EFSA Monitoring of chronic wasting disease (CWD) (IV)

EFSA Monitoring of chronic wasting disease (CWD) (IV)

Monitoring of chronic wasting disease (CWD) (IV)

Published: 17 April 2023 Adopted: 15 March 2023

EFSA Journal 2023;21(4):7936

DOI https://doi.org/10.2903/j.efsa.2023.7936

KEYWORDS chronic, wasting, CWD, surveillance, Europe, genotype

European Commission

QUESTION NUMBER EFSA‐Q‐2022‐00114

PANELS Biological Hazards

PANEL MEMBERS AT THE TIME OF ADOPTION

Konstantinos Koutsoumanis, Ana Allende, Avelino Alvarez‐Ordoñez, Declan Bolton, Sara Bover‐Cid, Marianne Chemaly, Robert Davies, Alessandra De Cesare, Lieve Herman, Friederike Hilbert, Roland Lindqvist, Maarten Nauta, Luisa Peixe, Panagiotis Skandamis, Elisabetta Suffredini, Marion Simmons and Giuseppe Ru.

CONTACT biohaz@efsa.europa.eu

Abstract

The European Commission requested an analysis of the Chronic Wasting Disease (CWD) monitoring programme in Norway, Sweden, Finland, Iceland, Estonia, Latvia, Lithuania and Poland (9 January 2017–28 February 2022). Thirteen cases were detected in reindeer, 15 in moose and 3 in red deer. They showed two phenotypes, distinguished by the presence or absence of detectable disease‐associated normal cellular prion protein (PrP) in lymphoreticular tissues. CWD was detected for the first time in Finland, Sweden and in other areas of Norway. In countries where the disease was not detected, the evidence was insufficient to rule out its presence altogether. Where cases were detected, the prevalence was below 1%. The data also suggest that the high‐risk target groups for surveillance should be revised, and ‘road kill’ removed. Data show that, in addition to differences in age and sex, there are differences in the prion protein gene (PRNP) genotypes between positive and negative wild reindeer. A stepwise framework has been proposed with expanded minimum background surveillance to be implemented in European countries with relevant cervid species. Additional surveillance may include ad hoc surveys for four different objectives, specific to countries with/without cases, focusing on parallel testing of obex and lymph nodes from adult cervids in high‐risk target groups, sustained over time, using sampling units and a data‐driven design prevalence. Criteria for assessing the probability of CWD presence have been outlined, based on the definition of the geographical area, an annual assessment of risk of introduction, sustained minimum background surveillance, training and engagement of stakeholders and a surveillance programme based on data‐driven parameters. All positive cases should be genotyped. Sample sizes for negative samples have been proposed to detect and estimate the frequency of PRNP polymorphisms. Double‐strand sequencing of the entire PRNP open reading frame should be undertaken for all selected samples, with data collated in a centralised collection system at EU level.

© European Food Safety Authority

Summary

Following the 3-year monitoring programme laid down by Regulation (EU) 2017/1972 that ended on 31 December 2020, and the extension of the intensified monitoring in Sweden until February 2022,the European Food Safety Authority (EFSA) was asked by the European Commission to deliver its scientific opinion by 31 March 2023 on the following terms of reference (ToRs): (1) To analyse the results of the monitoring programme carried out in Norway, Sweden, Finland, Iceland, Estonia, Latvia, Lithuania and Poland between 1 September 2017 and 28 February 2022, and in particular, to assess if the two objectives as set in the 2017 EFSA opinion on CWD in cervids have been met. (2) To describe any new knowledge on the epidemiology of CWD in EU/EEA countries. (3) To recommend, if considered appropriate, future CWD monitoring activities for the EU based on an assessment of the epidemiological situation. (4) Based on what is known about the epidemiology of CWD in EU/EEA countries, to describe the criteria relevant for considering an area not to be infected with CWD. (5) To provide the design of a genotyping protocol for positive samples, and for the negative samples of the 3-year monitoring programme stored as per point 3.3, section III.A of Annex III of Regulation (EC) No999/2001, specifying which negative samples should be genotyped, the codons of the PRNP gene to be genotyped and recommending genotyping assay/s for the implementation of the requirement by the NRLs.

The data used in this assessment comprises the surveillance data submitted to EFSA and validated and published every year in the EU summary reports for TSE, and the additional data and information provided by Finland, Norway and Sweden. Following the description of the surveillance data, the intensified surveillance conducted in countries with cases and the caseload with the estimation of observed prevalence, the minimum detectable prevalence at country, species and at primary sample unit (PSU) level was calculated. Scenario-tree modelling was applied to combine samples from low-and high-risk target groups to estimate the surveillance system sensitivity (SSe) for detecting CWD in a cervid species for a two-stage sampling system in a country (PSU and within PSU). New knowledge on the epidemiology of CWD since the last published EFSA opinion on CWD was in part collated from relevant scientific literature and in part based on the outcome of the surveillance data analysed within this mandate. To inform a stepwise approach to the design of future monitoring activities, a SWOT analysis of the past mandatory surveillance (2018–2020) was conducted. Since developing criteria for considering an area not to be infected were judged not possible, criteria for the estimation of the probability of occurrence in a determined geographical area were developed. Finally, country- and species-specific samples size for genotyping was calculated for detecting new polymorphisms or estimating the frequency of known polymorphisms in the PRNP, and to test their association with susceptibility/resistance to CWD.

The eight countries tested a total of 155,660 cervids older than 12 months of age and of unknown age during the mandate period (1 September 2017–28 February 2022), distributed as follows: Estonia (2,153), Finland (3,457), Iceland (304), Latvia (3,087), Lithuania (3,784), Norway (130,836), Poland (3,505) and Sweden (8,534). The hunted and slaughtered fit for human consumption (HSHC) group was the most frequently tested overall in terms of number of animals (83.8%) in six of the eight countries, with road kills (RK) being the most tested high-risk target group in Finland and Poland. Roe deer was the most tested species in three MS (Estonia, Latvia and Poland). Through this statutory surveillance, prion diseases in cervids were detected for the first time in two countries (Finland and Sweden). During the mandate period, a total of 31 cases were confirmed in three countries (Finland, Norway and Sweden): 13 reindeer, 15 moose and 3 red deer. Sweden and Finland intensified the surveillance in and around areas where CWD cases were found (‘affected areas’), testing both high-and low-risk target groups to increase overall probability of disease detection. The observed prevalence among HSHC in affected areas was 0.05% (95% CI 0.006–0.17%) in moose, 0.16% (0.04–0.57%) in red deer and 0.23% (95% CI: 0.12–0.4%) in reindeer. The observed prevalence was more than 10 times higher among moose and red deer submitted from high-risk target groups, emphasising the value of high-risk target groups to detect new CWD cases in these species. The results of the analyses revealed that RK should not be considered a high-risk target group.

The two objectives of CWD surveillance (detect disease and estimate prevalence) have partially been met, given the high variability in the implementation of surveillance at country, species, management systems and PSU level. As a result, for countries that conducted surveillance during the mandate period without finding CWD, the evidence is not sufficient to rule out the possibility of CWD being present. For those countries with cases detected, the observed prevalence is associated with uncertainty due to the sample-based monitoring.

Calculations of the minimum detectable prevalence at country level showed that it was possible to detect a prevalence in the general population (all species for the entire mandate period) close to the 0.1% referred to in the 2017 EFSA opinion. There is high variability between countries and between species within countries. The attained minimum detectable prevalence among high-risk animals was10% or lower in only 15.3% of the monitored PSU. The result of the scenario tree modelling showed that the overall estimated sensitivity for all cervids except wild reindeer was 95% or greater to detect CWD in Norway, Sweden and Poland considering a scenario of a minimum 5% prevalence and a relative risk (RR) of 5 for high-risk animals compared to HSHC. For a scenario of a minimum 1% prevalence, only Poland (96%) and Norway (94%) reached~95% sensitivity with their applied monitoring programme. In the analysis by species for RR of 5, only the following scenarios reached 95% sensitivity or higher to detect CWD: for semi-domesticated reindeer, Norway and Finland for 5%or higher prevalence and Norway for 1% or higher prevalence; for moose, Norway for 5% or higher prevalence; for roe deer, Norway and Poland for 5% or higher prevalence and Poland for 1% or higher prevalence. The rest of the assessed combinations (country/species) did not reach 95% sensitivity.

CWD has been detected in new areas within Norway but also in Sweden and Finland, and in a new species (red deer). The data showed two main disease phenotypes, distinguished by the presence(Ly+) or absence (Ly-) of detectable disease-associated PrP in the lymphoreticular tissues, in addition to that seen in the central nervous system. So far, the Ly+ phenotype has been observed only in wild reindeer and the Ly- only in moose and red deer. It was considered that these differences in tissue distribution could affect the transmissibility of the disease under field conditions and they were therefore considered as separate entities for the purpose of this epidemiological analysis.

In general, the geographic distribution of CWD Ly- in affected countries is patchy, while the CWD Ly+ was clustered in distribution. In areas where CWD has been found, the observed prevalence was low (<1%), although the prevalence in adult males above 2 years old was 1.5% in the first area where CWD Ly+ was detected in wild reindeer in Norway. The prevalence of CWD Ly+ increases with age and males were more likely to have the disease than females, while cases of CWD Ly- were mainly in old (>10 years) females (only a single male). Initial published data revealed genetic variations in Norwegian reindeer with some PRNP-alleles more frequently present in CWD Ly+ cases.

In the future, it is proposed that a minimum sustained background surveillance effort with a dedicated infrastructure and a good system for obtaining samples and testing should be available in every country. This effort should be focused on the testing of samples from relevant cervid species in high-risk target groups (clinical/sick(showing abnormal behaviour, locomotor disturbances or otherwise poor health) (SUS), fallen/culled (individuals found dead or killed for health/age reasons) (FC), hunted/slaughtered but declared unfit for human consumption (HSNHC)), systematically or opportunistically acquired. Beyond the minimum surveillance described above, specific surveillance activities can be implemented depending on the objectives set by risk managers. Common features of the proposed surveillance activities for four possible identified objectives are specific surveillance design for countries with/without previously detected cases of CWD, collection and testing of both retropharyngeal lymph node and brainstem samples; testing animals over 2 years of age if possible; maximising the sensitivity by prioritising the sampling of cervids from ‘high-risk’ target groups within each selected area and management system; consider a sustained rolling time frame for accumulating surveillance data; divide the area/region/country into sampling units based on the epidemiological and management knowledge of cervid populations present; and, in areas where disease is still undetected, set design prevalence based on the findings of this report or on new epidemiological data when available.

The criteria proposed for assessing the probability of CWD presence rather than ‘for considering an area non-infected with CWD’ include the definition of the geographical area by setting spatial boundaries; the annual assessment of the risk of introduction of CWD into the area to inform the surveillance design; a minimum sustained background surveillance with a dedicated infrastructure and a good system for obtaining samples and testing as described in ToR3; training and engagement of stakeholders, and an ‘output based’ surveillance programme based on data-driven input parameters.

All positive cases should be genotyped. Sample sizes for negative samples, ranging between 76 and145 at country and species level, have been proposed to detect and estimate the frequency of PRNP polymorphisms and to ascertain susceptibility association. Double strand sequencing the entire PRNP open reading frame should be undertaken for all selected samples. A centralised data collection system at EU level is required, allowing the collation and extraction of data for analysis, containing the complete coding sequence of the animal PRNP in standard format, and the metadata associated with each sampled animal.

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3.3.3.5. Public health considerations

Given the uncertainty about the zoonotic potential of European CWD isolates, a country may choose to take measures to prevent/minimise human exposure via the food chain. Systematic testing cannot be considered a surveillance strategy for data collection, but it is a risk mitigation measure. The EFSA opinion on chronic wasting disease III (EFSA BIOHAZ Panel, 2019) recommended the measures listed below to reduce/eliminate human exposure to CWD:

•Systematic testing: only allowing human consumption of meat, meat products and offal sourced from animals that have been tested negative for CWD.

•Targeted measures: prohibition of harvesting/hunting susceptible species or the introduction of compulsory testing of animals before human consumption in/from declared infected premises/areas (e.g. a farm, or a surveillance PSU (Section3.6), region, country, etc.).

•Systematic removal of high-risk tissues from all cervids intended for human consumption with no requirement for testing.

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Abstract

www.efsa.europa.eu/en/efsajournal/pub/7936

See full report; 

https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2023.7936

Norway More tests are needed to determine the occurrence of CWD

More tests are needed to determine the occurrence of CWD

Published 12.04.2023

In 2022, 17,584 deer animals were tested for scabies. The Veterinary Institute and NINA have collaborated to carry out the practical work on behalf of Mattilsynet and Miljødirektoratet.

Skrantesjuke (Chronic Wasting Disease – CWD) was detected in Norway in the spring of 2016, and since then a lot of resources have been spent on mapping.

-The aim is to map the occurrence and geographical distribution of classic and atypical skrantesjuke. This is necessary so that the administration can implement good measures to limit or combat this disease, says senior researcher and CWD coordinator at the Veterinary Institute Jørn Våge.

- The extensive surveillance means that we now know with reasonable certainty that CWD is not widespread with a high incidence among reindeer in Norway, while it is highly probable that infected animals are found on the Hardangervidda. Further testing is absolutely crucial in order to map the possible low incidence of the disease, he says.

In the monitoring program for the mapping and monitoring of scrapie disease (CWD) in 2022, it is stated that in the period 2016-2022, classic CWD is detected in 21 wild reindeer and atypical/sporadic CWD is detected in 11 moose and three deer.

https://www.vetinst.no/nyheter/fleire-prover-avgjerande-for-a-kartleggja-forekomst-av-cwd

Scientific Opinion

Update on chronic wasting disease (CWD) III

EFSA Panel on Biological Hazards (BIOHAZ), Kostas Koutsoumanis, Ana Allende, Avelino Alvarez-Ordoňez, Declan Bolton, Sara Bover-Cid … See all authors 

First published: 11 November 2019 https://doi.org/10.2903/j.efsa.2019.5863Citations: 3

Correspondence: biohaz@efsa.europa.eu

Requestor: European Commission

Question number: EFSA-Q-2018-00763

Panel members: Kostas Koutsoumanis, Ana Allende, Avelino Alvarez-Ordoñez, Declan Bolton, Sara Bover-Cid, Marianne Chemaly, Robert Davies, Alessandra De Cesare, Lieve Herman, Friederike Hilbert, Roland Lindqvist, Maarten Nauta, Luisa Peixe, Giuseppe Ru, Marion M. Simmons, Panagiotis Skandamis and Elisabetta Suffredini.

Acknowledgements: The EFSA Panel on Biological Hazards (BIOHAZ) wishes to acknowledge all European and North American researchers who provided data and information for this scientific output, and Maria Francesca Iulietto for her contributions to the draft.

Adopted: 26 September 2019

This article was originally published on the EFSA website www.efsa.europa.eu on 7 November 2019

Abstract

The European Commission asked EFSA for a Scientific Opinion: to revise the state of knowledge about the differences between the chronic wasting disease (CWD) strains found in North America (NA) and Europe and within Europe; to review new scientific evidence on the zoonotic potential of CWD and to provide recommendations to address the potential risks and to identify risk factors for the spread of CWD in the European Union. Full characterisation of European isolates is being pursued, whereas most NA CWD isolates have not been characterised in this way. The differing surveillance programmes in these continents result in biases in the types of cases that can be detected. Preliminary data support the contention that the CWD strains identified in Europe and NA are different and suggest the presence of strain diversity in European cervids. Current data do not allow any conclusion on the implications of strain diversity on transmissibility, pathogenesis or prevalence. Available data do not allow any conclusion on the zoonotic potential of NA or European CWD isolates. The risk of CWD to humans through consumption of meat cannot be directly assessed. At individual level, consumers of meat, meat products and offal derived from CWD-infected cervids will be exposed to the CWD agent(s). Measures to reduce human dietary exposure could be applied, but exclusion from the food chain of whole carcasses of infected animals would be required to eliminate exposure. Based on NA experiences, all the risk factors identified for the spread of CWD may be associated with animals accumulating infectivity in both the peripheral tissues and the central nervous system. A subset of risk factors is relevant for infected animals without involvement of peripheral tissues. All the risk factors should be taken into account due to the potential co-localisation of animals presenting with different disease phenotypes.

Summary

In 2018, the European Food Safety Authority (EFSA) was asked by the European Commission to deliver a Scientific Opinion on three Terms of Reference (ToRs) related to chronic wasting disease (CWD): (ToR1) to revise the state of knowledge, considering new scientific data, about the differences: a) between the strains found in different species in North America (NA) and in Europe and b) between the strains found so far in moose, reindeer and red deer in Europe, with the main emphasis on transmissibility (transmission paths), pathogenicity and prevalence of the different strains and susceptibility of the different species/genotypes; (ToR2) to revise the new scientific evidence on the zoonotic potential of CWD, to assess the risk of transmission to humans through the consumption of fresh meat, meat products and offal of cervids and to provide recommendations on possible additional control measures to address the risks identified; and (ToR3) to identify risk factors that can facilitate the spread of CWD in the European Union given the current situation of the disease.

To source the relevant data, the extensive literature searches used in the previous recent EFSA Scientific Opinions on CWD (EFSA BIOHAZ Panel, 2017, 2018) were updated. In addition, research groups known to be conducting bioassay transmission studies and molecular/biochemical studies (protein misfolding cyclic amplification (PMCA), real-time quaking-induced conversion (RT-QuIC), PrPres typing, conformational stability, proteinase K (PK) resistance, etc.) to characterise and the transmissibility of CWD field isolates from cases confirmed in Norway/Finland and in NA were asked to provide additional pre-publication data. Regarding risk factors, the strength of the evidence for the causal role of each factor was appraised, based on study design and a score-based ranking from the strongest evidence (intervention studies) to the weakest (theoretical biological plausibility) was applied.

Full characterisation of European isolates is being pursued through the collection of data on host species spectrum and genotype, clinical presentation, histopathology, immunopathology, tissue distribution, pathogenesis, the biochemical properties of the PrPSc, and bioassay through experimental transmission to a wide range of rodent models, whereas most NA CWD isolates have not been fully characterised in this way. However, preliminary data support the contention that the CWD strains identified to date in Europe and NA are different and suggest the presence of strain diversity in the European cervid population. The origin(s) of CWD in Europe remains unknown, and while it is clear that the disease identified in the reindeer in Nordfjella is contagious, the nature of the prion disease in the other species (the European moose and red deer) is still to be established.

NA CWD has been transmitted experimentally to cattle and sheep, but with incomplete attack rates. The species barrier appears higher for pigs, although challenged animals can support low-level prion amplification. Experimental transmission to transgenic (tg) mice and other rodent models shows some difference in the host ranges of different isolates but, particularly for the European isolates, many bioassays are still ongoing and data are not yet available. The number of strains, the strain diversity, the prevalence and the potential host range of disease in both NA and Europe CWD may all be underestimated.

In vitro studies suggest that CWD isolates derived from experimentally challenged reindeer, and elk with a specific PRNP polymorphism (132 MM) would present an intermediate potential of conversion of human PrP to disease-associated PrPSc. Some studies have shown that exposure to some NA CWD isolates can result in the conversion of human PrP in vitro and that some NA CWD isolates can transmit disease efficiently to squirrel monkeys. However, in vivo studies performed with humanised mice and macaques are considered to be the most pertinent models of human susceptibility and there is conflicting evidence on the transmissibility of NA CWD isolates in these models.

Epidemiological studies suffer from many methodological limitations and logistic constraints and some of them are still ongoing in NA but, until now, there is no epidemiological evidence of NA CWD causing disease in humans. The risk to humans through consumption of meat, meat products and offal derived from CWD-infected cervids cannot be directly assessed. At individual level, consumers of meat, meat products and offal derived from CWD-infected cervids will be exposed to the CWD agent(s). At the population level, the probability of exposure via consumption of venison depends on the prevalence of CWD agent(s) in each of the species that are consumed (reindeer, moose, red deer), which is not known. Preliminary testing of animals intended for human consumption with removal of any carcases that test positive, or the removal of high-risk tissues from cervids intended for human consumption, or the combination of these measures, would reduce the probability of dietary exposure of humans to the CWD agent(s). The prohibition of harvesting/hunting susceptible species in infected premises/areas could also be considered as a preventive measure.

Current EU legislation requires a 3-year monitoring programme for CWD from 1 January 2018 to 31 December 2020 to be implemented in six Member States (MSs) that have a wild and/or farmed and/or semi-domesticated population of moose and/or reindeer: Estonia, Finland, Latvia, Lithuania, Poland and Sweden. In 2018, the six MS tested a total 5,110 cervids, of which 4,674 (91.5%) were wild animals, mostly roe deer and red deer, and 436 (8.5%) were captive, farmed or semi-domesticated, with more than half of them being semi-domesticated reindeer tested in Finland. Over 59% of all cervids tested were from healthy hunted/slaughtered fit for human consumption animals, whose probability of disease is lower than that of sick animals, road kills or fallen stock. Up to 20 September 2019, 28 cases have been reported in Europe: 19 wild reindeer, 4 moose and one red deer in Norway, one moose in Finland and three moose in Sweden.

Using data from the NA CWD experience, 13 groups of risk factors have been identified based on their biological plausibility to spread CWD. Some of these are supported by epidemiological evidence from NA CWD studies with variable strength of evidence after applying the score-based ranking, while others remain hypothetical:

Natural movement of live wild deer from infected areas,

Man-mediated movement of live farmed/free-ranging deer from infected areas,

Failure to separate live farmed and free-ranging deer,

High deer density,

Species-specific social organisation,

Sex-related behaviours,

Natural or man-mediated animal aggregation,

Consumption of forage grown on contaminated soil,

Fallen stock or inappropriate disposal of carcasses and slaughter by-products,

Movement of other animals (working dogs, scavengers, predators),

Transfer of inanimate vehicles of contamination (fomites),

Environmental persistence of prions,

Host genetics.

All the identified risk factors may contribute to the spread of the disease when it is associated with the accumulation of infectivity in peripheral tissues, a host phenotype that is compatible with a contagious disease. A subset of risk factors (1, 2, 6, 8, 9, 10, 11, 12 and 13) is relevant to cases of disease that do not involve peripheral accumulation of infectivity and are therefore less contagious or non-contagious and may contribute to the spread of the disease mainly via environmental contamination following death. Some risk factors are man-mediated and are considered preventable. Their management could contribute to the decrease in the theoretical risk of spread of CWD. The potential co-localisation of cervid species and disease phenotypes mean that all the identified groups of risk factors should be taken into account when considering interventions.

It is recommended: (1) to document PRNP gene polymorphisms (nature, distribution and frequency) in European cervid populations; (2) to collate data to fully characterise European CWD isolates and collect data on new cases that may arise in Europe, together with NA positive control material; (3) to collect data on tissue distribution in naturally infected animals and, ideally, through experimental pathogenesis studies in cervid species; (4) and to maximise the effectiveness of the current compulsory surveillance by aiming at testing primarily animals in the risk groups and increasing sample size per primary sampling unit (PSU) (up to 30 at risk animals).

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3.6.3 Concluding remarks

The identification of risk factors associated with the spread of the disease can only be based on the epidemiological and biological understanding of NA CWD outbreaks, although the biological characterisation of the European isolates indicates that they differ from the known NA strains. The presence/absence of detectable PrPSc in the peripheral tissues as well as the CNS of affected animals can be used as an indicator of the probability of natural spread and animals assigned to different categories based on their pathogenesis. Both infected carriers and passive carriers are thought to be involved in the dissemination of the agent.

Two distinct, hypothetical working scenarios have been built on these categories: a contagious form (Scenario 1) affecting animals which can spread infectivity through direct contacts with other animals and/or contamination of the environment and a non-contagious form (Scenario 2) that is transmitted through environmental contamination from infected carcasses or via human activity, scavengers or via the feed chain.

Thirteen main groups of risk factors have been identified based on their biological plausibility to spread CWD. Some of them are supported by epidemiological evidence from NA CWD.

The only risk factors for the spread of CWD affecting exclusively Scenario 1 refer to animal to animal contact which will increase through the aggregation of live animals, promoted, or not, by human activities.

All other risk factors refer to natural or man-mediated movement or translocation of wild or farmed/free ranging animals and to environmental contamination and the subsequent exposure of animals to contaminated soil or vegetation. This can occur at the point of contamination (close to a fallen animal) or following translocation of contaminated materials by humans or scavengers. These risks will also vary depending on the social structures of different species and by habitat and the geography of specific regions. In wild populations, the co-existence of different species makes it important to consider all the identified risk factors in both scenarios.

Risk reduction through intervention would be possible for some man-mediated risk factors, namely activities leading to the translocation or aggregation of both wild and farmed live animals, as well as fallen stock management and appropriate disposal of carcasses and slaughter by-products. Uncertainty analysis

The sources of uncertainty associated with the available data have been summarised in tabular format (Table 10), describing the nature or cause of the uncertainty. The factors contributing to the list of uncertainties are mostly missing or incomplete data on disease prevalence, pathogenesis and isolate characterisation, affecting the entire Opinion and the answers to all of the ToRs.

Table 10. Sources of uncertainty identified in the risk assessment

https://efsa.onlinelibrary.wiley.com/doi/full/10.2903/j.efsa.2019.5863

https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2019.5863

Scientific Opinion

Scientific opinion on chronic wasting disease (II)

EFSA Panel on Biological Hazards (BIOHAZ), Antonia Ricci, Ana Allende, Declan Bolton, Marianne Chemaly, Robert Davies, Pablo Salvador Fernández Escámez … See all authors 

First published: 17 January 2018 https://doi.org/10.2903/j.efsa.2018.5132Citations: 9

Correspondence: biohaz@efsa.europa.eu

Requestor: European Commission

Question number: EFSA-Q-2016-00411

Panel members: Antonia Ricci, Ana Allende, Declan Bolton, Marianne Chemaly, Robert Davies, Pablo Salvador Fernández Escámez, Rosina Gironés, Lieve Herman, Kostas Koutsoumanis, Roland Lindqvist, Birgit Nørrung, Lucy Robertson, Moez Sanaa, Panagiotis Skandamis, Emma Snary, Niko Speybroeck, Benno Ter Kuile, Giuseppe Ru, Marion Simmons, John Threlfall and Helene Wahlström.

Adopted: 6 December 2017

Abstract

The European Commission asked EFSA for a scientific opinion on chronic wasting disease in two parts. Part one, on surveillance, animal health risk-based measures and public health risks, was published in January 2017. This opinion (part two) addresses the remaining Terms of Reference, namely, ‘are the conclusions and recommendations in the EFSA opinion of June 2004 on diagnostic methods for chronic wasting disease still valid? If not, an update should be provided’, and ‘update the conclusions of the 2010 EFSA opinion on the results of the European Union survey on chronic wasting disease in cervids, as regards its occurrence in the cervid population in the European Union’. Data on the performance of authorised rapid tests in North America are not comprehensive, and are more limited than those available for the tests approved for statutory transmissible spongiform encephalopathies surveillance applications in cattle and sheep. There are no data directly comparing available rapid test performances in cervids. The experience in Norway shows that the Bio-Rad TeSeE™ SAP test, immunohistochemistry and western blotting have detected reindeer, moose and red deer cases. It was shown that testing both brainstem and lymphoid tissue from each animal increases the surveillance sensitivity. Shortcomings in the previous EU survey limited the reliability of inferences that could be made about the potential disease occurrence in Europe. Subsequently, testing activity in Europe was low, until the detection of the disease in Norway, triggering substantial testing efforts in that country. Available data neither support nor refute the conclusion that chronic wasting disease does not occur widely in the EU and do not preclude the possibility that the disease was present in Europe before the survey was conducted. It appears plausible that chronic wasting disease could have become established in Norway more than a decade ago.

Summary

In 2016, the European Food Safety Authority (EFSA) was asked by the European Commission to deliver a scientific opinion on three Terms of Reference (ToRs): (1) surveillance, (2) public health and (3) (animal health risk-based measures) by 31 December 2016. On 18 January 2017, EFSA published a scientific opinion on chronic wasting disease (CWD) in cervids addressing these three ToRs (EFSA BIOHAZ Panel, 2017a). Within the same mandate, EFSA was asked to deliver by 31 December 2017 a scientific opinion on the following ToR: (4) are the conclusions and recommendations in the EFSA opinion of June 2004 on diagnostic methods for CWD still valid? If not, an update should be provided, and (5) update the conclusions of the 2010 EFSA opinion on the results of the European Union (EU) survey on CWD in cervids, as regards the occurrence of CWD in the cervid population in the EU.

No formal validation of test performance equivalent to the existing EU requirements for tests used for statutory surveillance in cattle and sheep has been undertaken for cervid material. A qualitative evaluation of the suitability of the Bio-Rad and the IDEXX rapid tests (RT) commercially available for the diagnosis of CWD was carried out by means of literature review (both an ad hoc literature review on the diagnosis of CWD and the references retrieved by the search conducted for the 2017 Opinion (EFSA BIOHAZ Panel, 2017a)), the data provided by the manufacturers, and the knowledge and expertise of the Working Group (WG) members.

A review of the available approaches to the diagnosis of CWD including the considerations underpinning the selection of animals, tissues and diagnostic tests has been conducted, as well as a review of the different diagnostic methods applied for the detection of CWD, both in the context of large-scale surveillance and for research purposes. Screening tests and confirmatory diagnostic methods have been reviewed along with methods for classification of isolates based on data from confirmatory testing, bioassay in potential natural host species and bioassay in rodent models. Requirements for the validation of new diagnostic tests, in particular, the steps and different pathways as defined by the International Organization of Animal Health (OIE) for validation of tests for wild populations, were considered. A review of all the validation exercises of RT for the detection of bovine spongiform encephalopathy (BSE) and for the diagnosis of transmissible spongiform encephalopathies (TSE) in small ruminants conducted in the EU has been included for comparison with the data current available for the rapid tests presently used for the detection of CWD in North America.

Sensitive amplification methods, such as protein misfolding cyclic amplification (PMCA) and real-time quaking-induced conversion (rtQuiC) that are currently under development for in vivo screening, or for the detection of environmental contamination, are also considered, but they are not yet at a point in their development where they could be applied in a statutory surveillance context.

To demonstrate how the potential for patchy CWD distribution could complicate surveillance in a heterogeneous geographic area the size of Europe, historical and contemporary maps of CWD distribution in the 28 contiguous US states east of the Mississippi River, spanning ~ 2.5 M km2, were used. This area approximates the EU (28 Member States (MS): > 4.4 M km2) with respect to several ecological, epidemiological and jurisdictional features relevant to CWD surveillance in the context of ToR 5.

Data on surveillance in Europe in 2015, 2016 and 2017 were extracted from annual reports submitted by the MS, and from the background information provided by the European Commission, and included in the mandate and the European Commission database. Surveillance data from Norway for the period 1 January 2017–27 November 2017 have been provided by the Norwegian Veterinary Institute, upon request. These data were used together with historical surveillance data from five Colorado mule deer herds collected over 15–21 years to provide a temporal reference of the estimated prevalence in new incursions of CWD and potential time lags in ‘epidemic’ emergence. Data from North America were used to generate a composite epidemic curve, and data from a published model were graphed for comparison with the observed data. The point estimate of comparable survey data from Norwegian reindeer (Nordfjella 1 region) was also calculated.

The experience in Norway so far shows that the Bio-Rad RT (TeSeE™ SAP) has detected cases of CWD in reindeer, moose and red deer. It has also been shown that antibodies raised against the core or C-terminal parts of the prion protein used for immunohistochemistry (IHC) and western blot (WB) were able to detect these cases.

Developments in immunoblotting techniques have resulted in the ability to discriminate experimental BSE from CWD in red deer. However, there is only limited information on the biological and molecular characteristics that define different strains in the North American cervid population against which the EU isolates could be compared and classified.

The conclusions (1, 2, 3, 4) and recommendations (3, 5, 6, 7) of the 2004 EFSA opinion on diagnostic methods for CWD remain valid. The available formal data on the performance of authorised RT for the detection of CWD in cervids in North America are not comprehensive and are much more limited than those available for the detection of BSE in cattle and scrapie in sheep. The lack of sufficient positive reference samples Europe, and a current lack of information on the strain(s) that might be circulating, make the estimation of the diagnostic sensitivity (DSe) of any test unfeasible for cervid samples, and preclude the development of alternative tests for use in European TSE surveillance in cervids. No direct comparison of test performance (i.e. parallel testing on the same panel of samples) can be made from the data available so there is no possibility to identify any differences between the two RT available on the market.

The generation of positive control material for European CWD strain/s, as recommended in both the 2004 and 2010 EFSA opinions (EFSA, 2004a,b; EFSA BIOHAZ Panel, 2010), for example, by experimental inoculation of a range of cervid species would be useful but is very difficult to perform, and would raise a number of practical and welfare issues. It would require the maintenance of experimentally infected individuals from non-domesticated species in high biosafety facilities for a long period of time. In the absence of the specific pathogenesis data that such studies would provide and in the light of the results from the Norwegian surveillance, both brainstem and lymphoid tissue should be tested from each animal to improve sensitivity possible from collected material. The added sensitivity conferred by the testing of lymphoid tissues in addition to the brainstem is further corroborated by the experience from the testing conducted in Norway; three out of the eight positive reindeer were positive on lymphoid tissue only and five were positive in both brainstem and lymphoid tissue. Similarly, the Norwegian experience indicates that there was no detectable lymphoid involvement in the moose and red deer cases.

The tissue distribution of infectivity in some CWD-infected cervids is now known to extend beyond the central nervous system and lymphoid tissues. While the removal of these specific tissues from the food chain, as recommended in the 2004 Opinion, would reduce human dietary exposure to infectivity, exclusion from the food chain of the whole carcass of any infected animal would be required to eliminate human dietary exposure.

The conclusions (1, 2, 4, 5, 6) and all recommendations (1, 2, 3, 4) of the 2010 EFSA opinion remain valid. Shortcomings in the 2006–2010 EU CWD survey design and subsequent implementation limited the reliability of inferences that could be made about the potential occurrence of CWD in Europe. Despite the lack of substantial surveillance in the EU since that time, cases of CWD have now been detected in wild Norwegian reindeer, moose and red deer, confirming the long-held suspicion that at least some European cervid species are susceptible. Since the implementation of the 2006–2010 EU survey, testing activity has been low in Europe until the detection of CWD in Norway triggered a substantial testing effort in this country in 2016 and 2017. The surveillance programme proposed in the 2017 EFSA opinion supersedes the specifications of the EU-wide survey that was implemented following the recommendations of the 2004 EFSA opinion.

Current available data do not preclude the possibility that CWD was present in Norway and perhaps elsewhere in Europe before the 2006–2010 EU CWD survey was conducted, whether in epidemic form or not. Comparing the point estimates of CWD prevalence among ‘adult’ (> 1 year old) reindeer harvested in Nordfjella 1 in 2016 (0.97%, 95% C.I.: 0.2–2.8%) and for the period 1 January–27 November 2017 (0.68%, 95% CI: 0.22–1.6%) to the epidemic curve for mule deer in investigated herds in the US, it appears plausible that CWD could have become established in Norway more than a decade ago.

Adhering to contemporary surveillance recommendations (EFSA BIOHAZ Panel, 2017a), especially with respect to focusing sampling on high-risk individuals and developing a biologically meaningful spatial sampling framework relevant to the populations being monitored, with the aim of achieving set target sample sizes at the primary sampling unit level, should improve the reliability and value of data arising from renewed CWD surveillance efforts by some MS in coming years. The finding of the first case of CWD in red deer in Norway means that the surveillance scheme as in Reg. 999/2001, as amended, does not cover geographically all the MS in which red deer are present.

Further recommendations have been made, among them, the incorporation of sampling and testing for CWD into any wildlife health surveillance programmes, and the increase of awareness and dissemination of information about CWD in appropriate forums in the EU in order to improve the reporting of suspect cases. In addition, it is recommended to use only trained personnel for sample collection, and to avoid any test or detection method that uses antibodies for which the epitope is known to be polymorphic in cervids, unless successful binding in positive animals with those polymorphisms can be demonstrated. Residual samples, including relevant metadata, should be retained from all positive animals, and from as many tissues as possible, for isolate classification, future test evaluation, epidemiology or research purposes. Complementary studies should be conducted to identify any relevant differences influencing the epidemiology of the disease and to investigate the presence and frequency of potentially resistant alleles in the European cervid population. Finally, it is recommended to keep the performance of all currently applied tests, including those still being developed, under review and revise and update statutory testing protocols as new data become available.

snip...see full report;

https://efsa.onlinelibrary.wiley.com/doi/full/10.2903/j.efsa.2018.5132

https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2018.5132

Chronic wasting disease (CWD) in cervids

EFSA Panel on Biological Hazards (BIOHAZ), Antonia Ricci, Ana Allende, Declan Bolton, Marianne Chemaly, Robert Davies, Pablo Salvador Fernández Escámez … See all authors 

First published: 18 January 2017 https://doi.org/10.2903/j.efsa.2017.4667Citations: 21

Correspondence: biohaz@efsa.europa.eu

Requestor: European Commission

Question number: EFSA-Q-2016-00387

Panel members: Antonia Ricci, Ana Allende, Declan Bolton, Marianne Chemaly, Robert Davies, Pablo Salvador Fernández Escámez, Rosina Gironés, Lieve Herman, Kostas Koutsoumanis, Roland Lindqvist, Birgit Nørrung, Lucy Robertson, Moez Sanaa, Panagiotis Skandamis, Emma Snary, Niko Speybroeck, Benno Ter Kuile, Giuseppe Ru, Marion Simmons, John Threlfall and Helene Wahlström.

Acknowledgements: The Panel wishes to thank the members of the Working Group on a request for a scientific opinion on Chronic Wasting Disease (CWD) in cervids: Sylvie Benestad, Dolores Gavier-Widen, Michael W Miller, Giuseppe Ru, Marion Simmons, Glenn C Telling and Morten Tryland for the preparatory work on this scientific opinion, and Angel Ortiz Pelaez for the support provided to this scientific opinion. The Panel wishes to acknowledge all organisations and individuals that provided data for this scientific opinion.

Amendment: The following changes were made: (i) the following sentence was added to p. 22: “However, Tg mice expressing ovine PrP challenged with CWD have resulted in highly efficient, life-long asymptomatic replication of these prions in the spleen tissue, with brain involvement at late stage (Béringue et al., 2012)”; (ii) the reference to Béringue et al., 2012 was added to the Reference list and (iii) wapiti was added to the top of p.21. These changes do not materially affect the contents or outcome of this scientific output. To avoid confusion, the older version has been removed from the EFSA Journal, but is available on request, as is a version showing all the changes made.

Adopted: 2 December 2016

Abstract

In April and May of 2016, Norway confirmed two cases of chronic wasting disease (CWD) in a wild reindeer and a wild moose, respectively. In the light of this emerging issue, the European Commission requested EFSA to recommend surveillance activities and, if necessary, additional animal health risk-based measures to prevent the introduction of the disease and the spread into/within the EU, specifically Estonia, Finland, Iceland, Latvia, Lithuania, Norway, Poland and Sweden, and considering seven wild, semidomesticated and farmed cervid species (Eurasian tundra reindeer, Finnish (Eurasian) forest reindeer, moose, roe deer, white-tailed deer, red deer and fallow deer). It was also asked to assess any new evidence on possible public health risks related to CWD. A 3-year surveillance system is proposed, differing for farmed and wild or semidomesticated cervids, with a two-stage sampling programme at the farm/geographically based population unit level (random sampling) and individual level (convenience sampling targeting high-risk animals). The current derogations of Commission Implementing Decision (EU) 2016/1918 present a risk of introduction of CWD into the EU. Measures to prevent the spread of CWD within the EU are dependent upon the assumption that the disease is already present; this is currently unknown. The measures listed are intended to contain (limit the geographic extent of a focus) and/or to control (actively stabilise/reduce infection rates in an affected herd or population) the disease where it occurs. With regard to the zoonotic potential, the human species barrier for CWD prions does not appear to be absolute. These prions are present in the skeletal muscle and other edible tissues, so humans may consume infected material in enzootic areas. Epidemiological investigations carried out to date make no association between the occurrence of sporadic Creutzfeldt–Jakob disease in humans and exposure to CWD prions.

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Summary

In April and May of 2016, Norway confirmed two cases of chronic wasting disease (CWD) in a wild reindeer and a wild moose, respectively. This was the first time CWD had been detected in Europe and the first natural case in reindeer in the world. In the light of the sensitivity of this emerging issue, the European Food Safety Authority (EFSA) was asked by the European Commission to deliver its scientific opinion by 31 December 2016 on the following Terms of Reference (ToRs): (1) to provide recommendations on surveillance of cervid populations at the country level aimed at detecting CWD and/or estimating the prevalence of CWD in Norway, Sweden, Finland, Iceland, Estonia, Latvia and Poland, which are the European Union (EU) and European Economic Area (EEA) countries with reindeer and/or moose populations, depending on the level of prevalence which is wished to be detected; (2) has new evidence become available with regard to possible public health risks due to the occurrence of CWD in cervids since the publication of the 2011 joint EFSA/ECDC opinion? Does the natural exposure of consumers to cervid products originating from regions where CWD cases are detected represent a risk for public health? (3) EFSA is asked to recommend, if necessary, additional animal health risk-based measures to prevent the introduction of CWD into the EU cervid populations and to prevent its spread within the EU.

It was agreed with the requestor to also include Lithuania in the mandate, and to consider the following seven wild, semi domesticated and farmed cervids, namely Eurasian tundra reindeer (Rangifer tarandustarandus), Finnish (Eurasian) forest reindeer (Rangifer tarandus fennicus), moose (Eurasian/European elk) (Alces alces alces), roe deer (Capreolus capreolus), white-tailed deer (Odocoileus virginianus), reddeer (Cervus elaphus) and fallow deer (Dama dama).

The data used in this assessment have been sourced via different literature searches, looking at the new evidence from experimental studies examining the transmissibility of transmissible spongiform encephalopathies (TSE) agents to humans, as well as the epidemiology, surveillance and control of CWD. The figures on wild and hunted population abundance were obtained from different sources and represent various methodologies used for census and estimates. Surveillance data in Europe in 2015 have been extracted from annual reports submitted by the Member States (MS) and from the background information provided by the European Commission and included in the mandate.

It was agreed, due to limited time and resources, to carry out a qualitative evaluation by means of literature reviews based on the knowledge and expertise of the Working Group (WG) members. The experts in the WG selected relevant references starting from review papers, books chapters, non-peer-review papers known by the experts themselves or retrieved through non-systematic searches until the information of the subject was considered sufficient to undertake the assessment by the WG. The literature search was used to support the expert review of these areas, and additional scientific information known by the experts was also considered in the assessment.

The surveillance system proposed for the countries concerned is based on the experience of CWD surveillance in North America and the knowledge of the different structures and management systems of the cervid populations of some of the countries concerned, in particular Norway and Sweden. The aims of the proposed surveillance system are to detect disease in countries where CWD has not yet been detected using a predefined design prevalence and to estimate prevalence in areas where disease has been detected. It is intended to overcome the shortcomings highlighted in the evaluation of the previous surveillance programme implemented in Europe 2006–2010. A two-stage sampling programme is proposed based on the application of random sampling at the first stage (for wild/semi domesticated cervids the ‘primary sampling units’(PSU) will correspond to geographical areas containing cervid populations, whereas for farmed cervids they will correspond to farms) and the application of convenience sampling of high-risk animals within PSU (found dead, hunted or slaughtered animals considered not fit for human consumption, road/predator kills and animals killed because they are sick or in poor body condition and not fit for human consumption) of any of the selected species at the second stage.

With regard to the public health risks, there is currently no experimental model that encompasses all the potential host and agent variability required to assess zoonotic potential directly for any animal prion disease, including CWD. Although CWD has been experimentally transmitted to squirrel monkeys; in vivo transmission of CWD to other animal models including macaques and humanised mice has notyet been reported. CWD strains, their prevalence, host range and zoonotic potential remain incompletely understood. All currently available data pertaining to host range and human risk are derived from isolates obtained from North American cervid species, but preliminary evidence from the Norwegian CWD cases raises the possibility that European and North American isolates are different from each other.

There is no evidence of an absolute species barrier between CWD-affected cervids and humans. CWD prions are present in the skeletal muscle and other edible tissues, which means that humans may consume infected material in enzootic areas. However, from the epidemiological investigations carried out to date, no association has been made between the occurrence of sporadic Creutzfeldt–Jakob disease (sCJD) in humans and exposure to CWD.

Since it was concluded that the most likely pathway of introduction of CWD into the EU is the movement of live cervids, the current derogations of Article 2.2 of the Commission Implementing Decision (EU) 2016/1918 present a risk of introduction of CWD into the EU. The probability of introduction of CWD into and spread within the EU associated with the movement of live cervids for direct slaughter is considered to be lower than situations in which live animals are translocated for other purposes. Minimising movements of live cervids would reduce the probability of introduction of CWD into the EU. The use of natural cervid urine lures is considered to increase the probability of introduction of CWD into the EU. Compliance with recommendations included in awareness campaigns targeting both local Norwegian hunters, and hunters visiting Norway from (and returning to) other countries, would reduce the probability of introduction of CWD into the EU.

Measures to prevent the spread of CWD within the EU are dependent upon the assumption that the disease is already present in some part of the EU territory. At the time of writing this opinion, this is unknown. The uncertainties associated with the limited knowledge of the situation of CWD in the countries concerned could render any of the recommended measures inadequate or insufficient. Moreover, strategies to prevent the introduction and/or the spread of CWD must be based on a combination of measures.

A list of measures has been included. Some aim to reduce animal-to-animal contact and lower population densities; others relate to increasing the disease awareness among stakeholders, reducing environmental contamination and developing contingency plans, including quarantine and other measures in infected and management premises/areas. Specific prohibitions within Norway, affecting silage, feed and lichen to prevent the spread of CWD, have been mentioned.

Additional activities have been recommended, such as reviewing the design of the surveillance system after 1 year of implementation, conducting a risk assessment to estimate the likelihood of introduction of CWD into the EU, collecting data on the cervid populations in Europe, implementing an individual identification system and record-keeping of farmed and semidomesticated cervids together with an adhoc data collection system for surveillance. Any positive case detected through surveillance should be genotyped and characterised by strain typing, with representative material archived for future reference. It is also recommended that all or a representative subset of cervids tested negative by surveillance are genotyped. Consideration is given to implementing surveillance programmes in other MS. Chronic wasting disease (CWD) in cervids. 

http://www.efsa.europa.eu/efsajournal4EFSA Journal 2017;15(1):4667

https://efsa.onlinelibrary.wiley.com/doi/abs/10.2903/j.efsa.2017.4667

see full report

https://efsa.onlinelibrary.wiley.com/doi/epdf/10.2903/j.efsa.2017.4667

DEFRA 

Friday, December 14, 2012 

DEFRA U.K. What is the risk of Chronic Wasting Disease CWD being introduced into Great Britain? A Qualitative Risk Assessment October 2012 

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In the USA, under the Food and Drug Administration's BSE Feed Regulation (21 CFR 589.2000) most material (exceptions include milk, tallow, and gelatin) from deer and elk is prohibited for use in feed for ruminant animals. With regards to feed for non-ruminant animals, under FDA law, CWD positive deer may not be used for any animal feed or feed ingredients. For elk and deer considered at high risk for CWD, the FDA recommends that these animals do not enter the animal feed system. However, this recommendation is guidance and not a requirement by law. Animals considered at high risk for CWD include: 

1) animals from areas declared to be endemic for CWD and/or to be CWD eradication zones and 

2) deer and elk that at some time during the 60-month period prior to slaughter were in a captive herd that contained a CWD-positive animal. 

Therefore, in the USA, materials from cervids other than CWD positive animals may be used in animal feed and feed ingredients for non-ruminants. 

The amount of animal PAP that is of deer and/or elk origin imported from the USA to GB can not be determined, however, as it is not specified in TRACES. 

It may constitute a small percentage of the 8412 kilos of non-fish origin processed animal proteins that were imported from US into GB in 2011. 

Overall, therefore, it is considered there is a __greater than negligible risk___ that (nonruminant) animal feed and pet food containing deer and/or elk protein is imported into GB. 

There is uncertainty associated with this estimate given the lack of data on the amount of deer and/or elk protein possibly being imported in these products. 

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36% in 2007 (Almberg et al., 2011). In such areas, population declines of deer of up to 30 to 50% have been observed (Almberg et al., 2011). In areas of Colorado, the prevalence can be as high as 30% (EFSA, 2011). The clinical signs of CWD in affected adults are weight loss and behavioural changes that can span weeks or months (Williams, 2005). In addition, signs might include excessive salivation, behavioural alterations including a fixed stare and changes in interaction with other animals in the herd, and an altered stance (Williams, 2005). These signs are indistinguishable from cervids experimentally infected with bovine spongiform encephalopathy (BSE). Given this, if CWD was to be introduced into countries with BSE such as GB, for example, infected deer populations would need to be tested to differentiate if they were infected with CWD or BSE to minimise the risk of BSE entering the human food-chain via affected venison. snip..... The rate of transmission of CWD has been reported to be as high as 30% and can approach 100% among captive animals in endemic areas (Safar et al., 2008). 

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In summary, in endemic areas, there is a medium probability that the soil and surrounding environment is contaminated with CWD prions and in a bioavailable form. In rural areas where CWD has not been reported and deer are present, there is a greater than negligible risk the soil is contaminated with CWD prion. snip..... In summary, given the volume of tourists, hunters and servicemen moving between GB and North America, the probability of at least one person travelling to/from a CWD affected area and, in doing so, contaminating their clothing, footwear and/or equipment prior to arriving in GB is greater than negligible... For deer hunters, specifically, the risk is likely to be greater given the increased contact with deer and their environment. However, there is significant uncertainty associated with these estimates. 

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Therefore, it is considered that farmed and park deer may have a higher probability of exposure to CWD transferred to the environment than wild deer given the restricted habitat range and higher frequency of contact with tourists and returning GB residents. 

snip..... 

https://web.archive.org/web/20170404125557/http://webarchive.nationalarchives.gov.uk/20130822084033/http://www.defra.gov.uk/animal-diseases/files/qra_chronic-wasting-disease-121029.pdf 

snip...

Thursday, April 6, 2023 

WOAH OIE CHAPTER 11.4 . BOVINE SPONGIFORM ENCEPHALOPATHY Article 11.4.1. 

https://woahoie.blogspot.com/2023/04/woah-oie-chapter-114-bovine-spongiform.html

WEDNESDAY, MARCH 29, 2023 

The use of animal by-products in a circular bioeconomy: Time for a TSE road map 3? 

https://transmissiblespongiformencephalopathy.blogspot.com/2023/03/the-use-of-animal-by-products-in.htm

ZOONOSIS, ZOONOTIC TRANSMISSION OF CHRONIC WASTING DISEASE TO HUMANS, HAS IT ALREADY HAPPENED?

Transmission of cervid prions to humanized mice demonstrates the zoonotic potential of CWD

Samia Hannaoui1 · Irina Zemlyankina1 · Sheng Chun Chang1 · Maria Immaculata Arifin1 · Vincent Béringue2 · Debbie McKenzie3 · Hermann M. Schatzl1 · Sabine Gilch1

Received: 24 May 2022 / Revised: 5 August 2022 / Accepted: 7 August 2022 / Published online: 22 August 2022 © The Author(s) 2022

Abstract

Prions cause infectious and fatal neurodegenerative diseases in mammals. Chronic wasting disease (CWD), a prion disease of cervids, spreads efficiently among wild and farmed animals. Potential transmission to humans of CWD is a growing concern due to its increasing prevalence. Here, we provide evidence for a zoonotic potential of CWD prions, and its probable signature using mice expressing human prion protein (PrP) as an infection model. Inoculation of these mice with deer CWD isolates resulted in atypical clinical manifestation with prion seeding activity and efficient transmissible infectivity in the brain and, remarkably, in feces, but without classical neuropathological or Western blot appearances of prion diseases. Intriguingly, the protease-resistant PrP in the brain resembled that found in a familial human prion disease and was transmissible upon second passage. Our results suggest that CWD might infect humans, although the transmission barrier is likely higher compared to zoonotic transmission of cattle prions. Notably, our data suggest a different clinical presentation, prion signature, and tissue tropism, which causes challenges for detection by current diagnostic assays. Furthermore, the presence of infectious prions in feces is concerning because if this occurs in humans, it is a source for human-to-human transmission. These findings have strong implications for public health and CWD management.

Our results indicate that if CWD crosses the species-barrier to humans, it is unlikely to resemble the most common forms of human prion diseases with respect to clinical signs, tissue tropism and PrPSc signature. For instance, PrPSc in variable protease-sensitive prionopathy (VPSPr), a sporadic form of human prion disease, and in the genetic form Gerst-mann-Sträussler-Scheinker syndrome (GSS) is defined by an atypical PK-resistant PrPSc fragment that is non-glycosylated and truncated at both C- and N-termini, with a molecular weight between 6 and 8 kDa [24, 44–46]. These biochemical features are unique and distinctive from PrPSc (PrP27-30) found in most other human or animal prion disease. The atypical PrPSc signature detected in brain homogenate of tg650 mice #321 (1st passage) and #3063 (2nd passage), and the 7–8 kDa fragment (Figs. 2, 4) are very similar to that of GSS, both in terms of migration profile and the N-terminal cleavage site.

CWD in humans might remain subclinical but with PrPSc deposits in the brain with an unusual morphology that does not resemble the patterns usually seen in different prion diseases (e.g., mouse #328; Fig. 3), clinical with untraceable abnormal PrP (e.g., mouse #327) but still transmissible and uncovered upon subsequent pas-sage (e.g., mouse #3063; Fig. 4), or prions have other reservoirs than the usual ones, hence the presence of infectivity in feces (e.g., mouse #327) suggesting a potential for human-to-human transmission and a real iatrogenic risk that might be unrecognizable. Here, humanized mice inoculated with CWD deer isolates had an atypical onset of the disease with myoclonus (93.75%), before presenting typical clinical signs, generating prions that presented with either atypical biochemical signature (#321 and #3063), shed in feces (#327), or were undetectable by the classical detection methods. The fact that we could not establish a strong correlation between disease manifestation in 3tg650 mice inoculated with Wisc-1- or 116AG-CWD and the presence of abnormal PrP (Western blot, IHC or RT-QuIC) might be explained by the presence of heterogeneous prions in the brains of infected mice with different seeding properties in vitro. Indeed, such heterogeneity and distinct seeding activities and infectivity of abnormal PrP fragments was observed in VPSPr cases [20, 43]. We could not establish a correlation between the presence of PrPSc deposits in mouse #328 and the absence of resist-ant PrP in Western blot even though prions present in the brain of this animal seeded the conversion of rPrP sub-strate very well (Fig. 1). Mouse #321 was not available for IHC; therefore, we cannot draw a conclusion about abnormal PrP deposits in the brain of this animal, but we can conclude that there is a correlation between level of prion seeding activity (Fig. 1) and the detection of atypical PrPres fragments (Fig. 2) present in the brain of this animal. We can only speculate that transmission of Wisc-1 to tg650 mice generated prions with characteristics that render them less prone to be unveiled with conventional methods. However, 77.7% of these mice showed prion seeding activity despite the absence of PK-resistant PrPSc for most of them, suggesting that protease-sensitive prions could be involved in these transmissions like it was shown in human prion diseases [54]. Further analyses such as conformation-dependent immunoassay [54] and in vivo sub-passages will be required to resolve these questions.

The finding that infectious PrPSc was shed in fecal material of CWD-infected humanized mice and induced clinical disease, different tropism, and typical three banding pattern-PrPres in bank voles that is transmissible upon second passage is highly concerning for public health. The fact that this biochemical signature in bank voles resembles that of the Wisc-1 original deer isolate and is different from that of bvWisc-1, in the migration profile and the glyco-form-ratio, is valid evidence that these results are not a product of contamination in our study. If CWD in humans is found to be contagious and transmissible among humans, as it is in cervids [57], the spread of the dis-ease within humans might become endemic. In contrast to bank voles inoculated with fecal homogenates from mouse #327, so far, we could not detect a PK-resistant PrPSc fragment in the brain homogenates of fecal homogenate-inoculated tg650 mice. The presence of PrPres in these mice will allow us to determine if the molecular signature of hCWD prions from the brain (mouse #321 and #3063) vs feces are the same. Previously, Beringue et al. found that extraneural prions, compared to neural prions, helped more to over-come the species barrier to foreign prions, in addition, different strain types emerged from such serial transmission [11]. Our data also suggest that prions found in the periphery may hold higher zoonotic potential than prions found in neural tissues. In fact, upon second passage, 50% of the tg650 mice inoculated with fecal homogenates from mouse #327 had succumbed with terminal disease compared to only 20% of brain/spinal cord homogenates inoculated-tg650 mice suggesting that hCWD prions found in feces transmit disease more efficiently. Our results also suggest that epidemiological studies [25] may have missed sub-clinical and atypical infections that are/might be transmissible, undetected by the gold standard tests, i.e., Western blot, ELISA, and IHC.

Ideally, an oral inoculation of mice with CWD prions would have mimicked best the natural route of exposure to acquired prion diseases like CWD. However, in our case, an oral inoculation would have been impossible considering the lifespan of the rodent models. It took us an extended amount of time, over two years and a half, to have a better idea of the extent of CWD transmission in the tg650 model, even with intracerebral inoculation, usually the fastest way to induce prion disease in a rodent model. Taking this into consideration, our study is the strongest proof-of-principle that CWD might be transmissible to humans. The overall risk for zoonotic transmission of CWD is likely lower than that for BSE; however, rather than predicting the absolute zoonotic risk for CWD, our study indicates the possibility of atypical features in humans. Furthermore, our findings provide striking insights into how CWD might manifest in humans and the impact it may have on human health. We have used Wisc-1/CWD1, one of the most common CWD strains, notably white-tailed deer prions, which have been shown to be more prone to generate human prions in vitro [47]. This implies a high risk of exposure to this strain, e.g., through consumption or handling of infected carcasses, in contrast to rarer CWD strains, and therefore, an actual risk for human health. Fecal shedding of infectious prions, if it occurs in humans, is particularly concerning because of potential human-to-human transmission and adaptation of hCWD. Overall, our findings suggest that CWD surveil-lance in humans should encompass a wider spectrum of tissues/organs tested and include new criteria in the diagnosis of potential patients.

https://doi.org/10.1007/s00401-022-02482-9

https://link.springer.com/epdf/10.1007/s00401-022-02482-9?sharing_token=Vrc9lVnCx6nATJCPtcFbwPe4RwlQNchNByi7wbcMAY4BjPjkpxdzH-iFThODxSPKAK1QgQ-_2ZGPNoYIZyD3IDH8BQbGxEd1NHRwsyD-plz3_gnMpuGB-13C4DkxsBjdAJb3TEqBq0mBLdO-wl3E0flYPQbhh654WM_ijkAKt_U=&fbclid=IwAR0urg8a9eojZqpU6GePUgt5fQxsWqxsxwTzF_E_MktS3jf11wDxZP9Jy10

SEE A FEW HIGHLIGHTS;

''Here, we provide evidence for a zoonotic potential of CWD prions, and its probable signature using mice expressing human prion protein (PrP) as an infection model.''

''Furthermore, the presence of infectious prions in feces is concerning because if this occurs in humans, it is a source for human-to-human transmission.'' ''These findings have strong implications for public health and CWD management.''

''We demonstrate that this transgenic line was susceptible to infection with CWD prions and displayed a distinct leading clinical sign, an atypical PrPSc signature and unusual fecal shedding of infectious prions.'' 

''Importantly, these prions generated by the human PrP transgenic mice were transmissible upon passage.'' 

Our results are the first evidence of a zoonotic risk of CWD when using one of the most common CWD strains, Wisc-1/CWD1 for ''infection. ''

''Our findings strongly suggest that CWD should be regarded as an actual public health risk. Here, we use humanized mice to show that CWD prions can cross the species barrier to humans, and remarkably, infectious prions can be excreted in feces.''

''Indeed, such heterogeneity and distinct seeding activities and infectivity of abnormal PrP fragments was observed in VPSPr cases [20, 43].''

''This implies a high risk of exposure to this strain, e.g., through consumption or handling of infected carcasses, in contrast to rarer CWD strains, and therefore, an actual risk for human health.'' 

''Fecal shedding of infectious prions, if it occurs in humans, is particularly concerning because of potential human-to-human transmission and adaptation of hCWD.'' 

Overall, our findings suggest that CWD surveillance in humans should encompass a wider spectrum of tissues/organs tested and include new criteria in the diagnosis of potential patients.

***> PLEASE NOTE;

''Our results indicate that if CWD crosses the species-barrier to humans, it is unlikely to resemble the most common forms of human prion diseases with respect to clinical signs, tissue tropism and PrPSc signature. For instance, PrPSc in variable protease-sensitive prionopathy (VPSPr), a sporadic form of human prion disease, and in the genetic form Gerstmann-Sträussler-Scheinker syndrome (GSS) is defned by an atypical PK-resistant PrPSc fragment that is non-glycosylated and truncated at both C- and N-termini, with a molecular weight between 6 and 8 kDa [24, 44–46]. These biochemical features are unique and distinctive from PrPSc (PrP27-30) found in most other human or animal prion disease. The atypical PrPSc signature detected in brain homogenate of tg650 mice #321 (1st passage) and #3063 (2nd passage), and the 7–8 kDa fragment (Figs. 2, 4) are very similar to that of GSS, both in terms of migration profle and the N-terminal cleavage site.''

snip...

''CWD in humans might remain subclinical but with PrPSc deposits in the brain with an unusual morphology that does not resemble the patterns usually seen in different prion diseases (e.g., mouse #328; Fig. 3), clinical with untraceable abnormal PrP (e.g., mouse #327) but still transmissible and uncovered upon subsequent passage (e.g., mouse #3063; Fig. 4), or prions have other reservoirs than the usual ones, hence the presence of infectivity in feces (e.g., mouse #327) suggesting a potential for human-to-human transmission and a real iatrogenic risk that might be unrecognizable. Here, humanized mice inoculated with CWD deer isolates had an atypical onset of the disease with myoclonus (93.75%), before presenting typical clinical signs, generating prions that presented with either atypical biochemical signature (#321 and #3063), shed in feces (#327), or were undetectable by the classical detection methods. The fact that we could not establish a strong correlation between disease manifestation in tg650 mice inoculated with Wisc-1- or 116AG-CWD and the presence of abnormal PrP (Western blot, IHC or RTQuIC) might be explained by the presence of heterogeneous prions in the brains of infected mice with diferent seeding properties in vitro. Indeed, such heterogeneity and distinct seeding activities and infectivity of abnormal PrP fragments was observed in VPSPr cases [20, 43].''

VPSPr, GSS, and CWD zoonosis, concerns there from, where did i hear this concern before?

8. Even though human TSE‐exposure risk through consumption of game from European cervids can be assumed to be minor, if at all existing, no final conclusion can be drawn due to the overall lack of scientific data. In particular the US data do not clearly exclude the possibility of human (sporadic or familial) TSE development due to consumption of venison. The Working Group thus recognizes a potential risk to consumers if a TSE would be present in European cervids. It might be prudent considering appropriate measures to reduce such a risk, e.g. excluding tissues such as CNS and lymphoid tissues from the human food chain, which would greatly reduce any potential risk for consumers. However, it is stressed that currently, no data regarding a risk of TSE infections from cervid products are available.

https://efsa.onlinelibrary.wiley.com/doi/full/10.2903/j.efsa.2018.5132

SUNDAY, APRIL 09, 2023 

Transmission of cervid prions to humanized mice demonstrates the zoonotic potential of CWD 

https://chronic-wasting-disease.blogspot.com/2023/04/transmission-of-cervid-prions-to.html

PRION CONFERENCE 2022 ABSTRACTS CWD TSE PrP ZOONOSIS 

Transmission of prion infectivity from CWD-infected macaque tissues to rodent models demonstrates the zoonotic potential of chronic wasting disease.

Samia Hannaouia, Ginny Chenga, Wiebke Wemheuerb, Walter J. Schulz-Schaefferb, Sabine Gilcha, and Hermann M. Schätzla aDepartment of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine & Hotchkiss Brain Institute; University of Calgary, Calgary, Canada; bInstitute of Neuropathology, Medical Faculty, Saarland University, Homburg/Saar, Germany

Aims: Chronic wasting disease (CWD) is a prion disease of cervids. Its rapid geographic expansion, shedding of infectivity and persistence in the environment for many years are of concern for humans. Here, we provide the first evidence by transmission experiments to different transgenic mouse models and bank voles that Cynomolgus macaques inoculated via different routes with CWD-positive cervid tissues harbor infectious prions that elicit clinical disease in rodents.

Material and Methods: We used tissue materials from macaques inoculated with CWD to inoculate transgenic mice overexpressing cervid PrPCfollowed by transmission into bank voles. We used RT-QuIC, immunoblot and PET blot analysis to assess brains, spinal cords, and tissues of the gastrointestinal tract (GIT) for the presence of prions.

Results: Our results show that of the macaque materials that induced clinical disease in transgenic mice,73% were from the CNS (46% spinal cord and 27% brain), and 27% were from the spleen, although attack rates were low around 20%. Clinical mice did not display PK-resistant PrPSc(PrPres) in immunoblot, but showed low-levels of prion seeding activity. Transmission into bank voles from clinical transgenic mice led to a 100% attack rate with typical PrPressignature in immunoblot, which was different from that of voles inoculated directly with CWD or scrapie prions. High-level prion seeding activity in brain and spinal cord and PrPresdeposition in the brain were present. Remarkably, we also found prion seeding activity in GIT tissues of inoculated voles. Second passage in bank voles led to a 100% attack rate in voles inoculated with brain, spinal cord and small intestine material from first round animals, with PrPresin immunoblot, prion seeding activity, and PrPresdeposition in the brain. Shortened survival times indicate adaptation in the new host. This also shows that prions detected in GIT tissues are infectious and transmissible. Transmission of brain material from sick voles back to cervidized mice revealed transmission in these mice with a 100% attack rate, and interestingly, with different biochemical signature and distribution in the brain.

Conclusions: Our findings demonstrate that macaques, considered the best model for the zoonotic potential of prions, were infected upon CWD challenge, including oral one. The disease manifested as atypical in macaques and transgenic mice, but with infectivity present at all times, as unveiled in the bank vole model with an unusual tissue tropism.

Funded by: The National Institutes of Health, USA, and the Alberta Prion Research Institute/Alberta Innovates Canada. Grant number: 1R01NS121016-01; 201,600,023

Acknowledgement: We thank Umberto Agrimi, Istituto Superiore di Sanità, Rome, Italy, and Michael Beekes, Robert-Koch Institute Berlin, Germany, for providing the bank vole model. We thank the University of Calgary animal facility staff and Dr. Stephanie Anderson for animal care.

Transmission of Cervid Prions to Humanized Mice Demonstrates the Zoonotic Potential of CWD

Samia Hannaouia, Irina Zemlyankinaa, Sheng Chun Changa, Maria Immaculata Arifina, Vincent Béringueb, Debbie McKenziec, Hermann M. Schatzla, and Sabine Gilcha

aDepartment of Comparative Biology and Experimental Medicine, Faculty of Veterinary Medicine; Hotchkiss Brain Institute; University of Calgary, Calgary, Canada; bUniversité Paris-Saclay, INRAE, UVSQ, VIM, Jouy-en-Josas, France; cDepartment of Biological Sciences, Center for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada

Aims: Chronic wasting disease (CWD), a prion disease of cervids, spreads efficiently among wild and farmed animals. Potential transmission to humans of CWD is a growing concern due to its increasing prevalence. Here, we aimed to determine the zoonotic potential of CWD using a mouse model for human prion diseases.

Material and Methods: Transgenic mice overexpressing human PrPChomozygous for methionine at codon 129 (tg650) were inoculated intracerebrally with brain homogenates of white-tailed deer infected with Wisc-1/CWD1 or 116AG CWD strains. Mice were monitored for clinical signs and were euthanized at terminal disease. Brains were tested by RT-QuIC, western blot upon PK digestion, and immunohistochemistry; fecal homogenates were analyzed by RT-QuIC. Brain/spinal cord and fecal homogenates of CWD-inoculated tg650 mice were inoculated into tg650 mice or bank voles. Brain homogenates of bank voles inoculated with fecal homogenates of CWD-infected tg650 mice were used for second passage in bank voles.

Results: Here, we provide the strongest evidence supporting the zoonotic potential of CWD prions, and their possible phenotype in humans. Inoculation of mice expressing human PrPCwith deer CWD isolates (strains Wisc-1 and 116AG) resulted in atypical clinical manifestations in > 75% of the mice, with myoclonus as leading clinical sign. Most of tg650 brain homogenates were positive for seeding activity in RT-QuIC. Clinical disease and presentation was transmissible to tg650 mice and bank voles. Intriguingly, protease-resistant PrP in the brain of tg650 mice resembled that found in a familial human prion disease and was transmissible upon passage. Abnormal PrP aggregates upon infection with Wisc-1 were detectable in thalamus, hypothalamus, and midbrain/pons regions.

Unprecedented in human prion disease, feces of CWD-inoculated tg650 mice harbored prion seeding activity and infectious prions, as shown by inoculation of bank voles and tg650 with fecal homogenates.

Conclusions: This is the first evidence that CWD can infect humans and cause disease with a distinctive clinical presentation, signature, and tropism, which might be transmissible between humans while current diagnostic assays might fail to detect it. These findings have major implications for public health and CWD-management.

Funded by: We are grateful for financial support from the Natural Sciences and Engineering Research Council of Canada, the National Institutes of Health, Genome Canada, and the Alberta Prion Research Institute. SG is supported by the Canada Research Chairs program.

Acknowledgement: We thank Dr. Trent Bollinger, WCVM, University of Saskatchewan, Saskatoon, Canada, for providing brain tissue from the WTD-116AG isolate, Dr. Stéphane Haïk, ICM, Paris, France, for providing brain tissue from vCJD and sCJD cases, and Dr. Umberto Agrimi, Istituto Superiore di Sanità, Italy, for the bank vole model. We thank animal facility staff for animal care, Dr. Stephanie Anderson for veterinary oversight, and Yo-Ching Cheng for preparing recombinant PrP substrates. Thank you to Dr. Stephanie Booth and Jennifer Myskiw, Public Health Agency of Canada, Canada.

The chronic wasting disease agent from white-tailed deer is infectious to humanized mice after passage through raccoons

Eric Cassmanna, Xu Qib, Qingzhong Kongb, and Justin Greenleea

aNational Animal Disease Center, Agricultural Research Service, US Department of Agriculture, Ames, IA, USA bDepartments of Pathology, Neurology, National Center for Regenerative Medicine, and National Prion Disease Pathology Surveillance Center, Case Western Reserve University, Cleveland, Ohio, USA

Aims: Evaluate the zoonotic potential of the raccoon passaged chronic wasting disease (CWD) agent in humanized transgenic mice in comparison with the North American CWD agent from the original white-tailed deer host.

Material and Methods: Pooled brain material (GG96) from a CWD positive herd was used to oronasally inoculate two white-tailed deer with wild-type prion protein genotype and intracranially inoculate a raccoon. Brain homogenates (10% w/v) from the raccoon and the two white-tailed deer were used to intracranially inoculate separate groups of transgenic mice that express human prion protein with methionine (M) at codon 129 (Tg40h). Brains and spleens were collected from mice at experimental endpoints of clinical disease or approximately 700 days post-inoculation. Tissues were divided and homogenized or fixed in 10% buffered neutral formalin. Immunohistochemistry, enzyme immunoassay, and western blot were used to detect misfolded prion protein (PrPSc) in tissue.

Results: Humanized transgenic mice inoculated with the raccoon passaged CWD agent from white-tailed deer exhibited a 100% (12/12) attack rate with an average incubation period of 605 days. PrPScwas detected in brain tissue by enzyme immunoassay with an average optical density of 3.6/4.0 for positive brains. PrPScalso was detected in brain tissue by western blot and immunohistochemistry. No PrPScwas detected in the spleens of mice inoculated with the raccoon passaged CWD agent. Humanized mice inoculated with the CWD agent from white-tailed deer did not have detectable PrPScusing conventional immunoassay techniques.

Conclusions: The host range of the CWD agent from white-tailed deer was expanded in our experimental model after one passage through raccoons.

Funded by: This research was funded in its entirety by congressionally appropriated funds to the United States Department of Agriculture, Agricultural Research Service. The funders of the work did not influence study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Acknowledgement: We thank Quazetta Brown, Lexi Frese, Rylie Frese, Kevin Hassall, Leisa Mandell, and Trudy Tatum for providing excellent technical support to this project.

Stable and highly zoonotic cervid prion strain is possible

Manuel Camacho, Xu Qi, Liuting Qing, Sydney Smith, Jieji Hu, Wanyun Tao, Ignazio Cali, and Qingzhong Kong Department of Pathology, Case Western Reserve University, Cleveland, USA

Aims: Whether CWD prions can infect humans remains unclear despite the very substantial scale and long history of human exposure of CWD in some areas. Multiple in vitro conversion experiments and in vivo animal studies suggest that the CWD-to-human transmission barrier is not unbreakable. A major public health concern on CWD zoonosis is the emergence of highly zoonotic CWD strains. We aim to address the question of whether highly zoonotic CWD strains are possible.

Material and Methods: We inoculated a few sCJD brain samples into cervidized transgenic mice, which were intended as negative controls for bioassays of brain tissues from sCJD cases who had hunted or consumed vension from CWD-endemic states. Some of these mice became infected and their brain tissues were further examined by serial passages in humanized or cervidized mice.

Results: Passage of sCJDMM1 in transgenic mice expressing elk PrP (Tg12) resulted in a ‘cervidized’ CJD strain that we termed CJDElkPrP. We observed 100% transmission of CJDElkPrPin transgenic mice expressing human PrP (Tg40h). We passaged CJDElkPrPtwo more times in the Tg12 mice. We found that such second and third passage CJDElkPrPprions also led to 100% infection in the Tg40h mice. In contrast, we and others found zero or poor transmission of natural elk CWD isolates in humanized mice, despite that natural elk CWD isolates and CJDElkPrPshare the same elk PrP sequence.

Conclusions: Our data demonstrate that highly zoonotic cervid prion strains are not only possible but also can be stably maintained in cervids and that CWD zoonosis is prion strain-dependent.

Funded by: NIH

Grant number: R01NS052319, R01NS088604, R01NS109532

Acknowledgement: We want to thank the National Prion Disease Pathology Surveillance Center and Drs. Allen Jenny and Katherine O’Rourke for providing the sCJD samples and the CWD samples, respectively.

Adaptation of chronic wasting disease (CWD) prion strains in hosts with different PRNP genotypes

Camilo Duque Velasqueza,c, Elizabeth Triscotta,c, Chiye Kima,c, Diana Morenoa,c, Judd Aikenb,c, and Debbie McKenziea,c

aDepartment of Biological Science, University of Alberta, Edmonton, AB T6G 2G8, Canada; bDepartment of Agriculture, Food & Nutritional Science, University of Alberta, Edmonton, AB T6G 2G8, Canada; cCentre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, AB T6G 2M8, Canada

Aims: The contagious nature of CWD epizootics and the PrPCamino acid variation of cervids (and susceptible sympatric species) guarantee the expansion of prion conformational diversity and selective landscapes where new strains can arise. CWD strains can have novel transmission properties including altered host range that may increase zoonotic risk as circulating strains diversify and evolve. We are characterizing the host adaptability of characterized CWD strains as well as CWD isolates from different cervid species in various enzootic regions.

Material and Methods: Characterized CWD strains as well as a number of isolates from hunter-harvested deer were bioassayed in our rodent panel (transgenic mice expressing cervid alleles G96, S96 and H95-PrPC, elk PrPC, bovine PrPC, and both hamsters and non-transgenic laboratory mice). Strain characteristics were compared using computer based scoring of brain pathology (e.g. PrPCWDbrain distribution), western blot and protein misfolding cyclic amplification (PMCA).

Results: Transmission of various isolates resulted in the selection of strain mixtures in hosts expressing similar PrPC, particularly for polymorphic white-tailed deer and for Norwegian reindeer. As of the second passage, transmission of P153 moose prions from Norway has not resulted in emergence of strains with properties similar to any North American CWD strains in our taxonomic collection (Wisc-1, CWD2, H95+and 116AG).

Conclusions: Our data indicates polymorphic white-tailed deer can favor infection with more than one strain. Similar to transmission studies of Colorado CWD isolates from cervids expressing a single PrPCprimary structure, the isolate from Norway reindeer (V214) represents a strain mixture, suggesting intrinsic strain diversity in the Nordfjella epizootic. The diversity of CWD strains with distinct transmission characteristics represents a threat to wildlife, sympatric domestic animals and public health.

Funded by: Genome Canada and Genome Alberta (Alberta Prion Research Institute and Alberta Agriculture & Forestry); NSERC Grant number: #LSARP 10205; NSERC RGPIN-2017-05539

Acknowledgement: We would like to thank Margo Pybus (Alberta Environment and Parks) Trent Bollinger (University of Saskatchewan) for providing us with tissue samples from hunter-harvested deer and Sylvie Benestad for providing moose and reindeer samples.

Application of PMCA to understand CWD prion strains, species barrier and zoonotic potential

Sandra Pritzkowa, Damian Gorskia, Frank Ramireza, Fei Wanga, Glenn C. Tellingb, Justin J. Greenleec, Sylvie L. Benestadd, and Claudio Sotoa aDepartment of Neurology, University of Texas Medical School at Houston, Houston, Texas, USA; bDepartment of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, USA; cVirus and Prion Research Unit, United States Department of Agriculture, Ames, Iowa, USA; dNorwegian Veterinary Institute, OIE Reference Laboratory for CWD, Ås, Norway

Aims: Chronic wasting disease (CWD) is a prion disease affecting various species of cervids that continues to spread uncontrollably across North America and has recently been detected in Scandinavia (Norway, Sweden and Finland). The mechanisms responsible for the natural transmission of CWD are largely unknown. Furthermore, the risk of CWD transmission to other species, including humans, is also unknown and remains a dangerous enigma. In this study, we investigated the potential of CWD prions to infect several other animal species (sheep, cattle, pig, hamster, and mouse) including humans, by examining their capacity to convert the normal prion protein of distinct species in a PMCA reaction. Moreover, we also investigated whether the in vivo passage of CWD through intermediate species alters their capacity for zoonotic transmission, which may represent a major hazard to human health.

Material and Methods: For these studies, we used brain material from CWD-infected white-tailed deer (Odocoileus virginianus), elk (Cervus canadensis), and mule deer (Odocoileus hemionus) as species native to North America. We also used CWD-infected Moose (Alces alces), reindeer (Rangifer tarandus) and red deer (Cervus elaphus) as Norwegian cervids. We also used brains from cattle, sheep and pigs experimentally infected by CWD. To study interspecies-transmission and zoonotic potential, samples were tested via PMCA for the conversion of PrPCinto PrPScusing different combinations of inoculum and host species. Based on these analyses we estimated the spillover and zoonotic potential for different CWD isolates. We define and quantify spillover and zoonotic potential indices as the efficiency by which CWD prions sustain prion generation in vitro at the expense of normal prion proteins from various mammals and human, respectively.

Results: Our results show that prions from some cervid species, especially those found in Northern Europe, have a higher potential to transmit disease characteristics to other animals. Conversely, CWD-infected cervids originated in North America appear to have a greater potential to generate human PrPSc. We also found that in vivo transmission of CWD to cattle, but not to sheep or pigs substantially increases the ability of these prions to convert human PrPCby PMCA.

Conclusions: Our findings support the existence of different CWD prion strains with distinct spillover and zoonotic potentials. We also conclude that transmission of CWD to other animal species may increase the risk for CWD transmission to humans. Our studies may provide a tool to predict the array of animal species that a given CWD prion could affect and may contribute to understanding the risk of CWD for human health.

Funded by: National Institute of Health Grant number: P01 AI077774

Generation of human chronic wasting disease in transgenic mice

Zerui Wanga, Kefeng Qinb, Manuel V. Camachoa, Ignazio Cali a,c, Jue Yuana, Pingping Shena, Tricia Gillilanda, Syed Zahid Ali Shaha, Maria Gerasimenkoa, Michelle Tanga, Sarada Rajamanickama, Anika Yadatia, Lawrence B. Schonbergerd, Justin Greenleee, Qingzhong Konga,c, James A. Mastriannib, and Wen-Quan Zoua,c

aDepartment of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH, USA; bDepartment of Neurology and Center for Comprehensive Care and Research on Memory Disorders, the University of Chicago Pritzker School of Medicine, Chicago, USA; cNational Prion Disease Pathology Surveillance Center, Case Western Reserve University School of Medicine, Cleveland, OH 44106, USA; dDivision of High-Consequence Pathogens and Pathology, Centers for Disease Control and Prevention, 1600 Clifton Rd, Atlanta, GA, USA; eVirus and Prion Research Unit, National Animal Disease Center, USDA, Agricultural Research Service, 1920 Dayton Avenue, Ames, IA, USA

Aims: Chronic wasting disease (CWD) results from the accumulation of an infectious misfolded conformer (PrPSc) of cellular prion protein (PrPC) in the brains of deer and elk. It has been spreading rapidly throughout many regions of North America, exported inadvertently to South Korea, and more recently identified in Europe. Mad cow disease has caused variant Creutzfeldt-Jakob disease (vCJD) in humans and is currently the only known zoonotic prion disease. Whether CWD is transmissible to humans remains uncertain. The aims of our study were not only to confirm whether CWD prion isolates can convert human brain PrPCinto PrPScin vitro by serial protein misfolding cyclic amplification (sPMCA) but also to determine whether the sPMCA-induced CWD-derived human PrPScis infectious.

Material and Methods: Eight CWD prion isolates from 7 elks and 1 deer were used as the seeds while normal human brain homogenates containing either PrP-129 MM (n = 2) or PrP-129 VV (n = 1) were used as the substrates for sPMCA assay. A normal elk brain tissue sample was used as a negative control seed. Two lines of humanized transgenic (Tg) mice expressing either human PrP-129VV or −129 MM polymorphism were included for transmission studies to determine the infectivity of PMCA-amplified PrPSc. Wester blotting and immunohistochemistry and hematoxylin & eosin staining were used for determining PrPScand neuropathological changes of inoculated animals.

Results: We report here the generation of the first CWD-derived infectious human PrPScusing elk CWD PrPScto initiate conversion of human PrPCfrom normal human brain homogenates with PMCA in vitro. Western blotting with a human PrP selective antibody confirmed that the PMCA-generated protease-resistant PrPScwas derived from the human brain PrPCsubstrate. Two lines of humanized transgenic mice expressing human PrPCwith either Val or Met at the polymorphic codon 129 developed clinical prion disease following intracerebral inoculation with the PMCA-generated CWD-derived human PrPSc. Diseased mice exhibited distinct PrPScpatterns and neuropathological changes in the brain.

Conclusions: Our study, using PMCA and animal bioassays, provides the first evidence that CWD PrPSchas the potential to overcome the species barrier and directly convert human PrPCinto infectious PrPScthat can produce bona fide prion disease when inoculated into humanized transgenic mice.

Funded by: CJD Foundation and NIH

Mortality surveillance of persons potentially exposed to chronic wasting disease

R.A. Maddoxa, R.F. Klosb, L.R. Willb, S.N. Gibbons-Burgenerb, A. Mvilongoa, J.Y. Abramsa, B.S. Applebyc, L.B. Schonbergera, and E.D. Belaya aNational Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention (CDC), Atlanta, USA; bWisconsin Department of Health Services (WDHS), Division of Public Health, Madison, USA; cNational Prion Disease Pathology Surveillance Center (NPDPSC), Case Western Reserve University, Cleveland, USA

Aims: It is unknown whether chronic wasting disease (CWD), a prion disease of cervids, can infect people, but consumption of meat from infected animals would be the most likely route of transmission. Wisconsin Department of Health Services, Division of Public Health (WDHS) personnel maintain a database consisting of information collected from hunters who reported eating, or an intention to eat, venison from CWD-positive cervids. These data, collected since 2003, allow for the evaluation of causes of mortality in individuals potentially exposed to CWD.

Material and Methods: The WDHS database contains the name, date of birth, when available, year of CWD-positive deer harvest, and city and state of residence for each potentially exposed individual. The database also includes information on how the deer was processed (self-processed or by a commercial operator) and when applicable, names of others with whom the venison was shared. Duplicate entries (i.e., those who consumed venison from CWD-positive deer in multiple hunt years) are determined by first name, last name, and date of birth. All names in the database are cross-checked with reported cases of human prion disease in Wisconsin and cases in the National Prion Disease Pathology Surveillance Center (NPDPSC) diagnostic testing database. Persons with date of birth available are also cross-checked with prion disease decedents identified through restricted-use national multiple cause-of-death data via a data use agreement with the National Center for Health Statistics (NCHS).

Results: The database currently consists of 1561 records for hunt years 2003–2017 and 87 additional records for 2018–2019. Of these, 657 records have accompanying date of birth; 15 entries were removed as duplicates leaving 642 unique individuals. Of these individuals, 278 of 426 (66%) who ate venison from a CWD-positive deer and provided processing information reported self-processing. No matches were found among any persons in the database cross-checked with WDHS human prion disease surveillance data, NPDPSC data (February 2022 update), and NCHS data through 2020.

Conclusions: Because of the linkage of person and CWD-positive animal in the WDHS database, reviewing the cause of mortality in potentially exposed persons is possible. The number of individuals cross-checked so far is likely only a small percentage of those potentially exposed to CWD in Wisconsin, and many more years of vital status tracking are needed given an expected long incubation period should transmission to humans occur. Nevertheless, the findings of this ongoing review are thus far reassuring.

Prion disease incidence, United States, 2003–2020

R.A. Maddoxa, M.K. Persona, K. Kotobellib, A. Mvilongoa, B.S. Applebyb, L.B. Schonbergera, T.A. Hammetta, J.Y. Abramsa, and E.D. Belaya aNational Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention (CDC), Atlanta, USA; bNational Prion Disease Pathology Surveillance Center (NPDPSC), Case Western Reserve University, Cleveland, USA

Aims: Mortality data, in conjunction with neuropathological and genetic testing results, are used to estimate prion disease incidence in the United States.

Material and Methods: Prion disease decedents for 2003–2020 were identified from restricted-use U.S. national multiple cause-of-death data, via a data use agreement with the National Center for Health Statistics, and from the National Prion Disease Pathology Surveillance Center (NPDPSC) database. NPDPSC decedents with neuropathological or genetic test results positive for prion disease for whom no likely match was found in the NCHS multiple cause-of-death data were added as cases for incidence calculations, while those with negative neuropathology results but with cause-of-death data indicating prion disease were removed. Unmatched cases in the NPDPSC database lacking neuropathological testing but with a positive real-time quaking-induced conversion (RT-QuIC) test result were additionally assessed. Age-specific and age-adjusted average annual incidence rates were calculated from the combined data; the year 2000 as the standard population and the direct method were used for age-adjustment.

Results: A total of 7,921 decedents were identified as having prion disease during 2003–2020 for an age-adjusted average annual incidence of 1.2 per million population. The age-adjusted incidence between males and females (1.3 and 1.1 per million, respectively) differed significantly (p < 0.0001). The age-specific average annual incidence among those <55 and ≥55 years of age was 0.2 and 4.8 per million, respectively; incidence among those ≥65 was 6.1 per million. Eighteen cases were <30 years of age for an age-specific incidence of 8.0 per billion; only 6 of these very young cases were sporadic (3 sporadic CJD, 3 sporadic fatal insomnia), with the rest being familial (9), variant (2), or iatrogenic (1). The age-adjusted annual incidence for the most recent year of data, 2020, was 1.3 per million. However, assessment of RT-QuIC positive cases lacking neuropathology in the NPDPSC database suggested that approximately 20% more cases may have occurred in that year; the addition of a subset of these cases that had date of death information available (n = 44) increased the 2020 rate to 1.4 per million.

Conclusions: Mortality data supplemented with the results of neuropathological, CSF RT-QuIC, and genetic testing can be used to estimate prion disease incidence. However, the identification in the NPDPSC database of RT-QuIC-positive cases lacking date of death information suggests that this strategy may exclude a number of probable prion disease cases. Prion disease cases <30 years of age, especially those lacking a pathogenic mutation, continue to be very rare.

Shedding of Chronic Wasting Disease Prions in Multiple Excreta Throughout Disease Course in White-tailed Deer

Nathaniel D. Denkersa, Erin E. McNultya, Caitlyn N. Krafta, Amy V. Nallsa, Joseph A. Westricha, Wilfred Goldmannb, Candace K. Mathiasona, and Edward A. Hoovera

aPrion Research Center, College of Veterinary Medicine and Biological Sciences, Department of Microbiology, Immunology, and Pathology; Colorado State University, Fort Collins, CO, USA; bDivision of Infection and Immunity, The Roslin Institute and the Royal Dick School of Veterinary Studies, University of Edinburgh, Midlothian, UK

Aims: Chronic wasting disease (CWD) now infects cervids in South Korea, North America, and Scandinavia. CWD is unique in its efficient transmission and shedding of prions in body fluids throughout long course infections. Questions remain as to the magnitude of shedding and the route of prion acquisition. As CWD continues to expand, the need to better understand these facets of disease becomes more pertinent. The purpose of the studies described was to define the longitudinal shedding profile of CWD prions in urine, saliva, and feces throughout the course of infection in white-tailed deer.

Material and Methods: Twelve (12) white-tailed deer were inoculated with either 1 mg or 300ng of CWD. Urine, saliva, and feces were collected every 3-month post-inoculation (MPI) throughout the study duration. Cohorts were established based on PNRP genotype: codon 96 GG (n = 6) and alternate codons 96 GS (n = 5) & 103NT (n = 1). Urine and saliva were analyzed using iron-oxide magnetic extraction (IOME) and real-time quaking induced conversion (RT-QuIC)(IQ). Feces were subjected to IOME, followed by 4 rounds protein misfolding cyclic amplification (PMCA) with products analyzed by RT-QuIC (IPQ). To determine whether IPQ may be superior to IQ, a subset of urine and saliva were also tested by IPQ. Results were compared with clinical disease status.

Results: Within the 96 GG cohort, positive seeding activity was detected in feces from all deer (100%), in saliva from 5 of 6 (83%), and in urine from 4 of 6 (66%). Shedding in all excreta occurred at, or just after, the first positive tonsil biopsy result. In the 96 GS/103NT cohort, positive seeding activity could be detected in feces from 3 of 6 (50%) deer, saliva in 2 of 6 (33%), and urine in 1 of 6 (16%). Shedding in excreta was detected >5 months after the first tonsil positive result. Four of six 96 GG deer developed clinical signs of CWD, whereas only 2 of the 96 GS/103NT did. Shedding was more frequently detected in deer with clinical disease. The IPQ protocol did not significantly improve detection in saliva or urine samples, however, it significantly augmented detection in feces by eliminating non-specific background commonly experienced with IQ. Negative control samples remained negative in samples tested.

Conclusions: These studies demonstrate: (a) CWD prion excretion occurs throughout infection; (2) PRNP genotype (GG≫GS/NT) influences the excreta shedding; and (3) detection sensitivity in excreta can vary with different RT-QuIC protocols. These results provide a more complete perspective of prion shedding in deer during the course of CWD infection.

Funded by: National Institutes of Health (NIH)

Grant number: RO1-NS061902-09 R to EAH, PO1-AI077774 to EAH, and R01-AI112956-06 to CKM

Acknowledgement: We abundantly thank Sallie Dahmes at WASCO and David Osborn and Gino D’Angelo at the University of Georgia Warnell School of Forestry and Natural Resources for their long-standing support of this work through provision of the hand-raised, CWD-free, white-tailed deer used in these studies

Large-scale PMCA screening of retropharyngeal lymph nodes and in white-tailed deer and comparisons with ELISA and IHC: the Texas CWD study

Rebeca Benaventea, Paulina Sotoa, Mitch Lockwoodb, and Rodrigo Moralesa

aDepartment of Neurology, McGovern Medical School, University of Texas Health Science Center at Houston, Texas, USA; bTexas Park and Wildlife Department, Texas, USA

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy that affects various species of cervids, and both free-ranging and captive animals. Until now, CWD has been detected in 3 continents: North America, Europe, and Asia. CWD prevalence in some states may reach 30% of total animals. In Texas, the first case of CWD was reported in a free-range mule deer in Hudspeth and now it has been detected in additional 14 counties. Currently, the gold standard techniques used for CWD screening and detection are ELISA and immunohistochemistry (IHC) of obex and retropharyngeal lymph nodes (RPLN). Unfortunately, these methods are known for having a low diagnostic sensitivity. Hence, many CWD-infected animals at pre-symptomatic stages may be misdiagnosed. Two promising in vitro prion amplification techniques, including the real-time quaking-induced conversion (RT-QuIC) and the protein misfolding cyclic amplification (PMCA) have been used to diagnose CWD and other prion diseases in several tissues and bodily fluids. Considering the low cost and speed of RT-QuIC, two recent studies have communicated the potential of this technique to diagnose CWD prions in RPLN samples. Unfortunately, the data presented in these articles suggest that identification of CWD positive samples is comparable to the currently used ELISA and IHC protocols. Similar studies using the PMCA technique have not been reported.

Aims: Compare the CWD diagnostic potential of PMCA with ELISA and IHC in RPLN samples from captive and free-range white-tailed deer. Material and Methods: In this study we analyzed 1,003 RPLN from both free-ranging and captive white-tailed deer collected in Texas. Samples were interrogated with the PMCA technique for their content of CWD prions. PMCA data was compared with the results obtained through currently approved techniques.

Results: Our results show a 15-fold increase in CWD detection in free-range deer compared with ELISA. Our results unveil the presence of prion infected animals in Texas counties with no previous history of CWD. In the case of captive deer, we detected a 16% more CWD positive animals when compared with IHC. Interestingly, some of these positive samples displayed differences in their electroforetic mobilities, suggesting the presence of different prion strains within the State of Texas.

Conclusions: PMCA sensitivity is significantly higher than the current gold standards techniques IHC and ELISA and would be a good tool for rapid CWD screening.

Funded by: USDA

Grant number: AP20VSSPRS00C143

ATYPRION project: assessing the zoonotic potential of interspecies transmission of CWD isolates to livestock (preliminary results).

Enric Vidala,b, Juan Carlos Espinosac, Samanta Gilera,b, Montserrat Ordóñeza,b, Guillermo Canteroa,b, Vincent Béringued, Justin J. Greenleee, and Juan Maria Torresc

aUnitat mixta d’Investigació IRTA-UAB en Sanitat Animal. Centre de Recerca en Sanitat Animal (CReSA). Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, Catalonia; bIRTA. Programa de Sanitat Animal. Centre de Recerca en Sanitat Animal (CReSA). Campus de la Universitat Autònoma de Barcelona (UAB), Bellaterra, Catalonia; cCentro de Investigación en Sanidad Animal, CISA-INIA-CSIC, Valdeolmos, Madrid, Spain; dMolecular Virology and Immunology, French National Research Institute for Agriculture, Food and Environment (INRAE), Université Paris-Saclay, Jouy-en-Josas, France; eVirus and Prion Research Unit, National Animal Disease Center, ARS, United States Department of Agriculture, Ames, IA, USA

Aims: Since variant Creutzfeldt-Jackob disease was linked to the consumption of bovine spongiform encephalopathy prions, the study of the pathobiological features of animal prions, particularly their zoonotic potential, is of great concern to the scientific community and public health authorities. Furthermore, interspecies transmission of prions has been demonstrated as a putative evolutionary mechanism for prions, that can lead to the emergence of new features including the ability to infect humans. For instance, small ruminants’ atypical scrapie prions, when propagated in a bovine or porcine host, can shift to a classical BSE phenotype thus posing a potential risk in case of human exposure. So far, no hard evidence of zoonotic transmission of cervids’ chronic wasting disease (CWD) to humans has been published, however experimental transmission to bovine, ovine and caprine hosts has been achieved. Our goal is to investigate if, once passaged through these domestic species, CWD prions might become infectious to humans.

Material and Methods: Different CWD isolates experimentally adapted to cattle, sheep and goat (Hamir et al, 2005, 2006, 2007, Greenlee et al 2012) have been intracerebrally inoculated to transgenic mouse models expressing the human cellular prion protein either homozygous for methionine or valine at codon 129 (Tg340-Met129 and Tg362-Val129). Additionally, inocula obtained from experimental transmission of elk CWD to ovinized (Tg501) and bovinized (BoTg110) transgenic mice, as well as white-tailed deer CWD to BoTg110 mice, are currently being bioassayed in both human PrPCtransgenic models.

Results and conclusions: No evidence of transmission has been found on first passage for bovine adapted elk and mule deer CWD to none of the humanized models. The remaining bioassays are ongoing without showing clinical signs yet, as well as second passages for the negative 1stpassages.

Funded by: La Marató de TV3 foundation. Grant number: ATYPRION (201,821–30-31-32)

https://www.tandfonline.com/doi/full/10.1080/19336896.2022.2091286

Prion Conference 2018 Abstracts

P190 Human prion disease mortality rates by occurrence of chronic wasting disease in freeranging cervids, United States

Abrams JY (1), Maddox RA (1), Schonberger LB (1), Person MK (1), Appleby BS (2), Belay ED (1)

(1) Centers for Disease Control and Prevention (CDC), National Center for Emerging and Zoonotic Infectious Diseases, Atlanta, GA, USA (2) Case Western Reserve University, National Prion Disease Pathology Surveillance Center (NPDPSC), Cleveland, OH, USA.

Background

Chronic wasting disease (CWD) is a prion disease of deer and elk that has been identified in freeranging cervids in 23 US states. While there is currently no epidemiological evidence for zoonotic transmission through the consumption of contaminated venison, studies suggest the CWD agent can cross the species barrier in experimental models designed to closely mimic humans. We compared rates of human prion disease in states with and without CWD to examine the possibility of undetermined zoonotic transmission.

Methods

Death records from the National Center for Health Statistics, case records from the National Prion Disease Pathology Surveillance Center, and additional state case reports were combined to create a database of human prion disease cases from 2003-2015. Identification of CWD in each state was determined through reports of positive CWD tests by state wildlife agencies. Age- and race-adjusted mortality rates for human prion disease, excluding cases with known etiology, were determined for four categories of states based on CWD occurrence: highly endemic (>16 counties with CWD identified in free-ranging cervids); moderately endemic (3-10 counties with CWD); low endemic (1-2 counties with CWD); and no CWD states. States were counted as having no CWD until the year CWD was first identified. Analyses stratified by age, sex, and time period were also conducted to focus on subgroups for which zoonotic transmission would be more likely to be detected: cases <55 years old, male sex, and the latter half of the study (2010-2015).

Results

Highly endemic states had a higher rate of prion disease mortality compared to non-CWD states (rate ratio [RR]: 1.12, 95% confidence interval [CI] = 1.01 - 1.23), as did low endemic states (RR: 1.15, 95% CI = 1.04 - 1.27). Moderately endemic states did not have an elevated mortality rate (RR: 1.05, 95% CI = 0.93 - 1.17). In age-stratified analyses, prion disease mortality rates among the <55 year old population were elevated for moderately endemic states (RR: 1.57, 95% CI = 1.10 – 2.24) while mortality rates were elevated among those ≥55 for highly endemic states (RR: 1.13, 95% CI = 1.02 - 1.26) and low endemic states (RR: 1.16, 95% CI = 1.04 - 1.29). In other stratified analyses, prion disease mortality rates for males were only elevated for low endemic states (RR: 1.27, 95% CI = 1.10 - 1.48), and none of the categories of CWD-endemic states had elevated mortality rates for the latter time period (2010-2015).

Conclusions

While higher prion disease mortality rates in certain categories of states with CWD in free-ranging cervids were noted, additional stratified analyses did not reveal markedly elevated rates for potentially sensitive subgroups that would be suggestive of zoonotic transmission. Unknown confounding factors or other biases may explain state-by-state differences in prion disease mortality.

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P172 Peripheral Neuropathy in Patients with Prion Disease

Wang H(1), Cohen M(1), Appleby BS(1,2)

(1) University Hospitals Cleveland Medical Center, Cleveland, Ohio (2) National Prion Disease Pathology Surveillance Center, Cleveland, Ohio.

Prion disease is a fatal progressive neurodegenerative disease due to deposition of an abnormal protease-resistant isoform of prion protein. Typical symptoms include rapidly progressive dementia, myoclonus, visual disturbance and hallucinations. Interestingly, in patients with prion disease, the abnormal protein canould also be found in the peripheral nervous system. Case reports of prion deposition in peripheral nerves have been reported. Peripheral nerve involvement is thought to be uncommon; however, little is known about the exact prevalence and features of peripheral neuropathy in patients with prion disease.

We reviewed autopsy-proven prion cases from the National Prion Disease Pathology Surveillance Center that were diagnosed between September 2016 to March 2017. We collected information regarding prion protein diagnosis, demographics, comorbidities, clinical symptoms, physical exam, neuropathology, molecular subtype, genetics lab, brain MRI, image and EMG reports. Our study included 104 patients. Thirteen (12.5%) patients had either subjective symptoms or objective signs of peripheral neuropathy. Among these 13 patients, 3 had other known potential etiologies of peripheral neuropathy such as vitamin B12 deficiency or prior chemotherapy. Among 10 patients that had no other clear etiology, 3 (30%) had familial CJD. The most common sCJD subtype was MV1-2 (30%), followed by MM1-2 (20%). The Majority of cases wasere male (60%). Half of them had exposure to wild game. The most common subjective symptoms were tingling and/or numbness of distal extremities. The most common objective finding was diminished vibratory sensation in the feet. Half of them had an EMG with the findings ranging from fasciculations to axonal polyneuropathy or demyelinating polyneuropathy.

Our study provides an overview of the pattern of peripheral neuropathy in patients with prion disease. Among patients with peripheral neuropathy symptoms or signs, majority has polyneuropathy. It is important to document the baseline frequency of peripheral neuropathy in prion diseases as these symptoms may become important when conducting surveillance for potential novel zoonotic prion diseases.

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P177 PrP plaques in methionine homozygous Creutzfeldt-Jakob disease patients as a potential marker of iatrogenic transmission

Abrams JY (1), Schonberger LB (1), Cali I (2), Cohen Y (2), Blevins JE (2), Maddox RA (1), Belay ED (1), Appleby BS (2), Cohen ML (2)

(1) Centers for Disease Control and Prevention (CDC), National Center for Emerging and Zoonotic Infectious Diseases, Atlanta, GA, USA (2) Case Western Reserve University, National Prion Disease Pathology Surveillance Center (NPDPSC), Cleveland, OH, USA.

Background

Sporadic Creutzfeldt-Jakob disease (CJD) is widely believed to originate from de novo spontaneous conversion of normal prion protein (PrP) to its pathogenic form, but concern remains that some reported sporadic CJD cases may actually be caused by disease transmission via iatrogenic processes. For cases with methionine homozygosity (CJD-MM) at codon 129 of the PRNP gene, recent research has pointed to plaque-like PrP deposition as a potential marker of iatrogenic transmission for a subset of cases. This phenotype is theorized to originate from specific iatrogenic source CJD types that comprise roughly a quarter of known CJD cases.

Methods

We reviewed scientific literature for studies which described PrP plaques among CJD patients with known epidemiological links to iatrogenic transmission (receipt of cadaveric human grown hormone or dura mater), as well as in cases of reported sporadic CJD. The presence and description of plaques, along with CJD classification type and other contextual factors, were used to summarize the current evidence regarding plaques as a potential marker of iatrogenic transmission. In addition, 523 cases of reported sporadic CJD cases in the US from January 2013 through September 2017 were assessed for presence of PrP plaques.

Results

We identified four studies describing 52 total cases of CJD-MM among either dura mater recipients or growth hormone recipients, of which 30 were identified as having PrP plaques. While sporadic cases were not generally described as having plaques, we did identify case reports which described plaques among sporadic MM2 cases as well as case reports of plaques exclusively in white matter among sporadic MM1 cases. Among the 523 reported sporadic CJD cases, 0 of 366 MM1 cases had plaques, 2 of 48 MM2 cases had kuru plaques, and 4 of 109 MM1+2 cases had either kuru plaques or both kuru and florid plaques. Medical chart review of the six reported sporadic CJD cases with plaques did not reveal clinical histories suggestive of potential iatrogenic transmission.

Conclusions

PrP plaques occur much more frequently for iatrogenic CJD-MM cases compared to sporadic CJDMM cases. Plaques may indicate iatrogenic transmission for CJD-MM cases without a type 2 Western blot fragment. The study results suggest the absence of significant misclassifications of iatrogenic CJD as sporadic. To our knowledge, this study is the first to describe grey matter kuru plaques in apparently sporadic CJD-MM patients with a type 2 Western blot fragment.

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P180 Clinico-pathological analysis of human prion diseases in a brain bank series

Ximelis T (1), Aldecoa I (1,2), Molina-Porcel L (1,3), Grau-Rivera O (4), Ferrer I (5), Nos C (6), Gelpi E (1,7), Sánchez-Valle R (1,4)

(1) Neurological Tissue Bank of the Biobanc-Hospital ClÃnic-IDIBAPS, Barcelona, Spain (2) Pathological Service of Hospital ClÃnic de Barcelona, Barcelona, Spain (3) EAIA Trastorns Cognitius, Centre Emili Mira, Parc de Salut Mar, Barcelona, Spain (4) Department of Neurology of Hospital ClÃnic de Barcelona, Barcelona, Spain (5) Institute of Neuropathology, Hospital Universitari de Bellvitge, Hospitalet de Llobregat, Barcelona (6) General subdirectorate of Surveillance and Response to Emergencies in Public Health, Department of Public Health in Catalonia, Barcelona, Spain (7) Institute of Neurology, Medical University of Vienna, Vienna, Austria.

Background and objective:

The Neurological Tissue Bank (NTB) of the Hospital Clínic-Institut d‘Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain is the reference center in Catalonia for the neuropathological study of prion diseases in the region since 2001. The aim of this study is to analyse the characteristics of the confirmed prion diseases registered at the NTB during the last 15 years.

Methods:

We reviewed retrospectively all neuropathologically confirmed cases registered during the period January 2001 to December 2016.

Results:

176 cases (54,3% female, mean age: 67,5 years and age range: 25-86 years) of neuropathological confirmed prion diseases have been studied at the NTB. 152 cases corresponded to sporadic Creutzfeldt-Jakob disease (sCJD), 10 to genetic CJD, 10 to Fatal Familial Insomnia, 2 to GerstmannSträussler-Scheinker disease, and 2 cases to variably protease-sensitive prionopathy (VPSPr). Within sCJD subtypes the MM1 subtype was the most frequent, followed by the VV2 histotype.

Clinical and neuropathological diagnoses agreed in 166 cases (94%). The clinical diagnosis was not accurate in 10 patients with definite prion disease: 1 had a clinical diagnosis of Fronto-temporal dementia (FTD), 1 Niemann-Pick‘s disease, 1 Lewy Body‘s Disease, 2 Alzheimer‘s disease, 1 Cortico-basal syndrome and 2 undetermined dementia. Among patients with VPSPr, 1 had a clinical diagnosis of Amyotrophic lateral sclerosis (ALS) and the other one with FTD.

Concomitant pathologies are frequent in older age groups, mainly AD neuropathological changes were observed in these subjects.

Discussion:

A wide spectrum of human prion diseases have been identified in the NTB being the relative frequencies and main characteristics like other published series. There is a high rate of agreement between clinical and neuropathological diagnoses with prion diseases. These findings show the importance that public health has given to prion diseases during the past 15 years. Continuous surveillance of human prion disease allows identification of new emerging phenotypes. Brain tissue samples from these donors are available to the scientific community. For more information please visit:

http://www.clinicbiobanc.org/banc-teixits-neurologics/mostres/en_index.html

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P192 Prion amplification techniques for the rapid evaluation of surface decontamination procedures

Bruyere-Ostells L (1), Mayran C (1), Belondrade M (1), Boublik Y (2), Haïk S (3), Fournier-Wirth C (1), Nicot S (1), Bougard D (1)

(1) Pathogenesis and control of chronic infections, Etablissement Français du Sang, Inserm, Université de Montpellier, Montpellier, France. (2) Centre de Recherche en Biologie cellulaire de Montpellier, CNRS, Université de Montpellier, Montpellier, France. (3) Inserm U 1127, CNRS UMR 7225, Sorbonne Universités, UPMC Université Paris 06 UMR S 1127, Institut du Cerveau et de la Moelle épinière, ICM, Paris, France.

Aims:

Transmissible Spongiform Encephalopathies (TSE) or prion diseases are a group of incurable and always fatal neurodegenerative disorders including Creutzfeldt-Jakob diseases (CJD) in humans. These pathologies include sporadic (sCJD), genetic and acquired (variant CJD) forms. By the past, sCJD and vCJD were transmitted by different prion contaminated biological materials to patients resulting in more than 400 iatrogenic cases (iCJD). The atypical nature and the biochemical properties of the infectious agent, formed by abnormal prion protein or PrPTSE, make it particularly resistant to conventional decontamination procedures. In addition, PrPTSE is widely distributed throughout the organism before clinical onset in vCJD and can also be detected in some peripheral tissues in sporadic CJD. Risk of iatrogenic transmission of CJD by contaminated medical device remains thus a concern for healthcare facilities. Bioassay is the gold standard method to evaluate the efficacy of prion decontamination procedures but is time-consuming and expensive. Here, we propose to compare in vitro prion amplification techniques: Protein Misfolding Cyclic Amplification (PMCA) and Real-Time Quaking Induced Conversion (RT-QuIC) for the detection of residual prions on surface after decontamination.

Methods:

Stainless steel wires, by mimicking the surface of surgical instruments, were proposed as a carrier model of prions for inactivation studies. To determine the sensitivity of the two amplification techniques on wires (Surf-PMCA and Surf-QuIC), steel wires were therefore contaminated with serial dilutions of brain homogenates (BH) from a 263k infected hamster and from a patient with sCJD (MM1 subtype). We then compared the different standard decontamination procedures including partially and fully efficient treatments by detecting the residual seeding activity on 263K and sCJD contaminated wires. We completed our study by the evaluation of marketed reagents endorsed for prion decontamination.

Results:

The two amplification techniques can detect minute quantities of PrPTSE adsorbed onto a single wire. 8/8 wires contaminated with a 10-6 dilution of 263k BH and 1/6 with the 10-8 dilution are positive with Surf-PMCA. Similar performances were obtained with Surf-QuIC on 263K: 10/16 wires contaminated with 10-6 dilution and 1/8 wires contaminated with 10-8 dilution are positive. Regarding the human sCJD-MM1 prion, Surf-QuIC allows us to detect 16/16 wires contaminated with 10-6 dilutions and 14/16 with 10-7 . Results obtained after decontamination treatments are very similar between 263K and sCJD prions. Efficiency of marketed treatments to remove prions is lower than expected.

Conclusions:

Surf-PMCA and Surf-QuIC are very sensitive methods for the detection of prions on wires and could be applied to prion decontamination studies for rapid evaluation of new treatments. Sodium hypochlorite is the only product to efficiently remove seeding activity of both 263K and sCJD prions.

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WA2 Oral transmission of CWD into Cynomolgus macaques: signs of atypical disease, prion conversion and infectivity in macaques and bio-assayed transgenic mice

Schatzl HM (1, 2), Hannaoui S (1, 2), Cheng Y-C (1, 2), Gilch S (1, 2), Beekes M (3), SchulzSchaeffer W (4), Stahl-Hennig C (5) and Czub S (2, 6)

(1) University of Calgary, Calgary Prion Research Unit, Calgary, Canada (2) University of Calgary, Faculty of Veterinary Medicine, Calgary, Canada, (3) Robert Koch Institute, Berlin, Germany, (4) University of Homburg/Saar, Homburg, Germany, (5) German Primate Center, Goettingen, Germany, (6) Canadian Food Inspection Agency (CFIA), Lethbridge, Canada.

To date, BSE is the only example of interspecies transmission of an animal prion disease into humans. The potential zoonotic transmission of CWD is an alarming issue and was addressed by many groups using a variety of in vitro and in vivo experimental systems. Evidence from these studies indicated a substantial, if not absolute, species barrier, aligning with the absence of epidemiological evidence suggesting transmission into humans. Studies in non-human primates were not conclusive so far, with oral transmission into new-world monkeys and no transmission into old-world monkeys. Our consortium has challenged 18 Cynomolgus macaques with characterized CWD material, focusing on oral transmission with muscle tissue. Some macaques have orally received a total of 5 kg of muscle material over a period of 2 years. After 5-7 years of incubation time some animals showed clinical symptoms indicative of prion disease, and prion neuropathology and PrPSc deposition were found in spinal cord and brain of euthanized animals. PrPSc in immunoblot was weakly detected in some spinal cord materials and various tissues tested positive in RT-QuIC, including lymph node and spleen homogenates. To prove prion infectivity in the macaque tissues, we have intracerebrally inoculated 2 lines of transgenic mice, expressing either elk or human PrP. At least 3 TgElk mice, receiving tissues from 2 different macaques, showed clinical signs of a progressive prion disease and brains were positive in immunoblot and RT-QuIC. Tissues (brain, spinal cord and spleen) from these and preclinical mice are currently tested using various read-outs and by second passage in mice. Transgenic mice expressing human PrP were so far negative for clear clinical prion disease (some mice >300 days p.i.). In parallel, the same macaque materials are inoculated into bank voles. Taken together, there is strong evidence of transmissibility of CWD orally into macaques and from macaque tissues into transgenic mouse models, although with an incomplete attack rate. The clinical and pathological presentation in macaques was mostly atypical, with a strong emphasis on spinal cord pathology. Our ongoing studies will show whether the transmission of CWD into macaques and passage in transgenic mice represents a form of non-adaptive prion amplification, and whether macaque-adapted prions have the potential to infect mice expressing human PrP. The notion that CWD can be transmitted orally into both new-world and old-world non-human primates asks for a careful reevaluation of the zoonotic risk of CWD.

See also poster P103

***> The notion that CWD can be transmitted orally into both new-world and old-world non-human primates asks for a careful reevaluation of the zoonotic risk of CWD.

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WA16 Monitoring Potential CWD Transmission to Humans

Belay ED

Centers for Disease Control and Prevention (CDC), National Center for Emerging and Zoonotic Infectious Diseases, Atlanta, GA, USA.

The spread of chronic wasting disease (CWD) in animals has raised concerns about increasing human exposure to the CWD agent via hunting and venison consumption, potentially facilitating CWD transmission to humans. Several studies have explored this possibility, including limited epidemiologic studies, in vitro experiments, and laboratory studies using various types of animal models. Most human exposures to the CWD agent in the United States would be expected to occur in association with deer and elk hunting in CWD-endemic areas. The Centers for Disease Control and Prevention (CDC) collaborated with state health departments in Colorado, Wisconsin, and Wyoming to identify persons at risk of CWD exposure and to monitor their vital status over time. Databases were established of persons who hunted in Colorado and Wyoming and those who reported consumption of venison from deer that later tested positive in Wisconsin. Information from the databases is periodically cross-checked with mortality data to determine the vital status and causes of death for deceased persons. Long-term follow-up of these hunters is needed to assess their risk of development of a prion disease linked to CWD exposure.

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P166 Characterization of CJD strain profiles in venison consumers and non-consumers from Alberta and Saskatchewan

Stephanie Booth (1,2), Lise Lamoureux (1), Debra Sorensen (1), Jennifer L. Myskiw (1,2), Megan Klassen (1,2), Michael Coulthart (3), Valerie Sim (4)

(1) Zoonotic Diseases and Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg (2) Department of Medical Microbiology and Infectious Diseases, University of Manitoba, Winnipeg (3) Canadian CJD Surveillance System, Public Health Agency of Canada, Ottawa (4) Division of Neurology, Department of Medicine Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton.

Chronic wasting disease (CWD) is spreading rapidly through wild cervid populations in the Canadian provinces of Alberta and Saskatchewan. While this has implications for tourism and hunting, there is also concern over possible zoonotic transmission to humans who eat venison from infected deer. Whilst there is no evidence of any human cases of CWD to date, the Canadian CJD Surveillance System (CJDSS) in Canada is staying vigilant. When variant CJD occurred following exposure to BSE, the unique biochemical fingerprint of the pathologic PrP enabled a causal link to be confirmed. However, we cannot be sure what phenotype human CWD prions would present with, or indeed, whether this would be distinct from that see in sporadic CJD. Therefore we are undertaking a systematic analysis of the molecular diversity of CJD cases of individuals who resided in Alberta and Saskatchewan at their time of death comparing venison consumers and non-consumers, using a variety of clinical, imaging, pathological and biochemical markers. Our initial objective is to develop novel biochemical methodologies that will extend the baseline glycoform and genetic polymorphism typing that is already completed by the CJDSS. Firstly, we are reviewing MRI, EEG and pathology information from over 40 cases of CJD to select clinically affected areas for further investigation. Biochemical analysis will include assessment of the levels of protease sensitive and resistant prion protein, glycoform typing using 2D gel electrophoresis, testing seeding capabilities and kinetics of aggregation by quaking-induced conversion, and determining prion oligomer size distributions with asymmetric flow field fractionation with in-line light scattering. Progress and preliminary data will be presented. Ultimately, we intend to further define the relationship between PrP structure and disease phenotype and establish a baseline for the identification of future atypical CJD cases that may arise as a result of exposure to CWD.

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Source Prion Conference 2018 Abstracts

http://transmissiblespongiformencephalopathy.blogspot.com/2018/05/prion-2018-may-22-25-2018-santiago-de.html

http://chronic-wasting-disease.blogspot.com/2018/07/oral-transmission-of-cwd-into.html

http://prionconference.blogspot.com/2018/

Volume 24, Number 8—August 2018 Research Susceptibility of Human Prion Protein to Conversion by Chronic Wasting Disease Prions

Marcelo A. BarriaComments to Author , Adriana Libori, Gordon Mitchell, and Mark W. Head Author affiliations: National CJD Research and Surveillance Unit, University of Edinburgh, Edinburgh, Scotland, UK (M.A. Barria, A. Libori, M.W. Head); National and OIE Reference Laboratory for Scrapie and CWD, Canadian Food Inspection Agency, Ottawa, Ontario, Canada (G. Mitchell)

Abstract Chronic wasting disease (CWD) is a contagious and fatal neurodegenerative disease and a serious animal health issue for deer and elk in North America. The identification of the first cases of CWD among free-ranging reindeer and moose in Europe brings back into focus the unresolved issue of whether CWD can be zoonotic like bovine spongiform encephalopathy. We used a cell-free seeded protein misfolding assay to determine whether CWD prions from elk, white-tailed deer, and reindeer in North America can convert the human prion protein to the disease-associated form. We found that prions can convert, but the efficiency of conversion is affected by polymorphic variation in the cervid and human prion protein genes. In view of the similarity of reindeer, elk, and white-tailed deer in North America to reindeer, red deer, and roe deer, respectively, in Europe, a more comprehensive and thorough assessment of the zoonotic potential of CWD might be warranted.

snip...

Discussion Characterization of the transmission properties of CWD and evaluation of their zoonotic potential are important for public health purposes. Given that CWD affects several members of the family Cervidae, it seems reasonable to consider whether the zoonotic potential of CWD prions could be affected by factors such as CWD strain, cervid species, geographic location, and Prnp–PRNP polymorphic variation. We have previously used an in vitro conversion assay (PMCA) to investigate the susceptibility of the human PrP to conversion to its disease-associated form by several animal prion diseases, including CWD (15,16,22). The sensitivity of our molecular model for the detection of zoonotic conversion depends on the combination of 1) the action of proteinase K to degrade the abundant human PrPC that constitutes the substrate while only N terminally truncating any human PrPres produced and 2) the presence of the 3F4 epitope on human but not cervid PrP. In effect, this degree of sensitivity means that any human PrPres formed during the PMCA reaction can be detected down to the limit of Western blot sensitivity. In contrast, if other antibodies that detect both cervid and human PrP are used, such as 6H4, then newly formed human PrPres must be detected as a measurable increase in PrPres over the amount remaining in the reaction product from the cervid seed. Although best known for the efficient amplification of prions in research and diagnostic contexts, the variation of the PMCA method employed in our study is optimized for the definitive detection of zoonotic reaction products of inherently inefficient conversion reactions conducted across species barriers. By using this system, we previously made and reported the novel observation that elk CWD prions could convert human PrPC from human brain and could also convert recombinant human PrPC expressed in transgenic mice and eukaryotic cell cultures (15).

A previous publication suggested that mule deer PrPSc was unable to convert humanized transgenic substrate in PMCA assays (23) and required a further step of in vitro conditioning in deer substrate PMCA before it was able to cross the deer–human molecular barrier (24). However, prions from other species, such as elk (15) and reindeer affected by CWD, appear to be compatible with the human protein in a single round of amplification (as shown in our study). These observations suggest that different deer species affected by CWD could present differing degrees of the olecular compatibility with the normal form of human PrP.

The contribution of the polymorphism at codon 129 of the human PrP gene has been extensively studied and is recognized as a risk factor for Creutzfeldt-Jakob disease (4). In cervids, the equivalent codon corresponds to the position 132 encoding methionine or leucine. This polymorphism in the elk gene has been shown to play an important role in CWD susceptibility (25,26). We have investigated the effect of this cervid Prnp polymorphism on the conversion of the humanized transgenic substrate according to the variation in the equivalent PRNP codon 129 polymorphism. Interestingly, only the homologs methionine homozygous seed–substrate reactions could readily convert the human PrP, whereas the heterozygous elk PrPSc was unable to do so, even though comparable amounts of PrPres were used to seed the reaction. In addition, we observed only low levels of human PrPres formation in the reactions seeded with the homozygous methionine (132 MM) and the heterozygous (132 ML) seeds incubated with the other 2 human polymorphic substrates (129 MV and 129 VV). The presence of the amino acid leucine at position 132 of the elk Prnp gene has been attributed to a lower degree of prion conversion compared with methionine on the basis of experiments in mice made transgenic for these polymorphic variants (26). Considering the differences observed for the amplification of the homozygous human methionine substrate by the 2 polymorphic elk seeds (MM and ML), reappraisal of the susceptibility of human PrPC by the full range of cervid polymorphic variants affected by CWD would be warranted.

In light of the recent identification of the first cases of CWD in Europe in a free-ranging reindeer (R. tarandus) in Norway (2), we also decided to evaluate the in vitro conversion potential of CWD in 2 experimentally infected reindeer (18). Formation of human PrPres was readily detectable after a single round of PMCA, and in all 3 humanized polymorphic substrates (MM, MV, and VV). This finding suggests that CWD prions from reindeer could be more compatible with human PrPC generally and might therefore present a greater risk for zoonosis than, for example, CWD prions from white-tailed deer. A more comprehensive comparison of CWD in the affected species, coupled with the polymorphic variations in the human and deer PRNP–Prnp genes, in vivo and in vitro, will be required before firm conclusions can be drawn. Analysis of the Prnp sequence of the CWD reindeer in Norway was reported to be identical to the specimens used in our study (2). This finding raises the possibility of a direct comparison of zoonotic potential between CWD acquired in the wild and that produced in a controlled laboratory setting. (Table).

The prion hypothesis proposes that direct molecular interaction between PrPSc and PrPC is necessary for conversion and prion replication. Accordingly, polymorphic variants of the PrP of host and agent might play a role in determining compatibility and potential zoonotic risk. In this study, we have examined the capacity of the human PrPC to support in vitro conversion by elk, white-tailed deer, and reindeer CWD PrPSc. Our data confirm that elk CWD prions can convert the human PrPC, at least in vitro, and show that the homologous PRNP polymorphisms at codon 129 and 132 in humans and cervids affect conversion efficiency. Other species affected by CWD, particularly caribou or reindeer, also seem able to convert the human PrP. It will be important to determine whether other polymorphic variants found in other CWD-affected Cervidae or perhaps other factors (17) exert similar effects on the ability to convert human PrP and thus affect their zoonotic potential.

Dr. Barria is a research scientist working at the National CJD Research and Surveillance Unit, University of Edinburgh. His research has focused on understanding the molecular basis of a group of fatal neurologic disorders called prion diseases.

Acknowledgments We thank Aru Balachandran for originally providing cervid brain tissues, Abigail Diack and Jean Manson for providing mouse brain tissue, and James Ironside for his critical reading of the manuscript at an early stage.

This report is independent research commissioned and funded by the United Kingdom’s Department of Health Policy Research Programme and the Government of Scotland. The views expressed in this publication are those of the authors and not necessarily those of the Department of Health or the Government of Scotland.

Author contributions: The study was conceived and designed by M.A.B. and M.W.H. The experiments were conducted by M.A.B. and A.L. Chronic wasting disease brain specimens were provided by G.M. The manuscript was written by M.A.B. and M.W.H. All authors contributed to the editing and revision of the manuscript.

https://wwwnc.cdc.gov/eid/article/24/8/16-1888_article

https://www.ed.ac.uk/clinical-brain-sciences/news/news-jul-dec-2018/cwd-prions-human-conversion

Prion 2017 Conference Abstracts 

First evidence of intracranial and peroral transmission of Chronic Wasting Disease (CWD) into Cynomolgus macaques: a work in progress 

Stefanie Czub1, Walter Schulz-Schaeffer2, Christiane Stahl-Hennig3, Michael Beekes4, Hermann Schaetzl5 and Dirk Motzkus6 1 University of Calgary Faculty of Veterinary Medicine/Canadian Food Inspection Agency; 2Universitatsklinikum des Saarlandes und Medizinische Fakultat der Universitat des Saarlandes; 3 Deutsches Primaten Zentrum/Goettingen; 4 Robert-Koch-Institut Berlin; 5 University of Calgary Faculty of Veterinary Medicine; 6 presently: Boehringer Ingelheim Veterinary Research Center; previously: Deutsches Primaten Zentrum/Goettingen 

This is a progress report of a project which started in 2009. 

21 cynomolgus macaques were challenged with characterized CWD material from white-tailed deer (WTD) or elk by intracerebral (ic), oral, and skin exposure routes. 

Additional blood transfusion experiments are supposed to assess the CWD contamination risk of human blood product. 

Challenge materials originated from symptomatic cervids for ic, skin scarification and partially per oral routes (WTD brain). 

Challenge material for feeding of muscle derived from preclinical WTD and from preclinical macaques for blood transfusion experiments. 

We have confirmed that the CWD challenge material contained at least two different CWD agents (brain material) as well as CWD prions in muscle-associated nerves. 

Here we present first data on a group of animals either challenged ic with steel wires or per orally and sacrificed with incubation times ranging from 4.5 to 6.9 years at postmortem. 

Three animals displayed signs of mild clinical disease, including anxiety, apathy, ataxia and/or tremor. 

In four animals wasting was observed, two of those had confirmed diabetes. 

All animals have variable signs of prion neuropathology in spinal cords and brains and by supersensitive IHC, reaction was detected in spinal cord segments of all animals. 

Protein misfolding cyclic amplification (PMCA), real-time quaking-induced conversion (RT-QuiC) and PET-blot assays to further substantiate these findings are on the way, as well as bioassays in bank voles and transgenic mice. 

At present, a total of 10 animals are sacrificed and read-outs are ongoing. 

Preclinical incubation of the remaining macaques covers a range from 6.4 to 7.10 years. 

Based on the species barrier and an incubation time of > 5 years for BSE in macaques and about 10 years for scrapie in macaques, we expected an onset of clinical disease beyond 6 years post inoculation. 

PRION 2017 DECIPHERING NEURODEGENERATIVE DISORDERS ABSTRACTS REFERENCE 

https://cjdfoundation.org/files/pdf/CWD%20study%20oral%20transmission%20of%20CWD%20to%20primates.pdf

8. Even though human TSE‐exposure risk through consumption of game from European cervids can be assumed to be minor, if at all existing, no final conclusion can be drawn due to the overall lack of scientific data. In particular the US data do not clearly exclude the possibility of human (sporadic or familial) TSE development due to consumption of venison. The Working Group thus recognizes a potential risk to consumers if a TSE would be present in European cervids. It might be prudent considering appropriate measures to reduce such a risk, e.g. excluding tissues such as CNS and lymphoid tissues from the human food chain, which would greatly reduce any potential risk for consumers. However, it is stressed that currently, no data regarding a risk of TSE infections from cervid products are available.

https://efsa.onlinelibrary.wiley.com/doi/full/10.2903/j.efsa.2018.5132

SATURDAY, FEBRUARY 23, 2019 

Chronic Wasting Disease CWD TSE Prion and THE FEAST 2003 CDC an updated review of the science 2019

https://chronic-wasting-disease.blogspot.com/2019/02/chronic-wasting-disease-cwd-tse-prion.html

TUESDAY, NOVEMBER 04, 2014 

Six-year follow-up of a point-source exposure to CWD contaminated venison in an Upstate New York community: risk behaviours and health outcomes 2005–2011

Authors, though, acknowledged the study was limited in geography and sample size and so it couldn't draw a conclusion about the risk to humans. They recommended more study. Dr. Ermias Belay was the report's principal author but he said New York and Oneida County officials are following the proper course by not launching a study. "There's really nothing to monitor presently. No one's sick," Belay said, noting the disease's incubation period in deer and elk is measured in years. "

http://chronic-wasting-disease.blogspot.com/2014/11/six-year-follow-up-of-point-source.html 

Transmission Studies

Mule deer transmissions of CWD were by intracerebral inoculation and compared with natural cases {the following was written but with a single line marked through it ''first passage (by this route)}....TSS

resulted in a more rapidly progressive clinical disease with repeated episodes of synocopy ending in coma. One control animal became affected, it is believed through contamination of inoculum (?saline). Further CWD transmissions were carried out by Dick Marsh into ferret, mink and squirrel monkey. Transmission occurred in ALL of these species with the shortest incubation period in the ferret.

snip.... 

https://web.archive.org/web/20090506002237/http://www..bseinquiry.gov.uk/files/mb/m11b/tab01.pdf

Prion Infectivity in Fat of Deer with Chronic Wasting Disease▿ 

Brent Race#, Kimberly Meade-White#, Richard Race and Bruce Chesebro* + Author Affiliations

In mice, prion infectivity was recently detected in fat. Since ruminant fat is consumed by humans and fed to animals, we determined infectivity titers in fat from two CWD-infected deer. Deer fat devoid of muscle contained low levels of CWD infectivity and might be a risk factor for prion infection of other species. 

http://jvi.asm.org/content/83/18/9608.full 

Prions in Skeletal Muscles of Deer with Chronic Wasting Disease 

Here bioassays in transgenic mice expressing cervid prion protein revealed the presence of infectious prions in skeletal muscles of CWD-infected deer, demonstrating that humans consuming or handling meat from CWD-infected deer are at risk to prion exposure. 

http://science.sciencemag.org/content/311/5764/1117..long 

*** now, let’s see what the authors said about this casual link, personal communications years ago, and then the latest on the zoonotic potential from CWD to humans from the TOKYO PRION 2016 CONFERENCE.

see where it is stated NO STRONG evidence. so, does this mean there IS casual evidence ???? “Our conclusion stating that we found no strong evidence of CWD transmission to humans”

From: TSS 

Subject: CWD aka MAD DEER/ELK TO HUMANS ???

Date: September 30, 2002 at 7:06 am PST

From: "Belay, Ermias"

To: Cc: "Race, Richard (NIH)" ; ; "Belay, Ermias"

Sent: Monday, September 30, 2002 9:22 AM

Subject: RE: TO CDC AND NIH - PUB MED- 3 MORE DEATHS - CWD - YOUNG HUNTERS

Dear Sir/Madam,

In the Archives of Neurology you quoted (the abstract of which was attached to your email), we did not say CWD in humans will present like variant CJD.. That assumption would be wrong. I encourage you to read the whole article and call me if you have questions or need more clarification (phone: 404-639-3091). Also, we do not claim that "no-one has ever been infected with prion disease from eating venison." Our conclusion stating that we found no strong evidence of CWD transmission to humans in the article you quoted or in any other forum is limited to the patients we investigated.

Ermias Belay, M.D. Centers for Disease Control and Prevention

-----Original Message-----

From: Sent: Sunday, September 29, 2002 10:15 AM

To: rr26k@nih.gov; rrace@niaid.nih.gov; ebb8@CDC.GOV

Subject: TO CDC AND NIH - PUB MED- 3 MORE DEATHS - CWD - YOUNG HUNTERS

Sunday, November 10, 2002 6:26 PM .......snip........end..............TSS

Thursday, April 03, 2008

A prion disease of cervids: Chronic wasting disease 2008 1: Vet Res. 2008 Apr 3;39(4):41 A prion disease of cervids: Chronic wasting disease Sigurdson CJ.

snip...

*** twenty-seven CJD patients who regularly consumed venison were reported to the Surveillance Center***,

snip... full text ; 

http://chronic-wasting-disease.blogspot.com/2008/04/prion-disease-of-cervids-chronic.html 

> However, to date, no CWD infections have been reported in people. 

sporadic, spontaneous CJD, 85%+ of all human TSE, did not just happen. never in scientific literature has this been proven.

if one looks up the word sporadic or spontaneous at pubmed, you will get a laundry list of disease that are classified in such a way;

sporadic = 54,983 hits https://www.ncbi.nlm.nih.gov/pubmed/?term=sporadic

spontaneous = 325,650 hits https://www.ncbi.nlm.nih.gov/pubmed/?term=spontaneous

key word here is 'reported'. science has shown that CWD in humans will look like sporadic CJD. SO, how can one assume that CWD has not already transmitted to humans? they can't, and it's as simple as that. from all recorded science to date, CWD has already transmitted to humans, and it's being misdiagnosed as sporadic CJD. ...terry 

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

> However, to date, no CWD infections have been reported in people. 

key word here is ‘reported’. science has shown that CWD in humans will look like sporadic CJD. SO, how can one assume that CWD has not already transmitted to humans? they can’t, and it’s as simple as that. from all recorded science to date, CWD has already transmitted to humans, and it’s being misdiagnosed as sporadic CJD. …terry 

*** 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).*** 

http://www.tandfonline.com/doi/full/10.4161/pri.28124?src=recsys 

http://www.tandfonline.com/doi/pdf/10.4161/pri.28124?needAccess=true 

https://wwwnc.cdc.gov/eid/article/20/1/13-0858_article 

CWD TSE PRION AND ZOONOTIC, ZOONOSIS, POTENTIAL

Subject: Re: DEER SPONGIFORM ENCEPHALOPATHY SURVEY & HOUND STUDY 

Date: Fri, 18 Oct 2002 23:12:22 +0100 

From: Steve Dealler 

Reply-To: Bovine Spongiform Encephalopathy Organization: Netscape Online member 

To: BSE-L@ References: 

Dear Terry,

An excellent piece of review as this literature is desperately difficult to get back from Government sites.

What happened with the deer was that an association between deer meat eating and sporadic CJD was found in about 1993. The evidence was not great but did not disappear after several years of asking CJD cases what they had eaten. I think that the work into deer disease largely stopped because it was not helpful to the UK industry...and no specific cases were reported. Well, if you dont look adequately like they are in USA currenly then you wont find any!

Steve Dealler 

=============== 

https://caninespongiformencephalopathy.blogspot.com/2010/03/canine-spongiform-encephalopathy-aka.html

''The association between venison eating and risk of CJD shows similar pattern, with regular venison eating associated with a 9 FOLD INCREASE IN RISK OF CJD (p = 0.04).''

CREUTZFELDT JAKOB DISEASE SURVEILLANCE IN THE UNITED KINGDOM THIRD ANNUAL REPORT AUGUST 1994

Consumption of venison and veal was much less widespread among both cases and controls. For both of these meats there was evidence of a trend with increasing frequency of consumption being associated with increasing risk of CJD. (not nvCJD, but sporadic CJD...tss) 

These associations were largely unchanged when attention was restricted to pairs with data obtained from relatives. ...

Table 9 presents the results of an analysis of these data.

There is STRONG evidence of an association between ‘’regular’’ veal eating and risk of CJD (p = .0.01).

Individuals reported to eat veal on average at least once a year appear to be at 13 TIMES THE RISK of individuals who have never eaten veal.

There is, however, a very wide confidence interval around this estimate. There is no strong evidence that eating veal less than once per year is associated with increased risk of CJD (p = 0.51).

The association between venison eating and risk of CJD shows similar pattern, with regular venison eating associated with a 9 FOLD INCREASE IN RISK OF CJD (p = 0.04).

There is some evidence that risk of CJD INCREASES WITH INCREASING FREQUENCY OF LAMB EATING (p = 0.02).

The evidence for such an association between beef eating and CJD is weaker (p = 0.14). When only controls for whom a relative was interviewed are included, this evidence becomes a little STRONGER (p = 0.08).

snip...

It was found that when veal was included in the model with another exposure, the association between veal and CJD remained statistically significant (p = < 0.05 for all exposures), while the other exposures ceased to be statistically significant (p = > 0.05).

snip...

In conclusion, an analysis of dietary histories revealed statistical associations between various meats/animal products and INCREASED RISK OF CJD. When some account was taken of possible confounding, the association between VEAL EATING AND RISK OF CJD EMERGED AS THE STRONGEST OF THESE ASSOCIATIONS STATISTICALLY. ...

snip...

In the study in the USA, a range of foodstuffs were associated with an increased risk of CJD, including liver consumption which was associated with an apparent SIX-FOLD INCREASE IN THE RISK OF CJD. By comparing the data from 3 studies in relation to this particular dietary factor, the risk of liver consumption became non-significant with an odds ratio of 1.2 (PERSONAL COMMUNICATION, PROFESSOR A. HOFMAN. ERASMUS UNIVERSITY, ROTTERDAM). (???...TSS)

snip...see full report ;

http://web.archive.org/web/20090506050043/http://www.bseinquiry.gov.uk/files/yb/1994/08/00004001.pdf

http://web.archive.org/web/20090506050007/http://www.bseinquiry.gov.uk/files/yb/1994/10/00003001.pdf

http://web.archive.org/web/20090506050244/http://www.bseinquiry.gov.uk/files/yb/1994/07/00001001.pdf

Stephen Dealler is a consultant medical microbiologist deal@airtime.co.uk 

BSE Inquiry Steve Dealler

Management In Confidence

BSE: Private Submission of Bovine Brain Dealler

snip...see full text;

MONDAY, FEBRUARY 25, 2019

***> MAD DOGS AND ENGLISHMEN BSE, SCRAPIE, CWD, CJD, TSE PRION A REVIEW 2019

https://bseinquiry.blogspot.com/2019/02/mad-dogs-and-englishmen-bse-scrapie-cwd.html

***> ''The association between venison eating and risk of CJD shows similar pattern, with regular venison eating associated with a 9 FOLD INCREASE IN RISK OF CJD (p = 0.04).''

***> In conclusion, sensory symptoms and loss of reflexes in Gerstmann-Sträussler-Scheinker syndrome can be explained by neuropathological changes in the spinal cord. We conclude that the sensory symptoms and loss of lower limb reflexes in Gerstmann-Sträussler-Scheinker syndrome is due to pathology in the caudal spinal cord. <***

***> The clinical and pathological presentation in macaques was mostly atypical, with a strong emphasis on spinal cord pathology.<*** 

***> The notion that CWD can be transmitted orally into both new-world and old-world non-human primates asks for a careful reevaluation of the zoonotic risk of CWD. <***

***> All animals have variable signs of prion neuropathology in spinal cords and brains and by supersensitive IHC, reaction was detected in spinal cord segments of all animals.<*** 

***> In particular the US data do not clearly exclude the possibility of human (sporadic or familial) TSE development due to consumption of venison. The Working Group thus recognizes a potential risk to consumers if a TSE would be present in European cervids.'' Scientific opinion on chronic wasting disease (II) <***

https://familialcjdtseprion.blogspot.com/2019/02/cwd-gss-tse-prion-spinal-cord-confucius.html

***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.***

Even if the prevailing view is that sporadic CJD is due to the spontaneous formation of CJD prions, it remains possible that its apparent sporadic nature may, at least in part, result from our limited capacity to identify an environmental origin.

https://www.nature.com/articles/srep11573

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. 

============== 

PRION 2015 CONFERENCE

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5019500/

***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. 

http://www.tandfonline.com/doi/abs/10.1080/19336896.2016.1163048?journalCode=kprn20 

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

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. 

http://www.tandfonline.com/doi/abs/10.1080/19336896.2016.1163048?journalCode=kprn20

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. 

http://www.ars.usda.gov/research/publications/publications.htm?SEQ_NO_115=313160

https://efsaopinioncwd.blogspot.com/2022/04/efsa-eu-request-for-scientific-opinion.html

WEDNESDAY, MARCH 16, 2022 

SHEEP BY-PRODUCTS AND WHAT ABOUT Scrapie TSE PrP and Potential Zoonosis? 

https://transmissiblespongiformencephalopathy.blogspot.com/2022/03/sheep-by-products-and-what-about.html

SO, WHO'S UP FOR SOME MORE TSE PRION POKER, WHO'S ALL IN $$$ 

SO, ATYPICAL SCRAPIE ROUGHLY HAS 50 50 CHANCE ATYPICAL SCRAPIE IS CONTAGIOUS, AS NON-CONTAGIOUS, TAKE YOUR PICK, BUT I SAID IT LONG AGO WHEN USDA OIE ET AL MADE ATYPICAL SCRAPIE A LEGAL TRADING COMMODITY, I SAID YOUR PUTTING THE CART BEFORE THE HORSE, AND THAT'S EXACTLY WHAT THEY DID, and it's called in Texas, TEXAS TSE PRION HOLDEM POKER, WHO'S ALL IN $$$

***> AS is considered more likely (subjective probability range 50–66%) that AS is a non-contagious, rather than a contagious, disease.

https://efsa.onlinelibrary.wiley.com/doi/full/10.2903/j.efsa.2021.6686

Research Paper

Cellular prion protein distribution in the vomeronasal organ, parotid, and scent glands of white-tailed deer and mule deer

Anthony Ness, Aradhana Jacob, Kelsey Saboraki, Alicia Otero, Danielle Gushue, Diana Martinez Moreno, Melanie de Peña, Xinli Tang, Judd Aiken, Susan Lingle & Debbie McKenzie ORCID Icon show less

Pages 40-57 | Received 03 Feb 2022, Accepted 13 May 2022, Published online: 29 May 2022

Download citation

https://doi.org/10.1080/19336896.2022.2079888

https://www.tandfonline.com/doi/full/10.1080/19336896.2022.2079888

PIGS

Title: Prion infectivity detected in swine challenged with chronic wasting disease via the intracerebral or oral route

Author item MOORE, S - Orise Fellow item Kunkle, Robert item SMITH, JODI - Iowa State University item WEST-GREENLEE, M - Iowa State University item Greenlee, Justin Submitted to: Prion Publication Type: Abstract Only Publication Acceptance Date: 4/4/2016 Publication Date: N/A Citation: N/A

Interpretive Summary:

Technical Abstract: Chronic wasting disease (CWD) is a naturally-occurring, fatal neurodegenerative disease of North American cervids. The potential for swine to serve as a host for the agent of chronic wasting disease is unknown. In the US, feeding of ruminant by-products to ruminants is prohibited, but feeding of ruminant materials to swine, mink, and poultry still occurs. In addition, scavenging of CWD-affected cervid carcasses by feral pigs presents a potential risk for CWD exposure. The purpose of this study was to investigate the susceptibility of swine to the CWD agent following oral or intracranial experimental challenge. At 8 weeks of age, crossbred pigs were challenged by the intracranial route (n=20), oral route (n=19), or were left unchallenged (n=9). At approximately 6 months of age, the time at which commercial pigs reach market weight, half of the pigs in each group were culled (<6 month challenge groups). The remaining pigs (>6 month challenge groups) were allowed to incubate for up to 73 months post challenge (mpc). At death a complete necropsy examination was performed, including testing of tissues for misfolded prion protein (PrPcwd) by western blotting (WB), enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC). None of the pigs developed clinical signs consistent with prion disease. Four >6 month intracranially challenged pigs (survival times 45-73 mpc) were positive by ELISA, two were also positive by WB, and one was positive by IHC. One >6 month orally challenged pig (64 mpc) was positive by ELISA. To further investigate the potential for infectivity, brain tissue from selected pigs was bioassayed in mice expressing porcine PRNP. Tissue from the two WB-positive >6 month intracranially challenged pigs produced positive bioassay results, albeit with low attack rates and variable incubation periods. Interestingly, bioassay of material from the longest surviving >6 month orally challenged pig (72 mpc), which was negative for PrPcwd by all other tests, produced a positive bioassay result. Bioassay of material from additional animals is currently underway. This study demonstrates that pigs can serve as potential hosts for CWD, although with low attack rates and scant PrPcwd accumulation. 

Detection of infectivity in orally challenged pigs using mouse bioassay raises the possibility that naturally exposed pigs act as a reservoir of CWD infectivity, even though affected pigs do not develop overt clinical signs or readily detectable PrPcwd.

https://www.ars.usda.gov/research/publications/publication/?seqNo115=326166

Research Project: Pathobiology, Genetics, and Detection of Transmissible Spongiform Encephalopathies Location: Virus and Prion Research

Title: The agent of chronic wasting disease from pigs is infectious in transgenic mice expressing human PRNP 

Author item MOORE, S - Orise Fellow item Kokemuller, Robyn item WEST-GREENLEE, M - Iowa State University item BALKEMA-BUSCHMANN, ANNE - Friedrich-Loeffler-institut item GROSCHUP, MARTIN - Friedrich-Loeffler-institut item Greenlee, Justin Submitted to: Prion Publication Type: Abstract Only Publication Acceptance Date: 5/10/2018 Publication Date: 5/22/2018 Citation: Moore, S.J., Kokemuller, R.D., West-Greenlee, M.H., Balkema-Buschmann, A., Groschup, M.H., Greenlee, J.J. 2018. The agent of chronic wasting disease from pigs is infectious in transgenic mice expressing human PRNP. Prion 2018, Santiago de Compostela, Spain, May 22-25, 2018. Paper No. WA15, page 44.

Interpretive Summary:

 The successful transmission of pig-passaged CWD to Tg40 mice reported here suggests that passage of the CWD agent through pigs results in a change of the transmission characteristics which reduces the transmission barrier of Tg40 mice to the CWD agent. If this biological behavior is recapitulated in the original host species, passage of the CWD agent through pigs could potentially lead to increased pathogenicity of the CWD agent in humans.

https://www.ars.usda.gov/research/publications/publication/?seqNo115=353091

''These results indicating the susceptibility of pigs to sheep scrapie, coupled with the limitations of the current feed ban, indicates that a revision of the feed ban may be necessary to protect swine production and potentially human health.''

ARS researchers at Ames, Iowa conducted this experiment to test the susceptibility of swine to U.S. scrapie isolates by intracranial and oral inoculation. Necropsies were done on a subset of animals at approximately 6 months post inoculation (PI): the time the pigs were expected to reach market weight. Remaining pigs were maintained and monitored for clinical signs of transmissible spongiform encephalopathies (TSE) until study termination at 80 months PI or when removed due to intercurrent disease. Brain samples were examined by multiple diagnostic approaches, and for a subset of pigs in each inoculation group, bioassay in mice expressing porcine prion protein. At 6 months PI, no evidence of scrapie infection was noted by any diagnostic method. However, at 51 months of incubation or greater, 5 animals were positive by one or more diagnostic methods. Furthermore, positive bioassay results were obtained from all inoculated groups (oral and intracranial; market weight and end of study) suggesting that swine are potential hosts for the agent of scrapie. Although the current U.S. feed ban is based on keeping tissues from TSE infected cattle from contaminating animal feed, swine rations in the U.S. could contain animal derived components including materials from scrapie infected sheep and goats. These results indicating the susceptibility of pigs to sheep scrapie, coupled with the limitations of the current feed ban, indicates that a revision of the feed ban may be necessary to protect swine production and potentially human health.

https://www.ars.usda.gov/research/project/?accnNo=432011&fy=2017

see full report;

Research Project: Pathobiology, Genetics, and Detection of Transmissible Spongiform Encephalopathies Location: Virus and Prion Research

2017 Annual Report

Objectives

Objective 1: Investigate the mechanisms of protein misfolding in prion disease, including the genetic determinants of misfolding of the prion protein and the environmental influences on protein misfolding as it relates to prion diseases. Subobjective 1.A: Investigate the differences in the unfolded state of wild-type and disease associated prion proteins to better understand the mechanism of misfolding in genetic prion disease. Subobjective 1.B: Investigate the influence of metal ions on the misfolding of the prion protein in vitro to determine if environmental exposure to metal ions may alter disease progression. Objective 2: Investigate the pathobiology of prion strains in natural hosts, including the influence of prion source genotype on interspecies transmission and the pathobiology of atypical transmissible spongiform encephalopathies (TSEs). Subobjective 2.A: Investigate the pathobiology of atypical TSEs. Subobjective 2.B: Investigate the influence of prion source genotype on interspecies transmission. Objective 3: Investigate sampling methodologies for antemortem detection of prion disease, including the utility of blood sampling as a means to assess prion disease status of affected animals and the utility of environmental sampling for monitoring herd prion disease status. Subobjective 3.A: Investigate the utility of blood sampling as a means to assess prion disease status of affected animals. Subobjective 3.B: Investigate the utility of environmental sampling for monitoring herd prion disease status.

Approach

The studies will focus on three animal transmissible spongiform encephalopathy (TSE) agents found in the United States: bovine spongiform encephalopathy (BSE); scrapie of sheep and goats; and chronic wasting disease (CWD) of deer, elk, and moose. The research will address sites of protein folding and misfolding as it relates to prion disease, accumulation of misfolded protein in the host, routes of infection, and ante mortem diagnostics with an emphasis on controlled conditions and natural routes of infection. Techniques used will include spectroscopic monitoring of protein folding/misfolding, clinical exams, histopathology, immunohistochemistry, and biochemical analysis of proteins. The enhanced knowledge gained from this work will help understand the underlying mechanisms of prion disease and mitigate the potential for unrecognized epidemic expansions of these diseases in populations of animals that could either directly or indirectly affect food animals.

Progress Report

All 8 project plan milestones for FY17 were fully met. Research efforts directed toward meeting objective 1 of our project plan center around the production of recombinant prion protein from either bacteria or mammalian tissue culture systems and collection of thermodynamic data on the folding of the recombinant prion protein produced. Both bacterial and mammalian expression systems have been established. Thermodynamic data addressing the denatured state of wild-type and a disease associated variant of bovine prion protein has been collected and a manuscript is in preparation. In research pertaining to objective 2, all studies have been initiated and animals are under observation for the development of clinical signs. The animal studies for this objective are long term and will continue until onset of clinical signs. In vitro studies planned in parallel to the animals studies have similarly been initiated and are ongoing. Objective 3 of the project plan focuses on the detection of disease associated prion protein in body fluids and feces collected from a time course study of chronic wasting disease inoculated animals. At this time samples are being collected as planned and methods for analysis are under development.

Accomplishments

1. Showed that swine are potential hosts for the scrapie agent. A naturally occurring prion disease has not been recognized in swine, but the agent of bovine spongiform encephalopathy does transmit to swine by experimental routes. Swine are thought to have a robust species barrier when exposed to the naturally occurring prion diseases of other species, but the susceptibility of swine to the agent of sheep scrapie has not been thoroughly tested. ARS researchers at Ames, Iowa conducted this experiment to test the susceptibility of swine to U.S. scrapie isolates by intracranial and oral inoculation. Necropsies were done on a subset of animals at approximately 6 months post inoculation (PI): the time the pigs were expected to reach market weight. Remaining pigs were maintained and monitored for clinical signs of transmissible spongiform encephalopathies (TSE) until study termination at 80 months PI or when removed due to intercurrent disease. Brain samples were examined by multiple diagnostic approaches, and for a subset of pigs in each inoculation group, bioassay in mice expressing porcine prion protein. At 6 months PI, no evidence of scrapie infection was noted by any diagnostic method. However, at 51 months of incubation or greater, 5 animals were positive by one or more diagnostic methods. Furthermore, positive bioassay results were obtained from all inoculated groups (oral and intracranial; market weight and end of study) suggesting that swine are potential hosts for the agent of scrapie. Although the current U.S. feed ban is based on keeping tissues from TSE infected cattle from contaminating animal feed, swine rations in the U.S. could contain animal derived components including materials from scrapie infected sheep and goats. These results indicating the susceptibility of pigs to sheep scrapie, coupled with the limitations of the current feed ban, indicates that a revision of the feed ban may be necessary to protect swine production and potentially human health.

2. Determined that pigs naturally exposed to chronic wasting disease (CWD) may act as a reservoir of CWD infectivity. Chronic wasting disease is a naturally occurring, fatal, neurodegenerative disease of cervids. The potential for swine to serve as a host for the agent of CWD disease is unknown. The purpose of this study was to investigate the susceptibility of swine to the CWD agent following experimental oral or intracranial inoculation. Pigs were assigned to 1 of 3 groups: intracranially inoculated; orally inoculated; or non-inoculated. At market weight age, half of the pigs in each group were tested ('market weight' groups). The remaining pigs ('aged' groups) were allowed to incubate for up to 73 months post inoculation (MPI). Tissues collected at necropsy were examined for disease-associated prion protein (PrPSc) by multiple diagnostic methods. Brain samples from selected pigs were bioassayed in mice expressing porcine prion protein. Some pigs from each inoculated group were positive by one or more tests. Bioassay was positive in 4 out of 5 pigs assayed. Although only small amounts of PrPSc were detected using sensitive methods, this study demonstrates that pigs can serve as hosts for CWD. Detection of infectivity in orally inoculated pigs using mouse bioassay raises the possibility that naturally exposed pigs could act as a reservoir of CWD infectivity. Currently, swine rations in the U.S. could contain animal derived components including materials from deer or elk. In addition, feral swine could be exposed to infected carcasses in areas where CWD is present in wildlife populations. The current feed ban in the U.S. is based exclusively on keeping tissues from TSE infected cattle from entering animal feeds. These results indicating the susceptibility of pigs to CWD, coupled with the limitations of the current feed ban, indicates that a revision of the feed ban may be necessary to protect swine production and potentially human health.

3. Developed a method for amplification and discrimination of the 3 forms of BSE in cattle. The prion protein (PrP) is a protein that is the causative agent of transmissible spongiform encephalopathies (TSEs). The disease process involves conversion of the normal cellular PrP to a pathogenic misfolded conformation. This conversion process can be recreated in the lab using a misfolding amplification process known as real-time quaking induced conversion (RT-QuIC). RT-QuIC allows the detection of minute amounts of the abnormal infectious form of the prion protein by inducing misfolding in a supplied substrate. Although RT-QuIC has been successfully used to detect pathogenic PrP with substrates from a variety of host species, prior to this work bovine prion protein had not been proven for its practical uses for RT-QuIC. We demonstrated that prions from transmissible mink encephalopathy (TME) and BSE-infected cattle can be detected with using bovine prion proteins with RT-QuIC, and developed an RT-QuIC based approach to discriminate different forms of BSE. This rapid and robust method, both to detect and discriminate BSE types, is of importance as the economic implications for different types of BSE vary greatly.

Review Publications

snip...

https://www.ars.usda.gov/research/project/?accnNo=432011&fy=2017

cwd scrapie pigs oral routes 

***> However, at 51 months of incubation or greater, 5 animals were positive by one or more diagnostic methods. Furthermore, positive bioassay results were obtained from all inoculated groups (oral and intracranial; market weight and end of study) suggesting that swine are potential hosts for the agent of scrapie. <*** 

>*** Although the current U.S. feed ban is based on keeping tissues from TSE infected cattle from contaminating animal feed, swine rations in the U.S. could contain animal derived components including materials from scrapie infected sheep and goats. These results indicating the susceptibility of pigs to sheep scrapie, coupled with the limitations of the current feed ban, indicates that a revision of the feed ban may be necessary to protect swine production and potentially human health. <*** 

***> Results: PrPSc was not detected by EIA and IHC in any RPLNs. All tonsils and MLNs were negative by IHC, though the MLN from one pig in the oral <6 month group was positive by EIA. PrPSc was detected by QuIC in at least one of the lymphoid tissues examined in 5/6 pigs in the intracranial <6 months group, 6/7 intracranial >6 months group, 5/6 pigs in the oral <6 months group, and 4/6 oral >6 months group. Overall, the MLN was positive in 14/19 (74%) of samples examined, the RPLN in 8/18 (44%), and the tonsil in 10/25 (40%). 

***> Conclusions: This study demonstrates that PrPSc accumulates in lymphoid tissues from pigs challenged intracranially or orally with the CWD agent, and can be detected as early as 4 months after challenge. CWD-infected pigs rarely develop clinical disease and if they do, they do so after a long incubation period. This raises the possibility that CWD-infected pigs could shed prions into their environment long before they develop clinical disease. Furthermore, lymphoid tissues from CWD-infected pigs could present a potential source of CWD infectivity in the animal and human food chains. 

https://www.ars.usda.gov/research/publications/publication/?seqNo115=353091

https://www.ars.usda.gov/research/project/?accnNo=432011&fy=2017

https://www.ars.usda.gov/research/publications/publication/?seqNo115=337105

Conclusions: This study demonstrates that PrPSc accumulates in lymphoid tissues from pigs challenged intracranially or orally with the CWD agent, and can be detected as early as 4 months after challenge. CWD-infected pigs rarely develop clinical disease and if they do, they do so after a long incubation period. This raises the possibility that CWD-infected pigs could shed prions into their environment long before they develop clinical disease. Furthermore, lymphoid tissues from CWD-infected pigs could present a potential source of CWD infectivity in the animal and human food chains.

https://www.ars.usda.gov/research/publications/publication/?seqNo115=337105

CONFIDENTIAL

EXPERIMENTAL PORCINE SPONGIFORM ENCEPHALOPATHY

LINE TO TAKE

3. If questions on pharmaceuticals are raised at the Press conference, the suggested line to take is as follows:- 

 "There are no medicinal products licensed for use on the market which make use of UK-derived porcine tissues with which any hypothetical “high risk" ‘might be associated. The results of the recent experimental work at the CSM will be carefully examined by the CSM‘s Working Group on spongiform encephalopathy at its next meeting.

DO Hagger RM 1533 MT Ext 3201

http://web.archive.org/web/20030822054419/www.bseinquiry.gov.uk/files/yb/1990/09/21009001.pdf

While this clearly is a cause for concern we should not jump to the conclusion that this means that pigs will necessarily be infected by bone and meat meal fed by the oral route as is the case with cattle. ...

http://web.archive.org/web/20031026000118/www.bseinquiry.gov.uk/files/yb/1990/08/23004001.pdf

we cannot rule out the possibility that unrecognised subclinical spongiform encephalopathy could be present in British pigs though there is no evidence for this: only with parenteral/implantable pharmaceuticals/devices is the theoretical risk to humans of sufficient concern to consider any action.

http://web.archive.org/web/20030822031154/www.bseinquiry.gov.uk/files/yb/1990/09/10007001.pdf

May I, at the outset, reiterate that we should avoid dissemination of papers relating to this experimental finding to prevent premature release of the information. ...

http://web.archive.org/web/20030822052332/www.bseinquiry.gov.uk/files/yb/1990/09/11005001.pdf

3. It is particularly important that this information is not passed outside the Department, until Ministers have decided how they wish it to be handled. ...

http://web.archive.org/web/20030822052438/www.bseinquiry.gov.uk/files/yb/1990/09/12002001.pdf

But it would be easier for us if pharmaceuticals/devices are not directly mentioned at all. ...

http://web.archive.org/web/20030518170213/www.bseinquiry.gov.uk/files/yb/1990/09/13004001.pdf

Our records show that while some use is made of porcine materials in medicinal products, the only products which would appear to be in a hypothetically ''higher risk'' area are the adrenocorticotrophic hormone for which the source material comes from outside the United Kingdom, namely America China Sweden France and Germany. The products are manufactured by Ferring and Armour. A further product, ''Zenoderm Corium implant'' manufactured by Ethicon, makes use of porcine skin - which is not considered to be a ''high risk'' tissue, but one of its uses is described in the data sheet as ''in dural replacement''. This product is sourced from the United Kingdom.....

http://web.archive.org/web/20030822054419/www.bseinquiry.gov.uk/files/yb/1990/09/21009001.pdf

FRIDAY, APRIL 07, 2023 

Case report: Two clusters of Creutzfeldt-Jakob disease cases within 1 year in West Michigan 

https://creutzfeldt-jakob-disease.blogspot.com/2023/04/case-report-two-clusters-of-creutzfeldt.html

***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.***

Even if the prevailing view is that sporadic CJD is due to the spontaneous formation of CJD prions, it remains possible that its apparent sporadic nature may, at least in part, result from our limited capacity to identify an environmental origin.

https://www.nature.com/articles/srep11573 

MONDAY, APRIL 10, 2023 

Missouri MDC reports 117 new cases of CWD for 2022 surveillance year

https://chronic-wasting-disease.blogspot.com/2023/04/missouri-mdc-reports-117-new-cases-of.html

TUESDAY, APRIL 11, 2023 

Texas TAHC Chronic Wasting Disease Discovered in Deer Breeding Facilities in Frio and Hamilton Counties 

https://chronic-wasting-disease.blogspot.com/2023/04/texas-tahc-chronic-wasting-disease.html

FRIDAY, FEBRUARY 17, 2023 

TEXAS OVERVIEW OF STATE RESPONSE TO CHRONIC WASTING DISEASE CWD TSE PRION April 2019 a Review

https://chronic-wasting-disease.blogspot.com/2023/02/texas-overview-of-state-response-to.html

WEDNESDAY, APRIL 12, 2023 

North Carolina Chronic Wasting Disease Found in Cumberland and Wilkes Counties

https://chronic-wasting-disease.blogspot.com/2023/04/north-carolina-chronic-wasting-disease.html

SATURDAY, APRIL 08, 2023 

Idaho 24 animals tested positive for CWD from 442 animals removed in the Slate Creek management area within unit 14 

https://chronic-wasting-disease.blogspot.com/2023/04/idaho-24-animals-tested-positive-for.html

TUESDAY, FEBRUARY 28, 2023 

Michigan MDARD Captive CWD Positives depopulated and quarantined 2023 

https://chronic-wasting-disease.blogspot.com/2023/02/michigan-mdard-captive-cwd-positives.html

THURSDAY, FEBRUARY 23, 2023 

Wisconsin Chronic Wasting Disease CWD TSE Prion Update for 2022 To Date 1480 Positive Cases

https://chronic-wasting-disease.blogspot.com/2023/02/wisconsin-chronic-wasting-disease-cwd_23.html

FRIDAY, FEBRUARY 17, 2023 

Alabama Third Case of CWD Confirmed in Lauderdale County

https://chronic-wasting-disease.blogspot.com/2023/02/alabama-third-case-of-cwd-confirmed-in.html

TUESDAY, FEBRUARY 14, 2023 

Iowa CWD TSE Prion Surveillance 2022-2023 Season 65 Positive and 19 Suspect Positive

https://chronic-wasting-disease.blogspot.com/2023/02/iowa-cwd-tse-prion-surveillance-2022.html

FRIDAY, MARCH 24, 2023 

Mountain lions, Wolves, Coyotes, could help stop the spread of CWD TSE Prion in deer, WHERE STUPID MEETS THE ROAD! 

https://transmissiblespongiformencephalopathy.blogspot.com/2023/03/mountain-lions-wolves-coyotes-could.html

Saturday, April 9, 2022 

EFSA EU Request for a scientific opinion on the monitoring of Chronic Wasting Disease (CWD) EFSA-Q-2022-00114 M-2022-00040 Singeltary Submission 

https://efsaopinioncwd.blogspot.com/2022/04/efsa-eu-request-for-scientific-opinion.html

Terry S. Singeltary Sr., Bacliff, Texas USA 77518 flounder9@verizon.net