About twenty five years ago, the appearance in the UK of Bovine Spongiform Encephalopathy (BSE), quickly brought the previously obscure “prion diseases” to the spotlight. The ensuing health and food crises that spread throughout Europe had devastating consequences. In the UK alone, there were more than 36,000 farms directly affected by BSE and the transmission of BSE prions to humans via the food chain has caused over 200 people in Europe to die from variant Creutzfeldt-Jakob disease (vCJD)



Classical BSE now appears to be under control, although it persists at very low levels. Of note, research, including EU-funded research, has played a key role in this success: the origin of the infection was tracked to prions in Meat and Bone Meal (MBM); tests based on prion protein-specific antibodies were developed, allowing detection of infected animals; experimental investigation of transmission barriers between different species allowed a rational estimation of risks, etc. All this led to the implementation of rational and efficacious policies, such as the MBM ban to protect the animal feed chain, and the Special Risk Material (SRM) regulations to protect the human food chain.

In spite of this progress, prions are still a threat. Epidemiological re-assessment indicates that the 10 year incubation period separating the peaks of BSE and of the vCJD epidemics is probably too short. In addition, results from a large number of human tonsil (and appendix) analyses in the UK suggest that there may be a high number of asymptomatic individuals who are positive for the disease-associated conformer prion protein PrPSc. A likely scenario is therefore that all those with signs of infection in tissue could have infective blood posing the risk for transmission via blood products (confirmed in several cases). Altogether, these data clearly demonstrate the potential risk of a second wave of vCJD.

Recently, several reports on cases of “atypical” BSE in cattle may lead to a major new epidemic, particularly since we still do not understand all factors determining the species barrier. Ovine scrapie is another concern, because it could mask ovine BSE, presumably transmissible to humans. Scrapie is endemic and not likely to be eradicated soon, although current control measures are effective at greatly reducing disease incidence. Atypical forms, which may be spontaneous, are not affected by these control measures and this form of disease will persist in the global sheep population. The low prevalence of these disease forms makes effective surveillance very challenging. However, there is a clear risk attendant on ignoring these cases without an understanding of their possible zoonotic potential, particularly when most forms of human disease have no established aetiology.

In this “Interim position paper”, based on on-going research in the EU-funded project PRIORITY, we will highlight the state-of-the-art knowledge and point out scientific challenges and the major questions for research. Strategic objectives and priorities in Europe in the future in the research that aims to control, eliminate or eradicate the threat posed by prions to our food and health are also indicated.

The PRIORITY project has focused on 4 themes, namely, the structure, function, conversion and toxicity of prions; detection of prions; mechanisms of prion transmission and spreading and epidemiology of prion diseases. This interim paper summarizes the opinions/positions reached within these themes after the first two years of the project.


1. Prion structure, function, conversion and toxicity

 State of the art

The mechanisms for conversion of the normal, cellular PrPC to PrPSc as well as strain diversity and transmission barriers are structurally enciphered. Thus, it is essential to understand the structure of PrPSc in order to design methods to interfere with prion propagation and spread, but it can also be of diagnostic significance if alternative and/or earlier markers could be identified - especially in vivo.

The PrPSc forms double amyloid fibers made up of two intertwined fibrils, each 3-4 nm wide, with no regular pitch. Limited proteolysis studies indicate that PrPSc monomers that make up these fibers contain stretches of high resistance to PK (presumably -strands) interspersed with short stretches with a higher proteolytic susceptibility, presumably loops and turns. The C-terminal stretch (180-231) is the most PK resistant region.

 Wild-type PrPC converts to PrPSc in the sporadic forms of the disorders through an unknown mechanism. To unravel the early events in structural prion formation is of major importance since the conversion of PrPC to PrPSc is the central event in prion diseases. Hereditary prion diseases are associated with about forty point mutations of the gene coding for the PrP denominated PRNP. Most of the variants associated with these mutations are located in the globular domain of the protein.



The basic tenet of the prion theory, i.e. that protein misfolding can be faithfully propagated, is by now widely accepted. Moreover, novel data increasingly implicate similar "prionoid" principles in the pathogenesis of “proteinopathies” such as Alzheimer, Huntington and Parkinson disease. In sharp contrast with this fundamental understanding, and despite the development of many new tools for prion research, the most basic mechanistic details of how prions function and how they cause disease have remained largely obscure. The structural basis of prion strains and their interactions with the host cell, remain mysterious.

Major questions and scientific challenges

Many aspects of prion replication can be demonstrated in vitro in systems containing only PrPC and PrPSc. At first approximation, prion propagation can thus be reduced to a biophysical problem dealing with alternative conformations, amyloid structures, and conformational coercion. However, like other pathogens, prions maintain a complex, two- way relationship with the host cell. It is clear that prions propagate in their natural hosts much more efficiently that they do in vitro.

The host cell provides both the molecular species (such as PrPC) and the molecular mechanisms required for the prion propagation. Questions related to (i) the uptake of prions by the host cell and relevance of intracellular pathways for prion conversion, (ii) the influence of host cell signals and factors on prion "replication", (iii) the normal function of the prion protein and pathogenesis, i.e. mechanisms by which prions cause dysfunctions or damage to the neurons, and (iv) the transfer of prions to neighboring cells, remain vastly unsolved.

A major scientific challenge is also to better understand the existence of different prion strains and mechanisms behind the transmission barriers between animal species. Related to this is the question why some prions are more dangerous than others for humans. In addition, since sporadic prion diseases affect mainly aging people (average age of onset being around 65 years old) a challenge will be to investigate age-related factors that promote the sporadic diseases to develop. Another major effort to understand basic mechanisms of the disease has to be undertaken in order to develop adequate and early therapies for humans affected by the diseases. This input can only come from the scientific community because there is a clear lack of industrial investment to study and develop compounds for CJD affected individuals.


Strategic objectives and priorities in the future

  • More data of a higher resolution needed to understand the structural basis of prion strain transmission barriers, e.g. by NMR-based, deuterium exchange analyses of recombinant PrPSc.

  • Structural analysis of the various point mutations present in the globular domain of PrPSc can unveil common folding traits that may allow to a better understanding of the early conformational changes leading to the formation of monomeric PrPSc.

  • Analyses combining high resolution imaging tools and neurophysiology that leads to a better understanding of the function of the prion protein and its pathological isoforms.

  • Analyses of the cause of prion toxicity and identification of host cell-derived factors that are “partners in crime” can provide novel strategies aiming at blocking prion propagation and toxicity.

  • Development of new treatment strategies for individuals affected by CJD.


2. Prion detection

State of the art

Advantages are taken of technical advances to improve prion detection in body fluids as well as in soil and waste. Such tests are urgently needed to prevent spread and transmission of prions.

Opinions – Positions:

The emergence of in vitro amplification technologies (such as PMCA and QUIC) represents a real revolution for prion detection. These techniques display sufficient theoretical sensitivity to allow prion detection in the body fluids (such as blood) collected in affected individuals. However, at the moment they remain of limited robustness and the mechanisms analytical conditions which allow amplification of misfolded PrP remain largely unknown. Such issues are similar to those encountered when PCR was developed in the 80’s . Despite those initial difficulties PCR is now a basic lab technology.

Prions may be considered also as potential environmental contaminants and their stability in the environment, wastewater and soils must be evaluated as a requested parameter for developing risk assessment studies. Prions are extremely resistant to inactivation and it has been described that prions can survive in soil over years. In the last years, deposition of scrapie and chronic wasting disease (CWD) prions in the environment through biological fluids and/or faeces has been proved. Conversely, BSE can also be introduced anthropogenically by transporting infectious prions via landfill leach or slaughterhouse wastewater. Furthermore, there is the possibility of discharged contaminated urine, feces and blood from CJD patients. All this information suggests strongly that infectious prions enter to the environment, and could be transported via water and expose humans and animals to infectious prion diseases. Therefore, it is critical to evaluate the fate of infectious prions in the environment and the potential sources of contamination. 


Major questions and scientific challenges


A major scientific challenge is to develop better prion detection methods that can have applications in pharma screening, consumables testing, environmental monitoring (e. g. allowing re-population of previously affected farms), and in vivo diagnostics.


The behavior and stability of prions in the environment and wastewater have to be better defined and the efficiency of waste water treatments in the removal of prions be assessed.

Strategic objectives and priorities in the future

  • Improving the performances and robustness of in vitro prion amplification technology

  • Establishing a relationship between the presence of PrPSc as demonstrated in an environmental matrix by in vitro amplification methodology and the risk of prion transmission for an individual that would be exposed to such matrix.

  • Redefining the techniques available to optimize the detection of prions in the divers environmental matrices with validated protocols.

  • Water samples impacted by infected animal excreta and waste water must be analyzed for the potential role in the transmission of prion diseases, producing data on the potential dissemination of prions in these areas.


 3. Prion transmission and spreading

State of the art

Insights on mechanisms by which prions enter the brain to induce a neurodegenerative diseases and exit an organism through body fluids, as well as on and how factors such as host age and inflammation affect these processes, are essential for assessment of prion transmission and pathogenesis.




We still do not know the precise cellular mechanisms by which prion infections get in or out of an animal, or how to detect/control either of these. Knowledge on these processes will assist both diagnostic and therapeutic intervention strategies.


Major questions and scientific challenges


By which cellular mechanisms do prions propagate from the periphery to the central nervous system to cause disease and how are they released into body fluids for spread into the surrounding?


Are species barriers to prions “rigid/absolute” and related to the prion strain, or can they at the individual host level be affected by host variables such as other infections and inflammatory disorders, and age?


Another important question is whether milk presents a risk for spread of prions.


Strategic objectives and priorities in the future


  • Identifying the organelle(s) and molecules involved in cell to cell prion spreading and release in the body fluids

  • Better understanding of when to target diagnostic/therapeutic strategies based on age/species.

  • Development of host cell-directed interventions to prevent propagation and spread of prions.

  • Which decontamination procedures should be implemented in clinical practice?

4. Prion epidemiology


State of the art

A better understanding on the way in which different strains of prions are spreading between animals and human beings, and the environmental factors that modulate such spreading is essential to design methods to prevent spread of prions within the communities. Crucial to prevent spread of prions are also improved methods for decontamination and disposal of animal waste as well as assessment of prions in waste water and soils.

Considerable efforts have been made by the EU during recent years by implementing a rigorous regimen to control prion infections in cattle, sheep and goat. An array of regulations such as the introduction of the feed ban, an effective surveillance and monitoring system, the destruction of SRM and establishing culling strategies by member state authorities had a significant impact on the decrease of numbers of incidences and the spread of the disease. Undoubtedly, these measures have reduced the number of BSE cases detected in the EU from 2,167 in 2001 (15 member states) to 65 cases in 2009 and 43 cases in 2010 and 11 cases (Sept. 2011) in 2011 in 27 member states.

Deposition of scrapie and CWD prions in the environment occurs through biological fluids and/or faeces. Data depict a scenario where prions may accumulate in the environment due to direct shedding from pre-clinical animals and remain infectious in soil and water for periods of time long enough to permit transmission to susceptible individuals. Although the scenario for BSE could not be completely the same (BSE prions are hardly detectable in extraneural tissues and are essentially restricted to the CNS), deposition of BSE prions in the environment may occur due to burial of carcasses and mortalities, and to a lesser extent, through biosolids generated in water treatment plants processing infected animals, especially those being unaware of it. Presumably this scenario occurred during the BSE epidemics. Furthermore, there is the possibility of discharged contaminated urine, feces and blood from CJD or vCJD patients. The potential presence CWD in Europe has not been significantly investigated (EC report, Chronic Wasting Disease and tissues that might carry a risk for human and animal feed chains, 2003). In humans, several molecular defined disease subtypes have been described. However, the molecular basis and epidemiological significance of these so called sporadic disease subtypes are not understood.


Opinions -Positions:

Although still declining, BSE has not been eradicated so far and regarding presumably sporadic cases of BSE one might question if eradication is generally achievable.

Furthermore, sporadic cases of BSE appear to be significantly different from orally acquired BSE in many aspects. The most obvious differences in such atypical/sporadic BSE are the tendency for ages of diseased animals to be in the last third of the life span for cattle and a different phenotype of the prion protein. Most recently, two new case of non-classical BSE were diagnosed in Switzerland, a country where BSE had been seen last in 2006. The overall picture of atypical/sporadic BSE is even complicated by the fact that these two new cases of BSE appear to be not equivalent to so far known atypical cases in cattle. Cases of atypical scrapie in sheep and goats as well as BSE in sheep and goats might even further complicate the picture. The fact that in the years 2010 and 2011 (Sept. 2011) atypical scrapie by far outnumbers the cases of classical scrapie causes quite some concern.

A major point of concern is therefore the occurrence of atypical cases of BSE, which in light of the new types of atypical BSE in Switzerland may remain undetected. Especially the occurrence of atypical BSE in elderly cows with an extended pre-clinical phase poses a particular challenge. Even in classical BSE, depending on different testing scenarios, the European Food Safety Agency (EFSA) has published an Opinion indicating the possibility of missing BSE cases in healthy or at risk animals. In the consortium’s opinion the chance of spread of BSE within the cattle population can be regarded as negligible as long as the feed ban is still operative. Likewise, under the present regulatory regimens the exposure risk for humans is very low.

Prion diseases cannot be eradicated, especially the spontaneous diseases, and it is the opinion of the consortium that a continuous robust surveillance of both animal and human populations is required.


Major questions and scientific challenges

Although the epidemiology of atypical cases supports the hypothesis of a spontaneous origin, they can be experimentally transmitted and therefore present a risk. Also stability of these prions upon passage is not yet known – they may become more ‘infectious’ by passages. A major scientific challenge is therefore to understand basic biology and key components determining susceptibility and transmissibility.

More information about the survival of prions to inactivation treatments in wastewater treatment plants and the stability to the environmental factors is necessary. Results suggest that bacterial proteolysis of prions is strongly related to the stability of the prions. Further analysis would, thus, be necessary to understand if improvements to increase the biological inactivation are a real solution for prions inactivation in wastewater treatment plant.

Data have indicated that the inactivation of infectious BSE in the environment can not be estimated only by the detection of protease resistant PrPSc levels. Improved PrP markers to be used as target parameter must be defined considering infectivity.


Strategic objectives and priorities in the future

  • The existence of atypical prions, which were until now unknown, in cattle and small ruminants, and the new concept of “prionopathies” in humans clearly show that

appropriate prion agent surveillance should be maintained in animal and human population, and that surveillance tools for field surveillance should be developed according to the scientific progress.

  • Definition of suitable wastewater treatments that would reduce the possibility of prion dissemination in the environment.

  • Implementation of a study of a potential presence of CWD in Europe, included surveillance programs for the detection of CWD prions and studies of their behavior in the environment, such as the stability to environmental factor and to treatment in wastewater treatment plants.

  • Development of programmes for education and awareness within farming communities and vets/medics, in particular, as a frontline surveillance

  • Establishing continuous- molecular strains defined- surveillance of all forms of human prion diseases for early identification of atypical cases and potential outbreaks in humans.











5. Proposed Recommendations


a. The question of re-introduction of ruminant protein into the food-chain

The opinion of the members of PRIORITY is that the sustainment of an absolute feed ban for ruminant protein to ruminants is the essential requirement, especially since the impact of non-classical forms of scrapie in sheep and goats is not fully understood or cannot be fully estimated. Therefore, the consortium strongly recommends prohibiting re-introduction of processed ruminant protein into the feed-chain. Arguments in support of this opinion are:

  • the large (and still uncharacterized) diversity of prion agents that circulate in animal populations;

  • the uncertainties related to prion epidemiology in animal populations;

  • the unknown efficacy of industrial processes applied to reduce microbiological risk during processed animal protein (PAP) production on most prion agents;

  • the intrinsic capacity of prions to cross interspecies transmission barriers;

  • the lack of sensitive methodology for identifying cross contamination in food.

The consortium is also hesitant to introduce processed ruminant proteins into fish food considering the paucity of data on prion infections in fishes and sea animals, and the risk of establishing an environmental contamination of the oceans that cannot be controlled.


b. Atypical prion agents

Atypical prion agents will probably in the next future represent the dominant form of prion diseases. Type L atypical BSE has clear zoonotic potential. Similarly, there are now some data that seem to indicate that atypical scrapie agent can cross various species barriers. Moreover, the current EU policy for eradicating scrapie (genetic selection in affected flocks) is inefficient to prevent atypical scrapie. In that context it would appear valuable

  • to develop knowledge related to pathogenesis and inter-individual transmission of atypical prion agents in ruminants (both intraspecies and interspecies)

  • to investigate for potential PrP resistance allele to the infection by atypical prion agents

  • to improve the sensitivity of detection assay that are applied in the field towards this type of agent

  • to maintain a robust surveillance of both animal and human populations


c. Species transmission barriers

Intensified search for a molecular signature of the species barrier is recommended, since this barrier is a key for many important policy areas - risk assessment, proportional policies, the need for screening of human products and food.


d. Prion structure

Prion strain structural language will remain an important issue for public health for the foreseeable future. Understanding the structural basis for strains and the basis for adaptation of a strain to a new host will require continued fundamental research.


e. Detection and therapy

Early detection of prion infection, ideally at preclinical stage, will remain crucial for development of effective treatment strategies in humans affected by the disease.




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