Tuesday, July 04, 2017



P3 Genetic Susceptibility of Free-ranging Cervids to Chronic Wasting Disease in a Recently Infected Region of the United States

Mr. William Miller1, Dr. W. David Walter2

1Intercollege Graduate Degree Program in Ecology, Pennsylvania Cooperative Fish and Wildlife Research Unit, The Pennsylvania State University, University Park, United States, 2U.S. Geological Survey, Pennsylvania Cooperative Fish and Wildlife Research Unit, The Pennsylvania State University, University Park, United States

Aims: Uncommon polymorphisms within the prion gene of many cervids have been linked to reductions in perceived susceptibility to chronic wasting disease (CWD). Previous research suggests that nonsynonymous polymorphisms in the prion gene can lead to differential infection, prevalence, and mortality rates. These factors can affect the long-term sustainability of wild cervid populations considering they influence future demography, population genetics, and disease susceptibility. Understanding the underlying frequency and distribution of susceptible prion genotypes in areas of recent CWD infection, such as the eastern United States (USA) and Scandinavia, can provide insights into future epidemiology and infection intensity. This study assessed perceived genetic susceptibility for two sympatric cervid species (Odocoileus virginianus and Cervus canadensis) in a region of the eastern USA recently infected with CWD.

Methods: Prion gene sequences were determined from 159 O. virginianus and 256 C. canadensis. Sequence data were analyzed using program R, and genotype frequencies were compared to previous studies to determine whether infection risk was greater in this region versus the endemic area (western USA) and regions with higher CWD prevalence. Spatial interpolation was used to compare genotype frequencies among subpopulations.

Results: The study region exhibited greater frequencies of more susceptible genotypes than other regions, with differences of as much as 16.5% compared to free-ranging cervids in core endemic areas and 27.9% compared to other USA and Canadian populations. Both species, however, exhibited substantial spatial heterogeneity in prion genotype frequencies, with O. virginianus subpopulations ranging from 43.8% to 100% for all disease-associated loci and C. canadensis subpopulations ranging from 69.2% to 100%. Initial results suggested the presence of four novel single nucleotide polymorphisms (SNPs) in O. virginianus, two of which were nonsynonymous.

Conclusions: Genotype frequencies indicate that the study region may be more susceptible to CWD than endemic areas, although infection has yet to reach C. canadensis in this region. Prion gene variability among subpopulations indicated that perceived susceptibility within regions may not be uniform. This suggests that fine-scale patterns of prion gene heterogeneity likely exist and may be important in disease epidemiology. Furthermore, initial results indicated that novel nonsynonymous SNPs may be present within these populations providing direction for future research to determine their association with CWD status. These results highlight the importance of identifying prion gene variation and genotype frequencies for disease-associated loci in areas of recent CWD infection or areas at risk of infection, such as the eastern USA and Europe.


P6 Pathways of prion spread during early chronic wasting disease in deer

Dr Clare Hoover1, Ms Kristen Davenport1, Dr Davin Henderson1, Dr Nathaniel Denkers1, Dr Candace Mathiason1, Dr Claudio Soto2, Dr Mark Zabel1, Dr Edward Hoover1

1Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, United States, 2Department of Neurology, University of Texas, Houston, United States

Aims: Among prion infections, two scenarios of prion spread are generally observed: (a) early lymphoid tissue replication or (b) direct neuroinvasion without substantial prior lymphoid amplification. In nature, cervids are infected with chronic wasting disease by oral and nasal mucosal exposure, and studies of early CWD pathogenesis have implicated pharyngeal lymphoid tissue as the earliest sites of prion accumulation. However, knowledge of chronological events of prion spread during early CWD infection remains incomplete.

Methods: To investigate early CWD pathogenesis, we exposed white-tailed deer to CWD prions by mucosal routes and performed serial necropsies during the initial four months post-exposure to assess PrP-CWD tissue distribution. Because prion burdens during the initial stages of infection can be low, real-time quaking-induced conversion (RT-QuIC), end-point quaking-induced conversion (EP-QuIC) and tyramide-signal amplification immunohistochemistry (TSA-IHC) methods were used to enhance PrP-CWD detection.

Results: Although PrP-CWD was not detected by either method in the initial days (1 and 3) post-exposure, we observed PrP-CWD seeding activity and follicular immunoreactivity in oropharyngeal lymphoid tissues (tonsil, retropharyngeal, mandibular, and parotid lymph nodes) at 1 and 2 months post-exposure (MPE). At 3 MPE, PrP-CWD replication had expanded to all systemic lymphoid tissues. By 4 MPE, the PrP-CWD burden in all lymphoid tissues had increased, and approached levels observed in terminal disease, yet there was no evidence of nervous system invasion. EP-QuIC evaluation of all necropsy tissues collected at 4 MPE identified prion seeding activity in parotid and mandibular salivary glands, in addition to all systemic lymphoid tissues.

Conclusions: These results identify the oropharynx as the first site of CWD prion entry and that the initial phase of prion amplification occurs in the oropharyngeal lymphoid tissues followed by rapid dissemination to systemic lymphoid tissues. This lymphoid replication phase appears to precede neuroinvasion or the establishment of a chronic sub-clinical carrier state. The identification of CWD prion seeding activity in salivary glands suggests that saliva is the earliest source of excreted prions and environmental contamination.


P22 Managing towards CWD resistance in Rocky Mountain elk (Cervus elaphus) using antemortem rectal biopsy testing and pedigree reconstruction

DVM, PhD Nicholas Haley1, PhD Davin Henderson2, Ms. Sara Wyckoff1, DVM, MS, DACVP Aaron Lehmkuhl3, DVM, PhD, DACVP Bruce Thomsen3, DVM, PhD, DACVP Edward Hoover2, DVM Dan Love4, MS Ed Kline4

1Department of Microbiology and Immunology, Midwestern University, Glendale, United States, 2Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, United States, 3USDA, APHIS, VS, STAS, NVSL, Ames, United States, 4Colorado Department of Agriculture, Denver, United States

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) affecting members of the cervid family which has been reported in 24 states and 2 Canadian provinces, as well as the Republic of South Korea and most recently Norway. The disease has been found with increasing frequency in both farmed and free-ranging cervids – efficiently transmitting within both groups. Management of CWD infected herds has historically involved herd depopulation in the case of farmed animals and herd reduction in the case of wild deer and elk. In CWD endemic areas, where prevalence rates in farmed deer and elk mirror those found in wild cervids, the appropriateness of alternative management strategies for farmed animals has not been examined. Our aims were to evaluate the practicality and sustainability of managing CWD in a closed elk herd, consisting of approximately 500 animals on 4000 acres, with CWD prevalence rates approaching 20%. Our primary objective was to implement a test-and-cull strategy relying on rectal biopsies as well as conventional and experimental diagnostic approaches. Secondly, we sought to correlate CWD status with genetic information, including Prnp allele and microsatellite analysis, to better understand the epidemiology of this disease and develop a rational management approach for this herd.

By comparing biopsy test results from both IHC and RT-QuIC, we worked with the ranch owners to develop an appropriate culling strategy for positive animals.
Genetic information was gathered using Prnp sequencing and microsatellite analysis, which was used collectively to assemble a herd pedigree.

CWD-infected animals were identified by both IHC and RT-QuIC, and were culled according to management directives. A herd pedigree was assembled which may allow us to identify animals with higher likelihoods of acquiring CWD based on familial history of CWD; a likelihood which will be quantitatively captured as the Relative Index of Susceptibility to CWD (RISC). Similar to an estimated breeding value, this figure may be used to select for and against individuals for breeding based on that history of susceptibility.

This project represents a unique opportunity to collect valuable information on CWD diagnostics and epidemiology, and may eventually prove useful in identifying additional genetic elements which contribute to CWD resistance in the natural host. We aim to continue with yearly sampling and data collection over the length of the proposed 5 year study to identify the effects of our management strategies on CWD prevalence, herd genetics, and reproductive success.


P34 The Role of Traditional Knowledge in the Monitoring and Management of Chronic Wasting Disease in Northern Alberta

Arlana Bennett1, Principle Investigator Brenda Parlee1
1University Of Alberta, Edmonton, Canada

To date, efforts and research surrounding Chronic Wasting Disease (CWD) prevalence and management in Alberta have primarily focused on economic impacts, proliferation of CWD, modeling of disease distribution, and questionably effective management practices. Due to the frequency and spread of CWD cases in Alberta, a national CWD control strategy was proposed which briefly highlighted the need to include First Nations people as key stakeholders, and as key contributors to the process of managing CWD in wild cervid populations.

The aim of this research is to determine how the local and traditional knowledge (TK) of First Nations hunters could inform the monitoring and management of cervids (deer, moose and elk specifically). This will be achieved by drawing on successful examples of wildlife co-management agreements in Canada, conceptualizing hunter/elder oral testimony, and analyzing one-on-one interviews with various individuals currently involved in the Provincial CWD Program Review Committee, and CWD research in Alberta.

Methods of data collection and analysis include: one-on-one interviews, transcription and analysis of previously conducted hunter/elder oral testimony from various community members in the Treaty 8 area of Northern Alberta, and a comparative analysis of wildlife co-management agreements across Canada, which are intended to support a “best practices” approach for developing a co-management arrangement with Treaty 8 First Nations.

A preliminary review of the literature finds very little evidence of First Nations consultation both from the Federal and Provincial governments, and members of the CWD Program Review Committee. Furthermore, there is a clear and demonstrated inability of the CWD program to limit the increase of CWD across Alberta and into subsequent cervid populations. Prior research with Treaty 8 hunters and elders indicates that communities continue to rely on cervids to support food security while the health and range of cervids is impacted by local resource development, climate change, and migratory shifts. As well, due to the changes in cervid health, Treaty 8 hunters are concerned about the health of cervids and are opting to travel longer distances to avoid locations with unhealthy animals. Further results into the likelihood of a co-management agreement between Treaty 8 First nations and the Federal/Provincial governments is forthcoming.

Results forthcoming.


P52 NaPTA/RT-QUIC detection of PrPCWD in saliva and urine of CWD-infected cervids and TgElk mice

Hyun Joo Sohn1, Kyung Je Park1, Gordon Mitchell2, In Soon Roh1, Hyo Jin Kim1, Hae Eun Kang1
1Foreign Animal Disease Division Animal And Plant Quarantine Agency(QIA), Gimcheon, South Korea, 2Canadian Food Inspection Agency, Ottawa, Canada

Aims: Chronic wasting disease(CWD) is the only prion disease affecting free-ranging animals, reported in North America, South Korea and Norway. CWD agents are shed in saliva, urine and feces which most likely contribute to the horizontal transmission between cervid species.The development of amplification-based seeding assays have been instrumental in the detection of low levels of prions in clinical samples. Using NaPTA precipitation and real-time quaking-induced conversion(NaPTA/RT-QUIC), we established a ultrasensitive detection method for PrPCWD in the saliva and urine of CWD affected cervids. Also we performed the longitudinal study to detect PrPCWD from ,in the CWD-infected, sequentially sampled transgenic mice overexpressing elk prion protein(TgElk mice).

Methods: Five saliva and two urine samples from CWD-infected cervids at the terminal stage of disease, and 28 urine samples from sequentially sampled CWD-infected TgElk mice (TgElk CWD) were stored at -80℃. 100uL of each sample was mixed with 10uL 2.8% sodium phosphotungustic acid (NaPTA) and incubated for 1hr at 37℃ with shaking at 1,350 rpm. Samples were centrifuged for 30min at 16,100 g. The pellet was resuspended in 10uL of 0.1% SDS/PBS for 30min at 55℃. RT-QUIC reactions were set up in 96-well clear bottom optic plates and consisted of 98uL RT-QUIC buffer [final concentrations of 1XPBS, 1mM EDTA, 10uM Thioflavin, 300mM NaCl buffer and 0.1mg/ml recombinant hamster recombinant protein(23-231) and 2uL of sample. The RT-QUIC assay was performed on a FLUOstar Omega fluorescence plate reader that was preheated to 55℃ for 60hr with 1min shaking at 700rpm followed by 1min incubation.

Results: NaPTA/RT-QUIC was applied to measure PrPCWD in urine samples collected on every 15days from 30dpi to 120dpi when CWD infected TgElk mice reached terminal stage. . and dpi typicallyCWD in PrPCWD in the urine in TgElk CWD was detectable in early stages(30 and 45dpi), disappeared during the intermediate stages of infection(60 and 75dpi) and reached the highest levels at 90dpi. PrPCWD was also detectable in late and terminal stages(120dpi). In addition, PrPCWD was detected in terminal urine samples from two sika deer(experimental cases) and terminal saliva samples from five cervids were also observed to consistently yield positive results by the NaPTA/RT-QUIC assay.

Conclusions: We demonstrate that CWD prions can be detected by NaPTA/RT-QUIC in the saliva and urine of TgElk mice, red deer and sika deer at the early and terminal stages of disease. Our method appears to be a very useful technique for both diagnosis and surveillance of CWD


P54 Molecular Dynamics Simulations of Cervid Prion Protein Variants to Asses Protein Stability and Susceptibility Towards Chronic Wasting Disease

Sara Amidian1,2, PhD Samia Hannaoui3, PhD Lyudmyla Dorosh4,5, PhD Maria Stepanova4,5, PhD Sabine Gilch3, PhD Holger Wille1,2

1Centre for Prions and Protein Folding Diseases, University of Alberta, Edmonton, Canada, 2Dept. of Biochemistry, University of Alberta, Edmonton, Canada, 3Dept. of Ecosystem and Public Health, Calgary Prion Research Unit, University of Calgary, Calgary, Canada, 4National Institute for Nanotechnology, University of Alberta, Edmonton, Canada, 5Dept. of Electrical and Computer Engineering, University of Alberta, Edmonton, Canada

Chronic Wasting Disease (CWD) is the most contagious member of the prion diseases and is efficiently transmitted between and within cervid species (deer, elk, moose, and reindeer). CWD is spreading uncontrollably in the United States and Canada, and has recently been detected in Europe.
In order to better understand the stability of different prion protein variants from various cervid species, we conduct molecular dynamics (MD) simulations with the aim of providing a link between atomic scale protein structure and stability data and susceptibility to misfolding. Here, the effect of a A116G polymorphism in the white tail deer (WTD) prion protein is evaluated by performing molecular dynamics simulations and subsequent analyses.

Method: Molecular Dynamics Simulations
The initial model for the WTD prion protein (residues 112-233) was generated using the crystal structure of recombinant deer prion protein (PDB: 4YXH) as a template. The wild type (WT) and the A116G systems were subjected to minimizations, equilibrations, and production MD simulations using the Gromacs package. In total, three independent simulations were performed for 50 ns for each system.

Results: The structure of the A116G prion protein is less stable than the WT form
The A116G polymorphism has a higher root mean square deviation (RMSD) and higher root mean square fluctuation (RMSF) values compared to the WT protein. This indicates that the structure of the A116G protein is less stable and has larger structural fluctuations. In addition, the radius of gyration (Rg), which is representative of the protein’s folding behaviour reveals that A116G has larger Rg values, indicating that the structure of the WT protein is more compactly folded and thus hypothetically more stable. Also, an increase in the solvent accessible surface area of the A116G variant suggests that the polymorphism renders its structure more solvent exposed.

According to our MD results, it is apparent that the structure of the WTD prion protein carrying the A116G polymorphism is less stable than the WT form. In order to explore the structural dynamics and stabilities of cervid prion protein polymorphisms, we will perform further MD simulations by adding the GPI (glycosyl phosphatidyl inositol) moiety to the prion protein and anchoring it into a membrane, bringing the simulations closer to physiological conditions.


P58 Evidence of multiple strains of chronic wasting disease in wild type mice

Mrs Aileen Boyle1, Dr Moira Bruce2, Mr Christopher Plinston1, Dr Elizabeth Williams3, Dr Jurgen Richt4, Prof Nora Hunter1, Prof Jean Manson1, Dr Abigail Diack1

1The Roslin Institute, Easter Bush, United Kingdom, 2Neuropathogenesis Unit, Institute for Animal Health, Edinburgh, United Kingdom, 3University of Wyoming, Laramie, USA, 4Agricultural Research Service, USDA, Ames, USA

Chronic wasting disease (CWD) is a highly infectious TSE affecting multiple species of cervids which continues to spread through the USA and Canada. More recently CWD has been identified in Norwegian moose and reindeer. In order to understand the spread of CWD between species and to assess its zoonotic potential, it is important to establish the diversity of strains in the field and how these vary both within and between species. Using well-defined strain typing panels of mice, we aimed to characterise American CWD isolates from the 1980s and 1990s in order to provide a baseline for further strain identification for North America and European CWD isolates.

Archival searches of The Roslin Institute TSE archive revealed six transmission studies in which wild type mouse panels (RIII, C57BL/6, VM and CVF1) were inoculated with American isolates from two mule deer, two elk and two white-tailed deer. One mule deer isolate was further passaged through VM mice. Strain characteristics including attack rates, TSE vacuolation and PrP deposition were re-analysed using stringent criteria.

This archival study provides preliminary evidence that multiple strains of CWD are present both within and between cervid species. All isolates transmitted on primary passage with variation apparent in attack rates and neuropathology dependent upon isolate/mouse strain combination. Evidence of two different strain phenotypes emerged; one arising in mule deer and one in both mule deer and white-tailed deer. Elk isolates gave inconclusive results due to low titre inoculum.

Serial passage of a mule deer sample in VM mice gave rise to a stable mouse passaged strain (409V). The characteristics of this strain include an incubation period of ~280 days, presence of PrP deposition predominantly in the form of plaques, and a low degree of vacuolation with no discernible pattern. Comparison of 409V characteristics with BSE and scrapie isolates passaged through VM mice revealed some similarities but nothing matching all characteristics.

We demonstrate that multiple strains of CWD have been present in the USA since the 1980s. We further hypothesise that the diversity of CWD strains will have increased since these isolates were collected. Production and characterisation of a mouse-adapted strain revealed marked differences between CWD and known natural or experimental UK TSE strains. Our data supports the multi-strain hypothesis for CWD and provides a platform for assessing similarities and differences in CWD strains worldwide, particularly in newly affected countries.


P64 Destabilizing polymorphism in cervid prion protein hydrophobic core dictates conversion efficiency, prion conformation and adaptation

Dr Samia Hannaoui1, Sara Amidian2, Yo Ching Cheng1, Camilo Duque-Velasquez2, Sampson Law1, Prof Glenn Telling3, Dr Debbie McKenzie2, Dr Holger Wille2, Dr Sabine Gilch1

1Calgary Prion Research Unit, Faculty of Veterinary Medicine, University of Calgary, Calgary, Canada, 2Center for Prions and Protein Folding Diseases, University of Alberta, Emonton, Canada, 3Prion Research Center, Colorado State University, Fort Collins, USA

Chronic wasting disease (CWD) is the most contagious member of the prion diseases effecting free-ranging and farmed animals. It is efficiently transmitted from animal to animal within and between cervid species. CWD affects elk, mule deer, white tailed deer (WTD), moose and reindeer. The disease is endemic in North-America and has been recently recognized in Europe. Although the PrP primary structure is highly conserved among cervids, the disease phenotype can be modulated by species-specific polymorphisms in the prion protein gene (Prnp). To gain insights on how molecular determinants can impact the generation and adaptation of new strains, we investigated the impact of the poorly described WTD polymorphism at residue 116 (A->G).

We used techniques to assess the biochemical properties of CWD prions such as proteinase K resistance, guanidine denaturation and glycosylation ratio. Cell-free conversion model (real-time quaking induced conversion (RT-QuIC)), primary neuronal cell cultures model (CGN) and mouse bioassays (tg(CerPrPM132)1536+/+) were used to verify the infectivity of CWD-WTD prions in vitro and in vivo.

We found that heterozygosity at codon 116 (A/G), compared to the WT (AA), results in CWD prions with i) lower conformational stability, ii) lower seeding activity in RT-QuIC, iii) reduced infectivity in CGN, iv) prolonged incubation time in tg(CerPrP132M)1536+/+ transgenic mice overexpressing wt deer PrP. Biochemical features were retained upon first passage in transgenic mice. Secondary passage of mWTD-116AG in tg(CerPrP132M)1536+/+ transgenic mice resulted in significantly shortened incubation times compared to the first passage, notably despite identical biochemical hallmarks of original and mouse-passaged WTD-116AG prions. Upon secondary passage mice succumbed to disease even faster than those inoculated with mouse-passaged WTD-WT (116AA) prions. Moreover, recombinant PrP (rPrP)-116G was more efficiently converted by both CWD-WTD isolates than the rPrP-116A when used as a substrate in RT-QuIC. This can be explained by our findings employing molecular dynamics simulations which revealed that the structure of PrP-116G is less stable and exhibits more structural fluctuation and signs of disruption than PrP-116A, as well as an increased solvent-accessible surface area.

We conclude that a single amino acid substitution in the hydrophobic core region affects the structural stability of PrPC. This, results in a CWD prions, WTD-116AG, with a lower conformational stability having a trend to adaptation to new hosts which can be cervid and non cervid species.


P78 Host Range of Chronic Wasting Disease (CWD) Isolates

Elizabeth Triscott1, Camilo Duque Velásquez1, Allen Herbst1, Trent Bollinger2, Judd M. Aiken1, Debbie McKenzie1

1Centre for Prions and Protein Folding Diseases, University Of Alberta, Edmonton, Canada, 2Canadian Cooperative Wildlife Health Centre, Western College of Veterinary Medicine, University of Saskatchewan, Saskatoon, Canada

Aims: Chronic wasting disease (CWD), an infectious prion disease of cervids, continues to expand its geographic range and increase in prevalence in North America. Given the diversity of cervid species in Canada’s CWD zone, we hypothesize that CWD agents are modified by transmission between free-ranging cervids with different PrPC primary structures, resulting in diversification of CWD agents. These agents could then have expanded or different host ranges.

Methods: To test this hypothesis, we explored the interspecies transmissibility of eleven CWD isolates from wild, hunter-harvested white-tailed deer and mule deer from western Saskatchewan. These isolates were biochemically characterized to define PrPCWD protease stability and molecular weight. CWD isolates were also inoculated into tg33 mice, which express deer Q95G96 (wt) PrPC, and Syrian Golden hamsters.

Results: There were no overt differences between these deer prions upon passage into tg33 mice. These isolates, however, varied in their ability to transmit into Syrian Golden hamsters, a model for interspecies transmission. Of the five white-tailed deer isolates, four induced the accumulation PrPRES in hamster brains after at two years post-infection. Only three of six mule deer isolates resulted in deposition of PrPRES after transmission in hamsters. Clinical disease was observed in two of the isolates, both originating in white-tailed deer. The biochemical characteristics of these hamster-passaged isolates were characterized and compared.

Conclusions: These data indicate that, while these isolates appear to have similar properties following passages into transgenic mice the most common cervid PRNP genotype, they are variable with respect to their ability to transmit to other species (hamsters). This suggests that variation in prion isolates from naturally infected cervids may broaden the host range of CWD agents upon interspecies transmission.


P79 Atypical PrPSc retains high infectivity after protease K treatment

Dr Ilaria Vanni1, DVM Laura Pirisinu1, Mr Stefano Marcon1, Dr Michele Angelo Di Bari1, DVM Claudia D'Agostino1, Dr Joaquin Castilla2,3, MD Pierluigi Gambetti4, DVM Romolo Nonno1, DVM Umberto Agrimi1

1Istituto Superiore Di Sanità, Department of Veterinary Public Health and Food Safety, Rome, Italy, 2CIC bioGUNE, Parque Tecnológico de Bizkaia, Derio, Spain, 3IKERBASQUE, Bilbao, Spain, 4Case Western Reserve University, Department of Pathology, Cleveland, United States of America

Aims: Latest studies reported that prion diseases associated with PrPSc characterized by an unusual protease resistant core (PrPres) cleaved at both N- and C-termini, are transmissible in rodent models (1,2). Although these studies suggested that atypical prions are associated with true infectivity similarly to classical prions, definite proof of the infectious role of these unusual PrPres aggregates is still missing.
To pursue this issue, we focused on human and experimental prion diseases associated with atypical PrPSc, respectively the Gerstmann-Sträussler-Scheinker (GSS) disease associated with the A117V PrP mutation and the neurological disease spontaneously developed by transgenic mice expressing wt bank vole PrP (TgL1).

Methods: Frozen brain tissues were obtained from human or experimental prion diseases associated with classical PrPSc (sCJD-MM1 and vole-adapted CWD) or atypical PrPSc (GSS with the A117V mutation and spontaneously sick TgL1 mice). Brain homogenates were either treated with PK (20 μg/ml, 37°C for 1 hour) or left untreated. PK-treated or untreated homogenates were then endpoint titrated by bioassay in bank voles. Infectivity titers were calculated as ID₅₀ U/g by the Spearman and Karber method.

Results: Overall, atypical prion diseases showed infectious titers as high, or even higher, than classical prions. Notably, GSS-A117V showed the highest infectious titer (10<9 .3="">), followed by vole-adapted CWD (10<8 .4="">), spontaneously sick TgL1 (10<7 .6="">) and sCJD MM1 (10<5 .6="">). After PK treatment, ID₅₀ U/g were 10<9 .0=""> for GSS-A117V, 10<8 .1=""> for CWD, 10<8 .4=""> for TgL1 and 10<5 .9=""> for sCJD MM1. The variation of the infectious titres after PK treatment was within the -0.3-to-+0.8 log range. Thus, in both classical and atypical prions PK treatment did not substantially affected the infectious titer.

Conclusions: Our study reveals that prion diseases characterized by atypical PrPSc exhibit high infectious titers and that such infectivity is retained upon PK treatment, as it happens for classical PrPSc. Notably, these data argue against the hypothesis of “protease sensitive” forms of PrPSc as being the misfolded conformers responsible for the abovementioned infectivity. Furthermore, our results highlight that the C-terminal region of PrPSc is dispensable for infectivity, thus suggesting that PrPres aggregates containing exclusively the central PrP domain, i.e. composed of PrP peptides spanning aa ̴90-150, could be considered fully competent “miniprions”. Efforts towards the purification of these aggregates are underway.


1. Pirisinu et al. Sci Rep. 2016; 6:20443.

2. Asante et al. PLoS Pathog. 2013; 9(9):e1003643.


109 Differential Effects of Divalent Cations on Elk PrPC Fibril Formation and Stability

Dr. Daniel Samorodnitsky1, Dr. Eric Nicholson1
1US Department Of Agriculture, Ames, United States

In this study, we examined the ability of a variety of divalent cations to influence the stability and in vitro conversion of two variants of truncated PrPC from an elk background (L/M132, 93-234, equivalent to human 90-231).

Misfolding of the normally folded prion protein of mammals (PrPC) into infectious fibrils causes a variety of different diseases, from scrapie in sheep to bovine spongiform encephalopathy in cattle to chronic wasting disease (CWD) in deer and elk. The misfolded form of PrPC, termed PrPSc, interacts with PrPC to create more PrPSc. This process is not clearly defined but is evidently affected by the presence and interactions of numerous cofactors, both biotic and abiotic. This includes the presence of nucleic acids, lipids, glycosylation patterns, pH, and the presence of ions.
PrPC has been shown to act as a copper-binding protein in vivo, though it also binds to other divalents as well. The significance of this binding action has not been conclusively elucidated. Previous reports have shown that metal binding sites occur throughout the N-terminal region of PrPC. However, the 90-231 fragment of human PrPC binds copper with sub-nanomolar affinity as well. Other cations like manganese have also been shown to affect PrPC abundance in a transcript-independent fashion.

Examined PrPC stability by thermal denaturation circular dichroism. Examined fibril formation via Real Time Quaking-Induced Conversion (RT-Quic).

We find that both copper and manganese de-stabilize PrPC. However, we also find that PrPC L132 exhibits a greater degree of divalent cation-induced destabilization in comparison to M132. This stands in contrast to the finding that the occurrence of leucine at position 132 confers resistance to CWD, while M132 is susceptible. However, in vitro conversion of PrPC is suppressed by the presence of both copper and manganese, in both L132 and M132 backgrounds.

This report demonstrates the complex importance of ionic character on PrPC folding pathway selection on the route to PrPSc formation.


P128 No cases of Chronic wasting disease from an ongoing monitoring program in Italy

Mrs Daniela Meloni1, Mrs Maria Gabriella Perrotta2, Mr Francesco Ingravalle1, Mrs Eleonora Aiassa1, Mrs Maria Concetta Cavarretta1, Miss Daniela Loprevite1, Mr Luca Nocilla1, Mrs Maria Mazza1, Miss Katia Varello1, Mrs Luna Montesion1, Dr Maria Cristina Bona1, Mrs Cristiana Maurella1, Mr Simone Peletto1, Mr Pier Luigi Acutis1, Mrs Elena Bozzetta1, Mr Giuseppe Ru1

1Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Turin, Italy, 2Ministry of Health, Rome, Italy

Chronic Wasting Disease (CWD) is known as a Northern American problem of deer, elk and moose. In March 2016 the first case of CWD was detected in Europe in a free-ranging reindeer in Southern Norway.. Subsequently, the disease has been confirmed in a Norwegian moose and, by August, the cases in the two species were summing up to five. The incursion of CWD in Europe is of concern due to its possible spread and impact on cervids. Here we describe the results of the first three-month period of Italian monitoring aimed at detecting the disease.

A national monitoring activity has been launched during the 2016 hunting season and involves adult red deer, roe deer and fallow deer. Convenience sampling of wild or farmed deer is targeting the following streams: found dead, road kills, hunted or slaughtered animals because sick or in poor body condition. As the idea is to test as much animals as possible to minimize the achievable design prevalence, no sample sizes or deadlines were set in advance. Both the brain stem and the retropharyngeal lymph nodes were to be collected from each animal and sent to the Italian national reference laboratory for TSEs in Torino. Rapid testing is carried out using the Herd Check IDEXX BSE-Scrapie Antigen EIA test whereas IHC, IH and Western blot analyses are used as confirmatory tests. A subset of samples, representative by species and geographical origin, is submitted to genotyping. Relevant data (species, sampling date, location and streams) have been collected for each animal.

Since the end of October 2016 samples from 92 cervids have been collected from nine Northern or Central Italian Regions. A final negative result was obtained for 81 animals (52 roe deer, 27 red deer and 2 fallow deer). Roe deer were mostly from road kills whereas a large proportion of red deer were from the found dead stream. Red deer and fallow deer carried wild type whereas 6 roe deer showed known polymorphisms.

The initial monitoring activity has not shown any evidence of the disease. However, based on the small number of tested animals, the sensitivity of the surveillance system is still limited. The origin, the incidence and the risk factors associate to CWD in Europe are currently not known, but the need for carrying out both effective surveillance and risk assessment should be identified as an European priority to safeguard wildlife.


P132 Resistance-associated deer prion protein polymorphisms limit the peripheral PrPCWD deposition

Ms Alicia Otero1, Dr Chad Johnson2, Dr Allen Herbst3,5, Mr Camilo Duque-Velasquez3,5, Dr Rosa Bolea1, Dr Juan José Badiola1, Dr Judd Aiken3,5, Dr Debbie Mckenzie4,5

1Centro De Encefalopatías Y Enfermedades Transmisibles Emergentes, Universidad de Zaragoza, Zaragoza, Spain, 2Department of Medicine, School of Medicine and Public Health, University of Wisconsin, Madison, USA, 3Department of Agriculture, Food and Nutritional Science, University of Alberta, Edmonton, Canada, 4Department of Biological Sciences, University of Alberta, Edmonton, Canada, 5Centre for Prions and Protein Folding Diseases, Edmonton, Canada

Among prion diseases, chronic wasting disease (CWD) is the only known spongiform encephalopathy affecting both captive and free-ranging animals. Horizontal transmission by direct animal interactions and via persistence of infectivity in the environment greatly hinders control and eradication of this disease. In addition, CWD infectivity has been detected in excreta (saliva, urine and feces) from infected cervids (Haley et al., 2011; Henderson et al., 2015). Cervid PrPC primary structures play a key role in CWD susceptibility resulting in extended incubation periods and affecting the propagation of CWD strains (Johnson et al., 2011; Duque Velasquez et al., 2015). Our main aim was to determine if there are substantial differences in the deposition pattern of PrPCWD between different PRNP genotypes of white-tailed deer infected by oral route.

We characterized, by immunohistochemical analysis, the distribution of abnormal prion protein (PrPCWD) aggregates in central nervous system and peripheral organs from Wisc1 orally inoculated white-tailed deer expressing four different PRNP genotypes: Q95G96/Q95G96 (wt/wt), S96/wt, H95/wt and H95/S96 (Johnson et al., 2011).

Although all deer genotypes show similar PrPCWD deposition intensity in the brain, distinct morphological patterns of PrPCWD were observed in the cerebellum. Interestingly, we observed that deer carrying the H95-PRNP allele accumulated less PrPCWD in peripheral organs, especially in tissues related to excreta production. Although PrPCWD was observed in several tissues, no PrPCWD deposition was detected in the skeletal muscle of any of the deer genotypes.

Our data suggest that, in this experimental transmission of Wisc1 prions, expression of H95-PrPC limited peripheral accumulation. Conversely, infected S96/wt and wt/wt deer presented with similar PrPCWD peripheral distribution patterns at the terminal stage of disease.


P143 Toward estimating sub-clinical human prion prevalence: Development of high-throughput amplification and detection methodologies.

Jeffrey Christiansen1, Sam Mellentine1, Clare Hoover1, Patrick Bosque2, James Ironside3, Edward Hoover1, Candace Mathiason1

1Colorado State University, Fort Collins, United States, 2University of Colorado School of Medicine, Aurora, United States, 3University of Edinburgh , Edinburgh, Scotland

Aims: The ability to determine the prevalence of subclinical diseases is of utmost importance to public health management. In the case of variant Creutzfeldt-Jakob disease (vCJD), determining subclinical prevalence has proven difficult. Foremost, the tissue tropism and time-course of abnormal prion protein (PrP) accumulation is not well characterized during the protracted asymptomatic phase of disease, making conclusive identification difficult. A recent study conducted in the UK identified 1 in 2000 subclinical vCJD carriers by assessing archived paraffin-embedded appendix tissues by immunohistochemistry (IHC). While vCJD exemplifies the dangers of TSE zoonosis, the unknown zoonotic potential of CWD and atypical scrapie cannot be neglected. Compounding the difficulty in diagnosing new zoonotic TSEs is the unknown clinical presentation and potential to manifest in an asymptomatic carrier state.

Methods: Detection of prions, especially during the asymptomatic phase of disease, has proven to be challenging and requires time consuming and expensive methods such as bioassay, IHC and Western blotting. The real time-quaking induced conversion assay (RT-QuIC) permits the amplification of low levels of prion seeding activity through cycles of shaking recombinant PrP substrate with a prion seed. The generation of nascent amyloid fibrils is measured in real-time through incorporation of thioflavin T, in a 96-well plate format for high-throughput.

Results: We assessed archived human biopsy tissues from CWD endemic and non-endemic regions for the presence of abnormal PrP by RT-QuIC. Formalin-fixed, paraffin-embedded histology blocks were processed to extract tissue homogenates. Our study focused on analyzing appendix biopsy samples due to their previous use in surveillance studies and accessibility, but we also show the methodology can be applied to additional lymphoreticular tissues such as tonsils, spleen and lymph nodes. We compared 100 appendix biopsies from patients residing within the chronic wasting disease (CWD) endemic region to 100 appendix biopsies from patients residing outside the CWD endemic region.

Conclusions: Our method offers an opportunity to identify abnormal PrP in archived biopsy specimens from health centers across the country to perform retrospective analysis on a statistically significant number of anonymized samples. Identification of subclinical disease prevalence is critical to limiting the spread of human prion disease through transfusions and iatrogenic transfer.


P144 Environmental Contamination Assessment of CWD Shed in Excreta by RT-QuIC

Dr. Davin Henderson1, Ms. Joanne Tennant1, Dr. Nicholas Haley2, Dr. Nathaniel Denkers1, Dr. Candace Mathiason1, Dr. Edward Hoover1

1Prion Research Center. Department of Microbiology, Immunology and Pathology. Colorado State University, Fort Collins, United States, 2Midwestern State University, Glendale , United States
Chronic wasting disease affects free-ranging or captive populations of deer, elk and moose in the United States, Canada, Korea, and most recently Norwegian caribou and moose. CWD is unique in its ability to spread in wild populations, which likely occurs through environmental dissemination or direct contact with prions shed into bodily fluids or excreta. We have made progress to rapidly and cost-effectively detect CWD in urine and feces, which contribute substantially to environmental contamination.

Aims: 1) Using real-time quaking induced conversion (RT-QuIC) we will estimate environmental contamination via urine and feces by CWD positive animals throughout the disease course. 2) We will apply RT-QuIC to estimate CWD incidence in populations without animal capture and assess the persistence of prion seeding activity in feces under environmental conditions in the native range of cervid species.

Methods: CWD prions in fecal and urine samples will be concentrated via iron oxide mediated extraction (IOME) and assayed by RT-QuIC using temperature adjustment modifications to select increase to preserve sensitivity while minimizing specificity.

Results: We assayed longitudinal cohorts of whitetailed deer for prion shedding in feces and urine during the CWD disease course and determined shedding kinetics and consistency. We applied a quantitative approach to these data using a reference brain sample to determine the levels of CWD prions shed in feces and urine during the disease course.

Conclusions: In deer experimentally inoculated with CWD approximately 100 (Tg5037) cervid PrP mouse LD50 doses are shed in urine and ~90 (TG5037) mouse LD50 doses are shed in feces per day. The relationship between cerPrP transgenic mouse and cerivd LD50 doses remains to be determined. In the future, we will apply RT-QuIC detection of CWD in feces to estimate CWD incidence in populations without animal capture and to determine how long prion seeding activity remains active in fecal samples under potentially harsh environment conditions that exist in the native range of cervid species.


P152 Unique reactivity of cervid recombinant PrP for the detection of L-BSE and CWD by RT-QuIC

MC Akio Suzuki1, Dr. Takeshi Yamasaki1, Dr. Rie Hasebe1, Dr. Motohiro Horiuchi1
1Hokkaido University, Sapporo, Japan

Although Classical bovine spongiform encephalopathy (C-BSE) is now under the control, potential risk of atypical BSE in cattle, and chronic wasting disease (CWD) in cervids to public and other animal species remains unclear. To reveal potential existence of atypical BSE and CWD, we attempted to establish real-time quaking-induced conversion (RT-QuIC) that can detect small amount of L-type atypical BSE (L-BSE) and CWD in the presence of high concentration of brain tissue in the reaction.

Expression plasmids for full-length recombinant mouse and hamster PrP (rMoPrP, rHamPrP) were kindly provided by Dr. Atarashi R, Miyazaki Univ. Gene encoding full-length cervid and bovine PrP (rCerPrP and rBoPrP) were inserted into pET11a. Each plasmid was introduced into BL21(DE3)pLysS, and the cells were grown in auto-induction medium. Refolding and purification of rPrP were performed using Ni-NTA resin. Ten-percent brain homogenates (BH) of cattle and deer infected with C-, L-BSE and CWD were serially diluted with PBS or corresponding normal BH and used as seeds. The reaction buffer containing 10 mM Na-phosphate (pH 7.4), 500 mM NaCl and 0.001% SDS was used for rCerPrP, rBoPrP and rMoPrP, while that containing 350 mM NaCl without SDS was used for rHamPrP. All RT-QuIC reactions were performed at 37 ˚C.

Among four rPrPs, rMoPrP and rCerPrP could detect L-BSE prions in the highest concentration of BH (10-3). The detection range of PBS-diluted L-BSE using each rPrP was 10-3-10-7. Surprisingly, the detection sensitivity of L-BSE using rCerPrP was increased one log in the presence of 0.1% normal cattle BH in the reaction, whereas that of C-BSE was significantly decreased under the same condition. The detection range of PBS-diluted CWD using rMoPrP and rCerPrP was 10-3-10-7; however, the sensitivity in the detection of CWD prions using rCerPrP was enhanced one log in the presence of 0.1% normal deer BH in the reaction. These results indicate that rCerPrP is a good substrate for detecting L-BSE and CWD in the high concentration of brain tissue.

Cervid rPrP stably reacted with L-BSE and CWD even in the presence of highest concentration of BH, and the sensitivity was increased in the presence of normal BH in the reaction. Thus, RT-QuIC using rCerPrP is practical for L-BSE and CWD detection. Furthermore, the sensitivity of C-BSE was significantly decreased in the presence of normal BH, suggesting that RT-QuIC using rCerPrP is useful for the discrimination of L-BSE from C-BSE prions.


P155 Sensitive detection of PrPCWD in soil from CWD infected farm by PMCAb

Hyun Joo Sohn1, Kyung Je Park1, In Soon Roh1, Hyo Jin Kim1, Hoo Chang Park1, Director of division Hae Eun Kang1

1Foreign animal disease division, Animal And Plant Quarantine Agency(QIA), Gimcheon, South Korea

Aims: Chronic wasting disease (CWD) is the prion disease that is known spread horizontally. CWD has confirmed last in Republic of Korea in 2016 since first outbreak of CWD in 2001. The environmental reservoirs mediate the transmission of this disease. The significant levels of infectivity have been detected in the saliva, urine, and feces of TSE-infected animals. Using serial protein misfolding cyclic amplification with beads (sPMCAb), we developed a detection method for CWD PrPCWD in soil from CWD affected farm in 2010. We found to detect PrPCWD in soil from CWD infected farm, but not detect PrPCWD in soil of normal cervid farm in Korea. Our method appears to be a very useful technique for monitoring PrPCWD levels in environmental conditions.

Methods: There are total of three steps with two washing steps for PrPCWD extraction and one amplification step for PrPCWD detection from soils of natural CWD farms which have confirmed horizontal spread of CWD in 2010 and 2016. The first washing step consists of slow rotating to remove large impurities. The second washing step was detached PrPCWD from abnormal prion contaminated soil by strong vortex. The last step was PrPCWD amplification step using sPMCAb. Sonication was performed with a Misonix 4000 sonicator with amplitude set to level 70, generating an average output of 160W during each cycle. One round consisted of 56 cycles of 30 sec of sonication followed 10min of 37℃ incubation. The samples(20uL) after each round of amplification were mixed with proteinase K (200ug/ml) and incubated 37℃ for 1hr. Samples were separated by SDS-PAGE and transferred onto PVDF membrane. After blocking, the membrane was incubated for 1h with 1st antibody S1 anti rabbit serum (QIA, 1:3000) and developed with enhanced chemiluminescent detection system

Results: We had collected 35 soil samples from the four farms which were confirmed CWD and five trace farm in 2016. After three rounds of sPMCAb, we detected PrPCWD in 10 samples from areas which were positive animals habitat but not detect PrPCWD in soil of wild cervids habitat and normal cervid farm in Korea. These results suggest that our method can be a very useful tool for monitoring PrPCWD contamination in environments

Conclusions: This sPMCAb method using soil washing solution by slow rotating and vortex is effective extraction method of PrPCWD from CWD contaminated soils. The method developed in this study will be useful for assessment of PrPCWD levels in the contaminated soils.


P164 CWD environmental contamination: assay of CWD prion binding to soil analyzed by magnetic extraction and real-time conversion

Dr. Nathaniel Denkers1, Dr. Davin Henderson1, Dr. Shannon Bartelt-Hunt2, Dr. Jason Bartz3, Dr. Edward Hoover1

1Colorado State University, Fort Collins, United States, 2University of Nebraska-Lincoln, Omaha, United States, 3Creighton University, Omaha, United States

Chronic wasting disease (CWD) is unique in its facile spread in nature, thought in substantial part due to indirect transmission due to environmental exposure. In the present study, we examine the binding of CWD prions to silty clay loam (SCL) using rapid and semi-quantitative iron oxide magnetic extraction (IOME) / real-time quaking-induced conversion (RT-QuIC) methodology.

Graded amounts of SCL (6.25-100mg) were incubated with 1mL-serial dilutions (10-2-10-7) of CWD (+) or CWD (-) brain homogenate (BH) for 60 minutes on an end-over-end rocker. Samples were then centrifuged, 2.0uL of each supernatant reserved for analysis, and the remaining supernatants were subjected to IOME for 30 minutes. Beads were magnetically separated and 2.0uL of the IOME supernatant were removed for analysis. Beads were then re-suspended in 10uL-0.1% SDS, as were all reserved supernatants. Samples were seeded into 96-well RT-QuIC plates containing recombinant Syrian hamster prion PrP substrate and thioflavin T (ThT). Amyloid seeding activity (recorded as ThT fluorescence emission at 480 nm) over time was measured using a fluorimeter/shaker. Samples were considered positive if they crossed a threshold 5 standard deviations above baseline fluorescence. Sample reaction rates (RR) were calculated, averaged, and expressed as 1/time in minutes.

Concentrations of SCL (6.25, 12.5, and 25mg) bound and removed the majority of seeding activity from higher dilutions (10-6-10-7) of CWD+ brain homogenate (BH) in a concentration-dependent manner, as determined by seeding activity in residual supernatants and IOME samples. SCL (25 & 100mg) reduced CWD seeding activity between 30-100 % (quantified as reaction rates of each fraction) in 10-4-10-5 dilutions, but had little measureable effect when log10-fold higher CWD+ brain concentrations were used (i.e. 10-2 - 10-3 dilutions). Based on data from these assays, we estimated the prion binding capacity of SCL to be approximately 40ng/mg SCL.

Silty clay loam exhibits highly efficient prion binding, inferring a durable environmental reservoir and an efficient mechanism for indirect horizontal CWD transmission.


P166 CWD strains in “resistant” deer PRNP genotypes

Mr Camilo Duque Vélasquez1, PhD Allen Herbst1, Ms Chiye Kim1, Ms Tracy Haldiman2, Ms Chae Kim2, PhD Serene Wohlgemuth1, PhD David Westaway1, PhD Judd Aiken1, M.D. Jiri Safar2, PhD Debbie McKenzie1

1Center For Prions and Protein Folding Diseases, Edmonton, Canada, 2National Prion Disease Pathology Surveillance Center, Case Western Reserve University, Cleveland, United States of America

Chronic wasting disease (CWD) is a prion disease of free-ranging and captive cervid species in North America, South Korea, and Norway. The contagious nature of cervid prions and variable PrPC primary structures, which influence host susceptibility, can result in the emergence of novel CWD strains. Emergent strains may affect host range, detection, and spread dynamics, impacting disease surveillance and eradication programs.

To evaluate the effects of deer PrPC amino acid polymorphisms on the biochemical and conformational properties of PrPCWD.
To characterize the transmission properties of CWD allotypes in transgenic mice expressing different deer PrPC molecules.

Using western blot analysis, proteolysis assays, sedimentation velocity fractionation, conformation-dependent immunoassay (CDI), we assessed the properties of four deer CWD isolates composed of different PrP primary structures. These isolates were also passaged in transgenic mouse lines expressing cervid PRNP polymorphisms as well as into hamsters and C57BL/6 mice.

Two CWD strains (Wisc-1 and H95+) with different PrPCWD signatures and host ranges were identified. While hamsters and transgenic (Tg) mice expressing deer G96-PrPC were susceptible to Wisc-1, C57Bl/6 and Tg mice expressing deer S96-PrPC resisted the infection. In contrast, the H95+ strain resulted in clinical disease in both transgenic mouse lines (G96 and S96) as well as C57BL/6 mice but not in hamsters.Serial passage in the Tg mice revealed the co-existence of both strains in deer expressing H95/wt and H95/S96 PrPC. These findings indicate that, after inter-genotypic passage, CWD isolates are composed of heterogeneous prion conformers that are differentially selected depending on the host-PRNP genotype.Serial passage in C57BL/6 resulted in a reduction of the incubation periods indicating H95+ adaptation.Finally, to evaluate the involvement of H95-PrPC in the selection of CWD strains, we passaged these CWD allotypes in newly generated cervidized mice expressing H95, S96 or wildtype PRNP. Mice expressing H95-PrPC succumbed faster when infected with H95/wt and H95/S96 deer CWD allotypes than with S96/wt and wt/wt CWD allotypes. The proclivity of the H95+ deer prions to generate disease in these mice supports the involvement of the H95 polymorphism in the emergence of novel CWD strains.

Propagation of prions between cervids expressing distinct PrPC primary structures generates PrPCWD conformational heterogeneity affecting the host range of CWD.


P167 Passage of scrapie from white-tailed deer back to sheep results in a phenotype change

Veterinary Medical Officer Justin Greenlee1, Robyn Kokemuller1, Dr. Jo Moore1, Professor Heather West Greenlee2

1Virus and Prion Research Unit, National Animal Disease Center, USDA, ARS, Ames, United States, 2Department of Biomedical Sciences, Iowa State University College of Veterinary Medicine, Ames, United States

The US No. 13-7 scrapie isolate had a 100% attack rate in white-tailed deer (WTD) after intracranial or oronasal challenge. Samples from affected deer had two different western blot (WB) patterns: cerebrum samples had a scrapie-like profile, but brainstem samples had a CWD-like profile. In contrast, transmission of CWD from WTD to sheep by the intracranial route has a low attack rate, and to-date oronasal exposure has been unsuccessful. The purpose of this study was to determine if sheep are susceptible to oronasal challenge with the scrapie agent derived from WTD.

Suffolk lambs of PRNP genotypes VV136RR154QQ171, AA136RR154QQ171, and AV136RR154QR171 were challenged by the oronasal route with a 10% brain homogenate derived from either the cerebrum or the brainstem of scrapie-affected WTD. Upon development of clinical signs, sheep were killed and necropsied. Tissues were tested for the presence of misfolded prion protein (PrPSc) by enzyme immunoassay, WB, and immunohistochemistry.

Sheep challenged oronasally with WTD-derived scrapie developed disease only when the inoculum was prepared from the cerebrum that had a scrapie-like WB profile. The first sheep to develop clinical signs at approximately 29 months post inoculation (MPI) had the VRQ/VRQ genotype. Sheep of the ARQ/ARQ genotype also developed clinical signs, but at a mean of 52 MPI. At 62 MPI, none of the sheep inoculated with material from the deer brainstem have developed clinical disease.

The No. 13-7 scrapie inoculum used in the original deer experiment readily infects WTD and sheep of various genotypes (20 MPI for ARQ/ARQ and 27 MPI for VRQ/VRQ) by the oronasal route. When inoculum is made from different brain regions of No 13-7 scrapie-infected deer, sheep with the VRQ/VRQ genotype inoculated with homogenate from the cerebrum with the scrapie-like WB pattern are the first to develop clinical signs. This is in contrast to the original No. 13-7 inoculum that has a faster incubation period in sheep with the ARQ/ARQ genotype. Similar to experiments conducted with CWD, sheep oronasally inoculated with brainstem material from deer with a CWD-like molecular profile have no evidence of disease after 62 MPI. While scrapie is not known to occur in free-ranging populations of WTD, experimental cases are difficult to differentiate from CWD. This work raises the potential concern that scrapie infected deer could serve as a confounding factor to scrapie eradication programs as scrapie from deer seems to be transmissible to sheep by the oronasal route.


P172 Multigenerational transmission of CWD prions from mother to offspring

Erin Mcnulty1, MS Amy Nalls1, Dr Clare Hoover1, Dr Jenny Powers2, Dr Edward Hoover1, Dr Candace Mathiason1

1Colorado State University, Fort Collins, United States, 2National Park Services, Fort Collins, United States

Aims: Chronic wasting disease (CWD) continues to demonstrate geographic expansion, now found in captive and/or free-range cervid populations in North America, South Korea and Norway. While horizontal transmission is credited for much of the spread of CWD, few studies have monitored the transmission of this disease from mother-to-offspring.
CWD-infected muntjac dams are able to become pregnant, carry, deliver and rear offspring during the long asymptomatic phase of prion infection. We have demonstrated that CWD prions can be transmitted from mother to first-generation offspring leading to prion infection and subsequent development of TSE disease, and that transmission occurs during gestation (Nalls 2013). We have also observed fecundity in first-generation offspring. In fact, one first-generation female muntjac gave birth to two nonviable second-generation offspring. Tissues harvested from these nonviable second-generation offspring harbor protein misfolding cyclic amplification (PMCA) competent prions.
Recently we revealed PrPC seeding activity and infectious prions within the reproductive milieu (uterus, ovaries, placentomes, amniotic fluid) of CWD-infected Reeve’s muntjac dams by PMCA, real time quaking-induced conversion (RT-QuIC) and bioassay. The presence of CWD prions in the pregnancy microenvironment begs the question: Is it possible CWD is transmitted from one generation to the next via intrauterine or germline exposure to infectious prions?

Methods: To begin to address this question we assessed tissues harvested from full-term second-generation nonviable muntjac offspring for infectivity by mouse bioassay. Transgenic mice expressing the cervid prion protein, Tg(CerPrP-E226)5037+/- (n=6/cohort), were IP-inoculated with PMCA-amplified lung, mammary gland, kidney or uterus from nonviable 2nd generation muntjac offspring (n=2) born to a first generation dam (n=1), or PMCA-amplified age and tissue-matched negative control second-generation offspring (n=2). Mice from all cohorts were examined for prions by western blot and RTQuIC.

Results: All mice (n=20) inoculated with PMCA-amplified tissue from “gestational CWD-exposed” second-generation offspring developed signs consistent with TSE disease, including severe ataxia and weight loss between 209-373 days pi, and were confirmed CWD positive by western blot and RT-QuIC. Negative control mice (n=9) receiving PMCA-amplified negative age and tissue-matched homogenates remained healthy and TSE-free for the same duration. Studies have been initiated to further assess the relationship between prions in ovaries and CWD transmission.

Conclusions: Our data indicates that: (1) multigenerational transmission of infectious CWD prions from mother-to-offspring may be possible and (2) early and persistent exposure of the developing embryo to infectious CWD prions in the uterine microenvironment may help explain the facile transmission of CWD in the native host.


P174 Assessing the Role of Plants in Transmission of Chronic Wasting Disease

Ms. Kaitlyn Miedema1, Ms. Aimee Ortega1, Ms. Savannah Rocha1, Dr. Mark Zabel1
1Colorado State University, Department of Microbiology, Immunology and Pathology, Fort Collins , United States

Recent work has found both internal and external plant surfaces are able to bind and retain infectious prions, suggesting plants may play an important role in prion transmission. To expand on these findings, we exposed rice plants (Oryza sativa L., cultivar kitaake) to a relatively low dose of prions and examined the ability of plants to uptake prions. In addition to laboratory manipulations, we wanted to investigate if plants were contaminated with chronic wasting disease prions (PrPCWD) in a natural setting. Thus, plants were collected from two sites in Rocky Mountain National Park (RMNP) to test for PrPCWD on both external and internal plant structures. The goals of these experiments are to provide additional insight into the role of plants in PrPCWD transmission.

Rice plants were grown in a solution containing 1% of a brain homogenate from a terminally ill elk for either 2h, 24h or 72h. Tissues were collected by cutting the leaves, stem and roots with disposable razor blades. Tissues were homogenized to a 10% w/v homogenate and tested for PrPCWD by protein misfolding cyclic amplification (PMCA).
To investigate the potential role of plants in CWD transmission in RMNP, two sites were identified and plants collected using disposable spades. Soil was then carefully removed from roots and plants were individually bagged. In the lab, plant surfaces were washed and wash solution was tested for PrPCWD by PMCA. Plant tissues were then homogenized to a 10%w/v homogenate and tested for PrPCWD by PMCA.

After PMCA, we were able to detect PrPCWD in root and leaf tissue, but not in stem tissue, of experimentally exposed rice plants by 2h post exposure (hpe). At 24hpe, PrPCWD was detected in leaf, stem and root tissues after PMCA. 72hpe samples are being processed.
We detected PrPCWD from the surface of two plants at a single location in RMNP after 6 rounds of PMCA. None of the homogenized plant tissues were positive by PMCA.

From the results of the experimental manipulation studies, we conclude that rice plants are able to uptake prions when grown in water contaminated with PrPCWD. We conclude from our field studies that plant surfaces are contaminated with prions and are detectable by PMCA; however, our inability to detect PrPCWD from tissue homogenates suggests internal structures of these plants are not contaminated with PrPCWD. These results provide new insights into the role of plants in CWD transmission.


P175 Disease-associated prion protein detected in lymphoid tissues from pigs challenged with the agent of chronic wasting disease

Jo Moore1, Robert Kunkle1, Naveen Kondru2, Sireesha Manne2, Jodi Smith1,3, Anumantha Kanthasamy2, Heather West Greenlee2, Justin Greenlee1

1Virus and Prion Research Unit, National Animal Disease Center, USDA, Ames, United States, 2Department of Biomedical Sciences, Iowa State University College of Veterinary Medicine, Ames, United States, 3Department of Veterinary Pathology, Iowa State University College of Veterinary Medicine, Ames, USA, Ames, United States

Chronic wasting disease (CWD) is a naturally-occurring, fatal neurodegenerative disease of cervids. We previously demonstrated that disease-associated prion protein (PrPSc) can be detected in the brain and retina from pigs challenged intracranially or orally with the CWD agent. In that study, neurological signs consistent with prion disease were observed only in one pig: an intracranially challenged pig that was euthanized at 64 months post challenge. The purpose of this study was to use an antigen-capture immunoassay (EIA) and real-time quaking-induced conversion (RT-QuIC) to determine whether PrPSc is present in lymphoid tissues from pigs challenged with the CWD agent.

At 2 months of age, crossbred pigs were challenged by the intracranial route (n=20), oral route (n=19), or not challenged (n=9). At approximately 6 months after challenge, when pigs were of an equivalent weight to commercial market weight pigs, half of the pigs in each group were culled (<6 challenge="" groups="" months="" pigs="" remaining="" the="">6 months challenge groups) were allowed to incubate for up to 73 months post challenge. The retropharyngeal lymph node (RPLN) was screened for the presence of PrPSc by EIA and immunohistochemistry (IHC). The RPLN, palatine tonsil, and mesenteric lymph node (MLN) from 6-7 pigs per challenge group were also tested using EIA and RT-QuIC.

PrPSc was not detected by EIA or IHC in any RPLNs. All tonsils and MLNs were negative by IHC, though the MLN from one pig in the oral <6 5="" 6="" at="" by="" detected="" eia.="" examined="" group="" in="" intracranial="" least="" lymphoid="" months="" of="" one="" pigs="" positive="" prpsc="" rt-quic="" the="" tissues="" was="">6 months group, 5/6 pigs in the oral <6 4="" and="" group="" in="" months="" oral="" pigs="" the="">6 months group. The MLN was positive in 14/19 (74%) of samples examined, the RPLN in 8/18 (44%), and the tonsil in 10/25 (40%).

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 6 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.


P176 Infectious CWD prions at the fetal-maternal interface

Ms. Amy Nalls1, Ms. Erin McNulty1, Ms. Laura Pulscher1, Dr. Clare Hoover1, Dr. Edward Hoover1, Dr. Candace Mathiason1

1Colorado State University, Fort Collins, United States

Aims: Ample evidence exists for the trafficking of infectious agents across the placenta, often with grave outcomes to the developing fetus (i.e. zika, brucella, cytomegalovirus). While less studied, pregnancy-related transmissible spongiform encephalopathies (TSEs) have been implicated in several species, including humans.

Our previous work demonstrated that prions can be transferred from mother-to-offspring resulting in the development of clinical TSE disease in offspring born to CWD-infected muntjac dams (Nalls 2013). We also revealed PMCA-prion seeding activity in maternal and fetal tissues harvested in utero from muntjac dams at various stages of pregnancy and CWD infection. What remained unknown was whether the prions detected at the fetal-maternal interface were infectious. In addition, we were interested to determine if the ultrasensitive RT-QuIC methodology may enhance our ability to detect prion seeding activity within the pregnancy microenvironment.

We undertook this study to assess CWD infectivity and RT-QuIC PrPc seeding activity in in utero harvested— (1) female reproductive tissues and fluids associated with the pregnancy microenvironment; the ovary, uterus and birthing fluids, and (2) the semipermeable interface between cervid mother and fetus throughout pregnancy; the placentome.

Methods: RT-QuIC: A total of 12 replicates/sample of ovary (n=6), uterus (n=6), placentome (n=5-6 placentomes each from n=2 pregnancies) and birthing fluids (n=4) were analyzed. Bioassay: Transgenic mice expressing the cervid prion protein, Tg(CerPrP-E226)5037+/- (n=9/cohort), were IC-inoculated with 30μl 10% homogenate of uterus (n=2), placentome (n=2) or 5-fold concentrated birthing fluids (n=3).

Results: RT-QuIC: PrPC seeding activity was consistently detected in 5/6 ovary, 6/6 uterus, 9/11 placentomes, 2/2 amniotic and 0/2 allantoic fluids. Bioassay: Clinical TSE disease (ataxia, weight loss and stiff tail) was observed in mice inoculated with uterus, placentome and amniotic fluid, but not allantoic fluid between 180-343 day pi while negative control cohorts remained healthy. Bioassay mice were confirmed TSE positive: brain (uterus 7/8, placentome 8/8, amniotic fluid 1/9, allantoic fluid 0/9) and spleen (uterus 7/8, placentome 7/8, amniotic fluid 2/9, allantoic fluid 0/0) by western blot and RT-QuIC.

Conclusions: Here, using a native CWD susceptible host, we have— for the first time— demonstrated infectious prions in the cervid pregnancy microenvironment and placental structure at the fetal-maternal interface. These findings reveal a source of infectious prions that the developing fetus is exposed to long before the birthing process, maternal grooming, or encounter with contaminated environments. Thus suggesting that CWD mother-to-offspring transmission may contribute to the facile transmission of CWD and be underappreciated for all TSEs.


P178 Chronic Wasting Disease in European moose is associated with PrPSc features different from North American CWD

Dr Laura Pirisinu1, Dr Linh Tran2, Dr Gordon Mitchell3, Dr Aru Balachandran3, Dr Thierry Baron4, Dr Cristina Casalone5, Dr Michele Di Bari1, Dr Umberto Agrimi1, Dr Romolo Nonno1, Dr Sylvie Benestad2
1Department of Veterinary Public Health and Food Safety, Istituto Superiore di Sanità, Rome, Italy, 2Norwegian Veterinary Institute, Oslo, Norway, 3Canadian Food Inspection Agency, National and OIE Reference Laboratory for Scrapie and CWD, Ottawa Laboratory Fallowfield, Ottawa, Canada, 4Neurodegenerative Diseases Unit, ANSES - French Agency for Food, Environmental and Occupational Health & Safety, Lyon, France, 5Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Turin, Italy

In 2016, Chronic Wasting Disease (CWD) was detected for the first time in Europe in three wild Norwegian reindeer (Rangifer tarandus tarandus) and in two moose (Alces alces). The biochemical analysis and the immunohistochemical distribution of PrPSc from Norwegian reindeer revealed a pattern similar to North American (NA) isolates¹.
In this study, we studied the biochemical features of PrPSc from the two CWD cases in Norwegian moose.

Western blot (WB) analysis of PK-treated PrPSc (PrPres) from Norwegian moose and reindeer isolates was performed according to the ISS discriminatory WB protocol (used in BSE and scrapie Italian surveillance). PrPres fragments were determined by epitope mapping (SAF84, L42, 9A2, 12B2 mAbs), before and after deglycosylation. CWD isolates from Canadian cervids (wapiti, moose and white tailed deer) and a panel of small ruminant and bovine prion strains circulating in Europe were also analysed.

WB analysis with different mAbs showed that PrPres from both Norwegian moose samples was different from that usually associated with CWD in cervids. Indeed, their main C-terminal fragment had a MW lower than the other CWD isolates, and could be discriminated by the absence of the 12B2 epitope. Furthermore, while NA CWD PrPSc is composed of a single PrPres fragment, Norwegian moose samples had an additional C-terminal PrPres fragment of ~13 kDa (CTF13).
Among ovine TSEs, classical scrapie and Nor98 were discriminated from both Norwegian moose isolates, while CH1641 samples had molecular features partially overlapping with the moose, i.e. a low MW PrPres and the presence of CTF13. In contrast, moose PrPSc did not overlap with any bovine PrPSc. Indeed, the MW of moose PrPres was lower than H-BSE and similar to C-BSE and L-BSE PrPres, but the two bovine prions lacked additional PrPres fragments.

Unexpectedly, PrPSc from Norwegian moose revealed features substantially different from all other CWD isolates. The PrPSc pattern of Norwegian moose was also different from Canadian moose, suggesting that the variant PrPSc type observed does not simply reflect a host factor and could represent a new CWD strain. Furthermore, PrPSc of Norwegian moose can be easily discriminated from all BSE types, classical scrapie and Nor98, while showing significant overlapping only with CH1641.

Bioassay in voles will help to clarify whether the different PrPSc types observed reflect the presence of a new CWD strain in Norwegian moose, and its relationships with known animal TSEs.


¹Benestad et al. Vet Res (2016)47:88


P184 Ultrastructure And Three Dimensional Imaging Of Amyloid Deposits In Mouse Model Of Chronic Wasting Disease

Md, Phd Beata Sikorska1, PhD Christina Sigurdson2, PhD Emil Zielonka1, MD, PhD Pawel P. Liberski1
1Deaprtment of Molecular Pathology and Neuropathology, Medical University Of Lodz, Lodz, Poland, 2Department of Pathology UC San Diego, La Jolla, USA

Aims: Amyloid plaques are neuropathological hallmarks of several of human prion diseases. In animals they are observed in bovine amyloidotic spongiform encephalopathy (BASE) and chronic wasting disease (CWD). We used an animal model of chronic wasting disease to compare the morphology, ultrastructure and three dimensional structure of CWD plaques to those observed in human prion diseases.

Methods: We analysed brain specimens of Tga20 mice infected with mCWD. For electron microscopy, mice were perfused with paraformaldehyde and glutaraldehyde. For confocal laser microscopy we used formalin fixed and paraffin embedded specimens. Antibodies against PrP, CD68, Iba-1, GFAP and NFP were used for single and double stainings. We used visualization software to show 3D reconstruction of the misfolded protein aggregates.

Results: By electron microscopy, we observed three types of amyloid deposits: unicentric plaques similar to kuru plaques in human prion diseases, large areas of amyloid fibrils which did not form round “star-like” image but formed irregular shapes with radially spreading fibrils at the margin which we called “irregular plaques” and large amorphic aggregates of amyloid. Some of the large deposits were subpial whereas plaques and plaque-like structures were often located perivascularly. The plaques were totally devoid of dystrophic neuritic elements. However, they were invaded by activated microglial cells. By confocal laser imaging we were able to show the 3D structure of the irregular palques with neuronal processes surrounding the plaques or to some extent intermingled with the amyloid fibres within the plaque, yet these axons did not show features of neuritic dystrophy. Double staining with microglial markers showed
prominent microglial reaction around the plaques.

Conclusions:. Apart from amyloid plaques similar to kuru plaques in human prion diseases, in mCWD we observed two types of unique PrPSc depostits: 1. large irregular plaques which did not form round “star-like” appearance of kuru plaques and 2. large amorphic aggregates of amyloid. As opposed to plaques in vCJD and GSS neither the kuru-like mCWD plaques nor the irregular plaques contained dystrophic neurites.


P219 Frist 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; 2Universitätsklinikum des Saarlandes und Medizinische Fakultät der Universität 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.





Thursday, June 29, 2017 


THURSDAY, JUNE 22, 2017 

PRION 2017 CONFERENCE P120 Early preclinical detection of prions in blood of macaques peripherally infected with the variant CJD agent

THURSDAY, JUNE 22, 2017 

National Prion Disease Pathology Surveillance Center Cases Examined(1) (May 18, 2017)

2001 FDA CJD TSE Prion Singeltary Submission



*** 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).***

Monday, May 02, 2016

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

*** 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. 

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

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

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

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

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

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

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

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

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


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

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

*** 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.

Saturday, April 23, 2016 

PRION 2016 TOKYO Saturday, April 23, 2016 

SCRAPIE WS-01: Prion diseases in animals and zoonotic potential 2016 Prion. 10:S15-S21. 2016 ISSN: 1933-6896 printl 1933-690X online Taylor & Francis Prion 2016 Animal Prion Disease Workshop 

Abstracts WS-01: Prion diseases in animals and zoonotic potential 

Juan Maria Torres a, Olivier Andreoletti b, J uan-Carlos Espinosa a. Vincent Beringue c. Patricia Aguilar a, Natalia Fernandez-Borges a. and Alba Marin-Moreno a "Centro de Investigacion en Sanidad Animal ( CISA-INIA ). Valdeolmos, Madrid. Spain; b UMR INRA -ENVT 1225 Interactions Holes Agents Pathogenes. ENVT. Toulouse. France: "UR892. Virologie lmmunologie MolécuIaires, Jouy-en-Josas. France 

Dietary exposure to bovine spongiform encephalopathy (BSE) contaminated bovine tissues is considered as the origin of variant Creutzfeldt Jakob (vCJD) disease in human. To date, BSE agent is the only recognized zoonotic prion. Despite the variety of Transmissible Spongiform Encephalopathy (TSE) agents that have been circulating for centuries in farmed ruminants there is no apparent epidemiological link between exposure to ruminant products and the occurrence of other form of TSE in human like sporadic Creutzfeldt Jakob Disease (sCJD). However, the zoonotic potential of the diversity of circulating TSE agents has never been systematically assessed. The major issue in experimental assessment of TSEs zoonotic potential lies in the modeling of the ‘species barrier‘, the biological phenomenon that limits TSE agents’ propagation from a species to another. In the last decade, mice genetically engineered to express normal forms of the human prion protein has proved essential in studying human prions pathogenesis and modeling the capacity of TSEs to cross the human species barrier. To assess the zoonotic potential of prions circulating in farmed ruminants, we study their transmission ability in transgenic mice expressing human PrPC (HuPrP-Tg). Two lines of mice expressing different forms of the human PrPC (129Met or 129Val) are used to determine the role of the Met129Val dimorphism in susceptibility/resistance to the different agents. These transmission experiments confirm the ability of BSE prions to propagate in 129M- HuPrP-Tg mice and demonstrate that Met129 homozygotes may be susceptible to BSE in sheep or goat to a greater degree than the BSE agent in cattle and that these agents can convey molecular properties and neuropathological indistinguishable from vCJD. However homozygous 129V mice are resistant to all tested BSE derived prions independently of the originating species suggesting a higher transmission barrier for 129V-PrP variant. Transmission data also revealed that several scrapie prions propagate in HuPrP-Tg mice with efficiency comparable to that of cattle BSE. While the efficiency of transmission at primary passage was low, subsequent passages resulted in a highly virulent prion disease in both Met129 and Val129 mice. Transmission of the different scrapie isolates in these mice leads to the emergence of prion strain phenotypes that showed similar characteristics to those displayed by MM1 or VV2 sCJD prion. 

***These results demonstrate that scrapie prions have a zoonotic potential and raise new questions about the possible link between animal and 
human prions.

***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.***

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

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



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.


Spongiform Encephalopathy in Captive Wild ZOO BSE INQUIRY

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

Evidence That Transmissible Mink Encephalopathy Results from Feeding Infected Cattle
Over the next 8-10 weeks, approximately 40% of all the adult mink on the farm died from TME.
The rancher was a ''dead stock'' feeder using mostly (>95%) downer or dead dairy cattle...
In Confidence - Perceptions of unconventional slow virus diseases of animals in the USA - APRIL-MAY 1989 - G A H Wells
3. Prof. A. Robertson gave a brief account of BSE. The US approach was to accord it a very low profile indeed. Dr. A Thiermann showed the picture in the ''Independent'' with cattle being incinerated and thought this was a fanatical incident to be avoided in the US at all costs. ...
*** The potential impact of prion diseases on human health was greatly magnified by the recognition that interspecies transfer of BSE to humans by beef ingestion resulted in vCJD. While changes in animal feed constituents and slaughter practices appear to have curtailed vCJD, there is concern that CWD of free-ranging deer and elk in the U.S. might also cross the species barrier. Thus, consuming venison could be a source of human prion disease. Whether BSE and CWD represent interspecies scrapie transfer or are newly arisen prion diseases is unknown. Therefore, the possibility of transmission of prion disease through other food animals cannot be ruled out. There is evidence that vCJD can be transmitted through blood transfusion. There is likely a pool of unknown size of asymptomatic individuals infected with vCJD, ***

*** and there may be asymptomatic individuals infected with the CWD equivalent. 

*** These circumstances represent a potential threat to blood, blood products, and plasma supplies. 

THURSDAY, JUNE 22, 2017 

World Organisation for Animal Health (OIE) to establish liaison office in College Station, Texas

15 November 1999
British Medical Journal
vCJD in the USA * BSE in U.S.
2 January 2000
British Medical Journal
U.S. Scientist should be concerned with a CJD epidemic in the U.S., as well
Diagnosis and Reporting of Creutzfeldt-Jakob Disease
Singeltary, Sr et al. JAMA.2001; 285: 733-734. Vol. 285 No. 6, February 14, 2001 JAMA
Diagnosis and Reporting of Creutzfeldt-Jakob Disease
To the Editor: In their Research Letter, Dr Gibbons and colleagues1 reported that the annual US death rate due to Creutzfeldt-Jakob disease (CJD) has been stable since 1985. These estimates, however, are based only on reported cases, and do not include misdiagnosed or preclinical cases. It seems to me that misdiagnosis alone would drastically change these figures. An unknown number of persons with a diagnosis of Alzheimer disease in fact may have CJD, although only a small number of these patients receive the postmortem examination necessary to make this diagnosis. Furthermore, only a few states have made CJD reportable. Human and animal transmissible spongiform encephalopathies should be reportable nationwide and internationally.
Terry S. Singeltary, Sr Bacliff, Tex
1. Gibbons RV, Holman RC, Belay ED, Schonberger LB. Creutzfeldt-Jakob disease in the United States: 1979-1998. JAMA. 2000;284:2322-2323.
26 March 2003
Terry S. Singeltary, retired (medically) CJD WATCH
I lost my mother to hvCJD (Heidenhain Variant CJD). I would like to comment on the CDC's attempts to monitor the occurrence of emerging forms of CJD. Asante, Collinge et al [1] have reported that BSE transmission to the 129-methionine genotype can lead to an alternate phenotype that is indistinguishable from type 2 PrPSc, the commonest sporadic CJD. However, CJD and all human TSEs are not reportable nationally. CJD and all human TSEs must be made reportable in every state and internationally. I hope that the CDC does not continue to expect us to still believe that the 85%+ of all CJD cases which are sporadic are all spontaneous, without route/source. We have many TSEs in the USA in both animal and man. CWD in deer/elk is spreading rapidly and CWD does transmit to mink, ferret, cattle, and squirrel monkey by intracerebral inoculation. With the known incubation periods in other TSEs, oral transmission studies of CWD may take much longer. Every victim/family of CJD/TSEs should be asked about route and source of this agent. To prolong this will only spread the agent and needlessly expose others. In light of the findings of Asante and Collinge et al, there should be drastic measures to safeguard the medical and surgical arena from sporadic CJDs and all human TSEs. I only ponder how many sporadic CJDs in the USA are type 2 PrPSc?
re-Human Prion Diseases in the United States
Posted by flounder on 01 Jan 2010 at 18:11 GMT

Tracking spongiform encephalopathies in North America

Xavier Bosch

Published: August 2003

DOI: http://dx.doi.org/10.1016/S1473-3099(03)00715-1


“My name is Terry S Singeltary Sr, and I live in Bacliff, Texas. I lost my mom to hvCJD (Heidenhain variant CJD) and have been searching for answers ever since. What I have found is that we have not been told the truth. CWD in deer and elk is a small portion of a much bigger problem.”

49-year-old Singeltary is one of a number of people who have remained largely unsatisfied after being told that a close relative died from a rapidly progressive dementia compatible with spontaneous Creutzfeldt-Jakob disease (CJD). So he decided to gather hundreds of documents on transmissible spongiform encephalopathies (TSE) and realised that if Britons could get variant CJD from bovine spongiform encephalopathy (BSE), Americans might get a similar disorder from chronic wasting disease (CWD) the relative of mad cow disease seen among deer and elk in the USA. Although his feverish search did not lead him to the smoking gun linking CWD to a similar disease in North American people, it did uncover a largely disappointing situation.

Singeltary was greatly demoralised at the few attempts to monitor the occurrence of CJD and CWD in the USA.  Only a few states have made CJD reportable. Human and animal TSEs should be reportable nationwide and internationally, he complained in a letter to the Journal of the American Medical Association (JAMA 2003; 285: 733).  "I hope that the CDC does not continue to expect us to still believe that the 85% plus of all CJD cases which are sporadic are all spontaneous, without route or source."

Diagnosis and Reporting of Creutzfeldt-Jakob Disease
Singeltary, Sr et al. JAMA.2001; 285: 733-734. Vol. 285 No. 6, February 14, 2001 JAMA
Diagnosis and Reporting of Creutzfeldt-Jakob Disease
To the Editor: In their Research Letter, Dr Gibbons and colleagues1 reported that the annual US death rate due to Creutzfeldt-Jakob disease (CJD) has been stable since 1985. These estimates, however, are based only on reported cases, and do not include misdiagnosed or preclinical cases. It seems to me that misdiagnosis alone would drastically change these figures. An unknown number of persons with a diagnosis of Alzheimer disease in fact may have CJD, although only a small number of these patients receive the postmortem examination necessary to make this diagnosis. Furthermore, only a few states have made CJD reportable. Human and animal transmissible spongiform encephalopathies should be reportable nationwide and internationally.
Terry S. Singeltary, Sr Bacliff, Tex
1. Gibbons RV, Holman RC, Belay ED, Schonberger LB. Creutzfeldt-Jakob disease in the United States: 1979-1998. JAMA. 2000;284:2322-2323.

Until recently, CWD was thought to be confined to the wild in a small region in Colorado. But since early 2002, it has been reported in other areas, including Wisconsin, South Dakota, and the Canadian province of Saskatchewan. Indeed, the occurrence of CWD in states that were not endemic previously increased concern about a widespread outbreak and possible transmission to people and cattle.

To date, experimental studies have proven that the CWD agent can be transmitted to cattle by intracerebral inoculation and that it can cross the mucous membranes of the digestive tract to initiate infection in lymphoid tissue before invasion of the central nervous system. Yet the plausibility of CWD spreading to people has remained elusive.

Part of the problem seems to stem from the US surveillance system. CJD is only reported in those areas known to be endemic foci of CWD. Moreover, US authorities have been criticised for not having performed enough prionic tests in farm deer and elk.

Although in November last year the US Food and Drug Administration issued a directive to state public-health and agriculture officials prohibiting material from CWD-positive animals from being used as an ingredient in feed for any animal species, epidemiological control and research in the USA has been quite different from the situation in the UK and Europe regarding BSE.

"Getting data on TSEs in the USA from the government is like pulling teeth", Singeltary argues. "You get it when they want you to have it, and only what they want you to have."

Norman Foster, director of the Cognitive Disorders Clinic at the University of Michigan (Ann Arbor, MI, USA), says that  "current surveillance of prion disease in people in the USA is inadequate to detect whether CWD is occurring in human beings"; adding that, "the cases that we know about are reassuring, because they do not suggest the appearance of a new variant of CJD in the USA or atypical features in patients that might be exposed to CWD. However, until we establish a system that identifies and analyses a high proportion of suspected prion disease cases we will not know for sure". The USA should develop a system modelled on that established in the UK, he points out.

Ali Samii, a neurologist at Seattle VA Medical Center who recently reported the cases of three hunters "two of whom were friends" who died from pathologically confirmed CJD, says that "at present there are insufficient data to claim transmission of CWD into humans"; adding that "[only] by asking [the questions of venison consumption and deer/elk hunting] in every case can we collect suspect cases and look into the plausibility of transmission further". Samii argues that by making both doctors and hunters more aware of the possibility of prions spreading through eating venison, doctors treating hunters with dementia can consider a possible prion disease, and doctors treating CJD patients will know to ask whether they ate venison.

CDC spokesman Ermias Belay says that the CDC "will not be investigating the [Samii] cases because there is no evidence that the men ate CWD-infected meat". He notes that although "the likelihood of CWD jumping the species barrier to infect humans cannot be ruled out 100%" and that "[we] cannot be 100% sure that CWD does not exist in humans& the data seeking evidence of CWD transmission to humans have been very limited". 



Terry S. Singeltary Sr.


Post a Comment

Subscribe to Post Comments [Atom]

<< Home