Prion 2016 Animal Prion Disease Workshop Abstracts

WS-01: Prion diseases in animals and zoonotic potential

Juan Maria Torres^a, Olivier Andreolettib, Juan-Carlos Espinosa^a, Vincent Beringue^c, Patricia Aguilar^a, Natalia Fernandez-Borges^a, and Alba Marin-Moreno^a

^aCentro de Investigacion en Sanidad Animal (CISA-INIA), Valdeolmos, Madrid, Spain;

^bUMR INRA-ENVT 1225 Interactions Hotes Agents Pathògenes, ENVT, Toulouse, France;

^cUR892, Virologie Immunologie Moléculaires, Jouy-en-Josas, France

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

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

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

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

WS-02: Scrapie in swine: A diagnostic challenge

Justin J. Greenlee

Robert A. Kunkle

Jodi D. Smith

Heather W. Greenlee

National Animal Disease Center, US Dept. of Agricultrue, Agricultural Research Service, Ames, IA, USA

Iowa State University College of Veterinary Medicine, Ames, IA, USA

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.

Since swine can be fed rations containing ruminant derived components in the United States and many other countries, we conducted this experiment to test the susceptibility of swine to US scrapie isolates by intracranial and oral inoculation. Scrapie inoculum was a pooled 10% (w/v) homogenate derived from the brains of clinically ill sheep from the 4th passage of a serial passage study of the U.S scrapie agent (No. 13-7) through susceptible sheep that were homozygous ARQ at prion protein residues 136, 154, and 171, respectively. Pigs were inoculated intracranially (n = 19) with a single 0.75 ml dose or orally (n = 24) with 15 ml repeated on 4 consecutive days. Necropsies were done on a subset of animals at approximately 6 months post inoculation (PI), at the time the pigs were expected to reach market weight. Remaining pigs were maintained and monitored for clinical signs of TSE until study termination at 80 months PtdIns or when removed due to intercurrent disease (primarily lameness). Brain samples were examined by immunohistochemistry (IHC), western blot (WB), and enzyme-linked immunosorbent assay (ELISA). Brain tissue from a subset of pigs in each inoculation group was used for bioassay in mice expressing porcine PRNP.

At six-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 methods: IHC (n = 4), WB (n = 3), or ELISA (n = 5). Interestingly, positive bioassay results were obtained from all inoculated groups (oral and intracranial; market weight and end of study).

Swine inoculated with the agent of scrapie by the intracranial and oral routes do not accumulate abnormal prion protein (PrP^Sc) to a level detectable by IHC or WB by the time they reach typical market age and weight. However, strong support for the fact that swine are potential hosts for the agent of scrapie comes from positive bioassay from both intracranially and orally inoculated pigs and multiple diagnostic methods demonstrating abnormal prion protein in intracranially inoculated pigs with long incubation times.

WS-03: Epidemiology of chronic wasting disease in Korea

Hyun Joo Sohn

In Soon Roh

Hyo Jin Kim

Tae Young Suh

Kyung Je Park

Hoo Chang Park

Byounghan Kim

Foreign Animal Disease Division (FADD), Animal and Plant Quarantine Agency (QIA), Gimcheon, Korea

Transmissible spongiform encephalopathy (TSE) is a fatal neurodegenerative disorder, which is so-called as prion diseases due to the causative agents (PrP^Sc). TSEs are believed to be due to the template-directed accumulation of disease-associated prion protein, generally designated PrP^Sc. Based on export information of Chronic wasting disease (CWD) suspected elk from Canada to Korea, CWD surveilance program was initiated by the Ministry of Agriculture, Food and Rural Affairs (MAFRA) in 2001. CWD control measures included stamping out of all animals in the affected farm, and through cleaning and disinfection of the premises. In addition, nationwide clinical surveillance of Korean native cervid and improved measures to ensure reporting of CWD suspect cases were implemented. Total of 9 elks were found to be affected. CWD was designated as a notifiable disease under the Act for Prevention of Livestock Epidemics in 2002. Additional CWD cases– 12 elks and 2 elks – were diagnosed in 2004 and 2005. On 2010, 6 elks, 7 sika deer, one red deer and 5 cross-breeds were confirmed as positive. Further epidemiological investigations showed that these CWD outbreaks were linked to the importation of elks from Canada in 1994 based on circumstantial evidences. CWD is the prion disease that is known spread horizontally. The experimental studies have shown that PrP^CWD is capable of transmitting CWD through saliva and blood. We conducted sPMCA and animal biosassy using contaminated soils in the playground of farm 2 which considered horizontal transmission between cervid and have been confirmed infectious PrP^CWD. This result suggests PrP^CWD shedding in the CWD contaminated soil is progressive through the disease course.

Keywords: CWD, soil, sPMCA

WS-04: Ultra-sensitive detection of PrP^Sc of classical and atypical BSEs

Yuichi Murayama

Kentaro Masujin

Yoshifumi Iwamaru

Morikazu Imamura

Yuichi Matsuura

Kohtaro Miyazawa

Fumiko Ono

Hiroaki Shibata

Hiroyuki Okada

Takashi Yokoyama

Influenza/Prion Disease Research Center, National Institute of Animal Health, Tsukuba, Japan

Chiba Institute of Science Faculty of Risk and Crisis Management, Choshi, Japan

Tsukuba Primate Research Center, National Institutes of Biomedical Innovation, Health and Nutrition, Tsukuba, Japan

It is now possible to amplify PrP^Sc in vitro using the protein misfolding cyclic amplification (PMCA) technique. PMCA has been applied to the detection of bovine C-BSE PrP^Sc in cattle and macaques. We demonstrated the PrP^Sc was detectable in the bodily fluids such as saliva, cerebrospinal fluid (CSF) and blood as well as in various tissues of C-BSE prion-infected animals. However, the PMCA method developed for C-BSE PrP^Sc was not effective for amplification of Atypical BSE (A-BSE) PrP^Sc. PMCA is a powerful technique, however, it is usually necessary to optimize amplification conditions according to the characteristics of each prion to achieve good amplification. To establish an efficient method to amplify PrP^Sc derived from animals with A-BSE, we examined the effects of various additives on the PMCA reaction: protein denaturants or stabilizers such as guanidine, urea, polyamines, high molecular weight compounds, trehalose, and L-arginine and its derivatives. Among these additives, L-arginine and its derivatives were the most effective in enhancing the in vitro amplification of A-BSE PrP^Sc in the presence of polyanions. This highly efficient PMCA method suitable for A-BSE PrP^Sc amplification is capable of amplifying a very small amount of PrP^Sc from the CSF, saliva, urine and plasma of A-BSE-infected animals.

WS-05: RT-QuIC assays in humans and animals

Steven J. Collins

Shannon Sarros

The University of Melbourne, Melbourne, Victoria, Australia

Prion diseases (also known as transmissible spongiform encephalopathies) are neurodegenerative diseases affecting both humans and animal species. The phenotypic spectrum is broad and includes Creutzfeldt-Jakob disease (CJD) and its variant zoonotic form (vCJD) in humans, while in animals, scrapie of sheep and goats, bovine spongiform encephalopathy (mad cow disease) and chronic wasting disease (CWD) of deer, elk and moose are naturally occurring forms. Although a precise understanding remains elusive, transmission and pathogenesis appear causally linked to the misfolding of the normal or cellular form of the prion protein (PrP^C) into disease associated conformers (PrPD), the latter enriched in β-strand secondary structure and typically displaying limited solubility and relative protease resistance.

Over the past approximately 10 y 2 protein amplification techniques, the protein misfolding cyclic amplification (PMCA) assay and real time quaking induced conversion (RT-QuIC) assay have been developed and successfully deployed in prion biology across a range of scientific and clinical applications including generation of de novo prions, quantitation of prion infectivity and enhanced premortem diagnostic capacity. While PMCA utilises sonication to facilitate protein amplification RT-QuIC employs vigorous shaking to achieve this outcome, with both techniques sharing the ability to amplify miniscule quantities of PrPD seed present in various tissues and body fluids to levels detectable using routine biochemical methods. Particularly in relation to human prion disease, the improved biomarker specificity offered through direct amplification and detection of PrPD observed in early studies is arguably the greatest advantage of these techniques in comparison to other diagnostic investigations such as cerebrospinal fluid (CSF) tau protein detection and neuroimaging.

The enhanced specificity, verified as close to 100% for detection of PrPD in CSF, has spawned international collaborative initiatives to rigorously assess and validate the RT-QuIC for routine CSF clinical diagnostic purposes. In parallel with collaborative CSF validation studies there have been successful efforts to refine RT-QuIC technical aspects aimed at allowing use of more accessible body fluids or tissues such as urine, saliva and nasal brushings, promote higher sample throughput, shorten assay times and offer accurate quantification of PrPD even at levels below those detectable when employing animal bioassays. Animal studies, especially those in well characterized rodent models of prion disease, support the generic properties of the RT-QuIC assay for PrPD detection and underpin the broad utility of this assay for studying prion disease and the high likelihood of translatability and inter-convertibility of technical refinements for human and animal use.

WS-06: Pathology of TSEs

John Spiropoulos

Animal and Plant Health Agency/Pathology Department, Addlestone, Surrey, UK

Pathology is the study of the structural and functional changes produced by diseases. To achieve this pathologists employ various approaches. Gross pathology was not useful in TSEs, so the application of histopathology to detect vacuoles or plaques was the only means of confirming TSE disease for decades. The discovery of PrP led to the production of anti-PrP antibodies, and enabled the development of PrP detection techniques such as immunohistochemistry, Histo- or PET-blot that have developed in parallel with biochemical methods such as Western blot and ELISA. These methods offer greater sensitivity than histopathology in TSE detection and crucially they can be applied to analyze various phenotypic aspects of single TSE sources increasing the amount of data and offering higher discriminatory power. The combined dataset attributed to a single TSE source expresses to a large extent the disease phenotype of this TSE in the specified host. In the absence of an isolatable infectious agent, disease phenotype in TSEs remains important for defining prion strain, and pathology plays a pivotal role in the characterization and discrimination of specific host/strain combinations under field conditions. The principles, advantages and disadvantages of the various techniques will be mentioned and their combined application for the identification of TSEs in a host and characterization of prion strains will be demonstrated using examples of straightforward and novel challenging cases such as H- and L- type BSE in sheep. Pathology provides a methodology that can be developed through research projects and subsequently applied in surveillance projects to identify known and novel TSEs. This has key implications as it informs policy bodies about the robustness of current surveillance measures for monitoring the emergence of animal TSEs.

WS-08: Intra- and interspecies transmission of atypical BSE - What can we learn from it

Anne Balkema-Buschmann

Grit Priemer

Markus Keller

Bob Hills

Martin H. Groschup

Friedrich-Loeffler-Institut/Institute for Novel and Emerging Infectious Diseases, Greifswald - Insel Riems, Germany

Health Canada, Transmissible Spongiform Encephalopathy Secretariat, Ottawa, Ontario, Canada

More than 10 y after the initial description of atypical BSE cases of the H-type and L-type, the origin of these cases as well as the transmissibility to other species, including their zoonotic potential, are still not fully understood. Intracerebral challenge experiments have been performed with both atypical BSE forms in cattle, and the distribution of the abnormal prion protein and infectivity has been analyzed in a variety of tissues. This confirmed the general restriction to the central nervous system, as it had generally been acknowledged for classical BSE, although a slightly earlier and stronger involvement of the peripheral nervous system and the skeletal muscle was observed. However, data from cattle orally challenged with atypical BSE, which might mimic the natural situation, are not yet available. Challenge experiment performed in human PrP transgenic mice as well as in non-human primates by other workgroups however indicated a higher zoonotic potential of L-type BSE as compared to classical BSE, underlining the lack of available information about the human health risk associated to these novel BSE forms.

Previous challenge experiments in bovine and ovine PrP transgenic mouse lines had indicated that the TSE strain characteristics especially of L-BSE are not always stable and predictable, since in certain models, analysis of the recipient hosts revealed a classical BSE signature. In order to further elucidate the strain features of both atypical BSE forms, we performed challenge and subpassage experiments in a variety of transgenic and conventional mouse lines and found that the PrP gene sequence of the host seems to have a considerable impact on the TSE signature (H-BSE or C-BSE) determined in the recipient. The impact of these results on the most important remaining open question about the origin of atypical BSE cases will be discussed.

WS-09: Pathogenesis and transmission of classical and atypical BSEs in cattle

Cristina Casalone

Barbara Iulini

Elena Vallino Costassa

Cristiano Corona

Maria Mazza

Pierluigi Acutis

Cristiana Maurella

Daniela Meloni

Elena Bozzetta

Alessandra Pautasso

Lorenzo Capucci

Byron Caughey

Maria Puopolo

Marion Simmons

Gianluigi Zanusso

Maurizio Pocchiari

Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Turin, Italy

Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna, Brescia, Italy

National Institute for Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA

Istituto Superiore di Sanita', Roma, Italy

Animal and Plant Health Agency (APHA), Addlestone, Surrey, UK

University of Verona, Verona, Italy

Many mammalian species can be affected by prion diseases or transmissible spongiform encephalopathies (TSEs). Classical bovine spongiform encephalopathy (C-BSE) was the first prion disease recognized in cattle and it is the only known zoonotic prion disease, having caused variant Creutzfeldt-Jakob disease (vCJD) in humans. Based on the biochemical signatures of disease-associated prion protein (PrP^Sc), 2 distinct forms of atypical bovine spongiform encephalopathies (H-BSE and L-BSE) have been distinguished from C-BSE since 2004. To date there is no comprehensive information about the origin of Atypical BSEs (sporadic vs. acquired) and this influence the knowledge gathered from experimental studies that may, or may not, represent the real distribution of the agent in the bodies of naturally affected animals. Moreover, there are only very limited data available concerning the pathogenesis of both atypical BSE forms, as compared to C-BSE. Thus, precautions that are presently taken to minimize the risk of prion contamination of the food supply chain might not be effective as expected to prevent the spread of these recently recognized strains. Furthermore, in the last few years a number of experimental transmission studies of atypical strains in different animal hosts have been performed. The most recent data on classical and atypical BSEs studies concerning pathogenesis and transmissions in cattle will be summarized in this talk, highlighting the critical aspects of developing a reliable transmission experiment. Furthermore, animal bioassay allows the reliable determination of prion strains and infectivity distribution in body tissues, but a fast, practical and more sensitive in vitro test is essential for discrimination of prion strains. On this basis, we have developed RT-QuIC conditions for the detection of C-BSE and atypical L-BSE using brain samples from C-BSE and L-BSE from Italian cattle. Both BSE forms can be sensitively detected and distinguished using specific recombinant PrP substrates within the same test run. Therefore, a comparison between strain discrimination sensitivity achieved by the RT-QuIC assay and bioassays will be made. Taken together, the results obtained will be a factor in planning primary prevention interventions for public health, as well as for evaluating and revising current surveillance programs for animal TSEs.

WS-11: Evaluating the species barriers of TSEs with transgenic mouse models

Jean C. Manson

Abigail Diack

Rona Barron

James Alibhai

Pedro Piccardo

Enrico Canellotti

Mathhew Bishop

Robert Will

The Roslin Institute, University of Edinburgh, Roslin, Midlothian, UK

NCJDRSU, University of Edinburgh, Edinburgh, UK

The risk of transmission of Transmissible Spongiform Encephalopathies (TSE) between different species has been notoriously unpredictable because the mechanisms of transmission are not fully understood. Of particular concern is the zoonotic potential of the animal TSEs, and while a transmission barrier between species often appears to prevent zoonotic infection, BSE was able to cross this barrier and infect humans with devastating consequences for the individuals infected. Of current concern in the UK are the results of a series of appendix studies which estimate that 1 in 2000 of the population could be carrying vCJD infection. One goal of our research is therefore to be able to predict the risk of TSE transmission to other species allowing measures to be implemented to block the transmission routes.

The interaction between the incoming agent and the host defines the susceptibility of the host to a particular strain. That interaction is in part defined by the sequence of the host and agent PrP. The structure of host and agent PrP have also been proposed as major components of this barrier. This has lead to the generation of a multiplicity of mice carrying PrP gene sequences from different species and with specific mutations to study cross species transmissions of TSE agents. Mice carrying specific mutations in their PrP sequence have been shown to alter the species barrier but not necessarily in a predictable way, although mice carrying the PrP gene of the appropriate host are usually more susceptible to agent derived from that host. In vivo studies using transgenic mice with altered PrP glycosylation have shown that the glycosylation status of host PrP can have a significant impact on disease transmission between species. Studies in transgenic mice have also indicated that transmission of agents through a new animal species can alter their zoonotic potential.

However we still lack an understanding of the nature of infection and the host response to TSE agents. Many studies have recently started to address these issues, and in vivo studies have allowed time course studies of disease to investigate the early events in the disease process. These studies have revealed that PrP can accumulate without leading to neurotoxicity, that the infectious entity has still to be defined, and that the host glial response is an important component in defining the neurodegenerative process. Gaining further insight into the disease process will enhance our ability to predict and prevent cross species transmissions.