CWD PRION CONGRESS SEPTEMBER 8-11 2010
PRION 2010
International Prion Congress: From agent to disease September 8–11, 2010 Salzburg, Austria
PRION 2010 is the top Global Annual TSE Conference in prion research, following a sequence of PRION meetings that were originally organized by the EU Network of Excellence NeuroPrion. In this proud tradition, PRION 2010 covers all aspects of this fascinating scientific area. PRION 2010 is a meeting of greatest interest for neuroscientists, protein structural biologists, geneticists, medical specialists including neurologists, neuropathologists, hygiene experts and blood product providers, veterinarians, epidemiologists, laboratory technicians, industry developers, risk assessors and managers. An outstanding list of Plenary Lecture, Symposia and Workshop Speakers is complemented by the plethora of original input from Poster Presentations. Special consideration is given this year to two areas of major interest: the renewed discussion about the zoonotic potential of animal prion diseases, given the emergence of atypical BSE and scrapie strains, and the breakthrough work on synthetic prions by several groups simultaneously.
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PPo4-4:
Survival and Limited Spread of TSE Infectivity after Burial
Karen Fernie, Allister Smith and Robert A. Somerville The Roslin Institute and R(D)SVS; University of Edinburgh; Roslin, Scotland UK
Scrapie and chronic wasting disease probably spread via environmental routes, and there are also concerns about BSE infection remaining in the environment after carcass burial or waste 3disposal. In two demonstration experiments we are determining survival and migration of TSE infectivity when buried for up to five years, as an uncontained point source or within bovine heads. Firstly boluses of TSE infected mouse brain were buried in lysimeters containing either sandy or clay soil. Migration from the boluses is being assessed from soil cores taken over time. With the exception of a very small amount of infectivity found 25 cm from the bolus in sandy soil after 12 months, no other infectivity has been detected up to three years. Secondly, ten bovine heads were spiked with TSE infected mouse brain and buried in the two soil types. Pairs of heads have been exhumed annually and assessed for infectivity within and around them. After one year and after two years, infectivity was detected in most intracranial samples and in some of the soil samples taken from immediately surrounding the heads. The infectivity assays for the samples in and around the heads exhumed at years three and four are underway. These data show that TSE infectivity can survive burial for long periods but migrates slowly. Risk assessments should take into account the likely long survival rate when infected material has been buried.
The authors gratefully acknowledge funding from DEFRA.
PPo8-13:
Degradation of Pathogenic Prion Protein and Prion Infectivity by Lichens
Christopher J. Johnson,1 James P. Bennett,1 Steven M. Biro,1,2 Cynthia M. Rodriguez,1,2 Richard A. Bessen3 and Tonie E. Rocke1
1USGS National Wildlife Health Center; 2Department of Bacteriology; University of Wisconsin, Madison; 3Department of Veterinary Molecular Biology; Montana State University; Bozeman, MT USA
Key words: prion, lichen, bioassay, protease, degradation
Few biological systems have been identified that degrade the transmissible spongiform encephalopathy (TSE)-associated form of the prion protein (PrPTSE) and TSE infectivity. Stability of the TSE agent allows scrapie and chronic wasting disease agents to persist in the environment and cause disease for years. Naturally-occurring or engineered processes that reduce infectivity in the environment could aid in limiting environmental TSE transmission. We have previously identified that species of at least three lichens, unusual, symbiotic organisms formed from a fungus and photosynthetic partner, contain a serine protease capable of degrading PrPTSE under gentle conditions. We tested the hypothesis that lichen extracts from these three species reduce TSE infectivity by treating infected brain homogenate with extracts and examining infectivity in mice. We found lichen extracts diminished TSE infectious titer by factors of 100 to 1,000 and that reductions in infectivity were not well-correlated with the extent of PrPTSE degradation observed by immunoblotting. For example, treatment of brain homogenate with Cladonia rangiferina extract caused <100-fold activity="" after="" agents.="" also="" and="" anti-prion="" br="" but="" characterization="" cladonia="" closely="" clusters="" comparison="" data="" decrease="" degradation="" degrade="" do="" extract="" favors="" focus="" fold="" for="" forms="" genera="" has="" immunoreactivity="" in="" indicate="" infectious="" infectivity="" known="" lichen="" more="" necessarily="" not="" of="" on="" or="" our="" phylogeny="" prion-degrading="" protease="" prp="" prptse.="" prptse="" reduction="" related="" remaining="" rendered="" searches="" some="" species-specificity="" species="" suggesting="" that="" the="" those="" to="" treatment="" uninfectious="" usnea="" was="" which="" with="" within="" yielded="">
PPo8-14:
Enzymatic Digestion of Chronic Wasting Disease Prions Bound to Soil
Samuel E. Saunders,1 Jason C. Bartz,2 Kurt C. Vercauteren3 and Shannon L. Bartelt-Hunt1 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; 3USDA; Animal and Plant Health Inspection Service; Wildlife Services; National Wildlife Research Center; Fort Collins, CO USA
Chronic wasting disease (CWD) and sheep scrapie can be transmitted via indirect environmental routes, and it is known that soil can serve as a reservoir of prion infectivity. Given the strong interaction between the prion protein (PrP) and soil, we hypothesized that binding to soil enhances prion resistance to enzymatic digestion, thereby facilitating prion longevity in the environment and providing protection from host degradation. We characterized the performance of a commercially available subtilisin enzyme, the Prionzyme, to degrade soil-bound and unbound CWD and HY TME PrP as a function of pH, temperature, and treatment time. The subtilisin enzyme effectively degraded PrP adsorbed to a wide range of soils and soil minerals below the limits of detection. Signal loss occurred rapidly at high pH (12.5) and within 7 d under conditions representative of the natural environment (pH 7.4, 22°C). Serial PMCA of treated soil samples suggests a greater than 6-log decrease in infectious titer compared with controls. We observed no apparent difference in enzyme effectiveness between bound and unbound CWD PrP. Our results show that although adsorbed prions do retain relative resistance to enzymatic digestion compared with other brain homogenate proteins, they can be effectively degraded when bound to soil. Our results also suggest a topical application of a subtilisin enzyme solution may be an effective decontamination method to limit disease transmission via environmental ‘hot spots’ of prion infectivity.
PPo2-27:
Generation of a Novel form of Human PrPSc by Inter-species Transmission of Cervid Prions
Marcelo A. Barria,1 Glenn C. Telling,2 Pierluigi Gambetti,3 James A. Mastrianni4 and Claudio Soto1 1Mitchell Center for Alzheimer’s disease and related Brain disorders; Dept of Neurology; University of Texas Houston Medical School; Houston, TX USA; 2Dept of Microbiology, Immunology & Molecular Genetics and Neurology; Sanders Brown Center on Aging; University of Kentucky Medical Center; Lexington, KY USA; 3Institute of Pathology; Case western Reserve University; Cleveland, OH USA; 4Dept of Neurology; University of Chicago; Chicago, IL USA
Prion diseases are infectious neurodegenerative disorders affecting humans and animals that result from the conversion of normal prion protein (PrPC) into the misfolded and infectious prion (PrPSc). Chronic wasting disease (CWD) of cervids is a prion disorder of increasing prevalence within the United States that affects a large population of wild and captive deer and elk. CWD is highly contagious and its origin, mechanism of transmission and exact prevalence are currently unclear. The risk of transmission of CWD to humans is unknown. Defining that risk is of utmost importance, considering that people have been infected by animal prions, resulting in new fatal diseases. To study the possibility that human PrPC can be converted into the infectious form by CWD PrPSc we performed experiments using the Protein Misfolding Cyclic Amplification (PMCA) technique, which mimic in vitro the process of prion replication. Our results show that cervid PrPSc can induce the pathological conversion of human PrPC, but only after the CWD prion strain has been stabilized by successive passages in vitro or in vivo. Interestingly, this newly generated human PrPSc exhibits a distinct biochemical pattern that differs from any of the currently known forms of human PrPSc, indicating that it corresponds to a novel human prion strain. Our findings suggest that CWD prions have the capability to infect humans, and that this ability depends on CWD strain adaptation, implying that the risk for human health progressively increases with the spread of CWD among cervids.
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.
PPo2-7:
Biochemical and Biophysical Characterization of Different CWD Isolates
Martin L. Daus and Michael Beekes Robert Koch Institute; Berlin, Germany
Key words: CWD, strains, FT-IR, AFM
Chronic wasting disease (CWD) is one of three naturally occurring forms of prion disease. The other two are Creutzfeldt-Jakob disease in humans and scrapie in sheep. CWD is contagious and affects captive as well as free ranging cervids. As long as there is no definite answer of whether CWD can breach the species barrier to humans precautionary measures especially for the protection of consumers need to be considered. In principle, different strains of CWD may be associated with different risks of transmission to humans. Sophisticated strain differentiation as accomplished for other prion diseases has not yet been established for CWD. However, several different findings indicate that there exists more than one strain of CWD agent in cervids. We have analysed a set of CWD isolates from white-tailed deer and could detect at least two biochemically different forms of disease-associated prion protein PrPTSE. Limited proteolysis with different concentrations of proteinase K and/or after exposure of PrPTSE to different pH-values or concentrations of Guanidinium hydrochloride resulted in distinct isolate-specific digestion patterns. Our CWD isolates were also examined in protein misfolding cyclic amplification studies. This showed different conversion activities for those isolates that had displayed significantly different sensitivities to limited proteolysis by PK in the biochemical experiments described above. We further applied Fourier transform infrared spectroscopy in combination with atomic force microscopy. This confirmed structural differences in the PrPTSE of at least two disinct CWD isolates. The data presented here substantiate and expand previous reports on the existence of different CWD strains.
PPo2-22:
CWD Strain Emergence in Orally Inoculated White-tailed Deer (Odocoileus virginianus) with Different PRNP Genotypes
Camilo Duque-Velasquez,1 Chad Johnson,2 Allen Herbst,1 Judd Aiken1 and Debbie McKenzie1 1Centre for Prions and Protein Folding Diseases; University of Alberta; Edmonton, Alberta Canada; 2Department of Soil Science; University of Wisconsin; Madison, Wisconsin USA
Key words: CWD, strains, emergence
Chronic wasting disease (CWD) is a prion disease affecting captive and free-ranging cervids in North America. We have previously demonstrated that specific Prnp polymorphisms are linked to susceptibility/resistance to CWD infection in free-ranging white-tailed deer populations. The “wild-type” alleles (with glutamine at aa 95 and a Glycine at aa 96) were over-represented in the infected deer while the polymorphisms at aa 95 (Q95H) and 96 (G96S) were under-represented in the CWD-positive animals. Experimental oral infection of white-tailed deer with known Prnp genotypes (with inocula from CWD-positive wt/wt deer) confirmed this link between Prnp primary sequence and incubation period. All orally infected animals became clinically positive for CWD. The wt/wt had the shortest incubation period (693 dpi) and the Q95H/G96S the longest (1596 dpi). Brain homogenates prepared from clinically affected deer of each genotype were treated with proteinase K and resolved by western blot; differences in the glycosylation pattern and PK resistance were observed and are suggestive of different PrPSc isoforms. Subsequent experiments regarding biochemical properties like detergent solubility, structural stability, host range and the stability of these characteristics upon serial passages will allow us to further define potential CWD strain emergence in white-tailed deer.
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. 3Tissues 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.
PPo3-19:
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.
PPo3-26:
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.
PPo3-20:
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.
PPo3-30:
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%.
PPo3-35:
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.
PPo3-40: 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.
PPo8-14:
Enzymatic Digestion of Chronic Wasting Disease Prions Bound to Soil
Samuel E. Saunders,1 Jason C. Bartz,2 Kurt C. Vercauteren3 and Shannon L. Bartelt-Hunt1 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; 3USDA; Animal and Plant Health Inspection Service; Wildlife Services; National Wildlife Research Center; Fort Collins, CO USA
Chronic wasting disease (CWD) and sheep scrapie can be transmitted via indirect environmental routes, and it is known that soil can serve as a reservoir of prion infectivity. Given the strong interaction between the prion protein (PrP) and soil, we hypothesized that binding to soil enhances prion resistance to enzymatic digestion, thereby facilitating prion longevity in the environment and providing protection from host degradation. We characterized the performance of a commercially available subtilisin enzyme, the Prionzyme, to degrade soil-bound and unbound CWD and HY TME PrP as a function of pH, temperature, and treatment time. The subtilisin enzyme effectively degraded PrP adsorbed to a wide range of soils and soil minerals below the limits of detection. Signal loss occurred rapidly at high pH (12.5) and within 7 d under conditions representative of the natural environment (pH 7.4, 22°C). Serial PMCA of treated soil samples suggests a greater than 6-log decrease in infectious titer compared with controls. We observed no apparent difference in enzyme effectiveness between bound and unbound CWD PrP. Our results show that although adsorbed prions do retain relative resistance to enzymatic digestion compared with other brain homogenate proteins, they can be effectively degraded when bound to soil. Our results also suggest a topical application of a subtilisin enzyme solution may be an effective decontamination method to limit disease transmission via environmental ‘hot spots’ of prion infectivity.
PPo8-20:
The Anti-prion Activity of Soil Organic Compounds Humic and Fulvic Acids
Joanna Narkiewicz,1,2 Ai H.N. Tran,1 Gabriele Giachin,1 Liviana Leita2 and Giuseppe Legname1, 1Neurobiology Sector; Scuola Internazionale Superiore di Studi Avanzati; International School for Advanced Studies; Bonomea, Trieste Italy; 2Agricultural Research Council (CRA); Research Centre for Soil-Plant System; Trieste, Gorizia Italy
A notable feature of prion diseases, as scrapie in sheep and chronic wasting disease in mule deer and elk, is horizontal transmission between grazing animals, suggesting that contaminated environment may contribute significantly to disease transmission. Increasing evidence suggests that soil may present natural reservoir of prion infectivity. Recent studies have shown that prions may persist in contaminated soil and remain infectious for years. As the mechanism of prion retention and persistence in soil is unknown, it is necessary to understand which soil components may interact with prions and thus contribute to disease transmission. Several reports indicate that prion have potential to interact with soil minerals, however the contribution of soil organic fraction in adsorption to prions has been neglected. Here, we present strong evidence for soil humic substances (HS) interaction with prions. We show that two HS, classified as humic and fulvic acids, interact with recombinant prion proteins in vitro. Moreover, we show that both HS possess anti-prion activity, both in vivo and in vitro. Both compounds induced elimination of PrPSc from chronically scrapie-infected GT1 mouse hypothalamic cells (ScGT1) in a dose-dependent manner. ScGT1 cells treatment with HS at concentration of 20mg/mL eliminated more than 95% of PrPSc and did not affect cell viability. Moreover, both HS induced inhibition of prion fibril formation in vitro, as determined by thioflavin T assay. Our results suggest that HS may contribute significantly to prion inactivation in natural soil environments.
PPo8-21:
Detection of PrPCWD in Rocky Mountain Elk Feces Using Protein Misfolding Cyclic Amplification
Bruce E Pulford,1 Terry Spraker,1 Jenny Powers,2 Margaret Wild2 and Mark D. Zabel1 1Department of Microbiology; Immunology and Pathology; College of Veterinary Medicine and Biomedical Sciences; Colorado State University; 2Biological Resource Management Division; United States National Park Service; CO, USA
Key words: CWD, feces, PMCA, elk
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy affecting cervids, including mule and white-tailed deer (Odocoileus hemionus and virginianus), elk (Cervus elaphus nelsoni) and moose (Alces alces shirasi). The method of CWD transmission between hosts is unclear, though there is evidence that feces excreted by infected animals may play a role. Recently, CWD prions was detected in feces using bioassays in cervidized mice, which took many months to produce results. In this study, we use a more rapid procedure, protein misfolding cyclic amplification (PMCA), to test elk feces for the presence of PK-resistant cervid PrP (PrPCWD). Feces were collected from symptomatic and asymptomatic elk in several northern Colorado locations, homogenized, mixed with normal brain homogenate from Tg5037 mice (expressing cervid PrP) and subjected to up to 9 rounds of PMCA (1 round = 40 secs sonication/30 mins at 70% maximum power, 24 hours). Western blots were used to detect PrPCWD using BAR-224 anti-PrP antibody. Rectal and CNS tissue from the elk were IHC-labeled and examined for the presence of PrPCWD. Fecal samples from symptomatic and asymptomatic elk that tested positive by IHC showed characteristic PrPCWD bands on western blots following PMCA. In addition, PMCA detected PrPCWD in 25% of fecal samples from IHC-negative animals. These data suggest that PMCA may (1) prove useful as a non-invasive method to supplement or even replace IHC testing of cervids for CWD, and (2) identify additional asymptomatic carriers of CWD, the prevalence of which may be underestimated using IHC.
http://www.neuroprion.org/resources/pdf_docs/conferences/prion2010/prion_2010_programme.pdf
Greetings,
A disturbing study indeed, but even more disturbing, the fact that this very study shows the potential for transmission of the TSE agent into the wild of yet another species in the USA. Science has shown that the feline is most susceptible to the TSE agent. Will CWD be the demise of the mountain lions, cougars and such in the USA? How many have ever been tested in the USA? I recall there is a study taking place ;
Review A prion disease of cervids: Chronic wasting disease Christina J. Sigurdson et al ;
Mountain lion (Puma concolor) susceptibility to experimental feeding of CWD prions is currently under investigation (M. Miller and L. Wolfe, personal communication).
snip...see full text ;
http://chronic-wasting-disease.blogspot.com/2008/12/lions-and-prions-and-deer-demise.html
Thursday, December 25, 2008
Lions and Prions and Deer Demise
http://chronic-wasting-disease.blogspot.com/2008/12/lions-and-prions-and-deer-demise.html
http://chronic-wasting-disease.blogspot.com/2010/07/comments-sought-on-revised-plan-to.html
http://chronic-wasting-disease.blogspot.com/2009/09/experimental-oral-transmission-of.html
http://chronic-wasting-disease.blogspot.com/2009/08/susceptibilities-of-nonhuman-primates.html
http://chronic-wasting-disease.blogspot.com/
TSS
International Prion Congress: From agent to disease September 8–11, 2010 Salzburg, Austria
PRION 2010 is the top Global Annual TSE Conference in prion research, following a sequence of PRION meetings that were originally organized by the EU Network of Excellence NeuroPrion. In this proud tradition, PRION 2010 covers all aspects of this fascinating scientific area. PRION 2010 is a meeting of greatest interest for neuroscientists, protein structural biologists, geneticists, medical specialists including neurologists, neuropathologists, hygiene experts and blood product providers, veterinarians, epidemiologists, laboratory technicians, industry developers, risk assessors and managers. An outstanding list of Plenary Lecture, Symposia and Workshop Speakers is complemented by the plethora of original input from Poster Presentations. Special consideration is given this year to two areas of major interest: the renewed discussion about the zoonotic potential of animal prion diseases, given the emergence of atypical BSE and scrapie strains, and the breakthrough work on synthetic prions by several groups simultaneously.
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PPo4-4:
Survival and Limited Spread of TSE Infectivity after Burial
Karen Fernie, Allister Smith and Robert A. Somerville The Roslin Institute and R(D)SVS; University of Edinburgh; Roslin, Scotland UK
Scrapie and chronic wasting disease probably spread via environmental routes, and there are also concerns about BSE infection remaining in the environment after carcass burial or waste 3disposal. In two demonstration experiments we are determining survival and migration of TSE infectivity when buried for up to five years, as an uncontained point source or within bovine heads. Firstly boluses of TSE infected mouse brain were buried in lysimeters containing either sandy or clay soil. Migration from the boluses is being assessed from soil cores taken over time. With the exception of a very small amount of infectivity found 25 cm from the bolus in sandy soil after 12 months, no other infectivity has been detected up to three years. Secondly, ten bovine heads were spiked with TSE infected mouse brain and buried in the two soil types. Pairs of heads have been exhumed annually and assessed for infectivity within and around them. After one year and after two years, infectivity was detected in most intracranial samples and in some of the soil samples taken from immediately surrounding the heads. The infectivity assays for the samples in and around the heads exhumed at years three and four are underway. These data show that TSE infectivity can survive burial for long periods but migrates slowly. Risk assessments should take into account the likely long survival rate when infected material has been buried.
The authors gratefully acknowledge funding from DEFRA.
PPo8-13:
Degradation of Pathogenic Prion Protein and Prion Infectivity by Lichens
Christopher J. Johnson,1 James P. Bennett,1 Steven M. Biro,1,2 Cynthia M. Rodriguez,1,2 Richard A. Bessen3 and Tonie E. Rocke1
1USGS National Wildlife Health Center; 2Department of Bacteriology; University of Wisconsin, Madison; 3Department of Veterinary Molecular Biology; Montana State University; Bozeman, MT USA
Key words: prion, lichen, bioassay, protease, degradation
Few biological systems have been identified that degrade the transmissible spongiform encephalopathy (TSE)-associated form of the prion protein (PrPTSE) and TSE infectivity. Stability of the TSE agent allows scrapie and chronic wasting disease agents to persist in the environment and cause disease for years. Naturally-occurring or engineered processes that reduce infectivity in the environment could aid in limiting environmental TSE transmission. We have previously identified that species of at least three lichens, unusual, symbiotic organisms formed from a fungus and photosynthetic partner, contain a serine protease capable of degrading PrPTSE under gentle conditions. We tested the hypothesis that lichen extracts from these three species reduce TSE infectivity by treating infected brain homogenate with extracts and examining infectivity in mice. We found lichen extracts diminished TSE infectious titer by factors of 100 to 1,000 and that reductions in infectivity were not well-correlated with the extent of PrPTSE degradation observed by immunoblotting. For example, treatment of brain homogenate with Cladonia rangiferina extract caused <100-fold activity="" after="" agents.="" also="" and="" anti-prion="" br="" but="" characterization="" cladonia="" closely="" clusters="" comparison="" data="" decrease="" degradation="" degrade="" do="" extract="" favors="" focus="" fold="" for="" forms="" genera="" has="" immunoreactivity="" in="" indicate="" infectious="" infectivity="" known="" lichen="" more="" necessarily="" not="" of="" on="" or="" our="" phylogeny="" prion-degrading="" protease="" prp="" prptse.="" prptse="" reduction="" related="" remaining="" rendered="" searches="" some="" species-specificity="" species="" suggesting="" that="" the="" those="" to="" treatment="" uninfectious="" usnea="" was="" which="" with="" within="" yielded="">
PPo8-14:
Enzymatic Digestion of Chronic Wasting Disease Prions Bound to Soil
Samuel E. Saunders,1 Jason C. Bartz,2 Kurt C. Vercauteren3 and Shannon L. Bartelt-Hunt1 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; 3USDA; Animal and Plant Health Inspection Service; Wildlife Services; National Wildlife Research Center; Fort Collins, CO USA
Chronic wasting disease (CWD) and sheep scrapie can be transmitted via indirect environmental routes, and it is known that soil can serve as a reservoir of prion infectivity. Given the strong interaction between the prion protein (PrP) and soil, we hypothesized that binding to soil enhances prion resistance to enzymatic digestion, thereby facilitating prion longevity in the environment and providing protection from host degradation. We characterized the performance of a commercially available subtilisin enzyme, the Prionzyme, to degrade soil-bound and unbound CWD and HY TME PrP as a function of pH, temperature, and treatment time. The subtilisin enzyme effectively degraded PrP adsorbed to a wide range of soils and soil minerals below the limits of detection. Signal loss occurred rapidly at high pH (12.5) and within 7 d under conditions representative of the natural environment (pH 7.4, 22°C). Serial PMCA of treated soil samples suggests a greater than 6-log decrease in infectious titer compared with controls. We observed no apparent difference in enzyme effectiveness between bound and unbound CWD PrP. Our results show that although adsorbed prions do retain relative resistance to enzymatic digestion compared with other brain homogenate proteins, they can be effectively degraded when bound to soil. Our results also suggest a topical application of a subtilisin enzyme solution may be an effective decontamination method to limit disease transmission via environmental ‘hot spots’ of prion infectivity.
PPo2-27:
Generation of a Novel form of Human PrPSc by Inter-species Transmission of Cervid Prions
Marcelo A. Barria,1 Glenn C. Telling,2 Pierluigi Gambetti,3 James A. Mastrianni4 and Claudio Soto1 1Mitchell Center for Alzheimer’s disease and related Brain disorders; Dept of Neurology; University of Texas Houston Medical School; Houston, TX USA; 2Dept of Microbiology, Immunology & Molecular Genetics and Neurology; Sanders Brown Center on Aging; University of Kentucky Medical Center; Lexington, KY USA; 3Institute of Pathology; Case western Reserve University; Cleveland, OH USA; 4Dept of Neurology; University of Chicago; Chicago, IL USA
Prion diseases are infectious neurodegenerative disorders affecting humans and animals that result from the conversion of normal prion protein (PrPC) into the misfolded and infectious prion (PrPSc). Chronic wasting disease (CWD) of cervids is a prion disorder of increasing prevalence within the United States that affects a large population of wild and captive deer and elk. CWD is highly contagious and its origin, mechanism of transmission and exact prevalence are currently unclear. The risk of transmission of CWD to humans is unknown. Defining that risk is of utmost importance, considering that people have been infected by animal prions, resulting in new fatal diseases. To study the possibility that human PrPC can be converted into the infectious form by CWD PrPSc we performed experiments using the Protein Misfolding Cyclic Amplification (PMCA) technique, which mimic in vitro the process of prion replication. Our results show that cervid PrPSc can induce the pathological conversion of human PrPC, but only after the CWD prion strain has been stabilized by successive passages in vitro or in vivo. Interestingly, this newly generated human PrPSc exhibits a distinct biochemical pattern that differs from any of the currently known forms of human PrPSc, indicating that it corresponds to a novel human prion strain. Our findings suggest that CWD prions have the capability to infect humans, and that this ability depends on CWD strain adaptation, implying that the risk for human health progressively increases with the spread of CWD among cervids.
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.
PPo2-7:
Biochemical and Biophysical Characterization of Different CWD Isolates
Martin L. Daus and Michael Beekes Robert Koch Institute; Berlin, Germany
Key words: CWD, strains, FT-IR, AFM
Chronic wasting disease (CWD) is one of three naturally occurring forms of prion disease. The other two are Creutzfeldt-Jakob disease in humans and scrapie in sheep. CWD is contagious and affects captive as well as free ranging cervids. As long as there is no definite answer of whether CWD can breach the species barrier to humans precautionary measures especially for the protection of consumers need to be considered. In principle, different strains of CWD may be associated with different risks of transmission to humans. Sophisticated strain differentiation as accomplished for other prion diseases has not yet been established for CWD. However, several different findings indicate that there exists more than one strain of CWD agent in cervids. We have analysed a set of CWD isolates from white-tailed deer and could detect at least two biochemically different forms of disease-associated prion protein PrPTSE. Limited proteolysis with different concentrations of proteinase K and/or after exposure of PrPTSE to different pH-values or concentrations of Guanidinium hydrochloride resulted in distinct isolate-specific digestion patterns. Our CWD isolates were also examined in protein misfolding cyclic amplification studies. This showed different conversion activities for those isolates that had displayed significantly different sensitivities to limited proteolysis by PK in the biochemical experiments described above. We further applied Fourier transform infrared spectroscopy in combination with atomic force microscopy. This confirmed structural differences in the PrPTSE of at least two disinct CWD isolates. The data presented here substantiate and expand previous reports on the existence of different CWD strains.
PPo2-22:
CWD Strain Emergence in Orally Inoculated White-tailed Deer (Odocoileus virginianus) with Different PRNP Genotypes
Camilo Duque-Velasquez,1 Chad Johnson,2 Allen Herbst,1 Judd Aiken1 and Debbie McKenzie1 1Centre for Prions and Protein Folding Diseases; University of Alberta; Edmonton, Alberta Canada; 2Department of Soil Science; University of Wisconsin; Madison, Wisconsin USA
Key words: CWD, strains, emergence
Chronic wasting disease (CWD) is a prion disease affecting captive and free-ranging cervids in North America. We have previously demonstrated that specific Prnp polymorphisms are linked to susceptibility/resistance to CWD infection in free-ranging white-tailed deer populations. The “wild-type” alleles (with glutamine at aa 95 and a Glycine at aa 96) were over-represented in the infected deer while the polymorphisms at aa 95 (Q95H) and 96 (G96S) were under-represented in the CWD-positive animals. Experimental oral infection of white-tailed deer with known Prnp genotypes (with inocula from CWD-positive wt/wt deer) confirmed this link between Prnp primary sequence and incubation period. All orally infected animals became clinically positive for CWD. The wt/wt had the shortest incubation period (693 dpi) and the Q95H/G96S the longest (1596 dpi). Brain homogenates prepared from clinically affected deer of each genotype were treated with proteinase K and resolved by western blot; differences in the glycosylation pattern and PK resistance were observed and are suggestive of different PrPSc isoforms. Subsequent experiments regarding biochemical properties like detergent solubility, structural stability, host range and the stability of these characteristics upon serial passages will allow us to further define potential CWD strain emergence in white-tailed deer.
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. 3Tissues 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.
PPo3-19:
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.
PPo3-26:
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.
PPo3-20:
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.
PPo3-30:
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%.
PPo3-35:
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.
PPo3-40: 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.
PPo8-14:
Enzymatic Digestion of Chronic Wasting Disease Prions Bound to Soil
Samuel E. Saunders,1 Jason C. Bartz,2 Kurt C. Vercauteren3 and Shannon L. Bartelt-Hunt1 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; 3USDA; Animal and Plant Health Inspection Service; Wildlife Services; National Wildlife Research Center; Fort Collins, CO USA
Chronic wasting disease (CWD) and sheep scrapie can be transmitted via indirect environmental routes, and it is known that soil can serve as a reservoir of prion infectivity. Given the strong interaction between the prion protein (PrP) and soil, we hypothesized that binding to soil enhances prion resistance to enzymatic digestion, thereby facilitating prion longevity in the environment and providing protection from host degradation. We characterized the performance of a commercially available subtilisin enzyme, the Prionzyme, to degrade soil-bound and unbound CWD and HY TME PrP as a function of pH, temperature, and treatment time. The subtilisin enzyme effectively degraded PrP adsorbed to a wide range of soils and soil minerals below the limits of detection. Signal loss occurred rapidly at high pH (12.5) and within 7 d under conditions representative of the natural environment (pH 7.4, 22°C). Serial PMCA of treated soil samples suggests a greater than 6-log decrease in infectious titer compared with controls. We observed no apparent difference in enzyme effectiveness between bound and unbound CWD PrP. Our results show that although adsorbed prions do retain relative resistance to enzymatic digestion compared with other brain homogenate proteins, they can be effectively degraded when bound to soil. Our results also suggest a topical application of a subtilisin enzyme solution may be an effective decontamination method to limit disease transmission via environmental ‘hot spots’ of prion infectivity.
PPo8-20:
The Anti-prion Activity of Soil Organic Compounds Humic and Fulvic Acids
Joanna Narkiewicz,1,2 Ai H.N. Tran,1 Gabriele Giachin,1 Liviana Leita2 and Giuseppe Legname1, 1Neurobiology Sector; Scuola Internazionale Superiore di Studi Avanzati; International School for Advanced Studies; Bonomea, Trieste Italy; 2Agricultural Research Council (CRA); Research Centre for Soil-Plant System; Trieste, Gorizia Italy
A notable feature of prion diseases, as scrapie in sheep and chronic wasting disease in mule deer and elk, is horizontal transmission between grazing animals, suggesting that contaminated environment may contribute significantly to disease transmission. Increasing evidence suggests that soil may present natural reservoir of prion infectivity. Recent studies have shown that prions may persist in contaminated soil and remain infectious for years. As the mechanism of prion retention and persistence in soil is unknown, it is necessary to understand which soil components may interact with prions and thus contribute to disease transmission. Several reports indicate that prion have potential to interact with soil minerals, however the contribution of soil organic fraction in adsorption to prions has been neglected. Here, we present strong evidence for soil humic substances (HS) interaction with prions. We show that two HS, classified as humic and fulvic acids, interact with recombinant prion proteins in vitro. Moreover, we show that both HS possess anti-prion activity, both in vivo and in vitro. Both compounds induced elimination of PrPSc from chronically scrapie-infected GT1 mouse hypothalamic cells (ScGT1) in a dose-dependent manner. ScGT1 cells treatment with HS at concentration of 20mg/mL eliminated more than 95% of PrPSc and did not affect cell viability. Moreover, both HS induced inhibition of prion fibril formation in vitro, as determined by thioflavin T assay. Our results suggest that HS may contribute significantly to prion inactivation in natural soil environments.
PPo8-21:
Detection of PrPCWD in Rocky Mountain Elk Feces Using Protein Misfolding Cyclic Amplification
Bruce E Pulford,1 Terry Spraker,1 Jenny Powers,2 Margaret Wild2 and Mark D. Zabel1 1Department of Microbiology; Immunology and Pathology; College of Veterinary Medicine and Biomedical Sciences; Colorado State University; 2Biological Resource Management Division; United States National Park Service; CO, USA
Key words: CWD, feces, PMCA, elk
Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy affecting cervids, including mule and white-tailed deer (Odocoileus hemionus and virginianus), elk (Cervus elaphus nelsoni) and moose (Alces alces shirasi). The method of CWD transmission between hosts is unclear, though there is evidence that feces excreted by infected animals may play a role. Recently, CWD prions was detected in feces using bioassays in cervidized mice, which took many months to produce results. In this study, we use a more rapid procedure, protein misfolding cyclic amplification (PMCA), to test elk feces for the presence of PK-resistant cervid PrP (PrPCWD). Feces were collected from symptomatic and asymptomatic elk in several northern Colorado locations, homogenized, mixed with normal brain homogenate from Tg5037 mice (expressing cervid PrP) and subjected to up to 9 rounds of PMCA (1 round = 40 secs sonication/30 mins at 70% maximum power, 24 hours). Western blots were used to detect PrPCWD using BAR-224 anti-PrP antibody. Rectal and CNS tissue from the elk were IHC-labeled and examined for the presence of PrPCWD. Fecal samples from symptomatic and asymptomatic elk that tested positive by IHC showed characteristic PrPCWD bands on western blots following PMCA. In addition, PMCA detected PrPCWD in 25% of fecal samples from IHC-negative animals. These data suggest that PMCA may (1) prove useful as a non-invasive method to supplement or even replace IHC testing of cervids for CWD, and (2) identify additional asymptomatic carriers of CWD, the prevalence of which may be underestimated using IHC.
http://www.neuroprion.org/resources/pdf_docs/conferences/prion2010/prion_2010_programme.pdf
Greetings,
A disturbing study indeed, but even more disturbing, the fact that this very study shows the potential for transmission of the TSE agent into the wild of yet another species in the USA. Science has shown that the feline is most susceptible to the TSE agent. Will CWD be the demise of the mountain lions, cougars and such in the USA? How many have ever been tested in the USA? I recall there is a study taking place ;
Review A prion disease of cervids: Chronic wasting disease Christina J. Sigurdson et al ;
Mountain lion (Puma concolor) susceptibility to experimental feeding of CWD prions is currently under investigation (M. Miller and L. Wolfe, personal communication).
snip...see full text ;
http://chronic-wasting-disease.blogspot.com/2008/12/lions-and-prions-and-deer-demise.html
Thursday, December 25, 2008
Lions and Prions and Deer Demise
http://chronic-wasting-disease.blogspot.com/2008/12/lions-and-prions-and-deer-demise.html
http://chronic-wasting-disease.blogspot.com/2010/07/comments-sought-on-revised-plan-to.html
http://chronic-wasting-disease.blogspot.com/2009/09/experimental-oral-transmission-of.html
http://chronic-wasting-disease.blogspot.com/2009/08/susceptibilities-of-nonhuman-primates.html
http://chronic-wasting-disease.blogspot.com/
TSS
Labels: CWD, HUMAN TRANSMISSION, prion, SOIL, strains
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