PPo3-1: Variant CJD Strain Remains Stable after Secondary Transmission
Matthew T. Bishop,1 Abigail Diack,2 Enrico Cancellotti,2 Robert Will1 and Jean Manson2
1National CJD Surveillance Unit; University of Edinburgh; western General Hospital; Edinburgh, Scotland UK; 2The Roslin Institute; University of Edinburgh and R(D)SVS; Roslin Biocentre; Midlothian, Scotland UK
Key words: vCJD, blood transfusion, transgenic mice, transmission, strain
Introduction. Blood transfusion associated vCJD infection has been observed in three codon 129 MM and one MV genotype cases. Passage of the vCJD agent in humans may generate a more infectious strain leading to an increased threat to the population. To understand whether strain adaptation has occurred we inoculated a panel of wild-type and humanised transgenic mice with material from donor and blood recipient vCJD cases.
Results. There was evidence of transmission with all inocula except the brain from the MV genotype case that was devoid of TSE pathology. Incubation period data show similarities across all inocula. The lesion profile scores for TSE vacuolation were similar between; donor and recipients, inocula sources, and mouse lines. The presence of PrP-Sc also confirmed similarities between blood donor and recipient vCJD cases.
Methods. Transgenic and wild-type mice were inoculated with brain and spleen material from an MM genotype blood transfusion associated vCJD case, the MV blood transfusion associated preclinical vCJD case, and both vCJD blood donors.
Conclusions. (1) Spleen tissue from the MV preclinical vCJD blood recipient was transmissible, indicating that MV hosts can propagate the vCJD agent, and that significant levels of infectious agent are present in the spleen before CNS involvement. ‘Silent’ spread within the human population is therefore a possibility from these carriers. (2) As blood donor and recipient vCJD cases show similar transmission properties from both brain and spleen material we hypothesise that human-to-human infection via this route has not caused adaptation to a more infectious agent.
PPo3-2: Comparison of Lesion Profiles and PrPSc Deposition Patterns Following Inoculation with 301V via Oral, Intragastric and Intracerebral Routes
Chris Vickery, Katy Beck, John Spiropoulos and Stephen Hawkins
All Veterinary Laboratories Agency; UK
Background. It is widely regarded that individual mouse adapted TSE strains are distinguished based on reproducible incubation periods and vacoulation patterns unique to a specific mouse line by inoculation via a consistent route. To investigate if a change of route of administration can alter the strain properties in the mice we compared TSE transmission properties and strain stability in IM mice following serial titration of mouse adapted BSE (301V) via oral, intragastric (i.g.) and intracerebral (i.c.) routes.
Results. Analysis of lesion profiles demonstrated that the lesion profiles from the i.c. challenges were elevated compared to the i.g and oral routes although area G1 (medulla) was consistently high irrespective of route of administration. Within the same route of administration lesion profiles seem to be more stable between different dilutions. Mean incubation periods at the 10-1 dilution following oral or i.g. inoculations were prolonged significantly compared to the i.c route (263, 232 and 123 mean days post inoculation respectively).
Materials and Methods. Standardised 301V strain was inoculated into IM mice. Post mortem TSE diagnosis was confirmed by microscopy examination of haematoxylin and eosin (H&E) sections. Lesion profiles which are a graphical representation of vacuolar lesion intensity in specific neuroanatomical areas were constructed according to standard methodology.
Conclusion. Preliminary analysis demonstrated a variation in lesion profiles between i.c. against oral and intragastric routes suggesting that the route of inoculation could affect vacuolation intensity. Further work involves comparison of the three routes of challenge using IHC analysis.
Acknowledgement
Work funded by SC0042.
PPo3-3: Scant PrP-Sc in the Placentae of Goats with Naturally Acquired Scrapie
K.I. O’Rourke, D.A. Schneider, D. Zhuang and T. Truscott
Department of Agriculture; USA
Domestic goats (Capra hircus) are a natural and experimental host of scrapie and bovine spongiform encephalopathy and are susceptible to experimental infection with the agents of TSEs of deer and elk (chronic wasting disease) and humans (Creutzfeldt Jakob disease). No data are available on the potential shedding of the agent through the placenta, the presumed route of transmission of ovine scrapie. This paper provides data on PrPSc levels in the shed placenta of goats with naturally acquired scrapie and compares these findings to similar data from field cases of classical ovine scrapie. The study demonstrated that the distribution of PrPSc in intact placentomes collected at necropsy was similar to that observed in sheep. However, of the five naturally infected goats, only one animal had detectable levels of PrPSc in shed placental cotyledons. Progeny of some of these goats have developed scrapie in spite of the low and variable placental PrPSc levels in their fetal cotyledonary tissues.
PPo3-4: White-tailed Deer are Susceptible to Sheep Scrapie by Intracerebral Inoculation
Justin J. Greenlee, Jodi D. Smith and Robert A. Kunkle
Virus and Prion Research Unit; National Animal Disease Center; ARS; USDA; Ames; IA USA
Interspecies transmission studies afford the opportunity to better understand the potential host range and origins of prion diseases. The purpose of this experiment was to determine susceptibility of white-tailed deer to scrapie after intracerebral inoculation and to compare clinical signs and lesions to chronic wasting disease (CWD). Deer (n = 5) were inoculated with 1 ml of a 10% (wt/vol) brain homogenate derived from a sheep clinically affected with scrapie. Non-inoculated deer were maintained as negative controls. Deer were observed daily for clinical signs of disease and euthanized and necropsied when unequivocal signs of TSE were noted. One animal died 7 months post inoculation (PI) due to intercurrent disease. At that time, examination of tissue by IHC and WB were negative. However, deer necropsied at 15–22 months PI were positive for scrapie by IHC and WB. Tissues with PrPd immunoreactivity included brain (at levels of cerebrum, hippocampus, colliculus, cerebellum and brainstem), trigeminal ganglion, neurohypophysis, retina, spinal cord and various lymphoid tissues including tonsil, retropharyngeal and mesenteric lymph nodes, peyer’s patches and spleen. This work demonstrates for the first time that white-tailed deer are susceptible to sheep scrapie by intracerebral inoculation. To further test the susceptibility of white-tailed deer to scrapie these experiments will be repeated with a more natural route of inoculation.
PPo3-5: Reciprocal Relationship Between Shadoo and PrPSc in Prion Disease
Joel C. Watts,1 Kurt Giles1,2 and Stanley B. Prusiner1,2
1Institute for Neurodegenerative Diseases; and 2Department of Neurology; University of California; San Francisco, CA USA
Key words: shadoo, prion, transgenic mice, pathogenesis
The central event in prion diseases is the conformational change of PrPC into PrPSc, a partially protease-resistant and neurotoxic conformer. However, the mechanisms by which PrPSc causes neuronal dysfunction remain poorly understood. A recently discovered protein, Shadoo (Sho), resembles the flexibly disordered N-terminal domain of PrPC. Interestingly, Sho protein levels are strikingly reduced in the brains of mice infected with the RML strain of prions (Watts et al. EMBO J 2007; 26:4038–50). This implies that Sho levels may be linked to prion replication or reflect the presence of PrPSc in the brain. To test this hypothesis, we examined levels of Sho during prion infection using a variety of experimental systems. Sho protein levels were markedly decreased in the brains of mice, hamsters and voles following challenge with a divergent series of natural and experimental prion strains. Furthermore, Sho levels were decreased in the brains of prion-infected transgenic mice overexpressing Sho and in infected neuroblastoma cells. No diminution in Sho levels was observed in aged transgenic mice with high cerebral Aβ Levels or in mice propagating protease-sensitive prions. Time-course experiments revealed that Sho levels were inversely proportional to levels of protease-resistant PrPSc. In prion-infected tissue, multiple lines of evidence argue that Sho is targeted to the endosomal/lysosomal pathway for degradation by a PrPSc-mediated process. We conclude that Sho is the first non-PrP marker specific for prion disease in mice. Further studies using this paradigm may provide insight into the cellular pathways and systems subverted by PrPSc during prion disease.
PPo3-6: Early Decline of LamR1+ Blood Cell Counts in Macaques Orally Dosed with the Bovine Spongiform Encephalopathy (BSE)-inducing Agent
Barbara Yutzy,1 Walter-Schulz-Schaeffer,2 Kay-Martin Hanschman,1 Julia Kress,1 Cheick Coulibaly,1 Uwe Hahmann,3 Pär Bierke,4 Gerhard Hunsmann,3 Johannes Löwer1 and Edgar Holznagel1
1Paul-Ehrlich-Institut; Federal Agency for Vaccines and Biomedicines; Langen, Germany; 2Department of Neuropathology; August-University Göttingen; Germany; 3German Primate Centre; Göttingen, Germany; 4Swedish Institute for Disease Control; Solna/Stockholm, Sweden
Key words: non-integrin laminin receptor, cynomolgus monkeys, FACS analyses, quantitative PCR
There are two classes of laminin receptors: the integrin family (VLA1-6) and so-called non-integrin laminin receptors (e.g., Laminin receptor type 1, LamR1). LamR1 acts as a cellular receptor for PrPC and its disease-inducing variant.
Here, we show data from 24 cynomolgus monkeys, which were infected with the BSE-inducing agent, and 12 MOCK controls. Blood samples were collected monthly and analyzed for the expression of PrPC, VLA1-6 and LamR1 by FACS analyses and quantitative PCR. In parallel, lymphoid and CNS tissues were collected from monkeys that were sacrificed at regular intervals during the asymptomatic phase of infection or after the onset of clinical signs.
The fluorescence intensity of LamR1 significantly (p < 0.0001) decreased on all main blood lymphocyte subsets during the second year after infection and remained at a very low to negative level until the clinical phase (>4 yrs. p.i.), whereas no changes were detectable among the VLAs. This change was accompanied by an increase in the PrPC fluorescence on blood cells. However, LamR1 was detectable on blood cells from infected macaques when the routine staining protocol was modified. Moreover, the mRNA levels showed no evidence of a decrease (LamR1) or an increase (PrPC). PrPres deposits outside of the GALT system were detectable from 1 year p.i. onward.
These and further data indicate that the epitope recognized by the used anti-LamR1 antibody was masked by a not yet identified ligand.
Acknowledgement
The study was supported by an EU grant and by the German Federal Ministry of Health.
PPo3-7: Prion Transmission from Cervids to Humans is Strain-dependent
Qingzhong Kong, Shenghai Huang,*Fusong Chen, Michael Payne, Pierluigi Gambetti and Liuting Qing
Department of Pathology; Case western Reserve University; Cleveland, OH USA
*Current address: Nursing Informatics; Memorial Sloan-Kettering Cancer Center; New York, NY USA
Key words: CWD, strain, human transmission
Chronic wasting disease (CWD) is a widespread prion disease in cervids (deer and elk) in North America where significant human exposure to CWD is likely and zoonotic transmission of CWD is a concern. Current evidence indicates a strong barrier for transmission of the classical CWD strain to humans with the PrP-129MM genotype. A few recent reports suggest the presence of two or more CWD strains. What remain unknown is whether individuals with the PrP-129VV/MV genotypes are also resistant to the classical CWD strain and whether humans are resistant to all natural or adapted cervid prion strains. Here we report that a human prion strain that had adopted the cervid prion protein (PrP) sequence through passage in cervidized transgenic mice efficiently infected transgenic mice expressing human PrP, indicating that the species barrier from cervid to humans is prion strain-dependent and humans can be vulnerable to novel cervid prion strains. Preliminary results on CWD transmission in transgenic mice expressing human PrP-129V will also be discussed.
Acknowledgement
Supported by NINDS NS052319 and NIA AG14359.
PPo3-8: Assessing the Risk of Sheep BSE Transmission to Humans
Chris Plinston, Nora Hunter, Jim Foster, Patricia Hart, Jean C. Manson and Rona M. Barron
Neuropathogenesis Division; The Roslin Institute and R(D)SVS; University of Edinburgh; Edinburgh, Scotland UK
Key words: human, transgenic, sheep BSE,
Strain typing studies have shown that the same strain of TSE agent is responsible for BSE in cattle and vCJD in humans. However, transmission studies of cattle BSE to humans using transgenic mice expressing human PrP have shown limited transmissibility, suggesting the presence of a substantial transmission barrier. To investigate this transmission barrier further, gene targeted transgenic mice expressing human PrP with the codon 129 polymorphism (HuMM, HuMV and HuVV) have been challenged with cattle BSE and Experimental Sheep BSE (Exp-ShBSE). BSE has not been identified in sheep in the field, but sheep have been shown to susceptible to experimental infection. Mice inoculated with cattle BSE failed to show any evidence of disease transmission. However following inoculation with Exp-ShBSE, 18/23 HuMM transgenic mice showed positive TSE pathology (400–750 dpi) in the form of vacuolation and/or PrP deposition, targeted mainly to the thalamic region. While no clinical signs of disease were reported in any of the HuTg mice receiving Exp-ShBSE, positive clinical and pathological signs of disease were observed in both control 129/Ola mice and gene targeted bovine transgenic mice. Control lines showed similar pathology, and incubation time ratios with both cattle BSE and Exp-ShBSE, however Exp-ShBSE incubation times were shorter (∼70–80 days) than those observed following cattle BSE inoculation. These data suggest a difference in susceptibility of HuMM mice to the BSE agent following propagation in either cattle or sheep. Experiments to establish the titre of BSE in sheep brainstem are underway.
Acknowledgement
Funded by Defra SE1441
PPo3-9: Potential of Cell Substrates used for Production of Biologics to Propagate Transmissible Spongiform Encephalopathy (TSE) Agents: 5-year Update
P. Piccardo,1,* L. Cervenakova,2 I. Vasilyeva,2 O. Yakovleva,2 I. Bacik,1 J. Cervenak,1 L. Gregori,1 K. Pomeroy,1 L. Kurillova,1 C. McKenzie2 and D.M. Asher1
1Laboratory of Bacterial and TSE Agents; CBER; FDA; USA; 2J. Holland Laboratory; American Red Cross; USA
*Presenting Author
Key words: cell culture, animal models, biologics, prion, TSE-agent
Background. TSE agents have contaminated human-tissue-derived therapeutics and animal vaccines. Many biologics are prepared in cell cultures. Although most cultures studied resisted infection with TSE agents, a few were susceptible.
Objectives. We are investigating susceptibility of several cell lines to infection with TSE agents.
Results. We studied Vero, CHO, MDCK, HEK-393 and WI-38 cells. We also studied SH-SY5Y cells overexpressing wild-type PrP and mutant PrPs. Cells exposed to TSE agents were serially propagated for 30 passages and samples tested for TSE-associated PrP (PrPTSE) and infectivity by intracerebral inoculation into transgenic mice and squirrel monkeys (BSE-exposed cells only). No exposed cell substrate has transmitted TSE to mice or monkeys to date. No PrPTSE was found in any exposed cells after 30 passages. Known susceptible murine cells exposed to mouse-adapted scrapie agent as positive controls accumulated PrPTSE. Three monkeys inoculated with BSE reference material have developed TSE to date.
Discussion. To date, no candidate cell substrate exposed to 3 TSE agents accumulated PrPTSE or propagated a TSE agent. Squirrel monkeys provide a new model to study BSE pathogenesis.
Methods. We inoculated brain suspensions containing agents of bovine spongiform encephalopathy (BSE), variant Creutzfeldt-Jakob disease (vCJD) or sporadic CJD into several cell lines important in manufacture of biologics. Serial dilutions of the BSE reference material used as inoculum were also inoculated into mice and squirrel monkeys.
The findings and conclusions in this article have not been formally disseminated by the Food and Drug Administration and should not be construed to represent any Administration determination or policy.
Acknowledgements
Support. NIAID-NIH AI-4893-02/FDA 224-05-1307
PPo3-10: Analysis of Fecal Samples from Sheep Naturally Infected with Scrapie for PrPSc Using an Ultrasensitive in vitro Amplification Method
S.J. Everest,1 L. Howells,1 L. Thorne,1 K.C. Gough,2 B.C. Maddison,3 K. Bishop,3 C.A. Baker3 and L.A. Terry1
1Molecular Pathogenesis and Genetics; VLA; Addlestone, UK; 2School of Veterinary Medicine and Science; University of Nottingham Sutton; Bonington, UK; 3ADAS; Department of Biology; University of Leicester; Leicester, UK
Key words: natural scrapie, feces, environmental contamination, transmission
Introduction. Scrapie infection in sheep and goats and chronic wasting disease (CWD) in deer are transmitted horizontally in the field. It is believed that transmission occurs orally via contaminated environmental sources. However, the importance of individual sources of infectivity in spreading the disease environmentally is not known. Infectivity has been detected in the feces of rodent models of prion disease and deer experimentally infected with CWD. In this study we analyzed feces from sheep naturally infected with scrapie for the presence of prions by serial protein misfolding amplification (sPMCA).
Results. PrPSc was detected in the feces of sheep infected with scrapie in both the preclinical and clinical phases of the disease.
Materials and Methods. Faecal samples were collected from VRQ/VRQ sheep exposed from birth and naturally infected with scrapie at clinical end-point and at 9 months of age and from age-matched unexposed sheep. Prions were extracted using either a NaPTA or silicon dioxide enrichment procedure prior to sPMCA. The presence of PrPSc was confirmed by western blot or ligand-based immunoassay.
Conclusion. This study demonstrates that PrPSc can be detected in the feces of naturally infected VRQ/VRQ sheep with terminal scrapie and in VRQ/VRQ sheep exposed to scrapie several months prior to the onset of clinical disease. These findings indicate that feces are a source of prion shedding into the environment and have implications for restocking of pastures following depopulation of infected flocks.
PPo3-11: Blood Transmission Experiments in Primates: Squirrel Monkeys (the Baxter Study)
Paul Brown, James Ironside, Susan Gibson, Robert G. Will, Thomas R. Kreil and Christian Abee
Plasma and buffy coat samples from 2 sCJD and 3 vCJD cases were inoculated i.c. and i.v. into a total of 21 squirrel monkeys. Pooled brain from the 3 vCJD patients titered 106 LD50/g (i.c.). Whole blood from each of 4 monkeys inoculated with 10% vCJD brain homogenate was transfused i.v. to individual recipient monkeys at approximately 3-month intervals during the incubation and clinical stages of disease in the donor animals. Plasma, RBC’s, platelets, and purified leukocytes from 6 chimpanzees infected with either human sCJD or GSS were inoculated i.c. and i.v. into 12 monkeys. In the entire group of monkeys inoculated with blood or blood components, only a single neuropathologically-verified transmission occurred within a 5-year observation period, in an animal inoculated with leukocytes from a pair of GSS-infected chimpanzee.
Conclusions. In a primate model highly susceptible to TSE (the squirrel monkey), infectivity was not detected (<10 i.c. LD50/ml) in plasma or buffy coat from 2 sCJD and 3 vCJD patients, or in whole blood from 1st passage vCJD in monkeys. We did, however, detect infectivity in one monkey inoculated with purified leukocytes from chimpanzee-passaged GSS, and observed what appeared to be a ‘triggering’ of symptomatic disease by physiological stress in chimpanzees subjected to plasmapheresis under general anaesthesia.
PPo3-12: VLA Scrapie Endemic Nucleus Flock (RECTORY)
Hugh A. Simmons, Mike D. Dawson
Veterinary Laboratories Agency; Addlestone, Surrey UK
Key words: endemic infection flock
Following an on farm epidemiological study of scrapie in the 1990s selected sheep from study flocks were purchased and brought to VLA. They were kept as a single flock at grass and from 2001 onwards this flock was established as a research resource which modelled active natural infection. This flock (Rectory) provides access to naturally infected sheep of known genotype where the pathogenesis is well characterised and the opportunity for in-vivo lifetime sampling of animals until the development of disease. In addition to blood, the flock has provided the opportunity for evaluating various other sampling approaches to live diagnosis. The VLA has matched controls in the biosecure classical scrapie free (Rickwood) flock (Simmons et al. BMC 2009; 5:8). As well as diagnostic and pathogenesis studies the Rectory flock continues to be used to investigate modes of transmission. Techniques to do this include the introduction and use of sheep and lambs from the Rickwood flock. Recently published studies include the evaluation of exposure risks for natural transmission of scrapie (Dexter, et al. BMC 2009; 5:38) and feeding studies that showed milk is a significant source of infection. (Konold, et al. BMC 2008; 4:14). The Rectory flock and farm is also a source of samples from a scrapie infected environment. Its facilities are contributing to research into decontamination techniques and experiments investigating infectivity decay on pasture. Material from the Rectory and Rickwood flocks can be obtained through the VLA Archive. Research access to either flock is through the authors.
PPo3-13: Transcriptome Analyzes of Bovine Spongiform Encephalopathy
Y. Tang,1 W. Xiang,2 H. Kretzschmar2 and O. Windl1
1Molecular Pathogenesis & Genetics Department; Veterinary Laboratories Agency; New Haw, UK; 2Center for Neuropathology and Prion Research; Ludwig-Maximilians University; Munich, Germany
Key words: BSE pathogenesis, ER stress, transcriptome, gene expression, microarray
Introduction. Many pathogenic events associated with a disease trigger changes in gene expression. In order to detect these changes in BSE we used microarray analysis to study brainstem samples from BSE field cases and from experimentally infected BSE cattle (the time course study) and compared these data to non-infected control samples.
Results and conclusions. 161 genes were found to be differentially regulated in the time course study and several of them were associated with prion diseases (Tang et al. J. Virol 2009; 83:9464). The analysis showed that the most changes in gene expression occurred between negative controls and animals 21 month post inoculation rather than between the controls and the animals with confirmed BSE. In the study of BSE field cases, 230 genes were identified as the differentially regulated. Among these genes there were 16 genes associated with endoplasmic reticulum (ER) and many of them are involved in stress related response, suggesting that ER stress might be part of BSE pathogenesis. Clustering analysis showed that the gene lists generated from one study could be used as biomarkers to predict sample status of the other study.
Methods. The microarray analysis was carried out using Affymetrix Bovine Genome GeneChips, which contain 24,128 probe sets. The data were normalized and analyzed using the GeneSpring software. Differentially regulated genes were identified by ANOVA and a 2-fold change. The time course samples included pre-clinical animals 6, 21, 27, 36, 39 months following oral inoculation as well as animals with confirmed BSE.
PPo3-14: Oral Secretion of Prions in Sheep Naturally Exposed to Scrapie
Kevin C. Gough,1 Ben C. Maddison,2 Claire A. Baker,2 Helen C. Rees,2 Linda A. Terry,3 Leigh Thorne3 |and Susan J. Belworthy3
1Department of Veterinary Medicine and Science; University of Nottingham; Sutton Bonington; Loughborough, UK; 2ADAS-UK LTD; Department of Biology; University of Leicester; Leicester, UK; 3Veterinary Laboratories Agency; New Haw; Addlestone, Surrey UK
Key words: scrapie, transmission, oral, secretion
Epidemiological and experimental evidence suggest that prions are secreted/excreted from sheep infected with scrapie, however the detection of such prions is challenging. A proven technique is serial protein misfolding cyclic amplification (sPMCA), an in vitro technique which amplifies minute quantities of PrPSc. Here, sPMCA was applied to the detection of prions in oral secretions during preclinical ovine scrapie infections. Buccal swabs were obtained from preclinical scrapie-infected sheep and prions eluted, concentrated on silicon dioxide and then amplified by sPMCA. Data clearly demonstrated that prions are present in buccal samples from sheep with a VRQ/VRQ PRNP genotype during preclinical scrapie infection. The application of this technique to other VRQ heterozygous scrapie susceptible sheep, kept on a farm with a high incidence of scrapie is ongoing. This will provide comparative data between susceptible sheep thought to differ in prion distribution and LRS involvement in prion pathogenesis. These data describe the secretion of prions into the oral cavity of asymptomatic scrapie-infected sheep, findings with implications for the transmission and environmental dissemination of ovine scrapie and very likely other prion diseases.
PPo3-15: Effects of Solution Chemistry on Prion Protein Adsorption to Soil Minerals
Shannon L. Bartelt-Hunt,1 Samuel E. Saunders,1 Qi Yuan1 and Jason C. Bartz2
1Department of Civil Engineering; University of Nebraska-Lincoln; Peter Kiewit Institute; Omaha, Nebraska USA; 2Department of Medical Microbiology and Immunology; Creighton University; Omaha, Nebraska USA
Prions are known to adsorb to a wide range of soils and soil minerals and remain infectious, thereby facilitating a sustained presence of chronic wasting disease and scrapie. It is likely that the prion protein (PrP), entering the environment in complex, competitive matrices such as animal excreta or tissue, will interact with soil particles under a range of solution chemistries, from dilute solutions to ones highly-concentrated with a variety of salts and organic molecules. Given that solution chemistry can affect soil and protein properties, leading to varied adsorption outcomes, we investigated prion protein adsorption to soil minerals in a range of aqueous solutions, including phosphate buffered saline (DPBS), NaCl, CaCl2 and deionized water. Using infectious HY TME brain homogenate as the PrP source, we monitored bound and unbound PrP in batch reactors up to 6 months, and in addition, PMCA was performed on soil mineral samples to evaluate variance in replication ability with respect to adsorption solution chemistry. Our results demonstrate the ability of PrP to adsorb to soil minerals in a wide range of solution chemistries, and only subtle differences were seen in the amount of adsorbed PrP between buffers. This suggests that solution chemistry does not significantly affect PrP adsorption, possibly due to an overwhelming influence of the competitive organic matrix (i.e., brain tissue, urine, blood) co-entering the soil environment with PrP.
PPo3-16: Downregulation of PrPC Expression in CAD5 Cells Significantly Limits but Does Not Prevent Formation of PrPtse
Martin Panigaj, Olga Janouskova and Karel Holada
Institute of Immunology and Microbiology; 1st Faculty of Medicine; Charles University; Praque, Czech Republic
Key words: cell lines, PrPC, PrPtse, RNA interference
Expression of cellular prion protein (PrPC) is crucial for the propagation of its pathogenic isoform (PrPtse). However, little is known about the mechanism of PrPtse propagation in cells and its impact on cell pathophysiology. To study the effect of PrPC downregulation on prion infection in vitro we have established CAD5 cell lines with stably silenced expression of PrPC (LP1 and LP2) by retroviral vector expressing shRNA in context of miRNA. In comparison with control lines (LN and LMP) downregulation led to reduction of PrPC expression under 10% on a protein level as shown by quantitative flow cytometry. Control lines expressed around 500,000 molecules of PrPC and lines LP1 and LP2 expressed around 35,000 molecules of PrPc. Downregulation of PrPC expression was confirmed by western blot and quantitative RT-PCR. Infection of cells with RML brain homogenate led to significant suppression of de novo PrPtse formation in LP1 and LP2 cells. Cell blot assay estimated that the levels of PrPtse were at least 100 times lower than in control lines and remained under the detection limit of western blot. Both, the medium and cell lysate of RML infected LN and LMP cells were able to infect susceptible CAD5 cells. However, the same material from infected LP1 and LP2 cells did not transmitted the infection. Our data suggest that despite significant reduction in PrPtse formation the downregulation of PrPC expression under 10% of its normal level does not prevent infection of CAD5 cells by prions.
Ppo3-17: A Typical/Nor98 Scrapie Infectivity in Sheep Peripheral Tissues
Caroline Lacroux,1 Leonor Orge,2,* Sylvie L. Benestad,3 Vincent Beringue,4 Claire Litaise,1 Stéphanie Simon,5 Hugh Simmons,6 Séverine Lugan,1 Fabien Corbière,1 Pierrette Costes,1 Nathalie Morel,5 François Schelcher1 and Olivier Andréoletti1,*
1UMR INRA ENVT 1225; Interactions Hôte Agent Pathogène; Ecole Nationale Vétérinaire de Toulouse; Toulouse, France; 2Laboratório Nacional de Investigação Veterinária; Estrada de Benfica, Lisboa, Portugal; 3National Veterinary Institute; Postboks; Oslo, Norway; 4INRA UR892; Virologie Immunologie Moléculaires; INRA; Jouy-en-Josas; 5CEA; Service de Pharmacologie et d’Immunoanalyse; IBiTec-S; DSV; CEA/Saclay; Gif sur Yvette cedex, France; 6VLA Weybridge; ASU; New Haw; Addlestone, Surrey UK
Key words: atypical, scrapie, peripheral tissues infectivity
Atypical/Nor98 scrapie was first identified in 1998 in Norway. It is now considered as a world widespread disease of small ruminants and currently represents more than the half of the detected TSE cases in Europe. Atypical/Nor98 scrapie agent biology and pathogenesis in its natural host is still poorly understood. Conversely to BSE and other small ruminants TSE agents, the ARR PrP allele does not provide protection against the disease, making the genetic selection policy inefficient to eradicate it. Based on the absence of detectable abnormal PrPSc in peripheral tissues the human and animal exposure risk to this specific TSE agent has been considered as low.
In the present study we first demonstrated that infectivity can accumulate, even if no abnormal PrP is detectable, in lymphoid tissues, nerves and muscles from natural and experimental Atypical/Nor98 scrapie cases. We furthermore demonstrated that, in comparison to other TSE agents, samples containing massive amount of Atypical/Nor98 scrapie infectivity can remain PrPSc negative. This feature probably impacts our perception of Atypical/Nor98 scrapie prevalence and spreading in field population. We finally evaluated, in both Atypical/Nor98 and classical scrapie cases, the infectivity loads accumulating in peripheral tissues that currently enter unrestricted into the food chain. The obtained results indicate that dietary exposure risk to small ruminants TSE agents is much higher than commonly believed. This conclusion raises the question of the potential capacities such TSE agents to transmit in other species.
PPo3-18: A Possible Case of Maternal Transmission of the BSE Agent within Captive Cheetah Affected with Feline Spongiform Encephalopathy
Anna Bencsik, Sabine Debeer, Thierry Petit and Thierry Baron
Afssa; Unité ATNC; Lyon, France; Zoo de la Palmyre; Les Mathes, France
Key words: BSE, FSE, vertical transmission
Introduction. Feline spongiform encephalopathy (FSE) is considered to be related to bovine spongiform encephalopathy (BSE). It has been reported in domestic cats as well as in captive wild cats including cheetahs, first in the United Kingdom (UK) and then in other European countries. In France, several cases were described in cheetahs either imported from UK or born in France. Here we report details of two other FSE cases in captive cheetah. These cases are of particular interest since the 2nd case of FSE in a cheetah born in France, appears most likely due to maternal transmission.1
Results. Complete PrPd study showed the close likeness between the two cheetah cases. The TgOvPrP4 mouse brains infected with cattle BSE and cheetah FSE revealed similar vacuolar lesion profiles, PrPd brain mapping with occurrence of typical florid plaques.
Materials and Methods. Using immunohistochemistry (IHC), pathological form of PrP(PrPd) was analyzed in the brains and peripheral organs of these two cheetahs. Transmission studies to the TgOvPrP4 mouse line were also performed, for comparison with the transmission of cattle BSE. Lesion profiles of the infected transgenic mice were analyzed as well as type and brain distribution of PrPd.
Conclusion. Collectively, these data indicate that both FSE cases harbor the same strain of agent as the cattle BSE agent. Because this is most probably a case of maternal transmission of the disease, this new observation may have some impact on our knowledge of vertical transmission of BSE agent-linked TSEs such as in human variant Creutzfeldt Jakob disease.
References
1. Bencsik et al. PLoS One 2009; 4:6929.
PPo3-19: Detection of CWD Prions in Salivary and Urinary Tissues of Deer: Potential Mechanisms of Pathogenesis and Prion Shedding
Nicholas J. Haley,1 Candace K. Mathiason,1 Glenn C. Telling2 and Edward A. Hoover1
1Department of Microbiology, Immunology and Pathology; College of Veterinary Medicine and Biomedical Sciences; Colorado State University; Fort Collins, Colorado USA; 2Department of Molecular Biology and Genetics; University of Kentucky; Lexington, Kentucky USA
Key words: chronic wasting disease, transmission, PMCA, pathogenesis, excretion, urine, saliva, salivary gland, urinary bladder, kidney, blood
Saliva and urine are thought to play an important role in the transmission and pathogenesis of chronic wasting disease (CWD) in captive and free-ranging cervids. We have previously identified PrPCWD in a variety of excreta using serial PMCA (sPMCA) and bioassay; however the source of infectious prions in urine and saliva has yet to be identified. In the present study, we applied sPMCA to tissues associated with saliva and urine production and excretion in an effort to seek proximal sources of prion shedding. Oropharyngeal and urogenital tissues, along with blood and obex from CWD-exposed cervids (comprising over 300 individual samples) were analyzed blindly in duplicate and scored based on apparent CWD burden. PrPCWD was detected by three rounds of sPMCA in tissues associated with saliva and urine production and excretion, notably salivary gland and urinary bladder; whereas blood samples from the same animals and concurrent negative controls (n = 116 of 117) remained negative. Route of inoculation and CNS burden appeared to play an important role in terminal prion distribution, in that IV-inoculated animals and those with increasing CNS levels of PrPCWD had higher and more widely distributed accumulation in excretory tissues. PMCA identification of PrPCWD in oropharyngeal and urogenital tissues—in the absence of detection by conventional methods—may indicate the presence of protease-sensitive infectious prions in excretory tissues not revealed by assays employing PK digestion or other means to remove PrPC reactivity. Thus, evaluation of peripheral tissues via sPMCA may allow additional insights into prion transmission, trafficking and pathogenesis.
PPo3-20: Transmission and Adaptation of Chronic Wasting Disease to North American Voles
Christina M. Carlson,1,2 Jay R. Schneider,1 Dennis M. Heisey,1 Joel A. Pedersen2 and Christopher J. Johnson1
1Prion Research Laboratory; USGS National Wildlife Health Center; Madison, Wisconsin USA; 2Program in Cellular and Molecular Biology; University of Wisconsin; Madison, Wisconsin USA
We previously demonstrated efficient transmission of cervid chronic wasting disease (CWD) to four species of native North American cricetid rodents (which include hamsters, voles and New World rats and mice) via intracerebral inoculation: meadow vole (Microtus pennsylvanicus), red-backed vole (Myodes gapperi), white-footed mouse (Peromyscus leucopus), and deer mouse (Peromyscus maniculatus). Onset of clinical disease was faster and median survival times shorter in the two vole species than the two Peromyscus species. To investigate CWD adaptation in a new host, we performed five serial passages in meadow voles, starting with CWD positive white-tailed deer inoculum. Initial challenge resulted in a median survival time of 280 days, progressively shortening to 67 days by fifth passage. Western blot analysis demonstrated the presence of PrPTSE in brain tissue throughout all passages, and suggested stability of the glycosylation site occupancy ratios as diglycosylated > monoglycosylated > unglycosylated, despite ongoing adaptation. This glycosylation pattern was consistent with those observed in other cricetid rodents challenged with CWD, scrapie, or mouse-adapted 139A scrapie and contrasts with the Muridae (Old World rodents) 139A pattern of monoglycosylated>diglycosylated>unglycosylated. Similarly, the molecular mass of proteinase K-cleaved PrPTSE was indistinguishable throughout all passages, indicating adaptation may result in more subtle changes to the PrPTSE protein than can be resolved by western blotting. Immunohistochemical staining of brain sections revealed punctate PrPTSE staining and spongiosis, especially in the thalamus. The results of this study show that CWD can be intracerebrally transmitted and adapted to meadow voles, suggesting this species could be a potential bridge species or reservoir for CWD.
PPo3-21: Intra- and Inter-species Prion Transmission Results in Selection of Sheep Scrapie Strains
Takashi Yokoyama,1 Kentaro Masujin,1 Mary Jo Schmerr,2 Shu Yujing,1 Hiroyuki Okada,1 Yoshifumi Iwamaru,1 Morikazu Imamura,1 Yuichi Matsuura,1 Yuichi Murayama1 and Shirou Mohri1
1Prion Disease Research Center; National Institute of Animal Health; 2Iowa State University
Key words: scrapie, heterogeneity, sheep, strain, PrPres
Sheep scrapie is caused by multiple prion strains, which have been classified on the basis of their biological characteristics in inbred mice. The heterogeneity of natural scrapie prions in sheep flocks has not been clarified. In this study, we intravenously injected two sheep (Suffolk and Corriedale) with material from a natural case of sheep scrapie (Suffolk breed). These three sheep had identical prion protein (PrP) genotypes. The protease-resistant core of PrP (PrPres) in the experimental Suffolk sheep was similar to that in the original Suffolk sheep. In contrast, PrPres in the Corriedale sheep differed from the original PrPres but resembled the unusual scrapie strain, CH1641. This unusual PrPres was not detected in the original sheep. The PrPres distributions in the brain and peripheral tissues differed between the 2 challenged sheep. A transmission study in wild-type and TgBoPrP mice, which overexpress cattle PrP, led to the selection of different prion strains. The pathological features of prion diseases are thought to depend on the dominantly propagated strain. Our results indicate that prion strain selection occurs after both inter- and intraspecies transmission. The unusual scrapie prion was a “hidden” component in typical sheep scrapie.
PPo3-22: Detection of Environmentally Associated PrPSc on a Farm with Endemic Scrapie
Ben C. Maddison,1 Claire A. Baker,1 Helen C. Rees,1 Linda A. Terry,2 Leigh Thorne,2 Susan J. Belworthy2 and Kevin C. Gough3
1ADAS-UK LTD; Department of Biology; University of Leicester; Leicester, UK; 2Veterinary Laboratories Agency; Surry, KT UK; 3Department of Veterinary Medicine and Science; University of Nottingham; Sutton Bonington, Loughborough UK
Key words: scrapie, evironmental persistence, sPMCA
Ovine scrapie shows considerable horizontal transmission, yet the routes of transmission and specifically the role of fomites in transmission remain poorly defined. Here we present biochemical data demonstrating that on a scrapie-affected sheep farm, scrapie prion contamination is widespread. It was anticipated at the outset that if prions contaminate the environment that they would be there at extremely low levels, as such the most sensitive method available for the detection of PrPSc, serial Protein Misfolding Cyclic Amplification (sPMCA), was used in this study. We investigated the distribution of environmental scrapie prions by applying ovine sPMCA to samples taken from a range of surfaces that were accessible to animals and could be collected by use of a wetted foam swab. Prion was amplified by sPMCA from a number of these environmental swab samples including those taken from metal, plastic and wooden surfaces, both in the indoor and outdoor environment. At the time of sampling there had been no sheep contact with these areas for at least 20 days prior to sampling indicating that prions persist for at least this duration in the environment. These data implicate inanimate objects as environmental reservoirs of prion infectivity which are likely to contribute to disease transmission.
PPo3-23: A Common Strain of Agent is Present in Variant CJD Cases from Five Different Countries
Abigail B. Diack,1 Robert G. Will,2 Jean-Philippe Brandel,3,8 Stephane Haik,3,8 Fabrizio Tagliavini,4 Cornelia Van Duijn,5 Ermias D. Belay,6 Wun-Ju Shieh,6 Pierluigi Gambetti,7 Lawrence B. Schonberger6 and Jean C Manson1
1The Roslin Institute and R(D)SVS; University of Edinburgh; Roslin, Midlothian UK; 2National CJD Surveillance Unit; western General Hospital; Edinburgh, UK; 3APHPCellule Nationale de référence des maladies de Creutzfeldt-Jakob; Groupe Hospitalier Pitié-Salpêtrière; 47–83 boulevard de l’Hôpital; France; 4Istituto Nazionale Neurologico Carlo Besta; via Celoria; Milano, Italy; 5Department of Epidemiology and Biostatistics; Erasmus University Medical School; Rotterdam, The Netherlands; 6Centers for Disease Control and Prevention; Atlanta, GA USA; 7Department of Pathology; National Prion Disease Pathology Surveillance Center; Case western Reserve University; Cleveland, Ohio; 8Alzheimer’s and Prion Diseases; CRicm; Inserm UMRS 975; CNRS UMR 7225; UPMC-Paris; France
Key words: variant CJD, strains
Introduction. vCJD was first reported in the UK in 1996. Since then, 47 cases of vCJD have been reported in a number of countries outside the UK. It has been hypothesised that a common strain of agent is responsible for all cases of vCJD despite their geographical origin. In order to test this hypothesis, a strain typing panel of mice were inoculated with brain material from four non-UK cases of vCJD.
Results. Evidence of disease transmission was apparent in all four cases of vCJD. There was a very close similarity in incubation period data from MR mice in all cases. TSE vacuolation lesion profile scores in MR mice were found to be almost identical in each case. Incubation period and lesion profiles from each case were comparable to UK reference cases.
Materials and Methods. A panel of wild-type mice were inoculated with brain material (frontal cortex where possible) from French, Dutch, Italian and American cases of vCJD. In these cases, the individual may have been exposed to BSE either through UK exports or a period of residency in the UK.
Conclusion. Preliminary findings suggest that there are very strong similarities between the transmission properties of the four non-UK vCJD cases and reference UK cases. These results support the hypothesis that a single strain of infectious agent is responsible for all vCJD infections despite cases having different countries of residence.
PPo3-24: Fluid Homeostasis is Altered in Sheep with Scrapie
M.C. Garza,1 L. González,2 J.J. Badiola,1 M. Jeffrey2 and S. Sisó2
1Centro de Investigación Encefalopatías y Enfermedades Transmisibles Emergentes; Departamento de Patología Animal; Facultad de Veterinaria; Universidad de Zaragoza; Zaragoza, Spain; 2Department of Pathology (Lasswade); Veterinary Laboratories Agency; Pentlands Science Park; Penicuik, Scotland UK
Key words: vasopressin, oxytocin, sensory circumventricular organs, scrapie
Vasopressin and oxytocin are involved in the maintenance of the osmotic pressure of blood and vascular tone. Hypothalamic neurons are responsible for the synthesis of these hormones and their transport to extra-hypothalamic areas, the pituitary gland for their release in the blood stream, and the sensory circumventricular organs (CVOs) for sensing the concentration of water and electrolytes in the blood.
In sheep with scrapie, the CVOs have severe accumulation of the disease-associated prion protein (PrPd). The present study aims to investigate if, as a result of infection and/or in connection with PrPd accumulation, fluid homeostasis is altered in sheep clinically affected with scrapie. Immunohistochemistry for PrPd, vasopressin and oxytocin was carried out in six hypothalamic sections of brains from 17 sheep with scrapie and 8 negative controls.
The preliminary data show that the synthesis of vasopressin and oxytocin is not altered in scrapie-affected sheep. However, the topographical distribution and magnitude and types of immunolabelling revealed that abnormal transport to extra-hypothalamic regulatory centres might be impaired in affected animals. Complementary work is in progress in order to determine alterations of the expression of related signalling peptides and hormone receptors located within the CVOs.
PPo3-25: Transmission of TSEs to Transgenic Mice Overexpressing Ovine PrP(ARR)
P.C. Griffiths,1 J.M. Plater,1 D. Jayasena,1 A.C. Tout,1 A. Chave,1 J.L. Vilotte,2 M.J. Stack1 and O. Windl1
1Molecular Pathogenesis and Genetics Department; Veterinary Laboratories Agency; Addlestone, Surrey UK; 2Unite Mixte de Recherche “Genetique Animale et Biologie Integrative”; Animal Genetics Department; Institut National de la Recherche Agronomique; Jouy-en-Josas, France
Key words: transmission, TSE, atypical scrapie, classical scrapie, transgenic mice, ovine PrP(ARR) allele
Preparation of transgenic mouse models overexpressing ovine PrPs of different allotypes could lead to faster, more economical bioassays for detection of sheep TSEs. Overexpression models could also be more sensitive to TSE detection than wild-type mice and the natural host. In sheep, the ovine PrP(ARR) allele is recognised as being more resistant to infection with classical scrapie than other sheep PrP allotypes. However, a few naturally occurring cases of classical scrapie, as well as experimental transmission of BSE, have been described in sheep homozygous for PrP(ARR). The ovine PrP(ARR) allele has also shown susceptibility to atypical scrapie infection in the natural host. Here, we present preliminary findings of the attempted transmission of TSE agents to transgenic ovine PrP(ARR) mice. Tg[OvPrP(ARR)] mice were prepared by pronuclear microinjection of an ovine PrP transgene encoding amino acid residues alanine (A) at codon 136, and arginine (R) at codons 154 and 171. Expression of ovine PrP(ARR) in three generated mouse lines was transferred to a mouse PrP-ablated background, and the subsequent mice characterized for transgene copy number by Southern hybridisation and transgene expression levels by western blotting. In general, Tg[OvPrP(ARR)] mice demonstrated apparent resistance to TSE infection when challenged with classical and atypical scrapie from sheep and BSE from cattle. However, transmission of one atypical scrapie isolate, with a low attack rate, was shown in one transgenic mouse line by western blot detection of proteinase K-resistant PrPSc. We found that three classical scrapie isolates, containing different PrP genotypes, did not transmit to Tg[OvPrP(ARR)] mice.
PPo3-26: Identification of Renal Origin for CWD Urinary Prion Excretion in Deer
Davis M. Seelig,1 Nicholas J. Haley,1 Jan P. Langeveld and Edward A. Hoover1
1Colorado State University; Department of Microbiology, Immunology and Pathology; Fort Collins, CO USA; 2Central Institute for Animal Disease Control (CIDC-Lelystad); Lelystad, The Netherlands
Chronic wasting disease (CWD) is an efficiently transmitted prion disease of cervids. Although bioassays have confirmed the presence of infectious prions in urine and other body fluids of infected deer, origin and mechanisms of prion transfer to and shedding in excreta remains unknown. To address these questions, we have developed enhanced immunohistochemistry (IHC) methods employing tyramide signal amplification (TSA) on formalin-fixed, paraffin-embedded (FFPE) tissues of n = 20 CWD-infected white-tailed deer. Using these methods we have demonstrated PrPCWD present granular to clumped aggregates both within the cytoplasm of renal tubule cells and in the interstitium. Cytoplasmic PrPCWD aggregates were detected most commonly in proximal convoluted tubule epithelial cells. PrPCWD was not identified in the lower urinary tract (ureters or bladder) of any CWD-infected animal. In summary, we present evidence for PrPCWD accumulation within the renal tubule cells, which may identify a proximate tissue source and explain the manner by which infectious prions are excreted in the urine of infected deer, thereby leading to the high degree of direct and indirect horizontal transmission of chronic wasting disease.
PPo3-27: Clint1-mediated Clathrin-dependent Retrograde Transport is Involved in PrPSc Trafficking in Neuro2a Mouse Neuroblastoma Cells
Takeshi Yamasaki, Akio Suzuki and Motohiro Horiuchi
Laboratory of Veterinary Hygiene; Graduate School of Veterinary Medicine; Hokkaido University; Sapporo, Japan
Although propagation of prion in neurons is believed to be tightly associated with neuronal degeneration, the molecular mechanisms of prion propagation in cells have not been fully understood yet. We previously suggested the possibility that PrPSc in Neuro2a mouse neuroblastoma cells persistently infected with 22L prion strain is dynamically transported through peri-nuclear regions that are expected to be involved in endosomal recycling pathway. Therefore, we focused on retrograde transport from endosome to trans-Golgi network (TGN) which is one of the pathways involved in recycling of molecules. PrPSc and clathrin heavy chain (CHC) were fractionated in the same fraction by density gradient centrifugation. Furthermore, PrPSc was co-immunoprecipitated with CHC when the microsomal fractions were immunoprecipitated with anti-CHC antibody. However, pretreatment of the microsomal fraction with detergent abolished the co-immunoprecipitation. These results suggest that at least some part of PrPSc is associated with clathrin coated vesicles. In fact, PrPSc was co-localized with CHC and clathrin interactor 1 (Clint1), which is known to act as clathrin adaptor molecule that is involved in retrograde transport from endosome to TGN, at peri-nuclear regions. The knockdown of Clint 1 gene expression by small interfering RNA altered the intracellular distribution of PrPSc from peri-nuclear regions to peripheral regions. On the other hand, the amounts of proteinase K resistant PrP in cells were not changed by the downregulation of Clint1 expression. Our data raise the possibility that some part of PrPSc is transported from endosomes to peri-nuclear regions by Clint1-mediated clathrin-dependent mechanism.
PPo3-28: A Cell Culture Based Model to Simulate PrPSc Infection in Non-human Primates
Ann-Christin Schmädicke,1 Wiebke M. Wemheuer,2 Sabine Gilch,3 Sabine Borchert,1 Judith Montag,4 Ina Vorberg,3 Hermann M. Schätzl,3 Walter J. Schulz-Schaeffer2 and Dirk Motzkus1
1Unit of Infection Models; and 4Unit of Infection Biology; German Primate Centre; Göttingen, Germany; 2Neuropathology Unit; UMG; Göttingen, Germany; 3Department of Virology; Technical University; Munich, Germany
Key words: non-human primates, species barrier, BSE, cell culture model
Objectives. The project aims at establishing a cell culture-based system that recapitulates prion infection in vivo. Prion propagation in cell lines that are transgenic for macaque PrP will be used to simulate BSE-infection of non-human primates as a model.
Results. The PrPSc permissive cell line mafaHpL3-4 was inoculated with macaque-adapted BSE in 12 well format. Infected and control cells were passaged in triplicate for 29 days post inoculation (dpi). Samples consisting of 106 cells were taken at regular intervals at 7, 9, 11, 14, 16, 18, 21, 23, 25 and 29 dpi, respectively. Production of Proteinase K-resistant PrPSc after infection was assessed by western blot analysis and by membrane adsorption assay. Our analysis revealed that macaque-adapted BSE infection of mafaHpL3-4 cells can be detected as early as 11 dpi. Using an ultrasensitive membrane adsorption assay PrPSc was detectable in less than 2 x 104 cells.
Discussion. Reduction or replacement of animal experimentation has become a major goal in biomedical research. To date, the use of non-human primates for risk assessment studies of zoonotic prion diseases is still essential. We have produced a transgenic cell line that can be used to simulate prion infection. The markedly reduced time from inoculation to PrPSc accumulation, in conjunction with a high number of replicates provides a powerful tool to assist in vivo animal models, which could lead to the reduction of animal experiments.
PPo3-29: Reaction of Complement Factors Differs with Prion Strains in vitro and in vivo
Rie Hasebe, Motohiro Horiuchi and Byron Caughey
Laboratory of Veterinary Hygiene; Graduate School of Veterinary Medicine; Hokkaido University; Sapporo, Japan; Rocky Mountain Laboratories; National Institute of Allergy and Infectious Diseases; National Institute of Health; Hamilton, MT USA
Key words: complement factors, prion strain
Roles of complement factors in prion infection of the central nervous system remain unclear. In this study, we attempted to assess possible involvement of complement factors in neuropathogenesis of prion disease. When neuro2a (N2a) cells persistently infected with Chandler- and 22L-scrapie were cultured in the presence of normal mouse serum (NMS), the number of degenerating (Annexin V-positive) cells were increased both in Chandler- and 22L-infected N2a cells. Preincubation of NMS with anti-complement C1q, C3 and/or C9 antibodies reduced the number of Annexin V-positive cells in Chandler-infected N2a cells, while only anti-C3 antibodies were influential on 22L-infected cells. Membrane attack complex (MAC) was detected on Chandler-infected N2a cells, but not on 22L-infected cells. These results suggest that different complement components reacted with Chandler- and 22L-infected N2a cells. To assess potential differences in complement activation between these prion strains in vivo, we tested for C1q and C3 by immunohistochemistry in Chandler- and 22L-infected C57BL/6 mouse brains in the pre-clinical phase. Consistent with the reaction of complement factors to prion-infected N2a cells, C1q, but not C3, was detected in the vacuolar lesions of the thalamus in Chandler-infected mouse brains. In contrast, in the same lesions of 22L-infected mouse brains, C3 was detected but C1q signals were sometimes lacking. These results suggest that Chandler and 22L strains activate different complement components in vivo as well as in N2a cells.
PPo3-30: Immunohistochemical and Biochemical Characteristics of BSE and CWD in Experimentally Infected European Red Deer (Cervus Elaphus)
Stuart Martin,1 Martin Jeffrey,1 Lorenzo González,1 Sílvia Sisó,1 Hugh Reid,2 Philip Steele,2 Mark Dagleish,2 Michael Stack,1 Melanie Chaplin,1 John Spiropoulos,1 Marion Simmons,1 Wilfred Goldmann3 and Aru Balachandran4
1Veterinary Laboratories Agency; Addlestone, Surrey UK; 2Moredun Research Institute, Penicuik, Midlothian, Scotland UK; 3Neuropathology Unit; Roslin, Scotland UK; 4Canadian Food Inspection Agency
Key words: red deer, BSE, CWD, IHC, WB, polymorphism, bioassay
Thirty-two deer were orally or intra-cerebrally dosed with homogenate from a pool of five BSE-positive bovine brains and negative control animals underwent identical procedures with sterile saline buffer. An extensive range of samples was tested by immunohistochemistry (IHC), western blot (WB, brainstem only) and mouse bioassay (2 positive deer). In the absence of clinical signs, none of the 12 orally-dosed deer culled after 6 or 12 months, nor 5 of 6 culled at the termination of the experiment (72 months), showed any evidence of abnormal PrP accumulation by IHC or WB. In contrast, all the 6 intra-cerebrally challenged and 1 of 6 orally dosed deer developed clinical disease at various times after infection. These deer showed widespread accumulation of disease specific PrP in the CNS, PNS and ENS but none in the LRS. Both IHC and WB features were similar to those of BSE in sheep, goats and cattle but unlike those seen in CWD in elk or scrapie in sheep. Analysis of the PrP ORF of all deer in the experiment identified a Q to E polymorphism at codon 226. Interestingly, the single deer that succumbed to oral BSE infection was the only QQ deer of the 6 allowed to develop clinical disease, suggesting that such polymorphism may influence the susceptibility of deer to oral BSE. Brain homogenates from positive deer were inoculated into panels of 20 Tg(cerPrP) 1536+/- mice, which developed neurological signs with an incubation period of 202–298 days post inoculation and attack rates of 90–95%.
PPo3-31: Inhibitors of Gastric Acid Secretion Increase the Risk of Prion Infection in Mice
Tom C. Martinsen,1,2 Sylvie L. Benestad,3 Torfinn Moldal,3 Kristin M. Aasaroed2 and Helge L. Waldum1,2
1Department of Cancer Research and Molecular Medicine; Faculty of Medicine; Norwegian University of Science and Technology (NTNU); 2Department of Medicine; St. Olav’s Hospital HF; Trondheim University Hospital; Norway; 3National Veterinary Institute; Oslo, Norway
Key words: gastric juice, scrapie, transmission, mice
Prions cause fatal transmissible degenerative encephalopathies in animals and man. The natural route of transmission is generally via contaminated food. As prions do not activate the immune system, non-immunological mechanisms of the gut may be the only defence against prion diseases. Gastric juice is a unique combination of hydrochloric acid and proteolytic enzyme pepsin. Its main function is to inactivate ingested microorganisms, and individuals with hypoacidity are more prone to infections. The aim of this study was to examine if drug induced inhibition of gastric acid secretion increases the rate of prion transmission.
Results. Thirteen out of 22 (59%) mice given omeprazole were PrPSc positive in their brain compared to six out of 24 (25%) mice in the control group (p < 0.035).
Material and Methods. Forty-six Tg338 mice (transgenic for the VRQ allele of ovine PrP) were given 1 ml of 0.05% sheep brain homogenates (Norwegian classical scrapie isolate) by gastric intubation. Twenty-two out of these animals were dosed with a proton pump inhibitor (omeprazole) 180 mg/kg subcutaneously twice a day (increasing gastric pH from 1.3 to 5.4). After 381 days the animals were sacrificed and their brains were examined for pathogenic prion protein (PrPSc) by ELISA and western blot.
Conclusion. Our study shows that normal gastric juice constitutes a significant defence against prion disease in mice. On the basis of this study, gastric hypochlorhydria, acquired or iatrogenic, would be expected to enhance the susceptibility to prion infection by the oral route.
PPo3-32: Nervous Dissemination of BSE in Orally Infected Goats
Frédéric Lantier,1,* Christine Hoffmann,2,* Patricia Berthon,1 Susanne Freyse,2 Isabelle Lantier,1 Anne Balkema-Buschmann,2 Christelle Rossignol,1 Kerstin Tauscher,2 Hervé Le Roux,1 Francis Barillet,3 Olivier Andréoletti4 and Martin H. Groschup2
*First authors
1INRA; IASP; Nouzilly, France; 2FLI-INEID; Riems, Germany; 3INRA; SAGA; Toulouse, France; 4INRA-ENVT; IHAP; Toulouse, France
Key words: BSE, goat, disease-associated PrP, pathogenesis
As the consumption of products from naturally BSE-infected goats represents a potential exposure risk to humans, we investigated tissue distribution of BSE in orally challenged goats of different PRNP genotypes. Healthy groups of three goats carrying three scrapie-susceptible PRNP genotypes [I142R211Q222/IRQ: wild type (WT), IRQ/IQQ: Q211 mutated (Q211) and IRQ/IRK: K222 mutated (K222)] were orally challenged with cattle (INRA) or goat (FLI) derived BSE and sequentially culled at 6, 12, 17 and 24–25 months post-inoculation (mpi). At necropsies, a large number of samples was taken for the detection of disease-associated PrP (PrPD) (IHC, ELISA, WB) and infectivity (transgenic bioassays).
First traces of PrPD in preclinical goats were detectable 12 mpi in the Ganglia coeliacum of a WT goat and in follicles of the ileocaecal junction in a Q211 goat. At 17 mpi, PrPD was localised in the ileal enteric nervous system of 2/3 WT goats and a Q211 goat. This goat also contained PrPD accumulation in the central nervous system (obex and thoracic spinal cord). Another Q211 goat showed PrPD in the obex only. First clinical symptoms were observed in 4 WT goats after 24–25 mpi, revealing a severe spongiform encephalopathy with a PrPD accumulation in the brain stem. Until now no PrPD was detectable in tonsils.
In this study incubation periods for BSE in goats were longer than for scrapie and are influenced by the PRNP genotypes. Pathogenic pathways, mainly limited to neuronal tissues, restrict the usefulness of lymphoid tissue biopsies for ante-mortem diagnosis of BSE-infected goats.
PPo3-33: The Role of Phagocytic Cells in Resistance to Scrapie in Sheep
Fiona Houston
Division of Animal Production and Public Health; Faculty of Veterinary Medicine; Glasgow, Scotland UK
The association between Prnp genotype and susceptibility or resistance to scrapie in sheep is well established, but the underlying mechanisms are not clearly understood. The hypothesis behind this study is that resistance to scrapie may be explained by more efficient degradation and clearance of PrPSc by phagocytic cells, e.g., macrophages and dendritic cells, in sheep with “resistant” genotypes. The ability of monocyte-derived macrophages and alveolar macrophages isolated from VRQ/VRQ and ARR/ARR sheep to degrade PrPSc was tested in an in vitro assay. The cells were incubated with scrapie-infected sheep brain homogenate, and the degradation of PrPSc assessed by transferring cells on to PVDF membranes at various time points following exposure, and immunostaining the proteinase K treated cells blots using a PrP-specific monoclonal antibody (SAF32). Preliminary results show that there is no difference in the rate of PrPSc degradation by macrophages from VRQ/VRQ and ARR/ARR sheep over 72 hours following exposure. In addition, Prnp genotype does not appear to have any effect on the phagocytic capacity of macrophages, as assessed by uptake of latex beads and fluorescently labelled bacteria. Confirmation of these results would suggest that alternative mechanisms play a more important role in resistance to scrapie in sheep.
PPo3-34: Prion Detection in Tissues and Body Fluids of Sheep Affected by Scrapie: A Comparison Between PMCA, Western-Blot and Bioassay
Gian Mario Cosseddu,1 Michele Di Bari,1 Philip Steele,2 Francesca Chianini,2 Luigi De Grossi,3 Umberto Agrimi,1 Marta Vascellari,4 Irini Fragkiadaki,4 Franco Mutinelli,4 Gabriele Vaccari1 and Romolo Nonno1
1Department of Public Veterinary Health and Food Safety; Istituto Superiore di Sanità; Via Regina Elena; Rome, Italy; 2Moredun Research Institue; Pentlands Science Park; Bush Loan; Penicuik, Midlothian UK; 3Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana; Via Appia Nuova; Roma, Italy; 4Istituto Zooprofilattico Sperimentale delle Venezie; Viale dell’Universita’; Legnaro, Padova Italy
Key words: scrapie, sheep, PMCA, bioassay
Aim of this study was to compare the ability of protein misfolding cyclic amplification (PMCA), western-blot and bank vole bioassay to detect scrapie prions in tissues and body fluids of sheep.
Ten-percent (w/v) homogenates of brain, kidney, semitendinosus muscle, sciatic nerve, prescapular lymph-node, oculomotor muscle, heart, tongue and salivary glands, together with a brain dilution curve from clinically affected sheep were submitted to PMCA using vole brain homogenate as substrate. Samples of saliva were also included in the study.
Western-blot revealed PrPSc up to the 10e-3 dilution of the brain curve. Positivity was also observed in lymph-nodes, salivary glands and sciatic nerve.
PMCA showed a higher sensitivity, being able to amplify PrPSc up to the 10e-5 brain dilution after 5 rounds. PrPSc amplification was obtained from salivary glands, prescapular lymph-node, sciatic nerve but also from kidney and oculomotor muscle, between 5 and 8 PMCA rounds. Moreover, 3 out 5 samples of saliva from infected sheep gave positive amplification after 8 rounds. Tongue, heart and semitendinosus muscle as well as negative controls remained negatives up to the 10th rounds, when the experiment was terminated.
The comparison between PMCA and vole bioassays gave consistent results. The same samples that were positive by PMCA produced disease in voles (with the exception of saliva samples the transmission of which started only recently). 10e-4 was the last brain dilution that produced disease in voles. In our conditions, PMCA is reliable and at least as much sensitive as vole bioassay for prion detection in sheep scrapie.
PPo3-35: Susceptibility of Domestic Cats to CWD Infection
Amy V. Nalls, Candace K. Mathiason, Nicholas J. Haley, Jeanette Hayes-Klug, Kelly R. Anderson, Davis M. Seelig, Dan S. Bucy, Susan L. Kraft and Edward A. Hoover
Colorado State University; Fort Collins, CO USA
Domestic and non-domestic cats have been shown to be susceptible to one prion disease, feline spongiform encephalopathy (FSE), thought to be transmitted through consumption of bovine spongiform encephalopathy (BSE) contaminated meat. Because domestic and free ranging felids scavenge cervid carcasses, including those in CWD affected areas, we evaluated the susceptibility of domestic cats to CWD infection experimentally. Groups of n = 5 cats each were inoculated either intracerebrally or orally with CWD deer brain homogenate. Between 40 and 43 months following IC inoculation, two cats developed mild but progressive symptoms including weight loss, anorexia, polydipsia, patterned motor behaviors, and ataxia—ultimately mandating euthanasia. Magnetic resonance imaging (MRI) on the brain of one of these animals (vs. two age-matched controls) performed just before euthanasia revealed increased ventricular system volume, more prominent sulci, and T2 hyperintensity deep in the white matter of the frontal hemisphere and in cortical grey distributed through the brain, likely representing inflammation or gliosis. PrPRES and widely distributed peri-neuronal vacuoles were demonstrated in the brains of both animals by immunodetection assays. No clinical signs of TSE have been detected in any of the 5 cats orally inoculated or the 2 remaining intracerebrally inoculated cats after 73 months pi. Although the limited data from this ongoing study must be considered preliminary, they raise the potential for cervid-to-feline transmission in nature.
PPo3-36: Inherited Prion Disease A117V is Transmissible to Transgenic Mice Expressing PRNP 117-valine
Emmanuel A. Asante, Jacqueline M. Linehan, Michelle Smidak, Sebastian Brandner and John Collinge
MRC Prion Unit and Department of Neurodegenerative Disease; UCL Institute of Neurology; Queen Square, London UK
Prion diseases are by definition transmissible, but some inherited prion diseases (IPDs) including Gerstmann-Straussler-Scheinker (GSS) disease associated with alanine to valine substitution at amino acid position 117 (A117V) on the human prion protein gene (PRNP) have not been shown to be transmissible experimentally and could represent prion proteinopathies than transmissible diseases. This IPD has a diverse phenotypic spectrum, even among members of the same family. The absence of transmission data, coupled with the inability to detect the pathological misfolded protein, PrPSc, in A117V GSS patients’ brains has led to the suggestion that pathology may be related to an aberrant topological form of PrP referred to as CtmPrP. We have therefore investigated the transmissibility of inherited prion disease A117V in appropriate transgenic models. Transgenic mice expressing human PrP 117V show for the first time that brain homogenates from IPD A117V patient brains are transmissible and can result in clinical disease and propagation of detectable full-length PrPSc. Although these 117V transgenic mice express the identical protein, there remains a substantial transmission barrier which manifests in low clinical attack rates and prolonged incubation periods. Iatrogenic and sporadic CJD isolates propagated readily in the 117V transgenic mice with the production of stable PrPSc. However, PrPSc produced from transmission of prions from IPD A117V patient brains were only transiently detectable. We conclude that previous difficulties in detecting PrPSc from A117V patients’ brains may be due to inherent instability and partial degradation of PrPSc to levels below the detection limit, but remaining sufficiently infectious for the successful transmission to 117V transgenic mice.
PPo3-37: Comparative Susceptibility of Sheep to Scrapie and BSE Might Explain Why the BSE Agent Does Not Circulate in the European Sheep Population
Romolo Nonno,1 Michele Di Bari,1 Claudia D’Agostino,1 Francesca Rosone,2 Stefano Marcon,1 Elena Esposito,1 Michela Conte,1 Barbara Chiappini,1 Nadia Palazzini,1 Geraldina Riccardi,1 Gabriele Vaccari,1 Luigi De Grossi2 and Umberto Agrimi1
1Istituto Superiore di Sanità; Rome, Italy; 2Istituto Zooprofilattico Sperimentale delle Regioni Lazio e Toscana; Rome, Italy
Sheep are susceptible to BSE and have been exposed to contaminated MBM. However, no sheep with natural BSE has been detected so far.
Here we compare the efficiency of transmission and the pathogenesis of scrapie and BSE in sheep. An Italian scrapie isolate was chosen because it targets the ARQ and AHQ alleles, similarly to BSE. Eight-to-ten months old, PrP-sequenced sheep (n = 210) were infected by the intracerebral (i.c.) or oral route.
By i.c., ARQ/ARQ sheep showed 100% attack rate and similar survival times following scrapie and BSE inoculation (460 and 480 dpi, respectively). ARR/ARR sheep were susceptible to BSE (5/7, 1,600 dpi), but not to scrapie (0/5, >2,800 dpi). Incubation times at primary transmission and second passage of BSE were very similar.
Despite the efficient i.c. transmission, sheep displayed a low susceptibility to BSE by the oral route. After >2,200 dpi, only 4/6 ARQ/ARQ (700 dpi) and 2/4 ARQ/AHQ (800 dpi) sheep succumbed to BSE, in contrast to the 100% attack rate observed with scrapie. Furthermore, scrapie induced PrPSc accumulation in the LRS from 2 mpi and in the CNS from 12 mpi onwards, while LRS and CNS were simultaneously positive starting from 13 mpi in BSE, with two sheep still negative at 17 and 20 mpi.
Overall, these findings indicate that adult sheep might be relatively resistant to oral BSE, possibly due to an inefficient lymphoid replication. This proposes that the exposure of sheep to contaminated MBM might have been insufficient to induce a sustainable infection under field conditions.
PPo3-38: Trafficking of Prion Proteins via Exosomes
Dana K. Thurm,1 Susanne Krasemann,1 Lars Redecke2 and Markus Glatzel1
1Institute of Neuropathology; University Medical Center Hamburg-Eppendorf; Germany; 2Joint Laboratory for Structural Biology of Infection and Inflammation of the Universities Hamburg and Lübeck; c/o DESY; Hamburg, Germany
Key words: prion protein, exosomes
Transmissible Spongiform Encephalopathies (TSEs) are neurodegenerative diseases; caused by a proteinaceous infectious particle of which a misfolded and beta-sheet rich isoform of the host encoded Prion Protein (PrPC) represents an essential component. This infectious conformer (PrPSc) mainly accumulates in the central nervous system as plaques and causes neuronal loss, astrocytic activation (gliosis) and spongiform change. Since PrPSc accumulates both intra and extracellularly, cell-to-cell transport is essential for disease progression.
Exosomes are small, nanometre-sized vesicles, released in the extracellular space when multivesicular bodies (MVBs) fuse with the plasma membrane. They arise from the inward budding from the limiting membrane of endosomes, contain cytosol and expose the extracellular domains of transmembrane proteins. The endosomal lipid bilayer has the same composition as the Plasma membrane of their donor cell and they can transport proteins or lipids to target cells.
As PrPC and potentially PrPSc are held in the Plasma membrane via a glycosylphosphatidyl-anchor they may be transported via exosomes.
We study trafficking of the Prion Protein via exosomes, looking at the following parameters:
1. Is the amount and composition (PrP amounts and types) of exosomes cell-defined?
2. Is the uptake of exosomes PrPC dependent?
PPo3-39: Experimental Bovine Spongiform Encephalopathy: Detection of PrPSc in the Small Intestine Relative to Exposure and Age
Michael J. Stack,1 Sarah J. Moore,2 Alberto Vidal-Diez,3 Mark E. Arnold,3 Elinor M. Jones,3 Yvonne I. Spencer,2 Paul Webb,2 John Spiropoulos,2 Peter Bellerby,2 Lisa Thurston,2 Julie Cooper,2 Melanie J. Chaplin,1 Linda A. Davis,1 Sharon Everitt,1 Raffaella Focosi-Snyman,1 Stephen A.C. Hawkins,2 Marion M. Simmons2 and Gerald A.H. Wells2
1Molecular Pathogenesis and Genetics Department; 2Pathology Department; and 3Centre for Epidemiology and Risk Analysis; Veterinary Laboratories Agency (VLA); New Haw, Addlestone, Surrey UK
European BSE controls decree destruction of all intestines from slaughtered cattle, but studies suggest that BSE infectivity is largely confined to the ileum. Relative to time post exposure this study investigates disease-associated prion protein (PrPSc) in the duodenum, jejunum and ileum of cattle exposed orally to a 1 g or 100 g dose of a titred BSE brainstem homogenate. Lymphoid follicles were counted and the frequency of affected follicles recorded. No PrPSc was detected in duodenum or jejunum of animals exposed to a 1 g dose, nor duodenum of animals receiving a 100 g dose. PrPSc was detected in lymphoid tissue of ileum of 1.0% animals receiving the 1 g dose and in jejunum and ileum of 13.8% and 45.5% respectively of animals receiving the 100 g dose. The frequency of PrPSc positive follicles was less than 1.5% per case and biochemical tests appeared less sensitive than immunohistochemistry. The probability of detecting lymphoid follicles in ileum declined with age and for the 100 g exposure the proportion of positive follicles increased while the proportion of positive animals decreased with age. Detection of PrPSc in neural tissue was rare. The results suggest that the jejunum and duodenum of BSE infected cattle contain orders of magnitude less BSE infectivity than ileum, irrespective of exposure dose. In animals receiving the low exposure dose, as in most natural cases of BSE, the rarity of PrPSc detection compared to high dose exposure, indicates a very low BSE risk from the jejunum and duodenum of cattle slaughtered for human consumption.
PPo3-40: Mother to Offspring Transmission of Chronic Wasting Disease
Candace K. Mathiason, Amy V. Nalls, Kelly Anderson, Jeanette Hayes-Klug, Nicholas Haley and Edward A. Hoover
Colorado State University, Department of Microbiology, Immunology and Pathology, Fort Collins, CO USA
Key words: chronic wasting disease, vertical transmission, muntjac deer
We have developed a new cervid model in small Asian muntjac deer (Muntiacus reevesi) to study potential modes of vertical transmission of chronic wasting disease (CWD) from mother to offspring. Eight of eight (8/8) muntjac doe orally infected with CWD tested PrPCWD lymphoid positive by 4 months post infection. Six fawns were born to these CWD-infected doe. Six fawns were born to 6 CWD-infected doe; 4 of the fawns were non-viable. The viable fawns have been monitored for CWD infection by immunohistochemistry and sPMCA performed on serial tonsil and rectal lymphoid tissue biopsies. PrPCWD has been detected in one fawn as early as 40 days of age. Moreover, sPMCA performed on rectal lymphoid tissue has yield positive results on another fawn at 10 days of age. In addition, sPMCA assays have also demonstrated amplifiable prions in maternal placental (caruncule) and mammary tissue of the dam.
Additional pregnancy related fluids and tissues from the doe as well as tissue from the nonviable fawns are currently being probed for the presence of CWD. In summary, we have employed the muntjac deer model, to demonstrate for the first time the transmission of CWD from mother to offspring. These studies provide the foundation to investigate the mechanisms and pathways of maternal prion transfer.