PLoS Pathog. 2023 Dec; 19(12): e1011815. Published online 2023 Dec 4. https://doi.org/10.1371/journal.ppat.1011815
PMCID: PMC10721168 PMID: 38048370
Disease phenotype of classical sheep scrapie is changed upon experimental passage through white-tailed deer
Robyn D. Kokemuller, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, 1 S. Jo Moore, Formal analysis, Investigation, Writing – original draft, Writing – review & editing, 1 Jifeng Bian, Formal analysis, Investigation, Methodology, Writing – review & editing, 1 M. Heather West Greenlee, Conceptualization, Investigation, Supervision, Writing – review & editing, 2 and Justin J. Greenlee, Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Project administration, Resources, Supervision, Writing – original draft, Writing – review & editing corresponding author 1 ,* Jason C. Bartz, Editor Author information Article notes Copyright and License information Associated Data Supplementary Materials Data Availability Statement
Abstract
Prion agents occur in strains that are encoded by the structure of the misfolded prion protein (PrPSc). Prion strains can influence disease phenotype and the potential for interspecies transmission. Little is known about the potential transmission of prions between sheep and deer. Previously, the classical US scrapie isolate (No.13-7) had a 100% attack rate in white-tailed deer after oronasal challenge. The purpose of this study was to test the susceptibility of sheep to challenge with the scrapie agent after passage through white-tailed deer (WTD scrapie). Lambs of various prion protein genotypes were oronasally challenged with WTD scrapie. Sheep were euthanized and necropsied upon development of clinical signs or at the end of the experiment (72 months post-inoculation). Enzyme immunoassay, western blot, and immunohistochemistry demonstrated PrPSc in 4 of 10 sheep with the fastest incubation occurring in VRQ/VRQ sheep, which contrasts the original No.13-7 inoculum with a faster incubation in ARQ/ARQ sheep. Shorter incubation periods in VRQ/VRQ sheep than ARQ/ARQ sheep after passage through deer was suggestive of a phenotype change, so comparisons were made in ovinized mice and with sheep with known strains of classical sheep scrapie: No. 13–7 and x-124 (that has a more rapid incubation in VRQ/VRQ sheep). After mouse bioassay, the WTD scrapie and x-124 isolates have similar incubation periods and PrPSc conformational stability that are markedly different than the original No. 13–7 inoculum. Furthermore, brain tissues of sheep with WTD scrapie and x-124 scrapie have similar patterns of immunoreactivity that are distinct from sheep with No. 13–7 scrapie. Multiple lines of evidence suggest a phenotype switch when No. 13–7 scrapie prions are passaged through deer. This represents one example of interspecies transmission of prions resulting in the emergence or selection of new strain properties that could confound disease eradication and control efforts.
Author summary Passage of the sheep-derived US No. 13–7 classical scrapie isolate through white-tailed deer results in a change in disease phenotype that is observed when the deer-passaged scrapie agent is inoculated back into sheep or ovinized mice. Upon passage back to sheep, the relationship between incubation period and sheep PRNP genotype is reversed from the original inoculum. Whereas inoculation with the original No.13-7 scrapie agent results in a shorter incubation period in sheep with the ARQ/ARQ genotype as compared to VRQ/VRQ sheep, the deer-passaged scrapie agent results in a shorter incubation period in VRQ/VRQ sheep. In addition, passage of the No.13-7 isolate through deer results in a change in the pattern of PrPSc deposition in the brain of affected sheep. Taken together with the results of bioassay and conformational stability assays this work supports emergence of strain properties different from the No. 13–7 inoculum and consistent with another classical scrapie strain called x-124. Interspecies transmission of the classical scrapie agent can result in a phenotype switch through emergence of new scrapie strain properties that could potentially expand the potential host range.
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In summary, this work demonstrates that interspecies transmission of prion isolates can result in the emergence of new strain properties that could alter the host range or require different management strategies to control disease spread.
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Detection of classical BSE prions in asymptomatic cows after inoculation with atypical/Nor98 scrapie
Marina Betancor1, Belén Marín1, Alicia Otero1#, Carlos Hedman1, Antonio Romero2, Tomás Barrio3, Eloisa Sevilla1, Jean Yves Douet3, Alvina Huor3, Juan José Badiola1, Olivier Andréoletti3, Rosa Bolea1.
1Centro de Encefalopatías y Enfermedades Transmisibles Emergentes, Facultad de Veterinaria, Universidad de Zaragoza, Instituto Agroalimentario de Aragón - IA2, 50013, Zaragoza, Spain. 2 Servicio de Cirugía y Medicina Equina, Hospital Veterinario, Universidad de Zaragoza, 50013, Zaragoza, Spain 3 UMR École Nationale Vétérinaire de Toulouse (ENVT) - Institut National pour l’Agriculture, l’Alimentation et l’Environnement (INRAE) - 1225 Interactions Hôtes Agents Pathogènes (IHAP), 31300 Toulouse, France.
Aims: The emergence of bovine spongiform encephalopathy (BSE) prions from atypical scrapie has been recently proved in rodent and swine models. This study aimed to assess whether the inoculation of atypical scrapie could induce BSE-like disease in cattle.
Materials and Methods: Four calves were intracerebrally challenged with atypical scrapie. Animals were euthanized without clinical signs of prion disease between 7.2 and 11.3 years post-inoculation and tested for the accumulation of prions by conventional techniques and protein misfolding cyclic amplification (PMCA).
Results: None of the bovines showed signs compatible with prion disease. In addition, all tested negative for PrPSc accumulation by immunohistochemistry and western blotting. However, an emergence of BSE-like prions was detected during in vitro propagation of brain samples from the inoculated animals.
Conclusions: These findings suggest that atypical scrapie may represent a potential source of BSE infection in cattle.
Funded by: This work was supported financially by the following Spanish and European Interreg grants: Ministerio de Ciencia, Innovación y Universidades (Spanish Government), cofunded by Agencia Estatal de Investigación and the European Union and POCTEFA, which was 65% co-financed by the European Regional Development Fund (ERDF) through the Interreg V-A Spain-France-Andorra program (POCTEFA 2014– 2020).
Grant number: n° PID2021-125398OB-I00, EFA148/16 REDPRION
Acknowledgement: The authors would like to thank Sandra Felices and Daniel Romanos for their excellent technical assistance. Authors would also like to acknowledge the use of Servicio General de Apoyo a la Investigación-SAI, Universidad de Zaragoza
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Evolution of Nor98/ Atypical scrapie by iterative propagation in a homologous ovine PrPC context
Sara Canoyra1, Alba Marín-Moreno1, Juan Carlos Espinosa1, Natalia Fernández-Borges1, Nuria Jerez-Garrido1, Sylvie L. Benestad2, Enric Vidal3, Leonor Orge4, Olivier Andreoletti5 and Juan María Torres1.
1Centro de Investigación en Sanidad Animal, CISA-INIA-CSIC, Madrid, Spain. 2Norwegian Veterinary Institute, Ås, Norway. 3Centre de Recerca en Sanitat Animal, Universitat Autònoma de Barcelona (UAB)–Institut de Recerca i Tecnologia Agroalimentàries, Barcelona, Spain. 4Laboratory of Pathology, National Institute for Agrarian and Veterinary Research, Oeiras, Portugal 5UMR Institut National de la Recherche Agronomique (INRA)/École Nationale Vétérinaire de Toulouse (ENVT), Interactions Hôtes Agents Pathogènes, Toulouse, France
Aims: Nor98/ Atypical scrapie (AS) is a prion disease that causes sporadic casesin sheep and goats. Previous studies have shown that the transmission of AS to otherspecies led to the emergence of new prion strains. In the bovine and porcine PrP, there has been reported the emergence of classical BSE prions (Huor et al., 2019, Espinosa et al., 2009, Marin et. al., 2021) and in the bank vole M109I-PrP context, a classical scrapie-like prion strain emerges(Pirisinu et al., 2022). In this study, we analysed the possible evolution of the AS prion within the same specie by modelling the transmission in a homologous ovine PrP context.
Materials and Methods: A panel of AS isolates with different genotypes and geographical origins both from sheep and goats were inoculated in the wild-type transgenic mice model (ARQ-PrP, Aguilar-Calvo et al., 2014).
Results: The isolates infect the ovine ARQ-PrP mice with homogeneous survival time and a complete attack rate. For several AS isolatesthe transmission led to the emergence of 19kDa (with BSE-like characteristics), 21kDa or atypical prions and mixtures of these agents.
Conclusions:
Iterative subpassages of AS isolates into transgenic mice carrying ovine PrP showed an emergence of classical prions during in vivo propagation. This could be caused by the coexistence of strains in the isolate or the evolution of the AS through propagation in the ovine PrP.
These results allow us to hypothesize whether atypical prions might be the origin of prion diversity, where atypical prions tend to acquire classical forms. These results are relevant to control the exposure of farmed animals and humans to AS.
Funded by: MCIN/AEI/ 10.13039/501100011033 Grant number: PID2019-105837RB-I00
Conformational shift as the evolutionary mechanism for classical BSE emergence from atypical scrapie
Sara Canoyra, Alba Marín-Moreno, Juan Carlos Espinosa, Natalia Fernández- Borges, and Juan María Torres
Centro de Investigación en Sanidad Animal, CISA-INIA-CSIC, Valdeolmos, Madrid, Spain
Aims: New prion strains emerge when the prion conformational characteristics change during intra- or cross-species transmission. There are two main theories, non-mutually exclusive, that could explain this phenomenon: the ‘deformed templating’ and the ‘conformational selection model’. According to the ‘deformed templating’ or mutation model, when the prion is unable to replicate in a new host there is a shift to a new PrPSc conformation. On the other hand, the ‘conformational selection’ theory postulates that prion isolates are a conglomerate of conformations and during cross-species transmission the species barrier acts as a filter.
In previous studies, we showed the emergence of the bovine spongiform encephalopathy agent (C-BSE) due to the transmission of atypical scrapie (AS) onto bovine PrP. This work will elucidate the evolutionary dichotomy in the AS transmission, providing supporting evidence on the hypothesis of the origin of the epidemic C-BSE prion from AS.
Material and Methods: A panel of AS isolates with different genotypes and geographical distribution was analyzed. To differentiate between AS and C-BSE two strain typing features were used: thermostability and PMCA propagation. The AS isolates underwent a heat treatment of 98°C during 2 h and were amplified in vitro by PMCA in bovine PrPC substrate. The templating activity with or without heat was determine after 10 amplification rounds by western blot characterization.
In addition, we analyzed an artificial mixture of AS and C-BSE generated by diluting C- BSE in a constant amount of AS.
Results: We observed a drastic loss in the C-BSE emergence due to the heat treatment. The AS is a thermolabile prion. Hence, the inactivation of the AS conformers with the ability to shift the conformation will slow down the emergence of the C-BSE.
In contrast, when we analyzed the artificial mixture C-BSE prions emerge even with the heat treatment. Therefore, if the AS isolates had contained a minoritarian C-BSE conformer (defended by the conformational selection model) the emergence wouldn’t have been affected by the heat.
Conclusions: Mutation is the main evolutionary mechanism responsible for the C-BSE emergence. The species barrier forces the shift to a possible structure (C-BSE in this case) in a thermodynamically unfavorable process.
This discovery reenforces the origin hypothesis of the epidemic C-BSE as a contact of the cattle with feed contaminated with AS. Where the AS will evolve shifting to a C-BSE stable conformation. This also has implications in the control of farmed animals and humans’ exposure to the AS.
Funded by:/Grant number: Project PID2019-105837RB-I00 MCIN/ AEI /10.13039/501,100,011,033 Fundación La Marató de TV3 Enfermedades
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The emergence of classical BSE from atypical/Nor98 scrapie
Alvina Huor, Juan Carlos Espinosa https://orcid.org/0000-0002-6719-9902, Enric Vidal https://orcid.org/0000-0002-4965-3286, +15, and Olivier Andreoletti
https://orcid.org/0000-0002-7369-6016 o.andreoletti@envt.fr Authors Info & Affiliations
Edited by Michael B. A. Oldstone, Scripps Research Institute, La Jolla, CA, and approved November 15, 2019 (received for review September 11, 2019) December 16, 2019
116 (52) 26853-26862
Significance
The origin of transmissible BSE in cattle remains unestablished. Sheep scrapie is a potential source of this known zoonotic. Here we investigated the capacity of sheep scrapie to propagate in bovine PrP transgenic mice. Unexpectedly, transmission of atypical but not classical scrapie in bovine PrP mice resulted in propagation of classical BSE prions. Detection of prion seeding activity by in vitro protein misfolding cyclic amplification demonstrated BSE prions in the original atypical scrapie isolates. BSE prion seeding activity was also detected in ovine PrP mice inoculated with limiting dilutions of atypical scrapie. Our data demonstrate that classical BSE prions can emerge during intra- and interspecies passage of atypical scrapie and provide an unprecedented insight into the evolution of mammalian prions.
Abstract
Atypical/Nor98 scrapie (AS) is a prion disease of small ruminants. Currently there are no efficient measures to control this form of prion disease, and, importantly, the zoonotic potential and the risk that AS might represent for other farmed animal species remains largely unknown. In this study, we investigated the capacity of AS to propagate in bovine PrP transgenic mice. Unexpectedly, the transmission of AS isolates originating from 5 different European countries to bovine PrP mice resulted in the propagation of the classical BSE (c-BSE) agent. Detection of prion seeding activity in vitro by protein misfolding cyclic amplification (PMCA) demonstrated that low levels of the c-BSE agent were present in the original AS isolates. C-BSE prion seeding activity was also detected in brain tissue of ovine PrP mice inoculated with limiting dilutions (endpoint titration) of ovine AS isolates. These results are consistent with the emergence and replication of c-BSE prions during the in vivo propagation of AS isolates in the natural host. These data also indicate that c-BSE prions, a known zonotic agent in humans, can emerge as a dominant prion strain during passage of AS between different species. These findings provide an unprecedented insight into the evolution of mammalian prion strain properties triggered by intra- and interspecies passage. From a public health perspective, the presence of c-BSE in AS isolates suggest that cattle exposure to small ruminant tissues and products could lead to new occurrences of c-BSE.
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Classical BSE was first recognized in 1984 and 1985 as a novel prion disease affecting cattle in the United Kingdom (40). Epidemiological data clearly established that the number of cases of c-BSE was amplified by the recycling of infected animal carcasses into cattle feed in the form of meat and bone meal (MBM) (41). Since bovine prion disease had not been recognized in cattle prior to the c-BSE epizootic and the disease is apparently noncontagious between cattle, several hypotheses were proposed to explain its emergence. These range from the spontaneous occurrence of c-BSE in cattle to the passage and adaptation of a prion originating from another species (42, 43). Our studies here that show the presence of c-BSE prions in AS isolates, combined with the demonstrated presence of AS in the United Kingdom long before the appearance of the c-BSE epizootic in cattle, suggests that the recycling of AS cases in MBM might be a source of bovine prion disease (20). In addition to its potential role in the initial emergence of c-BSE in cattle, the presence of c-BSE prions in natural cases of AS has current and direct implications for both the continued risk of this ovine prion disease to other farmed animals and for human exposure risks. The distribution of AS cases are widespread across the world (17–19). A recent retrospective analysis of surveillance data collected over a period exceeding 10 y in the European Union (EU) concluded that the prevalence of detected AS cases has remained relatively stable in the different member states, with between 2 and 6 positive cases per 10,000 tested animals per year. This implies that a substantial number of AS-infected animals could enter either the animal or human food chain each year (44, 45), and each case represents a potential source of exposure to the c-BSE agent for farmed animals (MBM derived from rendered small ruminants) and human consumers (consumption of healthy slaughtered animals), respectively. The epidemiological features of AS within the EU is likely to reflect the situation of the disease in other countries that breed and maintain small ruminants.
In Europe, the c-BSE crisis and the emergence of vCJD resulted in the implementation of a strong and coherent policy (EU regulation 999/2001) aimed at control and eradication of this animal prion disease. The total feed ban on the use of MBM in animal feed and the systematic retrieval from the food chain of ruminant tissues that have the potential to contain high levels of prion infectivity, so-called Specified Risk Material (SRM) measures, were instrumental for control of c-BSE in cattle and prevention of dietary human exposure to these bovine prions (46, 47). As a side effect, these measures also strongly limited the exposure of farmed animals and human consumers to the other TSE agents circulating in farmed animal species, including AS.
With the decline of the c-BSE epizootic in cattle and the combined increase in pressure from industry, EU authorities have begun to consider discontinuing certain TSE control measures. The abrogation of the SRM measures for small ruminants and the partial reauthorization of the use of processed animal protein, formerly known as MBM, in animal feed are part of the EU authorities’ agenda. Our observation of the presence of the c-BSE agent in AS-infected small ruminants suggests that modification of the TSE control measures could result in an increased risk of exposure to c-BSE prions for both animals and humans. Whether or not this exposure will result in further c-BSE transmission in cattle and/or humans remains an open and important question.
Monday, November 13, 2023
Food and Drug Administration's BSE Feed Regulation (21 CFR 589.2000) Singeltary Another Request for Update 2023
BSE prions propagate as either variant CJD-like or sporadic CJD-like prion strains in transgenic mice expressing human prion protein
Emmanuel A.Asante, Jacqueline M.Linehan, Melanie Desbruslais, Susan Joiner, Ian Gowland, Andrew L.Wood, Julie Welch, Andrew F.Hill, Sarah E.Lloyd, Jonathan D.F.Wadsworth and John Collinge1
MRC Prion Unit and Department of Neurodegenerative Disease, Institute of Neurology, University College, Queen Square, London WC1N 3BG, UK 1 Corresponding author e-mail: j.collinge@prion.ucl.ac.uk
Variant Creutzfeldt±Jakob disease (vCJD) has been recognized to date only in individuals homozygous for methionine at PRNP codon 129. Here we show that transgenic mice expressing human PrP methionine 129, inoculated with either bovine spongiform encephalopathy (BSE) or variant CJD prions, may develop the neuropathological and molecular phenotype of vCJD, consistent with these diseases being caused by the same prion strain. Surprisingly, however, BSE transmission to these transgenic mice, in addition to producing a vCJD-like phenotype, can also result in a distinct molecular phenotype that is indistinguishable from that of sporadic CJD with PrPSc type 2. These data suggest that more than one BSEderived prion strain might infect humans; it is therefore possible that some patients with a phenotype consistent with sporadic CJD may have a disease arising from BSE exposure.
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These studies further strengthen the evidence that vCJD is caused by a BSE-like prion strain. Also, remarkably, the key neuropathological hallmark of vCJD, the presence of abundant ¯orid PrP plaques, can be recapitulated on BSE or vCJD transmission to these mice. However, the most surprising aspect of the studies was the ®nding that an alternate pattern of disease can be induced in 129MM Tg35 mice from primary transmission of BSE, with a molecular phenotype indistinguishable from that of a subtype of sporadic CJD. This ®nding has important potential implications as it raises the possibility that some humans infected with BSE prions may develop a clinical disease indistinguishable from classical CJD associated with type 2 PrPSc. This is, in our experience, the commonest molecular sub-type of sporadic CJD. In this regard, it is of interest that the reported incidence of sporadic CJD has risen in the UK since the 1970s (Cousens et al., 1997). This has been attributed to improved case ascertainment, particularly as much of the rise is reported from elderly patients and similar rises in incidence were noted in other European countries without reported BSE (Will et al., 1998). However, it is now clear that BSE is present in many European countries, albeit at a much lower incidence than was seen in the UK. While improved ascertainment is likely to be a major factor in this rise, that some of these additional cases may be related to BSE exposure cannot be ruled out. It is of interest in this regard that a 2-fold increase in the reported incidence of sporadic CJD in 2001 has recently been reported for Switzerland, a country that had the highest incidence of cattle BSE in continental Europe between 1990 and 2002 (Glatzel et al., 2002). No epidemiological case±control studies with strati®cation of CJD cases by molecular sub-type have yet been reported. It will be important to review the incidence of sporadic CJD associated with PrPSc type 2 and other molecular subtypes in both BSE-affected and unaffected countries in the light of these ®ndings. If human BSE prion infection can result in propagation of type 2 PrPSc, it would be expected that such cases would be indistinguishable on clinical, pathological and molecular criteria from classical CJD. It may also be expected that such prions would behave biologically like those isolated from humans with sporadic CJD with type 2 PrPSc. The transmission properties of prions associated with type 2 PrPSc from BSE-inoculated 129MM Tg35 mice are being investigated by serial passage.
We consider these data inconsistent with contamination of some of the 129MM Tg35 mice with sporadic CJD prions. These transmission studies were performed according to rigorous biosafety protocols for preparation of inocula and both the inoculation and care of mice, which are all uniquely identi®ed by sub-cutaneous transponders. However, crucially, the same BSE inocula have been used on 129VV Tg152 and 129MM Tg45 mice, which are highly sensitive to sporadic CJD but in which such transmissions producing type 2 PrPSc were not observed. Furthermore, in an independent experiment, separate inbred lines of wild-type mice, which are highly resistant to sporadic CJD prions, also propagated two distinctive PrPSc types on challenge with either BSE or vCJD. No evidence of spontaneous prion disease or PrPSc has been seen in groups of uninoculated or mock-inoculated aged 129MM Tg35 mice.
While distinctive prion isolates have been derived from BSE passage in mice previously (designated 301C and 301V), these, in contrast to the data presented here, are propagated in mice expressing different prion proteins (Bruce et al., 1994). It is unclear whether our ®ndings indicate the existence of more than one prion strain in individual cattle with BSE, with selection and preferential replication of distinct strains by different hosts, or that `mutation' of a unitary BSE strain occurs in some types of host. Western blot analysis of single BSE isolates has not shown evidence of the presence of a proportion of monoglycosylated dominant PrPSc type in addition to the diglycosylated dominant pattern (data not shown). Extensive strain typing of large numbers of individual BSE-infected cattle either by biological or molecular methods has not been reported.
Presumably, the different genetic background of the different inbred mouse lines is crucial in determining which prion strain propagates on BSE inoculation. The transgenic mice described here have a mixed genetic background with contributions from FVB/N, C57BL/6 and 129Sv inbred lines; each mouse will therefore have a different genetic background. This may explain the differing response of individual 129MM Tg35 mice, and the difference between 129MM Tg35 and 129MM Tg45 mice, which are, like all transgenic lines, populations derived from single founders. Indeed, the consistent distinctive strain propagation in FVB and C57BL/6 versus SJL and RIIIS lines may allow mapping of genes relevant to strain selection and propagation, and these studies are in progress.
That different prion strains can be consistently isolated in different inbred mouse lines challenged with BSE prions argues that other species exposed to BSE may develop prion diseases that are not recognizable as being caused by the BSE strain by either biological or molecular strain typing methods. As with 129MM Tg35 mice, the prions replicating in such transmissions may be indistinguishable from naturally occurring prion strains. It remains of considerable concern whether BSE has transmitted to, and is being maintained in, European sheep flocks. Given the diversity of sheep breeds affected by scrapie, it has to be considered that some sheep might have become infected with BSE, but propagated a distinctive strain type indistinguishable from those of natural sheep scrapie.
WEDNESDAY, JANUARY 3, 2024
PROCEEDINGS ONE HUNDRED AND TWENTY SIXTH ANNUAL MEETING USAHA CWD, Scrapie, and BSE, October 2022 updated science 2024
PLEASE NOTE, CJD IS NOW 1 IN 5,000 GLOBALLY, COLLINGE ET AL 2023!
Professor John Collinge on tackling prion diseases, sCJD accounts for around 1 in 5000 deaths worldwide
MONDAY, SEPTEMBER 11, 2023
Professor John Collinge on tackling prion diseases “The best-known human prion disease is sporadic Creutzfeldt-Jakob disease (sCJD), a rapidly progressive dementia which accounts for around 1 in 5000 deaths worldwide.” There is accumulating evidence also for iatrogenic AD. Understanding prion biology, and in particular how propagation of prions leads to neurodegeneration, is therefore of central research importance in medicine.
MONDAY, DECEMBER 18, 2023
Change in Epidemiology of Creutzfeldt-Jakob Disease in the US, 2007-2020
TUESDAY, DECEMBER 12, 2023
CREUTZFELDT JAKOB DISEASE TSE PRION DISEASE UPDATE USA DECEMBER 2023
Terry S. Singeltary Sr.
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