Wednesday, March 15, 2017

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

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


"When considering the atypical L-BSE and H-BSE diseases of cattle, they have been assessed in both non-human primate and transgenic mouse bioassays (with mice transgenic for human PRNP) and both model systems indicate that H-BSE and L-BSE may have increased zoonotic potential compare with C-BSE." "The detection of all types of BSE is therefore of significant importance."

Research Paper

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

Matthew J. O‘Connor, Keith Bishop, Robert G. Workman, Ben C. Maddison & Kevin C. Gough

Pages 54-64 | Received 28 Sep 2016, Accepted 04 Nov 2016, Published online: 08 Feb 2017

Abstract

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

Keywords: atypical prions, bovine spongiform encephalopathy, H-type BSE, PMCA

snip...

RESULTS

The amplification of atypical H-BSE prions by sPMCA and subsequent analysis of the products was performed by varying up to 4 conditions: the source of substrate (including PrPC primary sequence), the sonication duration, the presence or absence of Teflon beads and the concentration of PK used to digest sPMCA products (Table 1). The first optimisation step looked at a single sonication time (40s) in the absence of Teflon beads using 4 substrates derived from bovine, ovine AHQ/AHQ (corresponding to codons 136, 151, and 170 in the PRNP gene respectively), ovine ARQ/ARQ and ovine VRQ/VRQ brains. Out of 2 replicate analyses for each substrate, only a single reaction with the bovine substrate gave detectable PrPSc triplet (Fig. 1A). Repeat analysis of these conditions with bovine substrate demonstrated that all 5 replicates gave low but detectable levels of PrPSc product (Fig. 1B). When sonication times of 30, and 20 s were used with bovine substrate, 30 s gave low levels of PrPSc in all 5 replicates and 20 s gave low levels of PrPSc in 2 out of 5 replicates. In the presence of Teflon beads with bovine substrate, all 3 sonication times failed to produce any PrPSc. The products of amplification of H-BSE in bovine substrate with all sonication times were digested with 5, 10 or 50 μg/ml. While weak PrPSc signals were detected on western blots with the highest protease concentration, all of these positive amplifications yielded higher levels of defined PrPSc triplet at the lower 2 concentrations over background undigested PrP (Fig. 1B and data not shown). It was noted that at these lower protease concentrations, a single band for substrate PrPC that was between the molecular weights for di- and mono-glycosylated PK-resistant PrPSc could be observed, as previously reported.17 The optimal conditions: bovine substrate, 40 s sonication times without Teflon beads and 10 μg/ml PK digestion of sPMCA products were used in all subsequent experiments. Three experimental isolates of H-BSE were available for analysis and all 3 amplified under these sPMCA conditions when analyzing 10 μl of a 10−2 dilution of brain homogenate (equivalent to 100 μg of brain material) with 5 out of 5 replicates amplified for each (Fig. 1B and C). Similar amplification of C-BSE consistently yielded PrPSc but 3 isolates of L-BSE were not amplified under these conditions (data not shown). Dilution series of H-BSE and C-BSE were analyzed (Table 2, Fig. 2). For C-BSE, 2 samples were analyzed and the limit of detection of PrPSc was when analyzing a 10−6 or 10−7 dilution of brain (equivalent to 10 or 1 ng of brain material). For H-BSE the limit of detection was at least as sensitive, PrPSc could be detected in the brain of 2 experimental isolates at 10−7 or 10−10 dilutions (equivalent to 1 ng or 1 pg of brain material). The analysis of 20 replicates that contained no PrPSc seed did not produce any detectable PrPSc triplet, only a single undigested PrPC band as described above (data not shown). It was clearly evident that amplification products from H-BSE seed were distinguishable from those from C-BSE seed across the range of seed concentrations used. The H-BSE and C-BSE products of sPMCA could be distinguished due to their PK-resistance by determining the ratio of PrPSc signal produced when digesting samples with 10 or 50 μg/ml PK (p = 0.008 using a 2-tailed, unpaired Student's t test; Fig. 3). The H-BSE and C-BSE products of amplification also differed in the molecular weight of the unglycosylated PrPSc band (p<0 .0001="" 2="" 4a="" 4c="" a="" analyzed="" and="" antibody="" b="" binding="" by="" fig.="" identified="" in="" level="" of="" p4="" p="0.0046;" ratio="" readily="" s="" span="" student="" t="" tailed="" test="" the="" to="" unpaired="" using="" values="" was="" which="">

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DISCUSSION

The detection of PrPSc is a surrogate marker for prion diseases and has formed the basis of veterinary screening tests for ruminants. C-BSE in cattle is the only zoonotic prion disease recorded to date and as such the testing of food production animals is driven by the requirement to detect the C-BSE agent and minimize its risk to human health. Screening tests applied to cattle use post mortem brain tissue samples from which to detect PrPSc and rely on the presence of relatively high levels of PrPSc in the caudal region of the brainstem. Such tests may not be sensitive enough to detect PrPSc present at low levels during relatively early stages of disease incubation. When considering the atypical L-BSE and H-BSE diseases of cattle, they have been assessed in both non-human primate and transgenic mouse bioassays (with mice transgenic for human PRNP) and both model systems indicate that H-BSE and L-BSE may have increased zoonotic potential compare with C-BSE.9,10,11The detection of all types of BSE is therefore of significant importance. Both L-BSE and H-BSE have been identified in mainly asymptomatic animals of a relatively advanced age, ∼12 y on average18 compare with the estimated incubation period for cattle naturally infected with C-BSE, which is 5.0–5.5 y.19,20 It has been suggested that atypical BSE types may be spontaneous diseases, therefore explaining the detection of PrPSc in mainly older animals.8 The presence of relatively low levels of PrPSc in atypical BSE cases, compared with C-BSE, is likely for most age groups of cattle. Therefore the detection of PrPSc by conventional biochemical tests used in routine surveillance would be more challenging in atypical BSE cases. Given the high zoonotic potential of atypical BSE types it is important to develop very high sensitivity tests to detect the concomitant PrPSc in routinely sampled tissue. Such tests would not only be applicable to routine surveillance testing but also to determine in vivo dissemination of atypical prions to inform risk assessments of bovine material entering the human food chain. To this end, several studies have looked to develop in vitro prion amplification assays to detect very low levels of atypical BSE prions. Murayama and coworkers used wild type mouse brain as sPMCA substrate supplemented with a cocktail of polyanions (polyadenylic acid salt (Poly-A), heparin and sodium polyphosphate) and L-arginine ethylester.16 The method included sPMCA amplifications over 280 hours and could detect L-BSE down to a 10−10 dilution of brain homogenate. The method could detect L-BSE in cerebrospinal fluid, saliva, urine and plasma from macaques experimentally infected with L BSE. Prion was detected in saliva and urine in preclinical samples from 1 of 2 infected animals; and in CSF samples taken preclinical in both animals.

Several studies have looked to use QuIC to amplify both L-BSE and H-BSE and have utilised recombinant hamster, sheep, human and bank vole PrP as substrates.15,21,22 Human and hamster PrP consisting of residues 23–231 and hamster PrP residues 90–231 were found to only amplify L-BSE but not C-BSE and could detect L-BSE down to 10−6 to 10−7 dilutions of brain homogenate, which they report is at least as sensitive as mouse bioassay21,22 and approximately 4 orders of magnitude more sensitive than commercial immunoassays.15 Using bank vole recombinant PrP as substrate allowed the amplification of H-, L- and C-BSE. H-BSE could be detected down to 10−8 to 10−9 dilution of brain homogenate and H-BSE and L-BSE could be detected in the CSF from clinically affected cows.15 

Masujin and coworkers propose a scheme to use QuIC to distinguish H- L- and C-BSE infections.15 The proposed method analyses samples simultaneously in bank vole and ovine ARR substrate on the same QuIC plate. C-BSE is only amplified in bank vole substrate and, for L-BSE and H-BSE, the comparative lag phases of amplification are compared for each substrate, with L-BSE having a relatively shorter lag phase compare with H-BSE in ARR substrate, and the reverse being the case in bank vole substrate. The test samples are run alongside positive control samples of each BSE type for comparison. The method was shown to be able to distinguish BSE types for dilutions between 10−3 and 10−5 of brain homogenate from clinically affected cows but could not differentiate BSE types at lower concentrations.

In summary, in vitro amplification methods have been established for L-BSE and H-BSE prions with higher sensitivity than transgenic mouse bioassay (∼10−5 dilution of brain homogenate). However, at present sPMCA can be used to only detect L-BSE and while the QuIC method can detect both L- and H-BSE, at lower levels of prion (< 10−5 dilution of brain homogenate) the BSE types cannot be distinguished.

Here, we developed a sPMCA method to detect H-BSE. The most optimal conditions used 40s sonication durations and bovine PrPC substrate. The assay amplified all isolates of H-BSE and C-BSE tested and could detect H-BSE down to 10−7 and 10−10 dilution of brain homogenate. L-BSE was not amplified under the conditions used and the H-BSE and C-BSE products of amplification could be readily distinguished by the molecular weight of their unglycosylated PrP product or by the relative PK-resistance of the PrPSc. The molecular weight differences between PrPSc from H-BSE and C-BSE is a trait seen in both the brain derived PrPSc and also the sPMCA products. It has also been previously reported that H-BSE has a lower PK resistance than C-BSE5 and this trait seems to be exaggerated in the sPMCA products. Importantly, the ability to identify H-BSE in sPMCA products was maintained across all of the dilution ranges of seed analyzed (down to 10−7 dilution of brain homogenate) indicating that the assay can detect and strain type very low levels of H-type BSE.

The present study describes a sPMCA method for the amplification of H-BSE PrPSc that has very high sensitivity and can distinguish H-BSE from other BSE types. Given these properties, the assay has the potential to describe the in vivo dissemination of H-BSE PrPSc in infected animals across the time-course of infection, in order to define tissues that harbour H-BSE prions. 

see full text ; 


 "When considering the atypical L-BSE and H-BSE diseases of cattle, they have been assessed in both non-human primate and transgenic mouse bioassays (with mice transgenic for human PRNP) and both model systems indicate that H-BSE and L-BSE may have increased zoonotic potential compare with C-BSE.9,10,11" "The detection of all types of BSE is therefore of significant importance." 

 PRICE OF PRION POKER GOES UP

*** SPONTANEOUS ATYPICAL BOVINE SPONGIFORM ENCEPHALOPATHY *** 

***Moreover, sporadic disease has never been observed in breeding colonies or primate research laboratories, most notably among hundreds of animals over several decades of study at the National Institutes of Health25, and in nearly twenty older animals continuously housed in our own facility.*** 


Primate Biol., 3, 47–50, 2016 www.primate-biol.net/3/47/2016/ doi:10.5194/pb-3-47-2016 © Author(s) 2016. CC

Attribution 3.0 License. 

Prions 

Walter Bodemer German Primate Center, Kellnerweg 4, 37077 Göttingen, Germany Correspondence to: Walter Bodemer (wbodemer@dpz.eu

Received: 15 June 2016 – Revised: 24 August 2016 – Accepted: 30 August 2016 – Published: 7 September 2016 

Abstract.

Prions gained widespread public and scientific interest in the year 2000. At that time, the human neurological Creutzfeldt–Jakob disease (CJD) was known. However, new CJD cases were diagnosed but they could not be ascribed to one of the classical CJD categories i.e. sporadic (sCJD), hereditary or acquired. Hence, they were classified as variant CJD (vCJD). Later on, experimental evidence suggested that vCJD was caused by prions postulated as unique novel infectious agents and, for example, responsible for bovine spongiform encephalopathy (BSE) also known as mad cow disease. The infection of humans by transmission of BSE prions also defined vCJD as a zoonotic disease. Prions, especially those associated with scrapie in sheep had been known for quite some time and misleadingly discussed as a slow virus. Therefore, this enigmatic pathogen and the transmission of this unusual infectious agent was a matter of a controversial scientific debate. An agent without nucleic acid did not follow the current dogma postulating DNA or RNA as inheritable information encoding molecules. Although numerous experimental results clearly demonstrated the infectious capacity of prions in several animal species, a model close to human was not readily available. Therefore, the use of rhesus monkeys (Macaca mulatta) served as a non-human primate model to elucidate prion infection under controlled experimental conditions. Not the least, transmission of BSE, human vCJD, and sCJD prions could be confirmed in our study. Any prion infection concomitant with progression of disease in humans, especially vCJD, could be analyzed only retrospectively and at late stages of disease. In contrast, the prion-infected rhesus monkeys were accessible before and after infection; the progression from early stage to late clinical stages – and eventually death of the animal–could be traced. Because of the phylogenetic proximity to humans, the rhesus monkey was superior to any rodent or other animal model. For these reasons an experimental approach had been conceived by J. Collinge in London and A. Aguzzi in Zurich and performed in a cooperative study with both research groups in the pathology unit of the German Primate Center (DPZ). The study in the DPZ lasted from 2001 until 2012. Our research in the pathology unit provided a temporal monitoring of how an initial prion infection develops eventually into disease; an approach that would have never been possible in humans since the time point of infection with prions from, for example, BSE is always unknown. Telemetry revealed a shift in sleep– wake cycles early on, long before behavioral changes or clinical symptoms appeared. Pathology confirmed nonneuronal tissue as hidden places where prions exist. The rhesus model also allowed first comparative studies of epigenetic modifications on RNA in peripheral blood and brain tissue collected from uninfected and prion infected animals. To conclude, our studies clearly demonstrated that this model is valid since progression to disease is almost identical to human CJD. 

Published by Copernicus Publications on behalf of the Deutsches Primatenzentrum GmbH (DPZ).

SNIP...

2 Methods and results 

2.1 Animals The reason to perform prion research in rhesus monkeys was to monitor infection and the temporal progression of prion infection in the rhesus monkey. In contrast to studies of human CJD cases, we could decide on the infectious dose. We also could control behavior immediately after prion inoculation and during the rather long time until animals died from the prion infection. Hidden places where prions might exist were found. Even epigenetic modifications on RNA could be detected. Taken together, these experimental approaches depended on animals. Using rhesus monkeys as a model system required thorough ethic reasoning and consultation with authorities before we actually turned to conduct the experiments. The Number of animals was limited just to fulfill statistical conditions. The individual health status was obtained and health care was provided throughout the study. The animals underwent daily inspection to monitor any changes in health and behavior. The experiments were conceived with the aim of reducing pain, suffering, and harm. Groups of animals were preferred in order to keep them in a social environment. The animals were originally kept in Vienna at Baxter and transferred to the German Primate Center (DPZ) in 2001. J. Collinge, A. Aguzzi, and C. Weissmann were the scientists who recommended this well-controlled prion infection study, and financial support was provided by an EU grant.To ensure statistical significance four groups consisting of four rhesus macaques each were formed: one uninfected control group, one group infected with BSE prions, one with vCJD prions, and one with sCJD prions. Health of animals, infection, and progression to disease was looked at in our pathology department and in cooperation with W. Schulz-Schaeffer at the UMG (University Medicine, Göttingen). Besides, neurologists from the UMG also observed the animals whenever clinical symptoms seemed to appear. This close observation and comparison with human CJD cases demonstrated how close clinical progression of human disease resembles the experimental infection in the non-human primate. 

2.2 Infection Infectious prions from brain tissue of one sCJD and one vCJD case (provided by J. Collinge) as well as BSE prions (from a “German” madcow case and provided by W. Schulz Schaeffer) were intraperitoneally administered into the rhesus monkeys. 

2.3 Monitoring of behavior and telemetry Early behavioral monitoring was carried out by the ethologists I. Machatschke and J. Dittami from Vienna University. Transmitters were used to record changes in the circadian rhythms. Body temperature, sleep–wake cycles, and activity profiles could be obtained over a time span of 2 years. Up to half a year after infection animals did not show any signs of prion infection. However, after 6 months and persisting for another few months some animals had some disturbances in circadian rhythms which disappeared and then never appeared again(I. Machatschke, personal communication,2006).For a rather long time of about 4–5 years animals seemed to be healthy. But then, almost all animals rapidly progressed to symptoms. Symptoms were highly similar or even identical to those seen in human CJD. 

2.4 Pathology Blood and necropsy specimens from the animals served as a valuable source to detect pathologically associated prion protein even in non-neuronal skeletal and cardiac tissue. These “hidden places” of prion pathology and replication were clearly demonstrated and extended our view where prions might spread within an organism. Not only leukocytes and neuronal tissue harbor abnormal prion protein isoforms but also other tissues can propagate prion protein isoforms leading to toxicity, cell degeneration, and eventually transmissible prions (Krasemann et al., 2010, 2013).

SNIP...

3 Conclusion 

Most importantly, early signs of an altered circadian rhythm, sleep–wake cycle, and activity and body temperature were recorded in prion-infected animals. This experimental approach would have never been feasible in studies with human CJD cases. After 4–6 years animals developed clinical symptoms highly similar to those typical for CJD. Clinicians confirmed how close the animal model and the human disease matched. Non-neuronal tissue like cardiac muscle and peripheral blood with abnormal, disease-related prion protein were detected in rhesus monkey tissues. 

Molecular changes in RNA from repetitive Alu and BC200 DNA elements were identified and found to be targets of epigenetic editing mechanisms active in prion disease. To conclude, our results with the rhesus monkey model for prion disease proved to be a valid model and increased our knowledge of pathogenic processes that are distinctive to prion disease.

SEE FULL TEXT ;


Saturday, April 23, 2016

PRION 2016 TOKYO

Saturday, April 23, 2016 

SCRAPIE WS-01: Prion diseases in animals and zoonotic potential 2016 

Prion. 10:S15-S21. 2016 ISSN: 1933-6896 printl 1933-690X online

Taylor & Francis

Prion 2016 Animal Prion Disease Workshop Abstracts

WS-01: Prion diseases in animals and zoonotic potential

Juan Maria Torres a, Olivier Andreoletti b, J uan-Carlos Espinosa a. Vincent Beringue c. Patricia Aguilar a,

Natalia Fernandez-Borges a. and Alba Marin-Moreno a

"Centro de Investigacion en Sanidad Animal ( CISA-INIA ). Valdeolmos, Madrid. Spain; b UMR INRA -ENVT 1225 Interactions Holes Agents Pathogenes. ENVT. Toulouse. France: "UR892. Virologie lmmunologie MolécuIaires, Jouy-en-Josas. France 

Dietary exposure to bovine spongiform encephalopathy (BSE) contaminated bovine tissues is considered as the origin of variant Creutzfeldt Jakob (vCJD) disease in human. To date, BSE agent is the only recognized zoonotic prion. Despite the variety of Transmissible Spongiform Encephalopathy (TSE) agents that have been circulating for centuries in farmed ruminants there is no apparent epidemiological link between exposure to ruminant products and the occurrence of other form of TSE in human like sporadic Creutzfeldt Jakob Disease (sCJD). However, the zoonotic potential of the diversity of circulating TSE agents has never been systematically assessed. The major issue in experimental assessment of TSEs zoonotic potential lies in the modeling of the ‘species barrier‘, the biological phenomenon that limits TSE agents’ propagation from a species to another. In the last decade, mice genetically engineered to express normal forms of the human prion protein has proved essential in studying human prions pathogenesis and modeling the capacity of TSEs to cross the human species barrier. 

To assess the zoonotic potential of prions circulating in farmed ruminants, we study their transmission ability in transgenic mice expressing human PrPC (HuPrP-Tg). Two lines of mice expressing different forms of the human PrPC (129Met or 129Val) are used to determine the role of the Met129Val dimorphism in susceptibility/resistance to the different agents.

These transmission experiments confirm the ability of BSE prions to propagate in 129M- HuPrP-Tg mice and demonstrate that Met129 homozygotes may be susceptible to BSE in sheep or goat to a greater degree than the BSE agent in cattle and that these agents can convey molecular properties and neuropathological indistinguishable from vCJD. However homozygous 129V mice are resistant to all tested BSE derived prions independently of the originating species suggesting a higher transmission barrier for 129V-PrP variant. 

Transmission data also revealed that several scrapie prions propagate in HuPrP-Tg mice with efficiency comparable to that of cattle BSE. While the efficiency of transmission at primary passage was low, subsequent passages resulted in a highly virulent prion disease in both Met129 and Val129 mice. Transmission of the different scrapie isolates in these mice leads to the emergence of prion strain phenotypes that showed similar characteristics to those displayed by MM1 or VV2 sCJD prion. These results demonstrate that scrapie prions have a zoonotic potential and raise new questions about the possible link between animal and human prions. 


why do we not want to do TSE transmission studies on chimpanzees $

5. A positive result from a chimpanzee challenged severly would likely create alarm in some circles even if the result could not be interpreted for man. I have a view that all these agents could be transmitted provided a large enough dose by appropriate routes was given and the animals kept long enough. Until the mechanisms of the species barrier are more clearly understood it might be best to retain that hypothesis. 

snip... 

R. BRADLEY


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

*** In complement to the recent demonstration that humanized mice are susceptible to scrapie, we report here the first observation of direct transmission of a natural classical scrapie isolate to a macaque after a 10-year incubation period. Neuropathologic examination revealed all of the features of a prion disease: spongiform change, neuronal loss, and accumulation of PrPres throughout the CNS. 

*** This observation strengthens the questioning of the harmlessness of scrapie to humans, at a time when protective measures for human and animal health are being dismantled and reduced as c-BSE is considered controlled and being eradicated. 

*** Our results underscore the importance of precautionary and protective measures and the necessity for long-term experimental transmission studies to assess the zoonotic potential of other animal prion strains.


SCRAPIE WS-01: Prion diseases in animals and zoonotic potential 2016

Prion. 10:S15-S21. 2016 ISSN: 1933-6896 printl 1933-690X online



O.05: Transmission of prions to primates after extended silent incubation periods: Implications for BSE and scrapie risk assessment in human populations 

Emmanuel Comoy, Jacqueline Mikol, Valerie Durand, Sophie Luccantoni, Evelyne Correia, Nathalie Lescoutra, Capucine Dehen, and Jean-Philippe Deslys Atomic Energy Commission; Fontenay-aux-Roses, France 

Prion diseases (PD) are the unique neurodegenerative proteinopathies reputed to be transmissible under field conditions since decades. The transmission of Bovine Spongiform Encephalopathy (BSE) to humans evidenced that an animal PD might be zoonotic under appropriate conditions. Contrarily, in the absence of obvious (epidemiological or experimental) elements supporting a transmission or genetic predispositions, PD, like the other proteinopathies, are reputed to occur spontaneously (atpical animal prion strains, sporadic CJD summing 80% of human prion cases). Non-human primate models provided the first evidences supporting the transmissibiity of human prion strains and the zoonotic potential of BSE. Among them, cynomolgus macaques brought major information for BSE risk assessment for human health (Chen, 2014), according to their phylogenetic proximity to humans and extended lifetime. We used this model to assess the zoonotic potential of other animal PD from bovine, ovine and cervid origins even after very long silent incubation periods. 

*** We recently observed the direct transmission of a natural classical scrapie isolate to macaque after a 10-year silent incubation period, 

***with features similar to some reported for human cases of sporadic CJD, albeit requiring fourfold long incubation than BSE. Scrapie, as recently evoked in humanized mice (Cassard, 2014), 

***is the third potentially zoonotic PD (with BSE and L-type BSE), 

***thus questioning the origin of human sporadic cases. We will present an updated panorama of our different transmission studies and discuss the implications of such extended incubation periods on risk assessment of animal PD for human health. 

===============

***thus questioning the origin of human sporadic cases*** 

***our findings suggest that possible transmission risk of H-type BSE to sheep and human. Bioassay will be required to determine whether the PMCA products are infectious to these animals. 


LOOKING FOR CWD IN HUMANS AS nvCJD or as an ATYPICAL CJD, LOOKING IN ALL THE WRONG PLACES $$$

*** These results would seem to suggest that CWD does indeed have zoonotic potential, at least as judged by the compatibility of CWD prions and their human PrPC target. Furthermore, extrapolation from this simple in vitro assay suggests that if zoonotic CWD occurred, it would most likely effect those of the PRNP codon 129-MM genotype and that the PrPres type would be similar to that found in the most common subtype of sCJD (MM1).***


*** The potential impact of prion diseases on human health was greatly magnified by the recognition that interspecies transfer of BSE to humans by beef ingestion resulted in vCJD. While changes in animal feed constituents and slaughter practices appear to have curtailed vCJD, there is concern that CWD of free-ranging deer and elk in the U.S. might also cross the species barrier. Thus, consuming venison could be a source of human prion disease. Whether BSE and CWD represent interspecies scrapie transfer or are newly arisen prion diseases is unknown. Therefore, the possibility of transmission of prion disease through other food animals cannot be ruled out. There is evidence that vCJD can be transmitted through blood transfusion. There is likely a pool of unknown size of asymptomatic individuals infected with vCJD, and there may be asymptomatic individuals infected with the CWD equivalent. These circumstances represent a potential threat to blood, blood products, and plasma supplies.


*** WDA 2016 NEW YORK *** 

We found that CWD adapts to a new host more readily than BSE and that human PrP was unexpectedly prone to misfolding by CWD prions. In addition, we investigated the role of specific regions of the bovine, deer and human PrP protein in resistance to conversion by prions from another species. We have concluded that the human protein has a region that confers unusual susceptibility to conversion by CWD prions. 

Student Presentations Session 2 

The species barriers and public health threat of CWD and BSE prions 

Ms. Kristen Davenport1, Dr. Davin Henderson1, Dr. Candace Mathiason1, Dr. Edward Hoover1 1Colorado State University 

Chronic wasting disease (CWD) is spreading rapidly through cervid populations in the USA. Bovine spongiform encephalopathy (BSE, mad cow disease) arose in the 1980s because cattle were fed recycled animal protein. These and other prion diseases are caused by abnormal folding of the normal prion protein (PrP) into a disease causing form (PrPd), which is pathogenic to nervous system cells and can cause subsequent PrP to misfold. CWD spreads among cervids very efficiently, but it has not yet infected humans. On the other hand, BSE was spread only when cattle consumed infected bovine or ovine tissue, but did infect humans and other species. The objective of this research is to understand the role of PrP structure in cross-species infection by CWD and BSE. To study the propensity of each species’ PrP to be induced to misfold by the presence of PrPd from verious species, we have used an in vitro system that permits detection of PrPd in real-time. We measured the conversion efficiency of various combinations of PrPd seeds and PrP substrate combinations. We observed the cross-species behavior of CWD and BSE, in addition to feline-adapted CWD and BSE. We found that CWD adapts to a new host more readily than BSE and that human PrP was unexpectedly prone to misfolding by CWD prions. In addition, we investigated the role of specific regions of the bovine, deer and human PrP protein in resistance to conversion by prions from another species. We have concluded that the human protein has a region that confers unusual susceptibility to conversion by CWD prions. CWD is unique among prion diseases in its rapid spread in natural populations. BSE prions are essentially unaltered upon passage to a new species, while CWD adapts to the new species. This adaptation has consequences for surveillance of humans exposed to CWD. 

Wildlife Disease Risk Communication Research Contributes to Wildlife Trust Administration Exploring perceptions about chronic wasting disease risks among wildlife and agriculture professionals and stakeholders


PRION 2016 TOKYO

Zoonotic Potential of CWD Prions: An Update

Ignazio Cali1, Liuting Qing1, Jue Yuan1, Shenghai Huang2, Diane Kofskey1,3, Nicholas Maurer1, Debbie McKenzie4, Jiri Safar1,3,5, Wenquan Zou1,3,5,6, Pierluigi Gambetti1, Qingzhong Kong1,5,6 1Department of Pathology, 3National Prion Disease Pathology Surveillance Center, 5Department of Neurology, 6National Center for Regenerative Medicine, Case Western Reserve University, Cleveland, OH 44106, USA. 4Department of Biological Sciences and Center for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Alberta, Canada, 2Encore Health Resources, 1331 Lamar St, Houston, TX 77010

Chronic wasting disease (CWD) is a widespread and highly transmissible prion disease in free-ranging and captive cervid species in North America. The zoonotic potential of CWD prions is a serious public health concern, but the susceptibility of human CNS and peripheral organs to CWD prions remains largely unresolved. We reported earlier that peripheral and CNS infections were detected in transgenic mice expressing human PrP129M or PrP129V. Here we will present an update on this project, including evidence for strain dependence and influence of cervid PrP polymorphisms on CWD zoonosis as well as the characteristics of experimental human CWD prions.

PRION 2016 TOKYO In Conjunction with Asia Pacific Prion Symposium 2016 PRION 2016 Tokyo Prion 2016



Monday, May 02, 2016

*** Zoonotic Potential of CWD Prions: An Update Prion 2016 Tokyo ***


MONDAY, MARCH 13, 2017

CHRONIC WASTING DISEASE CWD TSE PRION UDATE March 13, 2017



Thursday, December 08, 2016

USDA APHIS National Scrapie Eradication Program October 2016 Monthly Report Fiscal Year 2017 atypical NOR-98 Scrapie


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

*** In complement to the recent demonstration that humanized mice are susceptible to scrapie, we report here the first observation of direct transmission of a natural classical scrapie isolate to a macaque after a 10-year incubation period. Neuropathologic examination revealed all of the features of a prion disease: spongiform change, neuronal loss, and accumulation of PrPres throughout the CNS.

 *** This observation strengthens the questioning of the harmlessness of scrapie to humans, at a time when protective measures for human and animal health are being dismantled and reduced as c-BSE is considered controlled and being eradicated.

 *** Our results underscore the importance of precautionary and protective measures and the necessity for long-term experimental transmission studies to assess the zoonotic potential of other animal prion strains.


SCRAPIE WS-01: Prion diseases in animals and zoonotic potential 2016

Prion. 10:S15-S21. 2016 ISSN: 1933-6896 printl 1933-690X online



Tuesday, July 21, 2009

Transmissible mink encephalopathy - review of the etiology


Saturday, December 01, 2007

Phenotypic Similarity of Transmissible Mink Encephalopathy in Cattle and L-type Bovine Spongiform Encephalopathy in a Mouse Model


Sunday, December 10, 2006

Transmissible Mink Encephalopathy TME



Saturday, June 25, 2011

Transmissibility of BSE-L and Cattle-Adapted TME Prion Strain to Cynomolgus Macaque

"BSE-L in North America may have existed for decades"


Wednesday, April 25, 2012

4th MAD COW DISEASE U.S.A. CALIFORNIA ATYPICAL L-TYPE BSE 2012


Thursday, October 22, 2015

Former Ag Secretary Ann Veneman talks women in agriculture and we talk mad cow disease USDA and what really happened


Wednesday, December 21, 2016

TRANSMISSION, DIFFERENTIATION, AND PATHOBIOLOGY OF TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES 2016 ANNUAL REPORT ARS RESEARCH



Monday, January 2, 2017

Bovine Spongiform Encephalopathy Induces Misfolding of Alleged Prion-Resistant Species Cellular Prion Protein without Altering Its Pathobiological Features Articles, Neurobiology of Disease


Tuesday, September 06, 2016

A comparison of classical and H-type bovine spongiform encephalopathy associated with E211K prion protein polymorphism in wild type and EK211 cattle following intracranial inoculation


Saturday, July 23, 2016

BOVINE SPONGIFORM ENCEPHALOPATHY BSE TSE PRION SURVEILLANCE, TESTING, AND SRM REMOVAL UNITED STATE OF AMERICA UPDATE JULY 2016


Tuesday, July 26, 2016

Atypical Bovine Spongiform Encephalopathy BSE TSE Prion UPDATE JULY 2016


Monday, June 20, 2016

Specified Risk Materials SRMs BSE TSE Prion Program


Monday, January 09, 2017

Oral Transmission of L-Type Bovine Spongiform Encephalopathy Agent among Cattle CDC Volume 23, Number 2—February 2017



MONDAY, JANUARY 16, 2017

APHIS Bovine Spongiform Encephalopathy (BSE): Ongoing Surveillance Program Last Modified: Jan 5, 2017



 ***our findings suggest that possible transmission risk of H-type BSE to sheep and human. Bioassay will be required to determine whether the PMCA products are infectious to these animals.

P.86: Estimating the risk of transmission of BSE and scrapie to ruminants and humans by protein misfolding cyclic amplification

Morikazu Imamura, Naoko Tabeta, Yoshifumi Iwamaru, and Yuichi Murayama

National Institute of Animal Health; Tsukuba, Japan

To assess the risk of the transmission of ruminant prions to ruminants and humans at the molecular level, we investigated the ability of abnormal prion protein (PrPSc) of typical and atypical BSEs (L-type and H-type) and typical scrapie to convert normal prion protein (PrPC) from bovine, ovine, and human to proteinase K-resistant PrPSc-like form (PrPres) using serial protein misfolding cyclic amplification (PMCA).

Six rounds of serial PMCA was performed using 10% brain homogenates from transgenic mice expressing bovine, ovine or human PrPC in combination with PrPSc seed from typical and atypical BSE- or typical scrapie-infected brain homogenates from native host species. In the conventional PMCA, the conversion of PrPC to PrPres was observed only when the species of PrPC source and PrPSc seed matched. However, in the PMCA with supplements (digitonin, synthetic polyA and heparin), both bovine and ovine PrPC were converted by PrPSc from all tested prion strains. On the other hand, human PrPC was converted by PrPSc from typical and H-type BSE in this PMCA condition.

Although these results were not compatible with the previous reports describing the lack of transmissibility of H-type BSE to ovine and human transgenic mice, our findings suggest that possible transmission risk of H-type BSE to sheep and human. Bioassay will be required to determine whether the PMCA products are infectious to these animals.


P.170: Potential detection of oral transmission of H type atypical BSE in cattle using in vitro conversion

***P.170: Potential detection of oral transmission of H type atypical BSE in cattle using in vitro conversion

Sandor Dudas, John G Gray, Renee Clark, and Stefanie Czub Canadian Food Inspection Agency; Lethbridge, AB Canada

Keywords: Atypical BSE, oral transmission, RT-QuIC

The detection of bovine spongiform encephalopathy (BSE) has had a significant negative impact on the cattle industry worldwide. In response, governments took actions to prevent transmission and additional threats to animal health and food safety. While these measures seem to be effective for controlling classical BSE, the more recently discovered atypical BSE has presented a new challenge. To generate data for risk assessment and control measures, we have challenged cattle orally with atypical BSE to determine transmissibility and mis-folded prion (PrPSc) tissue distribution. Upon presentation of clinical symptoms, animals were euthanized and tested for characteristic histopathological changes as well as PrPSc deposition.

The H-type challenged animal displayed vacuolation exclusively in rostral brain areas but the L-type challenged animal showed no evidence thereof. To our surprise, neither of the animals euthanized, which were displaying clinical signs indicative of BSE, showed conclusive mis-folded prion accumulation in the brain or gut using standard molecular or immunohistochemical assays. To confirm presence or absence of prion infectivity, we employed an optimized real-time quaking induced conversion (RT-QuIC) assay developed at the Rocky Mountain Laboratory, Hamilton, USA.

Detection of PrPSc was unsuccessful for brain samples tests from the orally inoculated L type animal using the RT-QuIC. It is possible that these negative results were related to the tissue sampling locations or that type specific optimization is needed to detect PrPSc in this animal. We were however able to consistently detect the presence of mis-folded prions in the brain of the H-type inoculated animal. Considering the negative and inconclusive results with other PrPSc detection methods, positive results using the optimized RT-QuIC suggests the method is extremely sensitive for H-type BSE detection. This may be evidence of the first successful oral transmission of H type atypical BSE in cattle and additional investigation of samples from these animals are ongoing.




Monday, October 19, 2009 

Atypical BSE, BSE, and other human and animal TSE in North America Update October 19, 2009 

snip... 

 I ask Professor Kong ; 

Thursday, December 04, 2008 3:37 PM 

Subject: RE: re--Chronic Wating Disease (CWD) and Bovine Spongiform Encephalopathies (BSE): Public Health Risk Assessment 

''IS the h-BSE more virulent than typical BSE as well, or the same as cBSE, or less virulent than cBSE? just curious..... 

'' Professor Kong reply ; 

.....snip 

''As to the H-BSE, we do not have sufficient data to say one way or another, but we have found that H-BSE can infect humans. I hope we could publish these data once the study is complete. Thanks for your interest.'' 

Best regards, 

Qingzhong Kong, 

PhD Associate Professor

Department of Pathology Case Western Reserve University Cleveland, OH 44106 USA 

END...TSS


-------- Original Message -------- 

Subject: re-BSE prions propagate as either variant CJD-like or sporadic CJD 

Date: Thu, 28 Nov 2002 10:23:43 -0000 

From: "Asante, Emmanuel A" <e.asante@ic.ac.uk


Dear Terry,

I have been asked by Professor Collinge to respond to your request. I am a Senior Scientist in the MRC Prion Unit and the lead author on the paper. I have attached a pdf copy of the paper for your attention. Thank you for your interest in the paper.

In respect of your first question, the simple answer is, yes. As you will find in the paper, we have managed to associate the alternate phenotype to type 2 PrPSc, the commonest sporadic CJD. It is too early to be able to claim any further sub-classification in respect of Heidenhain variant CJD or Vicky Rimmer's version. It will take further studies, which are on-going, to establish if there are sub-types to our initial finding which we are now reporting. The main point of the paper is that, as well as leading to the expected new variant CJD phenotype, BSE transmission to the 129-methionine genotype can lead to an alternate phenotype which is indistinguishable from type 2 PrPSc.

I hope reading the paper will enlighten you more on the subject. If I can be of any further assistance please to not hesitate to ask. Best wishes.

Emmanuel Asante

<

____________________________________

Dr. Emmanuel A Asante MRC Prion Unit & Neurogenetics Dept. Imperial College School of Medicine (St. Mary's) Norfolk Place, LONDON W2 1PG Tel: +44 (0)20 7594 3794 Fax: +44 (0)20 7706 3272 email: e.asante@ic.ac.uk (until 9/12/02) New e-mail: e.asante@prion.ucl.ac.uk (active from now)

__________END...TSS________


Evidence That Transmissible Mink Encephalopathy Results from Feeding Infected Cattle

Over the next 8-10 weeks, approximately 40% of all the adult mink on the farm died from TME.

snip...

The rancher was a ''dead stock'' feeder using mostly (>95%) downer or dead dairy cattle...




In Confidence - Perceptions of unconventional slow virus diseases of animals in the USA - APRIL-MAY 1989 - G A H Wells

3. Prof. A. Robertson gave a brief account of BSE. The US approach was to accord it a very low profile indeed. Dr. A Thiermann showed the picture in the ''Independent'' with cattle being incinerated and thought this was a fanatical incident to be avoided in the US at all costs. ...


The occurrence of CWD must be viewed against the contest of the locations in which it occurred. It was an incidental and unwelcome complication of the respective wildlife research programmes. Despite it’s subsequent recognition as a new disease of cervids, therefore justifying direct investigation, no specific research funding was forthcoming. The USDA veiwed it as a wildlife problem and consequently not their province! ...page 26.


*** Spraker suggested an interesting explanation for the occurrence of CWD. The deer pens at the Foot Hills Campus were built some 30-40 years ago by a Dr. Bob Davis. At or abut that time, allegedly, some scrapie work was conducted at this site. When deer were introduced to the pens they occupied ground that had previously been occupied by sheep.



SPONTANEOUS ATYPICAL BOVINE SPONGIFORM ENCEPHALOPATHY

***Moreover, sporadic disease has never been observed in breeding colonies or primate research laboratories, most notably among hundreds of animals over several decades of study at the National Institutes of Health25, and in nearly twenty older animals continuously housed in our own facility.***


TUESDAY, JANUARY 17, 2017 

FDA PART 589 -- SUBSTANCES PROHIBITED FROM USE IN ANIMAL FOOD OR FEEDVIOLATIONS OFFICIAL ACTION INDICATED OAI UPDATE 2016 to 2017 BSE TSE PRION 




WEDNESDAY, JANUARY 18, 2017 

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



15 November 1999 

British Medical Journal 

vCJD in the USA * BSE in U.S. 


2 January 2000 

British Medical Journal 

U.S. Scientist should be concerned with a CJD epidemic in the U.S., as well 


26 March 2003 

Terry S. Singeltary, retired (medically) CJD WATCH 

I lost my mother to hvCJD (Heidenhain Variant CJD). I would like to comment on the CDC's attempts to monitor the occurrence of emerging forms of CJD. Asante, Collinge et al [1] have reported that BSE transmission to the 129-methionine genotype can lead to an alternate phenotype that is indistinguishable from type 2 PrPSc, the commonest sporadic CJD. However, CJD and all human TSEs are not reportable nationally. CJD and all human TSEs must be made reportable in every state and internationally. I hope that the CDC does not continue to expect us to still believe that the 85%+ of all CJD cases which are sporadic are all spontaneous, without route/source. We have many TSEs in the USA in both animal and man. CWD in deer/elk is spreading rapidly and CWD does transmit to mink, ferret, cattle, and squirrel monkey by intracerebral inoculation. With the known incubation periods in other TSEs, oral transmission studies of CWD may take much longer. Every victim/family of CJD/TSEs should be asked about route and source of this agent. To prolong this will only spread the agent and needlessly expose others. In light of the findings of Asante and Collinge et al, there should be drastic measures to safeguard the medical and surgical arena from sporadic CJDs and all human TSEs. I only ponder how many sporadic CJDs in the USA are type 2 PrPSc? 


RE: re-Human Prion Diseases in the United States part 2 flounder replied to flounder on 02 Jan 2010 at 21:26 GMT No competing interests declared. 

see full text ; 


*** Needless conflict ***

 Nature 485, 279–280 (17 May 2012) doi:10.1038/485279b

 Published online 16 May 2012

 Terry S. Singeltary Sr. said:

 I kindly wish to submit the following please ; 


Thursday, July 24, 2014 

*** Protocol for further laboratory investigations into the distribution of infectivity of Atypical BSE SCIENTIFIC REPORT OF EFSA New protocol for Atypical BSE investigations 


*** Singeltary reply ; 

Molecular, Biochemical and Genetic Characteristics of BSE in Canada Singeltary reply ; 


Monday, June 20, 2016 

Specified Risk Materials SRMs BSE TSE Prion Program 


Wednesday, July 15, 2015 

Additional BSE TSE prion testing detects pathologic lesion in unusual brain location and PrPsc by PMCA only, how many cases have we missed? 


***however in 1 C-type challenged animal, Prion 2015 Poster Abstracts S67 PrPsc was not detected using rapid tests for BSE. ***

Subsequent testing resulted in the detection of pathologic lesion in unusual brain location and PrPsc detection by PMCA only. IBNC Tauopathy or TSE Prion disease, it appears, no one is sure. 

Posted by flounder on 03 Jul 2015 at 16:53 GMT 



Terry S. Singeltary Sr.