Prion

ISSN: 1933-6896 (Print) 1933-690X (Online) Journal homepage: http://www.tandfonline.com/loi/kprn20

Managing prion disease risk To cite this article: (2011) Managing prion disease risk, Prion, 5:sup1, 123-145, DOI: 10.4161/ pri.15901 To link to this article: http://dx.doi.org/10.4161/pri.15901

Published online: 01 Apr 2011.

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Date: 19 January 2017, At: 01:35

PRION 2011: POSTER PRESENTATIONS

REVIEW

Prion 5: Supplement, 123-145; April/May/June 2011; © 2011 Landes Bioscience

Managing prion disease risks Risk.01: Lower Risk of Creutzfeldt-Jakob Disease in Pituitary Growth Hormone Recipients Initiating Treatment After 1977 Joseph Abrams,1,† Lawrence B. Schonberger,1 Ermias D. Belay,1 Ryan A. Maddox,1 Ellen W. Leschek,2 James L. Mills,2 Diane K. Wysowski3 and Judith E. Fradkin2 Centers for Disease Control and Prevention; Atlanta, GA USA; 2National Institutes of Health; Bethesda, MD USA; 3Food and Drug Administration; Rockville, MD USA † Presenting author; Email: [email protected]

1

Context. Creutzfeldt-Jakob disease (CJD) caused by contaminated cadaveric pituitary-derived human growth hormone (hGH) has been responsible for hundreds of deaths worldwide. Studies of US National Hormone and Pituitary Program (NHPP) hGH recipients have only found CJD in patients treated prior to 1977, when a new purification procedure with column chromatography was implemented for hGH extraction. Objective. To provide updated information on transmission of CJD to NHPP hGH recipients and determine if recipients of hGH produced after 1977 had a significantly lower CJD risk than pre-1977 recipients. Design. Data from the cohort of NHPP hGH recipients were studied. Risk of CJD adjusted for the duration of hGH treatment and years from first hGH treatment were calculated for pre-1977 and post-1977 recipients. Monte Carlo repeated sampling was utilized to estimate the proportion of post-1977 recipients with sufficient follow-up time for CJD infection to progress to CJD death. Main outcome measure. Use of likelihood functions to determine if the observed number of CJD cases in the post-1977 cohort was significantly fewer than expected if the CJD risk was equal to that of the pre-1977 cohort, controlling for treatment duration and follow-up time. Results. A total of 5570 patients was included in the analysis: 2099 in the pre-1977 cohort, 3471 in the post-1977 cohort. All 22 CJD cases (diagnosed from 1984 through 2009) occurred in the pre-1977 hGH recipients. Almost half (47.9%) of pre-1977 recipients had a treatment duration of ≥ 5 years compared to only 13.8% for post-1977 recipients. An estimated 28.2% of recipients in the post-1977 cohort were predicted to have sufficient follow-up time for CJD transmission to be detected. Based on the rates present in the pre-1977 cohort, the probability of observing no cases in the post-1977 cohort by chance alone was low (p = 0.0019). Conclusions. Risk of acquiring CJD was significantly lower for post-1977 NHPP hGH recipients than for pre-1977 recipients, suggesting that the new purification procedure in 1977 may have greatly reduced or eliminated CJD agent in hGH.

Risk.02: Proteomic Analysis of Ovine Sera to Identify Biomarkers Linked to Natural Scrapie Maria Mazza,1 Chiara Guglielmetti,1 Francesca Martucci,1 Marianna Pagano,1 Paola Marconi,2 Gianfranco Santagada,3 Pasquale Troiano,4 Maurizio Bruschi,5 Giovanni Candiano5 and Pier L. Acutis,1,† 1 Istituto Zooprofilattico del Piemonte, liguria e Valle d’Aosta; Torino, Italy; Istituto Zooprofilattico Sperimentale del Lazio e della Toscana; Firenze, Italy; 3 Istituto Zooprofilattico Sperimentale della Puglia e Basilicata; Matera, Italy; 4 Istituto Zooprofilattico Sperimentale della Puglia e Basilicata; Foggia, Italy; 5 Laboratory on Pathophysiology of Uremia Giannina Gaslini Children Hospital; Genova, Italy † Presenting author; Email: [email protected] 2

The diagnosis of scrapie and other transmissible spongiform encephalopathies (TSEs) is currently based on the post-mortem detection of the pathological isoform of the prion protein (PrPsc). The research of PrPsc in lymphoid tissue biopsies or biological fluids is not always a valid ante-mortem method to diagnose scrapie, so different studies were performed to identify alternative biomarkers in accessible tissues or body fluids. With this purpose we carried out a study on the sera of sheep with natural scrapie by two dimensional gel electrophoresis. Ovine serum samples were collected from four different Italian naturally scrapie-affected flocks. After rapid and confirmatory tests, 20 animals were chosen, ten positive susceptible and ten negative resistant or semi-resistant sheep, coupled by age, sex and breed. Serum samples were quantified and loaded on 17 cm pH310NL IPGs Strips. Second dimension was carried out on polyacrilamide gradient gels, which were stained with different methods. Images were compared by means of the Bionumerics 2D software (Applied Maths), assessing the presence or absence of spots and intensity variations, between paired samples and between the two groups of animals. At present, four positive and four negative serum samples were analyzed. Based on the preliminary data analysis no differences were found. The completion of the analysis of all the sampled animals, with higher resolution protocols, will clarify whether scrapie status can be detected by the identification of indirect markers.

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Risk.04: Demographic and Diagnostic Differences in Minorities with Sporadic Creutzfeldt-Jakob Disease

Risk.05: Sophisticated Equipments at the Service of the Prion Research Community

Brian S. Appleby,1,† Kristin K. Appleby2, 3 and Mitchell T. Wallin2

Fabien Aubry† and Valérie de Broglie

Johns Hopkins University School of Medicine; Baltimore, MD USA; 2Veterans Affairs Medical Center; Washington, DC USA; 3Parkinson’s and Movement Disorders Center of Maryland; Elkridge, MD USA † Presenting author; Email: [email protected]

†

1

Sporadic Creutzfeldt-Jakob disease (sCJD) is the most common form of human prion disease. Prior epidemiologic studies of CJD in the U.S.A have reported a lower age adjusted incidence rate for blacks compared to whites. The goal of this study was to explore possible demographic and diagnostic features that could explain this finding. Method. Previously collected data from Johns Hopkins and the Veterans Affairs Health Care System (VHS) between 19952007 were analyzed in this study following IRB approval. Only probable and definite cases of sCJD were included in the final analyses. Caucasian and Hispanic subjects were characterized as white and all other ethnicities were considered non-white in analyses. Chi-square analyses were used for categorical variables and Kaplan-Meier analyses were used for continuous variables. Results. 116 subjects [n = 100 (86.2%) Caucasian, n = 6 (5.2%) black, n = 6 (5.2%) Hispanic, and n = 4 (3.4%) other race] were included in this study. Age at disease onset differed significantly between whites (mean = 65.2 ± 0.89 years) and non-whites (mean = 57.8 ± 2.94 years) (Log Rank = 5.32, p = 0.021). The correct clinical diagnosis was determined more rapidly in non-whites (46.7 ± 19.5 days) compared to whites (197.3 ± 35.6 days) (Log Rank = 7.08, p = 0.008). Although tissue diagnosis did not differ significantly between groups, nonwhites were less likely to undergo autopsy (1/10, 10%) compared to whites (51/103, 49.5%) (Fisher’s exact test, 2-sided, p = 0.02). Conclusions. In this study population, non-whites had an earlier age at disease onset compared to whites. Non-whites also received the correct clinical diagnosis more quickly compared to whites and were less likely to undergo autopsy. Given these results, it is unclear if non-whites truly have a younger age at onset compared to whites as this may be the result of ascertainment bias. The much more rapid clinical diagnosis of non-whites suggests that a diagnosis of CJD was considered earlier in nonwhites who had an earlier age at disease onset. The decreased autopsy rate of non-whites is also concerning as this may falsely lower the estimated incidence of sCJD in non-whites in epidemiologic studies that rely heavily on neuropathologic data. Further studies examining the incidence and diagnostic process of sCJD in non-whites is needed. This study also demonstrates the importance of including patients that are diagnosed clinically and who do not undergo autopsies in CJD surveillance efforts.

Alliance Biosecure Foundation; Paris, France Presenting author; Email: [email protected]

In 2011, for the fifth consecutive year, the Alliance Biosecure Foundation is pleased to announce the opening of its new call of projects, selected by a Scientific Board which comprises of international experts. The highest priority will be given to research projects aimed at minimizing biological risk from prions. This year again, the Foundation Alliance Biosecure will makes available for use at a BSL-3 level (Neuroprion Research Platform, CEA, Paris) sophisticated equipments, adapted for the study of neurodegeneratives diseases. These equipments are composed of: (1) an influx cell sorter, with an enclosed decontaminable sort chamber, three lasers and 12 detectors, for the study of mammalians cells, blood cells, bacteria, yeast, virus, infected or not; (2) A nikon confocal microscope, with a thermostatic chamber for the study of living cells. Here, we present several results showing the interesting capabilities of these different devices. If you are interested to use one of these equipments or a combination for your studies, don’t hesitate to look for more information on the Foundation website (www.fondation-alliance-biosecure.org) or to contact Fabien Aubry (fabien.aubry@ cea.fr).

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Risk.06: Could National Mortality Registers Provide Reliable Indicators for Creutzfeldt-Jakob Disease Surveillance? A Study in France from the 2000-2008 Mortality Data Jean-Philippe Brandel,1, 5, 7,† Arlette Welaratne,1 Dominique Salomon,2, 7 Isabelle Capek,3 Véronique Vaillant,3 Albertine Aouba,4 Stéphane Haïk5, 7, 1 and Annick Alpérovitch2, 8 1 APHP Groupe Hospitalier Pitié Salpêtrière; Paris, France; 2INSERM U708 Neuroepidemiology; Paris, France; 3Institut de Veille Sanitaire; Saint Maurice, France; 4 INSERM CépiDc; Le Vésinet, France; 5INSERM UMR-S 975 Equipe maladie d’Alzheimermaladies à prions; Paris, France; 6CNRS UMR 7225; Paris, France; 7Université Pierre et Marie Curie-Paris; Paris, France † Presenting author; Email: [email protected]

Active surveillance of Creutzfeldt-Jakob disease, which has been implemented in European countries, requires important human resources and funding. As the epidemic of variant CJD due to the bovine spongiform encephalopathy agent is in decline, less intense surveillance systems, based on routine mortality or morbidity registers, could be considered. CJD data collected by the French national CJD surveillance centre were compared with CJD data registered in the national mortality statistics. From

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2000 to 2008, the two sources reported fairly similar numbers of CJD deaths (1188 and 1221 for the surveillance centre and the mortality register respectively). However, analysis of individual data showed important between-sources disagreements. At least 13% of CJD reported by the mortality register were false positive diagnoses and 21.6% of the CJD cases diagnosed by the surveillance centre were not registered as CJD in the national mortality statistics. For 126 deaths registered as CJD in the mortality statistics that had not been notified to the surveillance center, available data did not allow CJD diagnosis to be confirmed or excluded. One out of 22 variant CJD cases was not reported as having any type of CJD in the mortality statistics. Without further investigation, the conclusion could have been that CJD surveillance could be based on routinely collected mortality data. Considering the uncertainties on the evolution of the vCJD epidemics and the emergence of novel prion diseases in animals consumed by humans with zoonotic potential, these results support the idea that an active surveillance should be maintained to provide reliable data on future cases that may arise in next decades.

Risk.08: Estimating Community Dependence on Caribou for Meat in Northern Canadian Communities Angie Chiu,† Ellen Goddard and Brenda Parlee †

Risk.07: Prion Reduction of Blood Components by Affinity Filtration: Performance and Clinical Experience

University of Alberta; Edmonton, AB Canada Presenting author; Email: [email protected]

Chronic wasting disease (CWD) found in wild and domestic deer, elk, and moose in the US and Canada may potentially affect caribou (Rangifer tarandus) found in the northern regions of Canada. From the published literature, caribou is the most widely harvested and consumed animal in many communities. A major animal health risk such as chronic wasting disease may have significant impacts on the ability of households to secure an appropriate diet, which may depend on individual or community characteristics. Dietary recall data collected in 2005 by Sharma et al., 2009 and 2010, across four communities, two in the Inuvialuit Settlement Region and two in Nunavut, Canada, are used to assess the intake of caribou and other foods, both harvested and store-bought. The decision of whether to consume a particular food product and the share of expenditure on a particular product are modeled as a function of individual- and community-level variables. These communities represent socioeconomic diversity in the region, with two communities having high levels of industrial activity and two relatively low levels of development. Community population size ranges from about 800–3500 (Statistics Canada, 2006). Average employment rate is 53% (s.d. = 14.1), while average median income is $57,802 (s.d. 19068) (Statistics Canada, 2006). From the sample, community populations show ranges in percentage male from 42–50%, and the community employment rates vary from 5–51%. With respect to food availability, communities face distances varying from under 100 to over 200 kilometres to caribou harvesting sites. The communities studied have as many as seven retail food stores and as few as two. The econometric model estimates provide indicators of which factors strongly influence community status as a high- or low-caribou intake community. These results and the results of traditional harvest studies can be used as indicators of caribou dependence in other parts of Northern Canada, including the 31 communities in the Northwest Territories, 25 in Nunavut, 14 in Inuit Quebec and six in Labrador. Based on

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Patrick V. Gurgel1 and Steve J. Burton2,†

ProMetic BioSciences USA; Cary, NC USA; 2ProMetic BioSciences; Cambridge, UK † Presenting author; Email: [email protected]

1

complement activation, no coagulation activation, nor platelet activation. The use of the P-Capt filter caused a small loss of hemoglobin due to red cell entrapment within the volume of the filter. To date, clinical studies conducted by European blood services have shown no serious adverse reactions to the prion-reduced red cells and no new antibodies produced as a result of the introduction of the prion removal step. These results indicate that prion removal using affinity ligands is safe, does not affect the quality of the RBC product, and may be implemented to provide an additional layer of protection against vCJD transmission by blood components.

In view of the inherent difficulties in detecting vCJD prion in blood, prion removal is currently the only viable option for increasing blood safety with respect to transfusion transmission. In response, specific prion binding affinity resins (PrioClear™) have been developed and shown to remove over 4 logs of prion infectivity from human leucodepleted red blood cell concentrate spiked with 263K hamster brain homogenate. One of these resins was also effective in removing endogenous blood-borne TSE to below the limit of bioassay detection. These prion binding resins have the ability to bind TSE agents even in the presence of a 10E7 excess of other proteins. A PrioClear prion binding affinity resin forms the basis of the P-CaptTM device, manufactured by MacoPharma SA (Tourcoing, France). The device is designed to reduce prion infectivity from leucodepleted red blood cell concentrates (RBCs), and has been tested extensively by third parties in regards to its safety, efficacy, and effect on blood quality. The P-Capt filter has been shown to bind a wide range of mammalian prions and studies with 236K hamster brain infectivity demonstrate the filter has a prion binding capacity in excess of 10E7 log10 ID50 by western blot and 10E8 log10 ID50 by infectivity biosassay. Independent studies have shown that P-Capt filtered red cells caused no serious adverse events in healthy volunteers or recruited patients that received one or more units of leucodepleted, prionreduced RBCs. The RBCs maintained their expected shelf-life with observed hemolysis well within the acceptable limit, no

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the set of socio-economic and wildlife characteristics gathered for these other communities from the literature, communities may be categorized by being low- or high-caribou intake and therefore have higher- or lower vulnerability to possible changes in the caribou food supply due to CWD.

Tau and S-100B may be optimal choices for many sCJD case investigations. Reporting of quantitative assay results as well as combining Tau and S-100B could enhance the clinical utility of surveillance case definitions for sCJD. Risk.11: A Position Paper on the Relaxation of the Feed Ban in Europe

Risk.10: CSF Proteins and Diagnosis of Sporadic Creutzfeldt-Jakob Disease in Canada

Linda A. Detwiler,1,† Ulrich Sperling,2 Conrad Brunk,3 Adriano Aguzzi,4 Klaus Grunert5 and Philip Comer6

Michael B. Coulthart,1,† Gerard H. Jansen,2 Elina Olsen,3 Deborah L. Godal,1 Tim Connolly,3 Bernard C. Choi,3 Zheng Wang3 and Neil R. Cashman4

College of Veterinary Medicine; Mississippi State University; Millstone Township, NJ USA; 2The TAFS Forum; Bern, Switzerland; 3The University of Victoria; Victoria, BC Canada; 4Institute of Neuropathology; University Hospital of Zurich; Zurich, Switzerland; 5MAPP Centre for Research; Aarhus University; Aarhus, Denmark; 6 Det Norske Veritas; London, UK † Presenting author; Email: [email protected]

1

Public Health Agency of Canada; Winnipeg, MB Canada; 2University of Ottawa; Ottawa, ON Canada; 3Public Health Agency of Canada; Ottawa, ON Canada; 4 University of British Columbia; Vancouver, BC Canada † Presenting author; Email: [email protected]

1

Background. With its range of initial symptoms that may accompany other conditions, and a frequent need for timely diagnosis, sporadic Creutzfeldt-Jakob disease (sCJD) can present the clinician with significant challenges. Particularly widely employed for this purpose are assays for certain brain proteins in cerebrospinal fluid (CSF). Data are needed to better support systematic revision of diagnostic probabilities for sCJD on the basis of CSF protein assay results, in patient populations that also include diverse subacute encephalopathies eliciting a clinical suspicion of sCJD. Methods. CSF 14-3-3, total Tau and S-100B proteins were studied prospectively in 948 Canadian patients suspected of having sCJD, including 121 with autopsy-confirmed sCJD and 827 with probable non-CJD diagnoses. Various metrics of diagnostic accuracy including sensitivity, specificity, predictive values and likelihood ratios were estimated. Results. Estimated diagnostic accuracy for individual markers were mostly consistent with those of previously published studies at optimal cutoff thresholds for this study population (empirically defined for 14-3-3 immunoblot; 976 pg/mL for Tau; 2.5 ng/mL for S-100B). Sensitivity and specificity estimates respectively at these thresholds were 0.88 (95% CI, 0.81–0.93) and 0.71 (0.68–0.74) for 14-3-3; 0.90 (0.83–0.95) and 0.87 (0.85–0.90) for Tau; and 0.86 (0.78–0.91) and 0.86 (0.84–0.89) for S-100B; thus, the only outlier was 14-3-3 specificity (~0.7). Positive likelihood ratio (LR+) estimates were low to moderate: 3.0 (2.8–3.3) for 14-3-3; 7.1 (6.6–7.6) for Tau and 6.3 (5.8–6.8) for S-100B at optimal cutoff thresholds. Negative likelihood ratios were moderate: 0.17 (0.10–0.30) for 14-3-3; 0.12 (0.07–0.2) for Tau; and 0.17 (0.10–0.30) for S-100B. Interval LR estimates strengthened accuracy for patient subsets—for example, 31.4% of sCJD patients displayed extreme CSF Tau levels (>12 000 pg/mL), associated with an LR of 64.0 (23.3–175.9). Combining Tau and S-100B results, even at intermediate values, also enhanced accuracy; e.g., LR+ = 55.6 (20.1–153.7) with Tau > 5000 pg/mL and S-100B > 5.0 ng/mL. Conclusions. CSF Tau and S-100B show comparable or better diagnostic accuracy compared to 14-3-3 in a heterogeneous patient population with low average pre-test probability of sCJD.

Epidemiological evidence implicated contaminated rendered meat and bone meal as the source of the BSE epidemic in the UK, continental Europe, as well as a few other countries around the world. With the overall global decline of BSE cases, national governments are beginning to explore the possibility of relaxing some of the measures taken to bring the disease under control. The position paper examines the current scientific knowledge and other facets that may impact decisions regarding the feed bans. The paper was written and adopted by the members of the International Forum for Transmissible Animal Diseases and Food Safety (TAFS), a nonprofit Swiss Foundation. TAFS is an international platform created by a group of scientists, food industry experts, animal health regulators, epidemiologists, diagnosticians, food producers, and consumers. Its purpose is to establish and maintain lines of communication for the dissemination of reliable information to the public that can maintain confidence in the safety of food with regard to Transmissible Animal Diseases (TAD).

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Risk.12: Transmission of Atypical Italian sCJD Case to Humanized Mice Reveals a Novel Infectious Strain Roberta Galeno,1,† Marco Sbriccoli,1 Loredana Ingrosso,1 Silvia Graziano,1 Angelina Valanzano,1 Anna Poleggi,1 Angela De Pascalis,1 Anna Ladogana,1 Franco Cardone,1 Maria Puopolo,1 Gianluigi Zanusso2 and Maurizio Pocchiari1 Istituto Superiore di Sanità; Rome, Italy; 2University of Verona; Verona, Italy † Presenting author; Email: [email protected]

1

Sporadic Creutzfeldt-Jakob disease (sCJD) is a neurodegenerative prion disorder with uncertain etiology characterized by a typical combination of clinical symptoms, neuropathological lesions, and by the deposition of the pathological protein PrPTSE in the brain. The vast majority of patients affected by sCJD can be categorized according to the genotype at the polymorphic position

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129 of PrP (methionine or valine) and to the molecular mass of PrPTSE (type 1 or 2, corresponding to 21 or 19 kDa), yielding six possible combinations (MM1, MM2, VV1, VV2, MV1, and MV2) that associate with five clinico-pathological variants. Transmission studies of these sCJD subtypes into transgenic mice expressing the human prion protein allowed to identify four different infectious strains, which can partly explain the heterogeneity observed in sCJD patients.1 We recently described a novel molecular and pathological phenotype of sCJD (MV at position 129 of PrP), associated with an unprecedented electrophoretic pattern of PrPTSE characterized by the absence of the highly glycosylated isoform. In this work, we sought to characterize the prion strain associated with this atypical case by intracerebral inoculation into gene-targeted transgenic mice (HuTg) carrying the human PRNP gene with the three 129 genotype combinations. For comparison, three Italian sCJD cases heterozygous at position 129 of the prion protein, belonging to different subtypes (MV1, MV1/2, MV2), were transmitted to the same panel of transgenic mice. Survival times, attack rates, lesion profiles, and molecular analysis of the PrPTSE type recovered from mouse brains injected with the atypical case were compared with data from control animals. Mice inoculated with the atypical case displayed a restricted host tropism, with only a small number of VV animals that resulted PrPTSEpositive after an exceedingly long survival time. Interestingly, PrPTSE accumulated in brains from these mice lacks the diglycosylated band similar to that in sCJD inoculum, yet dissimilar to any other PrPTSE observed in HuTg mice by us and by other authors.1,2 Overall, these results strongly indicate that our atypical case associates with a new infectious strain of sCJD. Further investigations are needed to understand the possible connection with other human and animal prion diseases.

and the cattle industry. To find an inexpensive and environmentally responsible way to destroy prions and utilize SRM for valueadded byproducts, we investigated the thermophilic anaerobic digestion (TAD) process as an option. A 40% homogenate of brain tissue with a high titre of scrapie specific prion protein (PrPSc) was spiked into the lab-scale digesters with appropriate control groups. Digestion was processed at 550C and carried on for 90 days. Samples were taken at day 7, 11, 18, 26, 40, 60 and 90. Total protein was extracted, purified from TAD slurry, resolved by SDS -PAGE and detected using western blotting (WB) and the images were semi-quantified. Samples were also tested by ELISA. Biogas production from TAD with or without homogenate was measured using gas chromatography. The results demonstrated that PrPSc was eliminated in a timedependent manner during the TAD process while the cellular prion (PrPC ) was not detectable by day 7. PrPSc was degraded from day 7 to 26 in TAD group and TAD plus cellulose group; and not detected in TAD group at day 40 and in TAD plus cellulose group at day 26. The analysis showed that a 2.2 logs reduction of infectivity titre was achieved in TAD group and more than 3 logs achieved in the TAD plus cellulose group by day 26. A rapid degradation was observed during the first 11 days, coinciding with a peak biogas profile. ELISA testing further showed a time-dependent degradation. Biogas production and fuel value of biogas (ratio of methane over CO2) were significantly higher in TAD containing rich protein. Accumulated methane in TAD with infected as well as normal brain was 2.75-fold higher than that in TAD without protein-load during the 90 day’s course of digestion (p < 0.05). Bioassay in ovine transgenic mouse model is underway. Our study indicates that the TAD process is a biologically and environmentally friendly method to decontaminate prions and transform SRM into safe biogas and biofertilizer, which will generate additional revenues to the cattle industry and improve rural economy.

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Bishop MT, Will RG, Manson JC. Defining sporadic Creutzfeldt-Jakob disease strains and their transmission properties. Proc Natl Acad Sci USA 2010; 107:12005-10. 2. Bishop MT, Hart P, Aitchison L, Baybutt HN, Plinston C, Thomson V, et al. Predicting susceptibility and incubation time of human-to-human transmission of vCJD. Lancet Neurol 2006; 5:393-8.

Risk.13: Thermophilic Anaerobic Digestion Degrades Scrapie Prion Protein and Transforms Specified Risk Materials Into Bioenergy Tiejun T. Gao,1,† Yupin Tong,1 Lester Wong ,2 Xiaomei Li,4,1 Aru Balachandran,3 Nishandan Yogasingam,3 Gordon Mitchell,3 Evelyn Bowlby2 and John Wu2

Risk.14: Prion Detection During Anaerobic Digestion of Specified Risk Materials Using Protein Misfolding Cyclic Amplification (PMCA) and Immuno-Quantitative Real-Time PCR (iQ-RT-PCR) Brandon H. Gilroyed,1,† Tim Reuter,2 Shannon Braithwaite,3 Stefanie Czub,4 Catherine Graham,4 Aru Balachandran,5 Miodrag Belosevic,3 Norman Neumann3, 6 and Tim A. McAllister1

Highmark Renewables Research; Edmonton, AB Canada; 2Immunology-Virology Unit, Biology Section, Food Safety and Animal Health Division, Alberta Agriculture and Rural Development; Edmonton, AB Canada; 3Canadian Food Inspection Agency; Ottawa, ON Canada; 4Highmark Renewables Res; Edmonton, AB Canada † Presenting author; Email: [email protected]

Agriculture and Agri-Food Canada; Lethbridge, AB Canada; 2Alberta Agriculture and Rural Development; Lethbridge, AB Canada; 3University of Alberta; Edmonton, AB Canada; 4Canadian Food Inspection Agency; Lethbridge, AB Canada; 5Canadian Food Inspection Agency; Nepean, ON Canada; 6Alberta Provincial Labortory for Public Health; Edmonton, AB Canada † Presenting author; Email: [email protected]

Decontamination of bovine spongiform encephalopathy (BSE) prion and treatment of specified risk materials (SRM) are still a global challenge to life science researchers, food safety regulators

Background. The Canadian beef cattle industry annually generates approximately 250, 000 tonnes of specified risk materials (SRM) which are currently disposed of by rendering and

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landfilling. Both practices are neither economically or environmentally sound. Alternative disposal options are in demand. Anaerobic digestion (AD) of SRM is a potential treatment option which generates economic benefit through the production of renewable biogas energy and valuable fertilizer while diverting organic material away from landfills. Although there is significant organic matter degradation due to microbial activity during AD, the fate of prions is currently unknown. Objectives. Our objective was to assess prion biodegradation during anaerobic digestion using either PMCA or iQ-RT-PCR detection assays. Methods. Prion proteins (ovine scrapie or hamster 263k) were exposed to mesophilic laboratory scale AD for 30 days. Prions were either bound to polyvinylidene difluoride (PVDF) membranes, which acted as a vector for placement and retrieval of prions within the digester, or directly inoculated to AD as infectious brain homogenate (IBH). Prions bound to PVDF were assayed using either iQ-RT-PCR or PMCA. Where IBH was added directly to the anaerobic digester, PMCA was used to test the effluent for the presence of prions and provide a semi-quantitative estimate of prion inactivation. Results. Both iQ-RT-PCR and PMCA were able to detect PVDF-bound prions with great sensitivity prior to exposure to AD. However, after exposure to AD for as little as 1 h no signal was detectable using either assay. Experiments testing the ability of PMCA to detect prions added directly to the anaerobic digester as IBH are currently ongoing. Discussion. Although both iQ-RT-PCR and PMCA were capable of detecting prions bound to PVDF, brief exposure to AD inhibited both assays. Binding of non-target molecules within the AD matrix to the PVDF membranes likely masked the prions, making them unavailable for detection. A discussion on the efficacy of PMCA to detect prions directly added to the anaerobic digester will be presented.

Canadian field cases of BSE which had been previously classified as C-, L-, or H- type. The animals were monitored during incubation period, and clinical disease is described using a standardized examination protocol. Incubation period, description and progression of clinical signs was recorded and videotaped for objective evaluation. Results. All L- and H- type atypical BSE challenged animals began to display signs of clinical disease at approximately 11 months post inoculation, and disease progression was slow but constant until animals were euthanized. Clinical signs in all atypical BSE inoculated animals included hesitation at doors and gates, spontaneous muscle fasciculations and sensitivity to touch. Teeth grinding and excessive salivation are occasionally noted. Animals with L-type BSE are very anxious and show high levels of sensitivity to hand movement. One H-type animal shows periods of somnolence. Both H-type inoculated animals go down during handling and have difficulty rising and show sensitivity to movement around their head and neck area, but to a lesser degree than the L-type BSE inoculated animals. Interestingly, no locomotor abnormalities have been observed in either group. C-type challenged animals remain normal at approximately 18 months post inoculation. Clinical disease in C-type inoculated animals from a previous transmission study was typically slow and intermittently displayed during the initial stages and after a period of two to four months was more consistent and progressive. Clinical signs in C-type BSE were as previously reported in the literature. Discussion. The spectrum of clinical signs for all three types of BSE examined is similar. Incubation period is shorter for Hand L-type BSE as compared with C-type. Once clinical signs begin, progression is slow but relentless in atypical BSE, and more insidious in classical BSE. A summary of clinical signs presented in the three different types of BSE will be presented, and video of clinical disease will be displayed.

Risk.16: Clinical Disease in Cattle Experimentally Inoculated with All Types of BSE

Risk.17: Pathogenesis and Agent Distribution of Three Known Types of BSE

Catherine Graham,1,† Michel Levy,2 Ed Pajor,2 Garth McGregor,1 Rheana Flitton1 and Stefanie Czub1

Catherine Graham,1,† Bob Hills,2 Martin Groschup,3 Sandor Dudas1 and Stefanie Czub1

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1 Canadian Food Inspection Agency; Lethbridge, AB Canada; Faculty of Veterinary Medicine; University of Calgary; Calgary, AB Canada † Presenting author; Email: [email protected]

Canadian Food Inspection Agency; Lethbridge, AB Canada; 2Health Canada; Ottawa, ON Canada; 3Friedrich-Loeffler-Institut; Insel Riems, Germany † Presenting author; Email: [email protected]

1

Background. Classical, or C-type, bovine spongiform encephalopathy (BSE) has been extensively described in the literature. Recently, two novel forms of BSE, termed atypical BSE, have been reported in a number of countries. These new forms show differences in the biochemical characteristics of the prion protein and, where reported, tend to occur in aged animals but descriptions of clinical presentation are incomplete or absent. Materials and Methods. Female Hereford/Angus cross calves were intracranially challenged at approximately five months of age with 1 ml of a 10% brain homogenate originating from

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Objectives. To ascertain if the oral dose of bovine spongiform encephalopathy (BSE) infected material, scientifically established for clinically affected C-type BSE, is comparable to that for animals clinically affected with H-type and L-type BSE. To ascertain if an older bovine exposed to the BSE agent can become infected with a comparable oral dose used in other BSE C-type studies where only young calves, four to six months of age, were inoculated. To collect tissues, including those that are designated as specified risk material (SRM) for C-type BSE, to determine if the

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current list of SRM is a sufficient safeguard to protect against all BSE types. Materials and Methods. Inoculum. L- and H-type BSE inoculum (kindly provided by Dr. Martin Groschup Friedrich-Loeffler Institute) consisted of pooled brain material from animals intracerebrally inoculated with BSE as part of a pathogenesis study. C-type inoculum originated from a single animal intracerebrally inoculated with C-type BSE from a Canadian field case. Control inoculum consisted of brain material from a single animal confirmed to be negative for BSE. Total inoculum administered to animals was a single dose of 100g of brain material. Animals. Two separate groups were inoculated orally by oesophageal tube. Steer calves (Group 1) were inoculated at approximately five months of age. Three calves were inoculated with L- and H- type BSE, and one animal was similarly inoculated with BSE negative brain material. Yearling steers (Group 2) were inoculated at approximately 15 months of age. Three animals each were inoculated with the L-, H-, and C-type homogenates. One control animal was similarly inoculated with BSE negative brain material. The animals are observed daily by animal care staff and at least monthly by the animal care veterinarian. Blood samples are obtained monthly. Animals will be permitted to develop clinical disease, or will be euthanized after three years, and a wide variety of tissues will be sampled post mortem. Selected samples will be evaluated using multiple test methods including, but not limited to, immunohistochemistry, PET Blot, IDEXX EIA, and western blot. Results. Animals are currently 18 months (Group 1) or nine months (Group 2) post inoculation and are clinically normal at this time. Animals continue to be sampled and a wide variety of tissues and body fluids will be available for future studies.

macaque-adapted vCJD. In June 2010, we infected four cynomolgus macaques by the intravenous (1 ml) and intraperitoneal (2 ml) routes with 10% vCJD monkey brain suspensions obtained from CEA. Every two months, 70-90 ml of blood are collected from each infected animal, and blood is also obtained from two uninoculated control animals. Ten percent of the blood collected is reserved as whole blood and the remainder is processed into plasma, buffy coat and red blood cells. Each component is divided into aliquots and stored frozen at -80˚C. We expect this project to generate a large inventory of vCJD-infected and control macaque blood and blood components at various stages of infection. All animals remain asymptomatic nine months after inoculation. In parallel, we also initiated the biochemical characterization of the CEA macaque vCJD brain inoculum and compared it with WHO human sporadic CJD and vCJD candidate biological reference brain materials distributed by the National Institute for Biological Standards and Control (UK). We have begun titrations of infectivity in the human and macaque vCJD brain suspensions by serial dilutions and inoculation into two susceptible lines of wild-type mice (C57/BL and RIII). Samples of buffy coat from a macaque clinically ill with vCJD obtained from CEA were assayed by titration in C57/BL mice. Disclaimer. The findings and conclusions in this abstract have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Agency determination or policy. Support. This work is supported by a grant from Alliance BioSecure Foundation, France, and an award from the FDA.

©201 1L andesBi os c i enc e. Donotdi s t r i but e.

Risk.19: Development of P-CAPT® Filter for Reduction of Prion Infectivity and vCJD Transmission of Red Blood Cell Concentrates

Risk.18: Update on Macaque-Adapted vCJD Blood Panels

Chryslain Sumian,1 Patrick Gurgel,5,† Iwona Walicka,1 Nathalie Lescoutra-Etchegaray,3, 2 Luisa Gregori,6,# Julia Lathrop,4 Ruben Carbonell,7 Robert Rohwer7 and Steve Burton8

Kristy McDowell, Kitty Pomeroy, Julie Nemecek, Pedro Piccardo and David M. Asher Luisa Gregori† †

FDA/CBER/OBRR; Rockville, MD USA Presenting author; Email: [email protected]

Variant Creutzfeldt-Jakob Disease (vCJD) has been transmitted by blood transfusions and a plasma product. To date no validated test identifies vCJD-infected pre-syptomatic blood donors or contaminated blood donations. Candidate blood-based tests are in development, but vCJD-infected human blood samples are not available to validate the assays independently. We proposed, as an alternative to human blood, to prepare blood components from vCJD-infected and uninfected macaques to serve as reference standards for blood panels. Such materials should facilitate both development and validation candidate blood screening tests. In collaboration with the Institute of Emerging Diseases and Innovative Therapies, CEA, France, we started a program to obtain large quantities of blood from macaques infected with

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MacoPharma; Tourcoing, France; 2CEA, Prion Research Group, DSV/IMETI/SEPIA; Fontenay-aux-Roses, France; 3MacoPharma; Fontenay-aux-Roses, France; 4 Prolias; Washington, DC USA; 5ProMetic Life Sciences; Montréal, QC Canada; 6VAMC/ UM; Baltimore, MD USA; 7NCSU; Raleigh, NC USA; 8Prometic; Cambridge, UK # Present affiliation: CBER-FDA; Baltimore, MD USA; † Presenting author; Email: [email protected] 1

Background. Five patients in the UK have established the vCJD transmissibility of blood donated by asymptomatic carriers. Blood transfusion must urgently be safeguarded, and removal of prions from blood is one of the most promising strategies. The MacoPharma prion capture filter, P-Capt®, was developed in conjunction with Pathogen Removal and Diagnostic Technologies Inc. (PRDT) to remove infected prions from leucodepleted red blood cells concentrate (LD-RBCC) preventing vCJD blood transmission.

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Method. PRDT, a joint venture between ProMetic and the American Red Cross, identified ligands with strong and selective binding to the TSE causative agent. A panel of resins were shown to reduce brain-derived scrapie infectivity by 3–4 log10 from RBCC. One selected for endogenous challenge captured all detectable endogenous infectivity from LD scrapie-infected whole blood from hamsters as measured by bioassay. This resin was incorporated into the P-Capt filter (Macopharma), which was CE marked following toxicological, biocompatibility and hemocompatibility testing. A Volunteer Post-Market Surveillance Study for Neoantigenecity and RBC survival was carried out to demonstrate product safety. Operational validations have been performed by UK and Ireland blood services following their standard protocol. Results. The efficacy of removal has been demonstrated as >3 log10 reduction of exogenous brain spike infectivity in RBC, comparable to results obtained using the prion capture resin in column format confirming the broad utility of this affinity resin and consistency of prion capture; and >1.2 log10 in endogenous infectivity study. The P-Capt filter demonstrated no impact on RBC. No significant differences in RBC survival at 24 h were observed, no serious adverse events were reported and there were no indications of Neoantigen formation. Haematocompatibility and immunohematology data were confirmed by the blood services operational validations. Conclusion. The P-Capt is the only validated filter capable of removing the risk of transmission of vCJD through RBCC transfusion. Currently, the universal application of the filter represents the best strategy available to protect the blood supply from the second wave of vCJD transmission.

in the UK population. This situation has created a risk management issue, whereby healthcare providers have to prevent the potential transmission of prions through reusable instruments, while avoiding condemning costly instruments to destruction after they have been used on a patient deemed at risk of vCJD. A solution resides in better decontamination techniques to reduce the risk of iatrogenic infection. Cold atmospheric plasma (CAP) has demonstrated great efficiency at protein removal, and here we evaluate the impact of CAP on the infectivity of prion-contaminated surgical surfaces. Method. Pairs of wires were contaminated with brain homogenate preparations containing serial dilutions of the 263K prion strain in normal brain, and one wire of each pair was treated with CAP. The CAP-treated and control wires were then implanted intracerebrally in susceptible animals for an end-point titration infectivity assay. Result. Evidence so far suggests that treatment of the wires with a CAP narrow jet resulted in partial protein removal due to targeting issues. The infectivity study is still ongoing, though the latest data suggest that CAP also resulted in partial reduction of prion infectivity. Discussion. Only when the infectivity study is complete later this year will we be able to calculate the reduction in infectivity titre. This will be compared with the reduction in residual protein after treatment. Better performing CAP systems are being developed and we aim to achieve complete removal of protein contamination and correlate this with a reduction of prion infectivity to acceptable (i.e. undetectable) levels.

©201 1L andesBi os c i enc e. Donotdi s t r i but e.

Risk.21: Thirty-Year Review of Prion Disease Surveillance in the United States

Risk.20: Residual Protein Contamination and Prion Infectivity After Decontamination of Spiked Surgical Surfaces Using a Cold Atmospheric Plasma Jet

Robert C. Holman,1,† Ryan A. Maddox,1 Arianne M. Folkema,1 Arialdi M. Minino,2 Teresa A. Hammett,1 Kenneth D. Kochanek,2 James J. Sejvar,1 Ermias D. Belay1 and Lawrence B. Schonberger,1

Rodolphe Hervé,1,† Michael G. Kong,2 Emmanuel Comoy,3 Jean-Philippe Deslys3 and Bill Keevil1

CDC; Atlanta, GA USA; 2CDC; Hyattsville, MD USA † Presenting author; Email: [email protected]

1

University of Southampton; Southampton, UK; 2University of Loughborough; Loughborough, UK; 3CEA; Paris, France † Presenting author; Email: [email protected]

1

Background. Variant Creutzfeldt-Jakob disease (vCJD) appeared in the UK and other countries other ten years ago. Like a number of other transmissible spongiform encephalopathies (TSEs) affecting human, there is no cure available, and progressive and irreversible neurodegeneration leads ultimately to death. Infection is understood to be linked solely to the transmission of misfolded prion (the prion only hypothesis). Furthermore, it has become clear that prions bind strongly to the various surfaces of reusable surgical instruments and are extremely resilient to current decontamination procedures. There is also evidence that prions have a relatively wide tissue distribution in vCJD carriers in contrast with other TSEs, and the prevalence of vCJD is unclear, although recent surveys suggest it might be as high as 1 in 4000

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Background. With the emergence of bovine spongiform encephalopathy/variant Creutzfeldt-Jakob disease (vCJD) in the UK, the Centers for Disease Control and Prevention began utilizing national mortality data with additional surveillance mechanisms to monitor US occurrences of human prion disease. Objectives. To review US prion disease surveillance data. Methods. We analyzed national mortality data for prion disease deaths (a surrogate for CJD incidence) among US residents for the 30 year period, 1979–2008, augmenting and extending these data through 2010 with information from other surveillance mechanisms (e.g., national neuropathology surveillance). We calculated age-adjusted and age-specific death rates per million persons; race-specific rates used data available beginning 1981. We age-adjusted death rates to the standard projected US 2000 population.

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Results. A total of 7,615 deaths during 1979–2008 were identified for an average annual age-adjusted rate of 0.98 cases per million persons. The highest rate (1.15) was observed in 1997; the highest number of reported cases was in 2008 (348). By race, the rate (1.06) among whites, who constituted 95% of the cases, was significantly higher than among blacks (0.40), Asian/Pacific Islanders (0.63) and American Indians/Alaska Natives (0.42). The rate (4.0) among persons ≥55 years old was strikingly higher than the rate (0.14) among persons