments to declare BSE a notifiable disease are considered necessary. Numbers of specimens tested for BSE are still very small in most countries in Asia, indicating a pressing need to upgrade surveillance programmes by introducing modern (economically affordable) diagnostic methods and by conducting practical training courses on epidemiological surveillance systems. With the exception of Japan, very little work has been performed on scrapie in Asia although the disease has been routinely monitored in the People's Republic of China, India, Myanmar, Pakistan and Taipei China.

Methamphetamine increases Prion Protein and induces dopamine-dependent expression of protease resistant PrPsc.

PubMed

Ferrucci, M; Ryskalin, L; Biagioni, F; Gambardella, S; Busceti, C L; Falleni, A; Lazzeri, G; Fornai, F

2017-07-01

The cellular prion protein (PrPc) is physiologically expressed within selective brain areas of mammals. Alterations in the secondary structure of this protein lead to scrapie-like prion protein (PrPsc), which precipitates in the cell. PrPsc has been detected in infectious, inherited or sporadic neurodegenerative disorders. Prion protein metabolism is dependent on autophagy and ubiquitin proteasome. Despite not being fully elucidated, the physiological role of prion protein relates to chaperones which rescue cells under stressful conditions.Methamphetamine (METH) is a widely abused drug which produces oxidative stress in various brain areas causing mitochondrial alterations and protein misfolding. These effects produce a compensatory increase of chaperones while clogging cell clearing pathways. In the present study, we explored whether METH administration modifies the amount of PrPc. Since high levels of PrPc when the clearing systems are clogged may lead to its misfolding into PrPsc, we further tested whether METH exposure triggers the appearance of PrPsc. We analysed the effects of METH and dopamine administration in PC12 and striatal cells by using SDS-PAGE Coomassie blue, immune- histochemistry and immune-gold electron microscopy. To analyze whether METH administration produces PrPsc aggregates we used antibodies directed against PrP following exposure to proteinase K or sarkosyl which digest folded PrPc but misfolded PrPsc. We fond that METH triggers PrPsc aggregates in DA-containing cells while METH is not effective in primary striatal neurons which do not produce DA. In the latter cells exogenous DA is needed to trigger PrPsc accumulation similarly to what happens in DA containing cells under the effects of METH. The present findings, while fostering novel molecular mechanisms involving prion proteins, indicate that, cell pathology similar to prion disorders can be mimicked via a DA-dependent mechanism by a drug of abuse.

Chimeric elk/mouse prion proteins in transgenic mice.

PubMed

Tamgüney, Gültekin; Giles, Kurt; Oehler, Abby; Johnson, Natrina L; DeArmond, Stephen J; Prusiner, Stanley B

2013-02-01

Chronic wasting disease (CWD) of deer and elk is a highly communicable neurodegenerative disorder caused by prions. Investigations of CWD are hampered by slow bioassays in transgenic (Tg) mice. Towards the development of Tg mice that will be more susceptible to CWD prions, we created a series of chimeric elk/mouse transgenes that encode the N terminus of elk PrP (ElkPrP) up to residue Y168 and the C terminus of mouse PrP (MoPrP) beyond residue 169 (mouse numbering), designated Elk3M(SNIVVK). Between codons 169 and 219, six residues distinguish ElkPrP from MoPrP: N169S, T173N, V183I, I202V, I214V and R219K. Using chimeric elk/mouse PrP constructs, we generated 12 Tg mouse lines and determined incubation times after intracerebral inoculation with the mouse-passaged RML scrapie or Elk1P CWD prions. Unexpectedly, one Tg mouse line expressing Elk3M(SNIVVK) exhibited incubation times of <70 days when inoculated with RML prions; a second line had incubation times of <90 days. In contrast, mice expressing full-length ElkPrP had incubation periods of >250 days for RML prions. Tg(Elk3M,SNIVVK) mice were less susceptible to CWD prions than Tg(ElkPrP) mice. Changing three C-terminal mouse residues (202, 214 and 219) to those of elk doubled the incubation time for mouse RML prions and rendered the mice resistant to Elk1P CWD prions. Mutating an additional two residues from mouse to elk at codons 169 and 173 increased the incubation times for mouse prions to >300 days, but made the mice susceptible to CWD prions. Our findings highlight the role of C-terminal residues in PrP that control the susceptibility and replication of prions.

Conserved properties of human and bovine prion strains on transmission to guinea pigs

PubMed Central

Safar, Jiri G.; Giles, Kurt; Lessard, Pierre; Letessier, Frederic; Patel, Smita; Serban, Ana; DeArmond, Stephen J.; Prusiner, Stanley B.

2011-01-01

The first transmissions of human prion diseases to rodents used guinea pigs (Gps, Cavia porcellus). Later, transgenic (Tg) mice expressing human or chimeric human/mouse PrP replaced Gps, but the small size of the mouse limits some investigations. To investigate the fidelity of strain-specific prion transmission to Gps, we inoculated “type 1” and “type 2” prion strains into Gps: we measured the incubation times and determined the strain-specified size of the unglycosylated, protease-resistant (r) PrPSc fragment. Prions passaged once in Gps from cases of sporadic (s) Creutzfeldt–Jakob disease (CJD) and Gerstmann-Sträussler-Scheinker (GSS) disease caused by the P102L mutation were used as well as human prions from a variant (v) CJD case, bovine prions from bovine spongiform encephalopathy (BSE), and mouse-passaged scrapie prions. Variant CJD and BSE prions transmitted to all the inoculated Gps with incubation times of 367 ± 4 d and 436 ± 28 d, respectively. On second passage in Gps, vCJD and BSE prions caused disease in 287 ± 4 d and 310 ± 4 d, while sCJD and GSS prions transmitted in 237 ± 4 d and 279 ± 19 d, respectively. Although hamster Sc237 prions transmitted to 2 of 3 Gps after 574 and 792 d, mouse-passaged RML and 301V prion strains, the latter derived from BSE prions, failed to transmit disease to Gps. Those Gps inoculated with vCJD or BSE prions exhibited “type 2” unglycosylated, rPrPSc (19 kDa) while those receiving sCJD or GSS prions displayed “type 1” prions (21 kDa), as determined by Western blotting. Such strain-specific properties were maintained in Gps as well as mice expressing a chimeric human/mouse transgene. Gps may prove particularly useful in further studies of novel human prions such as those causing vCJD. PMID:21727894

Shaking Alone Induces De Novo Conversion of Recombinant Prion Proteins to β-Sheet Rich Oligomers and Fibrils

PubMed Central

Ladner-Keay, Carol L.; Griffith, Bethany J.; Wishart, David S.

2014-01-01

The formation of β-sheet rich prion oligomers and fibrils from native prion protein (PrP) is thought to be a key step in the development of prion diseases. Many methods are available to convert recombinant prion protein into β-sheet rich fibrils using various chemical denaturants (urea, SDS, GdnHCl), high temperature, phospholipids, or mildly acidic conditions (pH 4). Many of these methods also require shaking or another form of agitation to complete the conversion process. We have identified that shaking alone causes the conversion of recombinant PrP to β-sheet rich oligomers and fibrils at near physiological pH (pH 5.5 to pH 6.2) and temperature. This conversion does not require any denaturant, detergent, or any other chemical cofactor. Interestingly, this conversion does not occur when the water-air interface is eliminated in the shaken sample. We have analyzed shaking-induced conversion using circular dichroism, resolution enhanced native acidic gel electrophoresis (RENAGE), electron microscopy, Fourier transform infrared spectroscopy, thioflavin T fluorescence and proteinase K resistance. Our results show that shaking causes the formation of β-sheet rich oligomers with a population distribution ranging from octamers to dodecamers and that further shaking causes a transition to β-sheet fibrils. In addition, we show that shaking-induced conversion occurs for a wide range of full-length and truncated constructs of mouse, hamster and cervid prion proteins. We propose that this method of conversion provides a robust, reproducible and easily accessible model for scrapie-like amyloid formation, allowing the generation of milligram quantities of physiologically stable β-sheet rich oligomers and fibrils. These results may also have interesting implications regarding our understanding of prion conversion and propagation both within the brain and via techniques such as protein misfolding cyclic amplification (PMCA) and quaking induced conversion (QuIC). PMID:24892647

Detecting animal by-product intake using stable isotope ratio mass spectrometry (IRMS).

PubMed

da Silva, D A F; Biscola, N P; Dos Santos, L D; Sartori, M M P; Denadai, J C; da Silva, E T; Ducatti, C; Bicudo, S D; Barraviera, B; Ferreira, R S

2016-11-01

Sheep are used in many countries as food and for manufacturing bioproducts. However, when these animals consume animal by-products (ABP), which is widely prohibited, there is a risk of transmitting scrapie - a fatal prion disease in human beings. Therefore, it is essential to develop sensitive methods to detect previous ABP intake to select safe animals for producing biopharmaceuticals. We used stable isotope ratio mass spectrometry (IRMS) for 13 C and 15 N to trace animal proteins in the serum of three groups of sheep: 1 - received only vegetable protein (VP) for 89 days; 2 - received animal and vegetable protein (AVP); and 3 - received animal and vegetable protein with animal protein subsequently removed (AVPR). Groups 2 and 3 received diets with 30% bovine meat and bone meal (MBM) added to a vegetable diet (from days 16-89 in the AVP group and until day 49 in the AVPR group, when MBM was removed). The AVPR group showed 15 N equilibrium 5 days after MBM removal (54th day). Conversely, 15 N equilibrium in the AVP group occurred 22 days later (76th day). The half-life differed between these groups by 3.55 days. In the AVPR group, 15 N elimination required 53 days, which was similar to this isotope's incorporation time. Turnover was determined based on natural 15 N signatures. IRMS followed by turnover calculations was used to evaluate the time period for the incorporation and elimination of animal protein in sheep serum. The δ 13 C and δ 15 N values were used to track animal protein in the diet. This method is biologically and economically relevant for the veterinary field because it can track protein over time or make a point assessment of animal feed with high sensitivity and resolution, providing a low-cost analysis coupled with fast detection. Isotopic profiles could be measured throughout the experimental period, demonstrating the potential to use the method for traceability and certification assessments. Copyright © 2016 Elsevier Ltd. All rights reserved.

Different Molecular Mechanisms Mediate Direct or Glia-Dependent Prion Protein Fragment 90-231 Neurotoxic Effects in Cerebellar Granule Neurons.

PubMed

Thellung, Stefano; Gatta, Elena; Pellistri, Francesca; Villa, Valentina; Corsaro, Alessandro; Nizzari, Mario; Robello, Mauro; Florio, Tullio

2017-10-01

Glia over-stimulation associates with amyloid deposition contributing to the progression of central nervous system neurodegenerative disorders. Here we analyze the molecular mechanisms mediating microglia-dependent neurotoxicity induced by prion protein (PrP)90-231, an amyloidogenic polypeptide corresponding to the protease-resistant portion of the pathological prion protein scrapie (PrP Sc ). PrP90-231 neurotoxicity is enhanced by the presence of microglia within neuronal culture, and associated to a rapid neuronal [Ca ++ ] i increase. Indeed, while in "pure" cerebellar granule neuron cultures, PrP90-231 causes a delayed intracellular Ca ++ entry mediated by the activation of NMDA receptors; when neuron and glia are co-cultured, a transient increase of [Ca ++ ] i occurs within seconds after treatment in both granule neurons and glial cells, then followed by a delayed and sustained [Ca ++ ] i raise, associated with the induction of the expression of inducible nitric oxide synthase and phagocytic NADPH oxidase. [Ca ++ ] i fast increase in neurons is dependent on the activation of multiple pathways since it is not only inhibited by the blockade of voltage-gated channel activity and NMDA receptors but also prevented by the inhibition of nitric oxide and PGE 2 release from glial cells. Thus, Ca ++ homeostasis alteration, directly induced by PrP90-231 in cerebellar granule cells, requires the activation of NMDA receptors, but is greatly enhanced by soluble molecules released by activated glia. In glia-enriched cerebellar granule cultures, the activation of inducible nitric oxide (iNOS) and NADPH oxidase represents the main mechanism of toxicity since their pharmacological inhibition prevented PrP90-231 neurotoxicity, whereas NMDA blockade by D(-)-2-amino-5-phosphonopentanoic acid is ineffective; conversely, in pure cerebellar granule cultures, NMDA blockade but not iNOS inhibition strongly reduced PrP90-231 neurotoxicity. These data indicate that amyloidogenic peptides induce neurotoxic signals via both direct neuron interaction and glia activation through different mechanisms responsible of calcium homeostasis disruption in neurons and potentiating each other: the activation of excitotoxic pathways via NMDA receptors and the release of radical species that establish an oxidative milieu.

Removal of transmissible spongiform encephalopathy prion from large volumes of cell culture media supplemented with fetal bovine serum by using hollow fiber anion-exchange membrane chromatography.

PubMed

Chou, Ming Li; Bailey, Andy; Avory, Tiffany; Tanimoto, Junji; Burnouf, Thierry

2015-01-01

Cases of variant Creutzfeldt-Jakob disease in people who had consumed contaminated meat products from cattle with bovine spongiform encephalopathy emphasize the need for measures aimed at preventing the transmission of the pathogenic prion protein (PrPSc) from materials derived from cattle. Highly stringent scrutiny is required for fetal bovine serum (FBS), a growth-medium supplement used in the production of parenteral vaccines and therapeutic recombinant proteins and in the ex vivo expansion of stem cells for transplantation. One such approach is the implementation of manufacturing steps dedicated to removing PrPSc from materials containing FBS. We evaluated the use of the QyuSpeed D (QSD) adsorbent hollow-fiber anion-exchange chromatographic column (Asahi Kasei Medical, Tokyo, Japan) for the removal of PrPSc from cell culture media supplemented with FBS. We first established that QSD filtration had no adverse effect on the chemical composition of various types of culture media supplemented with 10% FBS or the growth and viability characteristics of human embryonic kidney (HEK293) cells, baby hamster kidney (BHK-21) cells, African green monkey kidney (Vero) cells, and Chinese hamster ovary (CHO-k1) cells propagated in the various culture-medium filtrates. We used a 0.6-mL QSD column for removing PrPSc from up to 1000 mL of Dulbecco's modified Eagle's medium containing 10% FBS previously spiked with the 263K strain of hamster-adapted scrapie. The Western blot analysis, validated alongside an infectivity assay, revealed that the level of PrPSc in the initial 200mL flow-through was reduced by 2.5 to > 3 log10, compared with that of the starting material. These results indicate that QSD filtration removes PrPSc from cell culture media containing 10% FBS, and demonstrate the ease with which QSD filtration can be implemented in at industrial-scale to improve the safety of vaccines, therapeutic recombinant proteins, and ex vivo expanded stem cells produced using growth media supplemented with FBS.

Removal of Transmissible Spongiform Encephalopathy Prion from Large Volumes of Cell Culture Media Supplemented with Fetal Bovine Serum by Using Hollow Fiber Anion-Exchange Membrane Chromatography

PubMed Central

Chou, Ming Li; Bailey, Andy; Avory, Tiffany; Tanimoto, Junji; Burnouf, Thierry

2015-01-01

Cases of variant Creutzfeldt-Jakob disease in people who had consumed contaminated meat products from cattle with bovine spongiform encephalopathy emphasize the need for measures aimed at preventing the transmission of the pathogenic prion protein (PrPSc) from materials derived from cattle. Highly stringent scrutiny is required for fetal bovine serum (FBS), a growth-medium supplement used in the production of parenteral vaccines and therapeutic recombinant proteins and in the ex vivo expansion of stem cells for transplantation. One such approach is the implementation of manufacturing steps dedicated to removing PrPSc from materials containing FBS. We evaluated the use of the QyuSpeed D (QSD) adsorbent hollow-fiber anion-exchange chromatographic column (Asahi Kasei Medical, Tokyo, Japan) for the removal of PrPSc from cell culture media supplemented with FBS. We first established that QSD filtration had no adverse effect on the chemical composition of various types of culture media supplemented with 10% FBS or the growth and viability characteristics of human embryonic kidney (HEK293) cells, baby hamster kidney (BHK-21) cells, African green monkey kidney (Vero) cells, and Chinese hamster ovary (CHO-k1) cells propagated in the various culture-medium filtrates. We used a 0.6-mL QSD column for removing PrPSc from up to 1000 mL of Dulbecco’s modified Eagle’s medium containing 10% FBS previously spiked with the 263K strain of hamster-adapted scrapie. The Western blot analysis, validated alongside an infectivity assay, revealed that the level of PrPSc in the initial 200mL flow-through was reduced by 2.5 to > 3 log10, compared with that of the starting material. These results indicate that QSD filtration removes PrPSc from cell culture media containing 10% FBS, and demonstrate the ease with which QSD filtration can be implemented in at industrial-scale to improve the safety of vaccines, therapeutic recombinant proteins, and ex vivo expanded stem cells produced using growth media supplemented with FBS. PMID:25874629

Protein Adhesion and Ion Substitution (on/in)to Minerals

NASA Astrophysics Data System (ADS)

Charlet, L.; Fernandez Martinez, A.; Chapron, Y.; Sahai, N.; Cuello, G.; Brendle, J.; Marichal, C.

2008-12-01

Arsenic and pathogenic prion protein-scrapie (PrPsc) are important contaminants which may soil and water for decades, unless they are removed by sorption. Two sorption mechanisms will be discussed, namely the organics (Prp and single aminoacid) adsorption on clay and the arsenic substitution in gypsum. The elucidation of these contrasted mechanisms will be shown to request complementary molecular-mechanical simulations with experimental spectroscopic investigations. As first example, structural studies performed at ILL/ESRF on As-doped gypsum (CaSO4 2H2O) using neutron and X-ray diffraction data and EXAFS were performed to determine how As fits into the bulk of gypsum structure. The combined Rietveld analysis of neutron and X-ray diffraction data shows an expansion of the unit cell volume proportional to the As concentration within the samples. to-sulfate substitution mechanisms were used as simulation starting hypotheses. DFT-based simulations (Mulliken analysis) were used to interpret charge distribution and to show that among the possible mechanisms, a sulphate substitution by either protonated, or fully deprotonated, arsenate ion, only the protonated arsenate substitution could best fit the EXAFS data. In the second example, we used Molecular Dynamics to understand the mechanism of strong binding of the pathogenic PrP peptide with clay mineral surfaces. We modeled only the infectious moiety, PrP92-138, of the whole PrPsc structure, with explicitly solvating water molecules in contact with the cleavage plane of pyrophillite, as a model for montmorillonite without any cationic substitution. Partial residual negative charges on the cleavage plane were balanced with K+ ions. The peptide anchored to the clay surface via up to 10 hydrogen bonds from lysine and histidine residues to oxygen atoms of the siloxane cavities, and a total adsorption energy of 3465 KJ.mol-1 was obtained. Our results were compared to the one obtained by chemical and thermal analysis, 23Na, 1H, 13C solid state NMR and MD computation on sorption of single lysine amino acid on model synthetic Na-montmorillonite. Our data provide further insight about interactions between lysine and montmorillonite which depend strongly on lysine concentration.

Metal Dyshomeostasis and Their Pathological Role in Prion and Prion-Like Diseases: The Basis for a Nutritional Approach

PubMed Central

Toni, Mattia; Massimino, Maria L.; De Mario, Agnese; Angiulli, Elisa; Spisni, Enzo

2017-01-01

Metal ions are key elements in organisms' life acting like cofactors of many enzymes but they can also be potentially dangerous for the cell participating in redox reactions that lead to the formation of reactive oxygen species (ROS). Any factor inducing or limiting a metal dyshomeostasis, ROS production and cell injury may contribute to the onset of neurodegenerative diseases or play a neuroprotective action. Transmissible spongiform encephalopathies (TSEs), also known as prion diseases, are a group of fatal neurodegenerative disorders affecting the central nervous system (CNS) of human and other mammalian species. The causative agent of TSEs is believed to be the scrapie prion protein PrPSc, the β sheet-rich pathogenic isoform produced by the conformational conversion of the α-helix-rich physiological isoform PrPC. The peculiarity of PrPSc is its ability to self-propagate in exponential fashion in cells and its tendency to precipitate in insoluble and protease-resistance amyloid aggregates leading to neuronal cell death. The expression “prion-like diseases” refers to a group of neurodegenerative diseases that share some neuropathological features with prion diseases such as the involvement of proteins (α-synuclein, amyloid β, and tau) able to precipitate producing amyloid deposits following conformational change. High social impact diseases such as Alzheimer's and Parkinson's belong to prion-like diseases. Accumulating evidence suggests that the exposure to environmental metals is a risk factor for the development of prion and prion-like diseases and that metal ions can directly bind to prion and prion-like proteins affecting the amount of amyloid aggregates. The diet, source of metal ions but also of natural antioxidant and chelating agents such as polyphenols, is an aspect to take into account in addressing the issue of neurodegeneration. Epidemiological data suggest that the Mediterranean diet, based on the abundant consumption of fresh vegetables and on low intake of meat, could play a preventive or delaying role in prion and prion-like neurodegenerative diseases. In this review, metal role in the onset of prion and prion-like diseases is dealt with from a nutritional, cellular, and molecular point of view. PMID:28154522

Reflections on a half-century in the field of transmissible spongiform encephalopathy.

PubMed

Brown, Paul

2009-01-01

The subject of transmissible spongiform encephalopathy may properly be said to have begun with the experimental transmission of scrapie by Cuillé and Chelle in 1936, although Creutzfeldt and Jakob had described the disease that bears their names in 1920-21. Thirty more years passed before the human disease was also shown to be transmissible, in 1966, and the following half century has seen the field move from classical biology to molecular biology and genetics, and from 'slow virus' to host-encoded 'prion' protein. Because nothing is more important to the research scientist than the process of seeing a problem and devising ways of solving it, and because we live and die by our publications, as much care should be given to these vehicles of our work and reputations as to the research itself. Four aspects have been chosen for comment: authorship, abbreviations, data presentation, and references. In addition to the 'science of research' there are several 'para-scientific' activities that may be categorized as 'the politics of research', which include administrative duties, committees (e.g., scientific meetings, grant organizations), journal/book editing, peer reviewing, and public relations Many young scientists are either unaware or dismissive of the importance of these 'scientific distractions', but their potential for influencing the direction of a field of research becomes increasingly evident as careers unfold. They are subject to uses and abuses, and some guidance and examples are given by way of illustration, particular attention being paid to the process of manuscript review which, because of its anonymity, is the most vulnerable to abuse. As public and government interest in prions wanes in parallel with the disappearance of iatrogenic and variant Creutzfeldt-Jakob disease, the flow of money to sustain research is in evident jeopardy. With an uncertain future, it nevertheless seems possible that one of two things may breathe new life into the field: either an unforeseen new outbreak of human disease will occur (as has happened in the past), or a cross-fertilization between prions and the larger family of protein misfolding diseases, especially Alzheimer's disease, will bear fruit. For obvious reasons, we should hope for the botanical alternative.

Distinct pathological phenotypes of Creutzfeldt-Jakob disease in recipients of prion-contaminated growth hormone.

PubMed

Cali, Ignazio; Miller, Cathleen J; Parisi, Joseph E; Geschwind, Michael D; Gambetti, Pierluigi; Schonberger, Lawrence B

2015-06-25

The present study compares the clinical, pathological and molecular features of a United States (US) case of growth hormone (GH)-associated Creutzfeldt-Jakob disease (GH-CJD) (index case) to those of two earlier referred US cases of GH-CJD and one case of dura mater (d)-associated CJD (dCJD). All iatrogenic CJD (iCJD) subjects were methionine (M) homozygous at codon 129 (129MM) of the prion protein (PrP) gene and had scrapie prion protein (PrP(Sc)) type 1 (iCJDMM1). The index subject presented with ataxia, weight loss and changes in the sleep pattern about 38 years after the midpoint of GH treatment. Autopsy examination revealed a neuropathological phenotype reminiscent of both sCJDMV2-K (a sporadic CJD subtype in subjects methionine/valine heterozygous at codon 129 with PrP(Sc) type 2 and the presence of kuru plaques) and variant CJD (vCJD). The two earlier cases of GH-CJDMM1 and the one of dCJDMM1 were associated with neuropathological phenotypes that differed from that of the index case mainly because they lacked PrP plaques. The phenotype of the earlier GH-CJDMM1 cases shared several, but not all, characteristics with sCJDMM1, whereas dCJDMM1 was phenotypically indistinguishable from sCJDMM1. Two distinct groups of dCJDMM1 have also been described in Japan based on clinical features, the presence or absence of PrP plaques and distinct PK-resistant PrP(Sc) (resPrP(Sc)) electrophoretic mobilities. The resPrP(Sc) electrophoretic mobility was, however, identical in our GH-CJDMM1 and dCJDMM1 cases, and matched that of sCJDMM1. Our study shows that receipt of prion-contaminated GH can lead to a prion disease with molecular features (129MM and PrP(Sc) type 2) and phenotypic characteristics that differ from those of sporadic prion disease (sCJDMM1), a difference that may reflect adaptation of "heterologous" prion strains to the 129MM background.

Solution structure of Syrian hamster prion protein rPrP(90-231).

PubMed

Liu, H; Farr-Jones, S; Ulyanov, N B; Llinas, M; Marqusee, S; Groth, D; Cohen, F E; Prusiner, S B; James, T L

1999-04-27

NMR has been used to refine the structure of Syrian hamster (SHa) prion protein rPrP(90-231), which is commensurate with the infectious protease-resistant core of the scrapie prion protein PrPSc. The structure of rPrP(90-231), refolded to resemble the normal cellular isoform PrPC spectroscopically and immunologically, has been studied using multidimensional NMR; initial results were published [James et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 10086-10091]. We now report refinement with better definition revealing important structural and dynamic features which can be related to biological observations pertinent to prion diseases. Structure refinement was based on 2778 unambiguously assigned nuclear Overhauser effect (NOE) connectivities, 297 ambiguous NOE restraints, and 63 scalar coupling constants (3JHNHa). The structure is represented by an ensemble of 25 best-scoring structures from 100 structures calculated using ARIA/X-PLOR and further refined with restrained molecular dynamics using the AMBER 4.1 force field with an explicit shell of water molecules. The rPrP(90-231) structure features a core domain (residues 125-228), with a backbone atomic root-mean-square deviation (RMSD) of 0.67 A, consisting of three alpha-helices (residues 144-154, 172-193, and 200-227) and two short antiparallel beta-strands (residues 129-131 and 161-163). The N-terminus (residues 90-119) is largely unstructured despite some sparse and weak medium-range NOEs implying the existence of bends or turns. The transition region between the core domain and flexible N-terminus, i.e., residues 113-128, consists of hydrophobic residues or glycines and does not adopt any regular secondary structure in aqueous solution. There are about 30 medium- and long-range NOEs within this hydrophobic cluster, so it clearly manifests structure. Multiple discrete conformations are evident, implying the possible existence of one or more metastable states, which may feature in conversion of PrPC to PrPSc. To obtain a more comprehensive picture of rPrP(90-231), dynamics have been studied using amide hydrogen-deuterium exchange and 15N NMR relaxation times (T1 and T2) and 15N{1H} NOE measurements. Comparison of the structure with previous reports suggests sequence-dependent features that may be reflected in a species barrier to prion disease transmission.

The dangers of disease transmission by artificial insemination and embryo transfer.

PubMed

Philpott, M

1993-01-01

This review summarizes the major infectious diseases of the three major agricultural species (cattle, sheep and pigs) and horses, and presents the evidence for and against the possibility of infectious agents being transmitted between animals via the venereal route or by the use of semen or early embryos in commercial artificial insemination (AI) or embryo transfer (ET). Cattle feature most prominently in the widespread distribution of frozen semen, and national and international organizations have set out guidelines to work towards disease-free bull studs with semen free from potential pathogens. With the control of major epizootic diseases, attention has been focused on such diseases as IBR, BVD and blue tongue, where clinical signs are rarely evident but the detection of virus in semen is of great importance. New information on the relevance of bacterial disease such as Mycobacterium paratuberculosis, campylobacteriosis and leptospirosis is reviewed, along with details of the mycoplasma and ureaplasma species of the bull's genital tract. Bovine spongiform encephalopathy (BSE) has attracted much research and semen is not regarded as a source of infection. New work on the pathogenesis of a number of diseases and the use of new biotechnology in diagnosis is included. The International Embryo Transfer Society (IETS) has encouraged a great deal of experimental work--much originating in Canada--on the risk of transmission of disease from donors to recipients via a 7-day-old blastocyst. There has been much success in demonstrating that with an approved protocol of handling the embryos, to date there is very little danger in disease transmission with both viruses and bacteria. The mycoplasma group appear more intractable and the role of BSE is still being evaluated. In sheep, scrapie, Brucella ovis infection and blue tongue feature in current work. In the pig there is a surge in international movement of pig semen, and Aujeszky's disease and the new so-called Blue Ear disease feature prominently. Much work is in progress on infectious agents likely to be found in the semen of stallions, with an expanding trade in the international movement of chilled and frozen semen. Equine embryo transfer experiments are hampered by the very limited number of embryos available. Reference is also made to the further risk of disease transmission by in vitro manipulated embryos.

Ecosystems supporting clusters of sporadic TSEs demonstrate excesses of the radical-generating divalent cation manganese and deficiencies of antioxidant co factors Cu, Se, Fe, Zn. Does a foreign cation substitution at prion protein's Cu domain initiate TSE?

PubMed

Purdey, M

2000-02-01

Analyses of food chains supporting isolated clusters of sporadic TSEs (CWD in N Colorado, scrapie in Iceland, CJD in Slovakia) demonstrate a consistent 2 1/2+ fold greater concentration of the pro-oxidant divalent cation, manganese (Mn), in relation to normal levels recorded in adjoining TSE-free localities. Deficiencies of the antioxidant co factors Cu/Se/Zn/Fe and Mg, P and Na were also consistently recorded in TSE foodchains. Similarities between the clinical/pathological profile of TSEs and Mn delayed psycho-neurotoxicity in miners are cited, and a novel theory generated which suggests that sporadic TSE results from early life dependence of TSE susceptible genotypes on ecosystems characterised by this specific pattern of mineral imbalance. Low Cu/Fe induces an excessive absorption of Mn in ruminants and an increased oxidation of Mn2+ into its pro oxidant species, Mn3+, which accumulates in mitochondria of CNS astrocytes in Mn SOD deficient genotypes. Deficiencies of scavenger co factors Cu/Zn/Se/Fe in the CNS permits Mn3+ initiated chain reactions of auto-oxidant mediated neuronal degeneration to proliferate, which, in turn, up-regulates the expression of the Cu-metalloprotein, prion protein (PrP). Once the rate of PrP turnover and its demand for Cu exceeds the already depleted supply of Cu within the CNS, PrP can no longer bind sufficient Cu to maintain its conformation. Mn3+ substitutes at the vacated Cu domain on PrP, thus priming up a latent capacity for lethal auto-oxidative activity to be carried along with PrP like a 'trojan horse'; where Mn 3+ serves as the integral 'infectious' transmissible component of the misfolded PrP-cation complex. The Mn overactivation of concanavalin A binding to glycoprotein and Mn-initiated autoxidation results in a diverse pathological profile involving receptor capping, aggregation/modification of CNS membrane/cytoskeletal proteins. TSE ensues. The BSE/nv CJD strain entails a 'synthetic' induction of the same CNS mineral disturbance, where 'in utero' exposure to Cu-chelating insecticides/Mn supplements accelerates the onset of a more virulent 'strain' of adolescent TSE. Copyright 2000 Harcourt Publishers Ltd.

Iatrogenic Creutzfeldt-Jakob disease with Amyloid-β pathology: an international study.

PubMed

Cali, Ignazio; Cohen, Mark L; Haїk, Stéphane; Parchi, Piero; Giaccone, Giorgio; Collins, Steven J; Kofskey, Diane; Wang, Han; McLean, Catriona A; Brandel, Jean-Philippe; Privat, Nicolas; Sazdovitch, Véronique; Duyckaerts, Charles; Kitamoto, Tetsuyuki; Belay, Ermias D; Maddox, Ryan A; Tagliavini, Fabrizio; Pocchiari, Maurizio; Leschek, Ellen; Appleby, Brian S; Safar, Jiri G; Schonberger, Lawrence B; Gambetti, Pierluigi

2018-01-08

The presence of pathology related to the deposition of amyloid-β (Aβ) has been recently reported in iatrogenic Creutzfeldt-Jakob disease (iCJD) acquired from inoculation of growth hormone (GH) extracted from human cadaveric pituitary gland or use of cadaveric dura mater (DM) grafts.To investigate this phenomenon further, a cohort of 27 iCJD cases - 21 with adequate number of histopathological sections - originating from Australia, France, Italy, and the Unites States, were examined by immunohistochemistry, amyloid staining, and Western blot analysis of the scrapie prion protein (PrP Sc ), and compared with age-group matched cases of sporadic CJD (sCJD), Alzheimer disease (AD) or free of neurodegenerative diseases (non-ND).Cases of iCJD and sCJD shared similar profiles of proteinase K-resistant PrP Sc with the exception of iCJD harboring the "MMi" phenotype. Cerebral amyloid angiopathy (CAA), either associated with, or free of, Thioflavin S-positive amyloid core plaques (CP), was observed in 52% of 21 cases of iCJD, which comprised 37.5% and 61.5% of the cases of GH- and DM-iCJD, respectively. If only cases younger than 54 years were considered, Aβ pathology affected 41%, 2% and 0% of iCJD, sCJD and non-ND, respectively. Despite the patients' younger age CAA was more severe in iCJD than sCJD, while Aβ diffuse plaques, in absence of Aβ CP, populated one third of sCJD. Aβ pathology was by far most severe in AD. Tau pathology was scanty in iCJD and sCJD.In conclusion, (i) despite the divergences in the use of cadaveric GH and DM products, our cases combined with previous studies showed remarkably similar iCJD and Aβ phenotypes indicating that the occurrence of Aβ pathology in iCJD is a widespread phenomenon, (ii) CAA emerges as the hallmark of the Aβ phenotype in iCJD since it is observed in nearly 90% of all iCJD with Aβ pathology reported to date including ours, and it is shared by GH- and DM-iCJD, (iii) although the contributions to Aβ pathology of other factors, including GH deficiency, cannot be discounted, our findings increase the mounting evidence that this pathology is acquired by a mechanism resembling that of prion diseases.

Increased infectivity of anchorless mouse scrapie prions in transgenic mice overexpressing human prion protein.

PubMed

Race, Brent; Phillips, Katie; Meade-White, Kimberly; Striebel, James; Chesebro, Bruce

2015-06-01

Prion protein (PrP) is found in all mammals, mostly as a glycoprotein anchored to the plasma membrane by a C-terminal glycosylphosphatidylinositol (GPI) linkage. Following prion infection, host protease-sensitive prion protein (PrPsen or PrPC) is converted into an abnormal, disease-associated, protease-resistant form (PrPres). Biochemical characteristics, such as the PrP amino acid sequence, and posttranslational modifications, such as glycosylation and GPI anchoring, can affect the transmissibility of prions as well as the biochemical properties of the PrPres generated. Previous in vivo studies on the effects of GPI anchoring on prion infectivity have not examined cross-species transmission. In this study, we tested the effect of lack of GPI anchoring on a species barrier model using mice expressing human PrP. In this model, anchorless 22L prions derived from tg44 mice were more infectious than 22L prions derived from C57BL/10 mice when tested in tg66 transgenic mice, which expressed wild-type anchored human PrP at 8- to 16-fold above normal. Thus, the lack of the GPI anchor on the PrPres from tg44 mice appeared to reduce the effect of the mouse-human PrP species barrier. In contrast, neither source of prions induced disease in tgRM transgenic mice, which expressed human PrP at 2- to 4-fold above normal. Prion protein (PrP) is found in all mammals, usually attached to cells by an anchor molecule called GPI. Following prion infection, PrP is converted into a disease-associated form (PrPres). While most prion diseases are species specific, this finding is not consistent, and species barriers differ in strength. The amino acid sequence of PrP varies among species, and this variability affects prion species barriers. However, other PrP modifications, including glycosylation and GPI anchoring, may also influence cross-species infectivity. We studied the effect of PrP GPI anchoring using a mouse-to-human species barrier model. Experiments showed that prions produced by mice expressing only anchorless PrP were more infectious than prions produced in mice expressing anchored PrP. Thus, the lack of the GPI anchor on prions reduced the effect of the mouse-human species barrier. Our results suggest that prion diseases that produce higher levels of anchorless PrP may pose an increased risk for cross-species infection. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Phthalocyanines as Molecular Scaffolds to Block Disease-Associated Protein Aggregation.

PubMed

Valiente-Gabioud, Ariel A; Miotto, Marco C; Chesta, María E; Lombardo, Verónica; Binolfi, Andres; Fernández, Claudio O

2016-05-17

The aggregation of proteins into toxic conformations plays a critical role in the development of different neurodegenerative diseases such as Alzheimer's disease (AD), Parkinson's disease (PD), and Creutzfled-Jakob's disease (CJD). These disorders share a common pathological mechanism that involves the formation of aggregated protein species including toxic oligomers and amyloid fibrils. The aggregation of alpha-synuclein (αS) in PD and the amyloid beta peptide (Aβ) and tau protein in AD results in neuronal death and disease onset. In the case of CJD, the misfolding of the physiological prion protein (PrP) induces a chain reaction that results in accumulation of particles that elicit brain damage. Currently, there is no preventive therapy for these diseases and the available therapeutic approaches are based on the treatment of the symptoms rather than the underlying causes of the disease. Accordingly, the aggregation pathway of these proteins represents a useful target for therapeutic intervention. Therefore, understanding the mechanism of amyloid formation and its inhibition is of high clinical importance. The design of small molecules that efficiently inhibit the aggregation process and/or neutralize its associated toxicity constitutes a promising tool for the development of therapeutic strategies against these disorders. In this accounts, we discuss current knowledge on the anti-amyloid activity of phthalocyanines and their potential use as drug candidates in neurodegeneration. These tetrapyrrolic compounds modulate the amyloid assembly of αS, tau, Aβ, and the PrP in vitro, and protect cells from the toxic effects of amyloid aggregates. In addition, in scrapie-infected mice, these compounds showed important prophylactic antiscrapie properties. The structural basis for the inhibitory effect of phthalocyanines on amyloid filament assembly relies on specific π-π interactions between the aromatic ring system of these molecules and aromatic residues in the amyloidogenic proteins. Analysis of the structure-activity relationship in phthalocyanines revealed that their anti-amyloid activity is highly dependent on the type of metal ion coordinated to the tetrapyrrolic system but is not sensitive to the number of peripheral charged substituents. The tendency of phthalocyanines to oligomerize (self-association) via aromatic-aromatic stacking interactions correlates precisely with their binding capabilities to target proteins and, more importantly, determines their efficiency as anti-amyloid agents. The ability to block different types of disease-associated protein aggregation raises the possibility that these cyclic tetrapyrrole compounds have a common mechanism of action to impair the formation of a variety of pathological aggregates. Because the structural and molecular basis for the anti-amyloid effects of these molecules is starting to emerge, combined efforts from the fields of structural, cellular, and animal biology will result critical for the rational design and discovery of new drugs for the treatment of amyloid related neurological disorders.

Molecular dynamics studies on the buffalo prion protein.

PubMed

Zhang, Jiapu; Wang, Feng; Chatterjee, Subhojyoti

2016-01-01

It was reported that buffalo is a low susceptibility species resisting to transmissible spongiform encephalopathies (TSEs) (same as rabbits, horses, and dogs). TSEs, also called prion diseases, are invariably fatal and highly infectious neurodegenerative diseases that affect a wide variety of species (except for rabbits, dogs, horses, and buffalo), manifesting as scrapie in sheep and goats; bovine spongiform encephalopathy (BSE or "mad-cow" disease) in cattle; chronic wasting disease in deer and elk; and Creutzfeldt-Jakob diseases, Gerstmann-Sträussler-Scheinker syndrome, fatal familial insomnia, and Kulu in humans etc. In molecular structures, these neurodegenerative diseases are caused by the conversion from a soluble normal cellular prion protein (PrP(C)), predominantly with α-helices, into insoluble abnormally folded infectious prions (PrP(Sc)), rich in β-sheets. In this article, we studied the molecular structure and structural dynamics of buffalo PrP(C) (BufPrP(C)), in order to understand the reason why buffalo is resistant to prion diseases. We first did molecular modeling of a homology structure constructed by one mutation at residue 143 from the NMR structure of bovine and cattle PrP(124-227); immediately we found that for BufPrP(C)(124-227), there are five hydrogen bonds (HBs) at Asn143, but at this position, bovine/cattle do not have such HBs. Same as that of rabbits, dogs, or horses, our molecular dynamics studies also revealed there is a strong salt bridge (SB) ASP178-ARG164 (O-N) keeping the β2-α2 loop linked in buffalo. We also found there is a very strong HB SER170-TYR218 linking this loop with the C-terminal end of α-helix H3. Other information, such as (i) there is a very strong SB HIS187-ARG156 (N-O) linking α-helices H2 and H1 (if mutation H187R is made at position 187, then the hydrophobic core of PrP(C) will be exposed (L.H. Zhong (2010). Exposure of hydrophobic core in human prion protein pathogenic mutant H187R. Journal of Biomolecular Structure and Dynamics 28(3), 355-361)), (ii) at D178, there is a HB Y169-D178 and a polar contact R164-D178 for BufPrP(C) instead of a polar contact Q168-D178 for bovine PrP(C) (C.J. Cheng, & V. Daggett. (2014). Molecular dynamics simulations capture the misfolding of the bovine prion protein at acidic pH. Biomolecules 4(1), 181-201), (iii) BufPrP(C) owns three 310 helices at 125-127, 152-156, and in the β2-α2 loop, respectively, and (iv) in the β2-α2 loop, there is a strong π-π stacking and a strong π-cation F175-Y169-R164.(N)NH2, has been discovered.

Is mad cow disease caused by a bacteria?

PubMed

Broxmeyer, L

2004-01-01

Transmissible spongioform enchephalopathies (TSE's), include bovine spongiform encephalopathy (also called BSE or "mad cow disease"), Creutzfeldt-Jakob disease (CJD) in humans, and scrapie in sheep. They remain a mystery, their cause hotly debated. But between 1994 and 1996, 12 people in England came down with CJD, the human form of mad cow, and all had eaten beef from suspect cows. Current mad cow diagnosis lies solely in the detection of late appearing "prions", an acronym for hypothesized, gene-less, misfolded proteins, somehow claimed to cause the disease. Yet laboratory preparations of prions contain other things, which could include unidentified bacteria or viruses. Furthermore, the rigors of prion purification alone, might, in and of themselves, have killed the causative virus or bacteria. Therefore, even if samples appear to infect animals, it is impossible to prove that prions are causative. Manuelidis found viral-like particles, which even when separated from prions, were responsible for spongiform STE's. Subsequently, Lasmezas's study showed that 55% of mice injected with cattle BSE, and who came down with disease, had no detectable prions. Still, incredibly, prions, are held as existing TSE dogma and Heino Dringer, who did pioneer work on their nature, candidly predicts "it will turn out that the prion concept is wrong." Many animals that die of spongiform TSE's never show evidence of misfolded proteins, and Dr. Frank Bastian, of Tulane, an authority, thinks the disorder is caused by the bacterial DNA he found in this group of diseases. Recently, Roels and Walravens isolated Mycobacterium bovis it from the brain of a cow with the clinical and histopathological signs of mad cow. Moreover, epidemiologic maps of the origins and peak incidence of BSE in the UK, suggestively match those of England's areas of highest bovine tuberculosis, the Southwest, where Britain's mad cow epidemic began. The neurotoxic potential for cow tuberculosis was shown in pre-1960 England, where one quarter of all tuberculous meningitis victims suffered from Mycobacterium bovis infection. And Harley's study showed pathology identical to "mad cow" from systemic M. bovis in cattle, causing a tuberculous spongiform encephalitis. In addition to M. bovis, Mycobacterium avium subspecies paratuberculosis (fowl tuberculosis) causes Johne's disease, a problem known and neglected in cattle and sheep for almost a century, and rapidly emerging as the disease of the new millennium. Not only has M. paratuberculosis been found in human Crohn's disease, but both Crohn's and Johne's both cross-react with the antigens of cattle paratuberculosis. Furthermore, central neurologic manifestations of Crohn's disease are not unknown. There is no known disease which better fits into what is occurring in Mad Cow and the spongiform enchephalopathies than bovine tuberculosis and its blood-brain barrier penetrating, virus-like, cell-wall-deficient forms. It is for these reasons that future research needs to be aimed in this direction. Copyright 2004 Elsevier Ltd.

Soil humic substances hinder the propagation of prions

NASA Astrophysics Data System (ADS)

Leita, Liviana; Giachin, Gabriele; Margon, Alja; Narkiewicz, Joanna; Legname, Giuseppe

2013-04-01

Prions are infectious pathogens causing fatal neurodegenerative disorders, known as transmissible spongiform encephalopathies (TSEs), or prion diseases, which affect different mammalian species. TSEs include scrapie in sheep, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in mule deer, elk, and moose (cervids), and Creutzfeldt-Jakob disease (CJD) in humans. The prominent, if not only, component of prions is a misfolded conformer (PrPSc) of a constitutive sialoglycoprotein, the cellular prion protein (PrPC). A notable feature of prion diseases is horizontal transmission between grazing animals, implying that contaminated soil may serve to propagate the disease. In this respect, it has been reported that grazing animals ingest from tens to hundreds grams of soil per day, either incidentally through the diet, or deliberately in answering salt needs, and that mule deer can develop CWD after grazing in locations that previously housed infected animals. Prions may enter the environment through different routes, including animal excreta and secreta which mainly contribute to soil contamination. Recent studies have proven that prions can be retained in soil, which could act as a carrier of infectivity even several years after the contamination. However, within the large spread of potentially infected lands, prion diseases have become endemic only in geographically limited regions. The reasons for this geographical distribution remain unknown, but it suggests a role of the different kinds of soil in either enhancing or attenuating prion infectivity. The extent of prion transmission from the contaminated environment is unknown. Several studies have tried to address the issue of prion interaction with soil, but, at the present, different approaches show several drawbacks and technical difficulties, as soil is a complex, multi-component system of both mineral and organic interacting substances. Most research has focused on the adsorption capacity of clay minerals; however the contribution of soil organic components in adsorption has so far been neglected, as they represent a minor soil fraction on a weight basis. Among organic molecules, humic substances (HSs) are natural polyanions that result among the most reactive compounds in the soil and possess the largest specific surface area. Humic substances make up a large portion of the dark matter in humus and consist of heterogeneous mixtures of transformed biomolecules exhibiting a supramolecular structure. HSs are classified as humic acids (HAs), which are soluble only in alkaline solutions, and fulvic acids (FAs), which are soluble in both alkaline and acid solutions. The amphiphilic characteristics confer to HAs and FAs great versatility to interact with xenobiotics and reasonably also with prion proteins and/or prions too, leading to the formation of adducts with peculiar chemical and biophysical characteristics, thus affecting the transport, fixation and toxicity of prion. Results from our chemical, biophysical and biochemical investigation will be presented and results on anti-prion activity exerted by HAs and FAs will be provided, thus suggesting that amendment of contaminated soil with humic substances could be a strategy to contrast prion diffusion.

Theoretical and computational studies in protein folding, design, and function

NASA Astrophysics Data System (ADS)

Morrissey, Michael Patrick

2000-10-01

In this work, simplified statistical models are used to understand an array of processes related to protein folding and design. In Part I, lattice models are utilized to test several theories about the statistical properties of protein-like systems. In Part II, sequence analysis and all-atom simulations are used to advance a novel theory for the behavior of a particular protein. Part I is divided into five chapters. In Chapter 2, a method of sequence design for model proteins, based on statistical mechanical first-principles, is developed. The cumulant design method uses a mean-field approximation to expand the free energy of a sequence in temperature. The method successfully designs sequences which fold to a target lattice structure at a specific temperature, a feat which was not possible using previous design methods. The next three chapters are computational studies of the double mutant cycle, which has been used experimentally to predict intra-protein interactions. Complete structure prediction is demonstrated for a model system using exhaustive, and also sub-exhaustive, double mutants. Nonadditivity of enthalpy, rather than of free energy, is proposed and demonstrated to be a superior marker for inter-residue contact. Next, a new double mutant protocol, called exchange mutation, is introduced. Although simple statistical arguments predict exchange mutation to be a more accurate contact predictor than standard mutant cycles, this hypothesis was not upheld in lattice simulations. Reasons for this inconsistency will be discussed. Finally, a multi-chain folding algorithm is introduced. Known as LINKS, this algorithm was developed to test a method of structure prediction which utilizes chain-break mutants. While structure prediction was not successful, LINKS should nevertheless be a useful tool for the study of protein-protein and protein-ligand interactions. The last chapter of Part I utilizes the lattice to explore the differences between standard folding, from the fully denatured state, and cotranslational folding, whereby one end of a protein is synthesized and released before the other. Cotranslational folding is shown to accelerate folding kinetics, particularly when the target backbone contains many local contacts. Additionally, cotranslation is shown capable of "guiding" a model protein into a metastable, local contact-rich state, despite the existence of a true native state of much lower energy. In Part II, a model is developed for the behavior of PrP, a unique mammalian protein which has been shown to possess two native states. The pathogenic "scrapie" state PrPSc, which has not been structurally characterized, is known to trigger conversion of the characterized endogenous conformation PrPC into additional PrPSc, Residues 144--153 are shown to form the most hydrophilic naturally occurring alpha-helix, out of a broad database with more than 10,000 candidates. The novel beta-nucleation model proposes that PrPSc, is not a distinct mono-molecular state, but is rather a beta-sheet-like aggregate centered around helix-1 components of multiple PrP molecules. The remainder of Part II uses molecular dynamics simulations to support the beta-nucleation hypothesis, and to propose a system of peptide ligands which may arrest the process of prion propagation.