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Find video protocols related to scientific articles indexed in Pubmed.
Chronic Wasting Disease of Cervids: Current Knowledge and Future Perspectives.
Annu Rev Anim Biosci
PUBLISHED: 11-12-2014
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A naturally occurring transmissible spongiform encephalopathy (TSE) of mule deer was first reported in Colorado and Wyoming in 1967 and has since spread to other members of the cervid family in 22 states, 2 Canadian provinces, and the Republic of Korea. Chronic wasting disease (CWD), caused by exposure to an abnormally folded isoform of the cellular prion protein, is characterized by progressive neurological disease in susceptible natural and experimental hosts and is ultimately fatal.CWDis thought to be transmitted horizontally in excreta and through contaminated environments, features common to scrapie of sheep, though rare among TSEs. Evolving detection methods have revealed multiple strains of CWD and with continued development may lead to an effective antemortem test. Managing the spread of CWD, through the development of a vaccine or environmental cleanup strategies, is an active area of interest. As such, CWD represents a unique challenge in the study of prion diseases. Expected final online publication date for the Annual Review of Animal Biosciences Volume 3 is February 15, 2015. Please see http://www.annualreviews.org/catalog/pubdates.aspx for revised estimates.
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Quantitative Assessment of Prion Infectivity in Tissues and Body Fluids by RT-QuIC.
J. Gen. Virol.
PUBLISHED: 10-12-2014
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Prions are amyloid-forming proteins that cause transmissible spongiform encephalopathies through a process involving the templated conversion of the normal cellular prion protein (PrPC) to a pathogenic misfolded conformation. Templated conversion has been modeled in several in vitro assays, including serial protein misfolding amplification (sPMCA), amyloid seeding, and real time quaking induced conversion (RT-QuIC). Because RT-QuIC measures formation of amyloid fibrils in real time, it can be used to estimate the rate of seeded conversion. Here we use samples from deer infected with chronic wasting disease (CWD) in RT-QuIC to show that serial dilution of prion seed is linearly related to the rate of amyloid formation over a range of 10-3 to 10-8 µg. We then used an amyloid formation rate standard curve derived from a bioassayed reference sample (CWD+ brain homogenate) to estimate the prion seed concentration and infectivity in tissues, body fluids and excreta. Using these methods we estimate that urine and saliva from CWD-infected deer contain between 1 and 5 LD50 per 10 ml, respectively. Thus, over the 1 to 2 year course of infection, a substantial environmental reservoir of CWD prion contamination accumulates.
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Structural effects of PrP polymorphisms on intra- and interspecies prion transmission.
Proc. Natl. Acad. Sci. U.S.A.
PUBLISHED: 07-17-2014
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Understanding the molecular parameters governing prion propagation is crucial for controlling these lethal, proteinaceous, and infectious neurodegenerative diseases. To explore the effects of prion protein (PrP) sequence and structural variations on intra- and interspecies transmission, we integrated studies in deer, a species naturally susceptible to chronic wasting disease (CWD), a burgeoning, contagious epidemic of uncertain origin and zoonotic potential, with structural and transgenic (Tg) mouse modeling and cell-free prion amplification. CWD properties were faithfully maintained in deer following passage through Tg mice expressing cognate PrP, and the influences of naturally occurring PrP polymorphisms on CWD susceptibility were accurately reproduced in Tg mice or cell-free systems. Although Tg mice also recapitulated susceptibility of deer to sheep prions, polymorphisms that provided protection against CWD had distinct and varied influences. Whereas substitutions at residues 95 and 96 in the unstructured region affected CWD propagation, their protective effects were overridden during replication of sheep prions in Tg mice and, in the case of residue 96, deer. The inhibitory effects on sheep prions of glutamate at residue 226 in elk PrP, compared with glutamine in deer PrP, and the protective effects of the phenylalanine for serine substitution at the adjacent residue 225, coincided with structural rearrangements in the globular domain affecting interaction between ?-helix 3 and the loop between ?2 and ?-helix 2. These structure-function analyses are consistent with previous structural investigations and confirm a role for plasticity of this tertiary structural epitope in the control of PrP conversion and strain propagation.
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Detection of chronic wasting disease in the lymph nodes of free-ranging cervids by real-time quaking-induced conversion.
J. Clin. Microbiol.
PUBLISHED: 06-23-2014
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Chronic wasting disease (CWD), a transmissible spongiform encephalopathy of deer, elk, and moose, is the only prion disease affecting free-ranging animals. Since the disease was first identified in northern Colorado and southern Wyoming in 1967, new epidemic foci of the disease have been identified in 20 additional states, as well as two Canadian provinces and the Republic of South Korea. Identification of CWD-affected animals currently requires postmortem analysis of brain or lymphoid tissues using immunohistochemistry (IHC) or an enzyme-linked immunosorbent assay (ELISA), with no practical way to evaluate potential strain types or to investigate the epidemiology of existing or novel foci of disease. Using a standardized real-time (RT)-quaking-induced conversion (QuIC) assay, a seeded amplification assay employing recombinant prion protein as a conversion substrate and thioflavin T (ThT) as an amyloid-binding fluorophore, we analyzed, in a blinded manner, 1,243 retropharyngeal lymph node samples from white-tailed deer, mule deer, and moose, collected in the field from areas with current or historic CWD endemicity. RT-QuIC results were then compared with those obtained by conventional IHC and ELISA, and amplification metrics using ThT and thioflavin S were examined in relation to the clinical history of the sampled deer. The results indicate that RT-QuIC is useful for both identifying CWD-infected animals and facilitating epidemiological studies in areas in which CWD is endemic or not endemic.
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Prion-seeding activity in cerebrospinal fluid of deer with chronic wasting disease.
PLoS ONE
PUBLISHED: 01-01-2013
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Transmissible spongiform encephalopathies (TSEs), or prion diseases, are a uniformly fatal family of neurodegenerative diseases in mammals that includes chronic wasting disease (CWD) of cervids. The early and ante-mortem identification of TSE-infected individuals using conventional western blotting or immunohistochemistry (IHC) has proven difficult, as the levels of infectious prions in readily obtainable samples, including blood and bodily fluids, are typically beyond the limits of detection. The development of amplification-based seeding assays has been instrumental in the detection of low levels of infectious prions in clinical samples. In the present study, we evaluated the cerebrospinal fluid (CSF) of CWD-exposed (n=44) and naïve (n=4) deer (n=48 total) for CWD prions (PrP(d)) using two amplification assays: serial protein misfolding cyclic amplification with polytetrafluoroethylene beads (sPMCAb) and real-time quaking induced conversion (RT-QuIC) employing a truncated Syrian hamster recombinant protein substrate. Samples were evaluated blindly in parallel with appropriate positive and negative controls. Results from amplification assays were compared to one another and to obex immunohistochemistry, and were correlated to available clinical histories including CWD inoculum source (e.g. saliva, blood), genotype, survival period, and duration of clinical signs. We found that both sPMCAb and RT-QuIC were capable of amplifying CWD prions from cervid CSF, and results correlated well with one another. Prion seeding activity in either assay was observed in approximately 50% of deer with PrP(d) detected by IHC in the obex region of the brain. Important predictors of amplification included duration of clinical signs and time of first tonsil biopsy positive results, and ultimately the levels of PrP(d) identified in the obex by IHC. Based on our findings, we expect that both sPMCAb and RT-QuIC may prove to be useful detection assays for the detection of prions in CSF.
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In vitro detection of prionemia in TSE-infected cervids and hamsters.
PLoS ONE
PUBLISHED: 01-01-2013
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Blood-borne transmission of infectious prions during the symptomatic and asymptomatic stages of disease occurs for both human and animal transmissible spongiform encephalopathies (TSEs). The geographical distribution of the cervid TSE, chronic wasting disease (CWD), continues to spread across North America and the prospective number of individuals harboring an asymptomatic infection of human variant Creutzfeldt-Jakob Disease (vCJD) in the United Kingdom has been projected to be ~1 in 3000 residents. Thus, it is important to monitor cervid and human blood products to ensure herd health and human safety. Current methods for detecting blood-associated prions rely primarily upon bioassay in laboratory animals. While bioassay provides high sensitivity and specificity, it requires many months, animals, and it is costly. Here we report modification of the real time quaking-induced conversion (RT-QuIC) assay to detect blood-borne prions in whole blood from prion-infected preclinical white-tailed deer, muntjac deer, and Syrian hamsters, attaining sensitivity of >90% while maintaining 100% specificity. Our results indicate that RT-QuIC methodology as modified can provide consistent and reliable detection of blood-borne prions in preclinical and symptomatic stages of two animal TSEs, offering promise for prionemia detection in other species, including humans.
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Rapid antemortem detection of CWD prions in deer saliva.
PLoS ONE
PUBLISHED: 01-01-2013
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Chronic wasting disease (CWD) is an efficiently transmitted prion disease of cervids, now identified in 22 United States, 2 Canadian provinces and Korea. One hallmark of CWD is the shedding of infectious prions in saliva, as demonstrated by bioassay in deer. It is also clear that the concentration of prions in saliva, blood, urine and feces is much lower than in the nervous system or lymphoid tissues. Rapid in vitro detection of CWD (and other) prions in body fluids and excreta has been problematic due to the sensitivity limits of direct assays (western blotting, ELISA) and the presence of inhibitors in these complex biological materials that hamper detection. Here we use real-time quaking induced conversion (RT-QuIC) to demonstrate CWD prions in both diluted and prion-enriched saliva samples from asymptomatic and symptomatic white-tailed deer. CWD prions were detected in 14 of 24 (58.3%) diluted saliva samples from CWD-exposed white-tailed deer, including 9 of 14 asymptomatic animals (64.2%). In addition, a phosphotungstic acid enrichment enhanced the RT-QuIC assay sensitivity, enabling detection in 19 of 24 (79.1%) of the above saliva samples. Bioassay in Tg[CerPrP] mice confirmed the presence of infectious prions in 2 of 2 RT-QuIC-positive saliva samples so examined. The modified RT-QuIC analysis described represents a non-invasive, rapid ante-mortem detection of prions in complex biologic fluids, excreta, or environmental samples as well as a tool for exploring prion trafficking, peripheralization, and dissemination.
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Mother to offspring transmission of chronic wasting disease in reeves muntjac deer.
PLoS ONE
PUBLISHED: 01-01-2013
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The horizontal transmission of prion diseases has been well characterized in bovine spongiform encephalopathy (BSE), chronic wasting disease (CWD) of deer and elk and scrapie of sheep, and has been regarded as the primary mode of transmission. Few studies have monitored the possibility of vertical transmission occurring within an infected mother during pregnancy. To study the potential for and pathway of vertical transmission of CWD in the native cervid species, we used a small cervid model-the polyestrous breeding, indoor maintainable, Reeves muntjac deer-and determined that the susceptibility and pathogenesis of CWD in these deer reproduce that in native mule and white-tailed deer. Moreover, we demonstrate here that CWD prions are transmitted from doe to fawn. Maternal CWD infection also appears to result in lower percentage of live birth offspring. In addition, evolving evidence from protein misfolding cyclic amplification (PMCA) assays on fetal tissues suggest that covert prion infection occurs in utero. Overall, our findings demonstrate that transmission of prions from mother to offspring can occur, and may be underestimated for all prion diseases.
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Intranasal inoculation of white-tailed deer (Odocoileus virginianus) with lyophilized chronic wasting disease prion particulate complexed to montmorillonite clay.
PLoS ONE
PUBLISHED: 01-01-2013
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Chronic wasting disease (CWD), the only known prion disease endemic in wildlife, is a persistent problem in both wild and captive North American cervid populations. This disease continues to spread and cases are found in new areas each year. Indirect transmission can occur via the environment and is thought to occur by the oral and/or intranasal route. Oral transmission has been experimentally demonstrated and although intranasal transmission has been postulated, it has not been tested in a natural host until recently. Prions have been shown to adsorb strongly to clay particles and upon oral inoculation the prion/clay combination exhibits increased infectivity in rodent models. Deer and elk undoubtedly and chronically inhale dust particles routinely while living in the landscape while foraging and rutting. We therefore hypothesized that dust represents a viable vehicle for intranasal CWD prion exposure. To test this hypothesis, CWD-positive brain homogenate was mixed with montmorillonite clay (Mte), lyophilized, pulverized and inoculated intranasally into white-tailed deer once a week for 6 weeks. Deer were euthanized at 95, 105, 120 and 175 days post final inoculation and tissues examined for CWD-associated prion proteins by immunohistochemistry. Our results demonstrate that CWD can be efficiently transmitted utilizing Mte particles as a prion carrier and intranasal exposure.
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Evidence for distinct chronic wasting disease (CWD) strains in experimental CWD in ferrets.
J. Gen. Virol.
PUBLISHED: 09-14-2011
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Chronic wasting disease (CWD) is an evolving prion disease of cervids (deer, elk and moose) that has been recognized in North America and Korea. Infection of non-cervid reservoir or transport species in nature is not reported. However, the ferret (Mustela putorius furo) is susceptible to CWD after experimental inoculation. Here, we report that infection of ferrets with either of two ferret CWD isolates by various routes of exposure has revealed biologically distinct strain-like properties distinguished by different clinical progression and survival period. The isolates of ferret CWD were also differentiated by the distribution of the infectious prion protein (PrP(CWD)) in the brain and periphery, and by the proteinase K sensitivity of PrP(CWD). These findings suggest that diversity in prion conformers exists in CWD-infected cervids.
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Alteration of the chronic wasting disease species barrier by in vitro prion amplification.
J. Virol.
PUBLISHED: 06-22-2011
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Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of cervids now detected in 19 states of the United States, three Canadian provinces, and South Korea. Whether noncervid species can be infected by CWD and thereby serve as reservoirs for the infection is not known. To investigate this issue, we previously used serial protein misfolding cyclic amplification (sPMCA) to demonstrate that CWD prions can amplify in brain homogenates from several species sympatric with cervids, including prairie voles (Microtus ochrogaster) and field mice (Peromyscus spp.). Here, we show that prairie voles are susceptible to mule deer CWD prions in vivo and that sPMCA amplification of CWD prions in vole brain enhances the infectivity of CWD for this species. Prairie voles inoculated with sPMCA products developed clinical signs of TSE disease approximately 300 days prior to, and more consistently than, those inoculated with CWD prions from deer brain. Moreover, the deposition patterns and biochemical properties of protease-resistant form of PrP (PrP(RES)) in the brains of affected voles differed from those in cervidized transgenic (CerPrP) mice infected with CWD. In addition, voles inoculated orally with sPMCA products developed clinical signs of TSE and were positive for PrP(RES) deposition, whereas those inoculated orally with deer-origin CWD prions did not. These results demonstrate that transspecies sPMCA of CWD prions can enhance the infectivity and adapt the host range of CWD prions and thereby may be useful to assess determinants of prion species barriers.
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Chronic wasting disease prion trafficking via the autonomic nervous system.
Am. J. Pathol.
PUBLISHED: 05-12-2011
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Chronic wasting disease (CWD) is a fatal spongiform encephalopathy that is efficiently transmitted among members of the mammalian family Cervidae, including deer, elk, and moose. Typical of prion diseases, CWD is characterized by the conversion of the native protease-sensitive protein PrP(C) to a protease-resistant isoform, denoted PrP(RES). In native species, spread of the disease likely results from the ingestion of prion-containing excreta, including urine, saliva, or feces. Although cervid prion protein-expressing transgenic [Tg(CerPrP)] mice have been shown to be effective surrogates of natural CWD, uncertainties remain regarding the mechanisms by which CWD prions traffic in vivo, including the manner by which CWD prions traffic from the gastrointestinal tract to the central nervous system. We used elk prion protein-expressing transgenic [Tg(CerPrP-E)] mice, infected by three different routes of inoculation, and tissue-based IHC to elucidate that centripetal and centrifugal CWD prion transit pathways involve cells and fibers of the autonomic nervous systems, including the enteric nervous system and central autonomic network. Moreover, we identified CWD PrP(RES) associated with the cell bodies and processes of enteric glial cells within the enteric nervous system of CWD-infected Tg(CerPrP-E) mice. The present findings demonstrate the importance of the peripheral and central autonomic networks in CWD neuroinvasion and neuropathogenesis and suggest that enteroglial cells may facilitate the shedding of prions via the intestinal tract.
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Detection of chronic wasting disease prions in salivary, urinary, and intestinal tissues of deer: potential mechanisms of prion shedding and transmission.
J. Virol.
PUBLISHED: 04-27-2011
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Efficient horizontal transmission is a signature trait of chronic wasting disease (CWD) in cervids. Infectious prions shed into excreta appear to play a key role in this facile transmission, as has been demonstrated by bioassays of cervid and transgenic species and serial protein misfolding cyclic amplification (sPMCA). However, the source(s) of infectious prions in these body fluids has yet to be identified. In the present study, we analyzed tissues proximate to saliva, urine, and fecal production by sPMCA in an attempt to elucidate this unique aspect of CWD pathogenesis. Oropharyngeal, urogenital, and gastrointestinal tissues along with blood and obex from CWD-exposed cervids (comprising 27 animals and >350 individual samples) were analyzed and scored based on the apparent relative CWD burden. PrP(CWD)-generating activity was detected in a range of tissues and was highest in the salivary gland, urinary bladder, and distal intestinal tract. In the same assays, blood from the same animals and unseeded normal brain homogenate controls (n = 116 of 117) remained negative. The PrP-converting activity in peripheral tissues varied from 10(-11)- to 10(0)-fold of that found in brain of the same animal. Deer with highest levels of PrP(CWD) amplification in the brain had higher and more widely disseminated prion amplification in excretory tissues. Interestingly, PrP(CWD) was not demonstrable in these excretory tissues by conventional Western blotting, suggesting a low prion burden or the presence of protease-sensitive infectious prions destroyed by harsh proteolytic treatments. These findings offer unique insights into the transmission of CWD in particular and prion infection and trafficking overall.
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Minor oral lesions facilitate transmission of chronic wasting disease.
J. Virol.
PUBLISHED: 11-17-2010
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While chronic wasting disease (CWD) prion transmission, entry, and trafficking remain incompletely elucidated, natural exposure of the oral and/or nasal mucous membranes seems certain. Cervids commonly sustain minor lesions on oral mucous membranes that could have an impact on susceptibility to prion infection. To explore this potential cofactor, we studied cohorts of cervid PrP transgenic mice with or without superficial abrasions on the lingual mucosa to determine whether minor oral mucosa lesions may enhance susceptibility to CWD infections. Results demonstrated that minor lingual abrasions substantially facilitate CWD transmission, revealing a cofactor that may be significant in cervids and perhaps other species.
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Prion strain mutation determined by prion protein conformational compatibility and primary structure.
Science
PUBLISHED: 05-13-2010
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Prions are infectious proteins composed of the abnormal disease-causing isoform PrPSc, which induces conformational conversion of the host-encoded normal cellular prion protein PrPC to additional PrPSc. The mechanism underlying prion strain mutation in the absence of nucleic acids remains unresolved. Additionally, the frequency of strains causing chronic wasting disease (CWD), a burgeoning prion epidemic of cervids, is unknown. Using susceptible transgenic mice, we identified two prevalent CWD strains with divergent biological properties but composed of PrPSc with indistinguishable biochemical characteristics. Although CWD transmissions indicated stable, independent strain propagation by elk PrPC, strain coexistence in the brains of deer and transgenic mice demonstrated unstable strain propagation by deer PrPC. The primary structures of deer and elk prion proteins differ at residue 226, which, in concert with PrPSc conformational compatibility, determines prion strain mutation in these cervids.
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Pathogenesis of chronic wasting disease in cervidized transgenic mice.
Am. J. Pathol.
PUBLISHED: 04-15-2010
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Chronic wasting disease (CWD) is a fatal, endemic prion disease of wild and captive cervids, including deer, elk, and moose. Typical of prion diseases, CWD is characterized by the conversion of the native, protease-sensitive protein PrP(C) to a protease-resistant isoform, denoted as PrP(RES). Here we have studied the expression of cervid PrP(C) and the pathogenesis of CWD infection in transgenic mice expressing the normal cervid prion protein (Tg[CerPrP] mice). Using tissue-based in situ immunohistochemistry protocols, we first identified cervid PrP(C) expression in the lymphoid, nervous, hemopoietic, endocrine, and certain epithelial tissues of Tg[CerPrP] mice. Tg[CerPrP] mice were then inoculated with CWD via one of four routes (intracerebral, intravenous, intraperitoneal, or oral); all groups developed spongiform encephalopathy, although the oral route required a larger infecting dose. Incubation periods were 184 +/- 13, 218 +/- 15, 200 +/- 7, and 350 +/- 27 days after inoculation, respectively. In longitudinal studies, we tracked the appearance of PrP(RES) in the brain, spleen, Peyers patches, lymph nodes, pancreatic islets of Langerhans, bone marrow, and salivary glands of preclinical and terminal mice. In addition, we documented horizontal transmission of CWD from inoculated mice and to un-inoculated cohabitant cage-mates. This work documents the multiroute susceptibility, pathogenesis, and lateral transmission of CWD infection in Tg[CerPrP] mice, affirming this model as a robust system to study this cervid transmissible spongiform encephalopathy.
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B cells and platelets harbor prion infectivity in the blood of deer infected with chronic wasting disease.
J. Virol.
PUBLISHED: 03-10-2010
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Substantial evidence for prion transmission via blood transfusion exists for many transmissible spongiform encephalopathy (TSE) diseases. Determining which cell phenotype(s) is responsible for trafficking infectivity has important implications for our understanding of the dissemination of prions, as well as their detection and elimination from blood products. We used bioassay studies of native white-tailed deer and transgenic cervidized mice to determine (i) if chronic wasting disease (CWD) blood infectivity is associated with the cellular versus the cell-free/plasma fraction of blood and (ii) in particular if B-cell (MAb 2-104(+)), platelet (CD41/61(+)), or CD14(+) monocyte blood cell phenotypes harbor infectious prions. All four deer transfused with the blood mononuclear cell fraction from CWD(+) donor deer became PrP(CWD) positive by 19 months postinoculation, whereas none of the four deer inoculated with cell-free plasma from the same source developed prion infection. All four of the deer injected with B cells and three of four deer receiving platelets from CWD(+) donor deer became PrP(CWD) positive in as little as 6 months postinoculation, whereas none of the four deer receiving blood CD14(+) monocytes developed evidence of CWD infection (immunohistochemistry and Western blot analysis) after 19 months of observation. Results of the Tg(CerPrP) mouse bioassays mirrored those of the native cervid host. These results indicate that CWD blood infectivity is cell associated and suggest a significant role for B cells and platelets in trafficking CWD infectivity in vivo and support earlier tissue-based studies associating putative follicular B cells with PrP(CWD). Localization of CWD infectivity with leukocyte subpopulations may aid in enhancing the sensitivity of blood-based diagnostic assays for CWD and other TSEs.
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PrP conformational transitions alter species preference of a PrP-specific antibody.
J. Biol. Chem.
PUBLISHED: 03-01-2010
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The epitope of the 3F4 antibody most commonly used in human prion disease diagnosis is believed to consist of residues Met-Lys-His-Met (MKHM) corresponding to human PrP-(109-112). This assumption is based mainly on the observation that 3F4 reacts with human and hamster PrP but not with PrP from mouse, sheep, and cervids, in which Met at residue 112 is replaced by Val. Here we report that, by brain histoblotting, 3F4 did not react with PrP of uninfected transgenic mice expressing elk PrP; however, it did show distinct immunoreactivity in transgenic mice infected with chronic wasting disease. Compared with human PrP, the 3F4 reactivity with the recombinant elk PrP was 2 orders of magnitude weaker, as indicated by both Western blotting and surface plasmon resonance. To investigate the molecular basis of these species- and conformer-dependent preferences of 3F4, the epitope was probed by peptide membrane array and antigen competition experiments. Remarkably, the 3F4 antibody did not react with MKHM but reacted strongly with KTNMK (corresponding to human PrP-(106-110)), a sequence that is also present in cervids, sheep, and cattle. 3F4 also reacted with elk PrP peptides containing KTNMKHV. We concluded that the minimal sequence for the 3F4 epitope consists of residues KTNMK, and the species- and conformer-dependent preferences of 3F4 arise largely from the interactions between Met(112) (human PrP) or Val(115) (cervid PrP) and adjacent residues.
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Aerosol and nasal transmission of chronic wasting disease in cervidized mice.
J. Gen. Virol.
PUBLISHED: 02-17-2010
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Little is known regarding the potential risk posed by aerosolized prions. Chronic wasting disease (CWD) is transmitted horizontally, almost surely by mucosal exposure, and CWD prions are present in saliva and urine of infected animals. However, whether CWD may be transmissible by the aerosol or nasal route is not known. To address this question, FVB mice transgenetically expressing the normal cervid PrP(C) protein [Tg(cerPrP) mice] were exposed to CWD prions by either nose-only aerosol exposure or by drop-wise instillation into the nostrils. Mice were monitored for signs of disease for up to 755 days post-inoculation (p.i.) and by examination of tissues for lesions and PrP(CWD) after necropsy. In particular, nasal mucosa, vomeronasal organ, lungs, lymphoid tissue and the brain were assessed for PrP(CWD) by Western blotting and immunohistochemistry. Six of seven aerosol-exposed Tg(cerPrP) mice developed clinical signs of neurological dysfunction mandating euthanasia between 411 and 749 days p.i. In all these mice, CWD infection was confirmed by detection of spongiform lesions and PrP(CWD) in the brain. Two of nine intranasally inoculated Tg(cerPrP) mice also developed transmissible spongiform encephalopathy associated with PrP(CWD) between 417 and 755 days p.i. No evidence of PrP(CWD) was detected in CWD-inoculated Tg(cerPrP) mice examined at pre-terminal time points. These results demonstrate that CWD can be transmitted by aerosol (as well as nasal) exposure and suggest that exposure via the respiratory system merits consideration for prion disease transmission and biosafety.
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Feline leukemia virus immunity induced by whole inactivated virus vaccination.
Vet. Immunol. Immunopathol.
PUBLISHED: 10-31-2009
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A fraction of cats exposed to feline leukemia virus (FeLV) effectively contain virus and resist persistent antigenemia/viremia. Using real-time PCR (qPCR) to quantitate circulating viral DNA levels, previously we detected persistent FeLV DNA in blood cells of non-antigenemic cats considered to have resisted FeLV challenge. In addition, previously we used RNA qPCR to quantitate circulating viral RNA levels and determined that the vast majority of viral DNA is transcriptionally active, even in the absence of antigenemia. A single comparison of all USDA-licensed commercially available FeLV vaccines using these modern sensitive methods has not been reported. To determine whether FeLV vaccination would prevent nucleic acid persistence, we assayed circulating viral DNA, RNA, antigen, infectious virus, and virus neutralizing (VN) antibody in vaccinated and unvaccinated cats challenged with infectious FeLV. We identified challenged vaccinates with undetectable antigenemia and viremia concomitant with persistent FeLV DNA and/or RNA. Moreover, these studies demonstrated that two whole inactivated virus (WIV) adjuvanted FeLV vaccines (Fort Dodge Animal Healths Fel-O-Vax Lv-K) and Schering-Plough Animal Healths FEVAXYN FeLV) provided effective protection against FeLV challenge. In nearly every recipient of these vaccines, neither viral DNA, RNA, antigen, nor infectious virus could be detected in blood after FeLV challenge. Interestingly, this effective viral containment occurred despite a weak to undetectable VN antibody response. The above findings reinforce the precept of FeLV infection as a unique model of effective retroviral immunity elicited by WIV vaccination, and as such holds valuable insights into retroviral immunoprevention and therapy.
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Utilizing the FIV model to understand dendritic cell dysfunction and the potential role of dendritic cell immunization in HIV infection.
Vet. Immunol. Immunopathol.
PUBLISHED: 10-14-2009
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Dendritic cells (DC) are potent antigen presenting cells which initiate and coordinate the immune response making them central targets of and attractive candidates for manipulation in chronic lentiviral infections. Emerging evidence suggests that DC immune function is disrupted during both acute and chronic infection with human immunodeficiency virus (HIV), simian immunodeficiency virus (SIV), and feline immunodeficiency virus (FIV). Despite some early promising data, the use of DC for lentiviral immunotherapy has not fulfilled its expected potential and has been complicated by the large number of variables involved in DC harvesting, purifying, and antigen loading. Pre-clinical studies aimed at identifying successful strategies for DC augmentation of current HIV treatment protocols are needed. Over the past two decades, the FIV model for HIV infection has increased the understanding of retroviral pathogenesis, and studies have begun using the FIV model to study DC dysfunction and DC-mediated immunotherapy. Careful consideration of the many variables involved in DC function and therapy should help develop protocols to explore the potential of DC vaccine-based therapies for lentiviral infection.
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Detection of sub-clinical CWD infection in conventional test-negative deer long after oral exposure to urine and feces from CWD+ deer.
PLoS ONE
PUBLISHED: 09-29-2009
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Chronic wasting disease (CWD) of cervids is a prion disease distinguished by high levels of transmissibility, wherein bodily fluids and excretions are thought to play an important role. Using cervid bioassay and established CWD detection methods, we have previously identified infectious prions in saliva and blood but not urine or feces of CWD+ donors. More recently, we identified very low concentrations of CWD prions in urine of deer by cervid PrP transgenic (Tg[CerPrP]) mouse bioassay and serial protein misfolding cyclic amplification (sPMCA). This finding led us to examine further our initial cervid bioassay experiments using sPMCA.
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Surveillance for transmissible spongiform encephalopathy in scavengers of white-tailed deer carcasses in the chronic wasting disease area of Wisconsin.
J. Toxicol. Environ. Health Part A
PUBLISHED: 08-22-2009
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Chronic wasting disease (CWD), a class of neurodegenerative transmissible spongiform encephalopathies (TSE) occurring in cervids, is found in a number of states and provinces across North America. Misfolded prions, the infectious agents of CWD, are deposited in the environment via carcass remains and excreta, and pose a threat of cross-species transmission. In this study tissues were tested from 812 representative mammalian scavengers, collected in the CWD-affected area of Wisconsin, for TSE infection using the IDEXX HerdChek enzyme-linked immunosorbent assay (ELISA). Only four of the collected mammals tested positive using the ELISA, but these were negative when tested by Western blot. While our sample sizes permitted high probabilities of detecting TSE assuming 1% population prevalence in several common scavengers (93%, 87%, and 87% for raccoons, opossums, and coyotes, respectively), insufficient sample sizes for other species precluded similar conclusions. One cannot rule out successful cross-species TSE transmission to scavengers, but the results suggest that such transmission is not frequent in the CWD-affected area of Wisconsin. The need for further surveillance of scavenger species, especially those known to be susceptible to TSE (e.g., cat, American mink, raccoon), is highlighted in both a field and laboratory setting.
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Detection of protease-resistant cervid prion protein in water from a CWD-endemic area.
Prion
PUBLISHED: 07-14-2009
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Chronic wasting disease (CWD) is the only known transmissible spongiform encephalopathy affecting free-ranging wildlife. Although the exact mode of natural transmission remains unknown, substantial evidence suggests that prions can persist in the environment, implicating components thereof as potential prion reservoirs and transmission vehicles.(1-4) CWD-positive animals may contribute to environmental prion load via decomposing carcasses and biological materials including saliva, blood, urine and feces.(5-7) Sensitivity limitations of conventional assays hamper evaluation of environmental prion loads in soil and water. Here we show the ability of serial protein misfolding cyclic amplification (sPMCA) to amplify a 1.3 x 10(-7) dilution of CWD-infected brain homogenate spiked into water samples, equivalent to approximately 5 x 10(7) protease resistant cervid prion protein (PrP(CWD)) monomers. We also detected PrP(CWD) in one of two environmental water samples from a CWD endemic area collected at a time of increased water runoff from melting winter snow pack, as well as in water samples obtained concurrently from the flocculation stage of water processing by the municipal water treatment facility. Bioassays indicated that the PrP(CWD) detected was below infectious levels. These data demonstrate detection of very low levels of PrP(CWD) in the environment by sPMCA and suggest persistence and accumulation of prions in the environment that may promote CWD transmission.
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Altered bone marrow dendritic cell cytokine production to toll-like receptor and CD40 ligation during chronic feline immunodeficiency virus infection.
Immunology
PUBLISHED: 06-17-2009
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Impaired dendritic cell (DC) function is thought to be central to human immunodeficiency virus-associated immunodeficiency. In this study, we examined the effect of chronic feline immunodeficiency virus (FIV) infection on DC cytokine production in response to microbial and T-cell stimulation. Cytokine production after either Toll-like receptor (TLR) or CD40 ligation in bone marrow-derived DCs (BM-DCs) was measured in naïve and chronically FIV-infected cats. The BM-DCs were stimulated with ligands to TLR-2, -3, -4, -7 and -9 or cocultured with 3T3 cells expressing feline CD40 ligand. Ligation of TLR-4 and TLR-9 in BM-DCs from infected cats resulted in a significant decrease in the ratio of interleukin-12 (IL-12) to IL-10. Conversely, TLR-7 ligation produced a significant increase in the IL-12 : IL-10 ratio in BM-DCs from infected cats. No difference was noted for TLR-3 ligation. RNA expression levels of TLR-2, -3, -4, -7 and -9 were not significantly altered by FIV infection. CD40 ligation significantly elevated both IL-10 and IL-12 messenger RNA production but did not alter the IL-12 : IL-10 ratio. Chronic FIV infection alters the ratio of immunoregulatory cytokines produced by BM-DCs in response to certain pathogen-derived signals, which is probably relevant to the increased risk of opportunistic infections seen in lentiviral infection.
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Chronic wasting disease prions in elk antler velvet.
Emerging Infect. Dis.
PUBLISHED: 05-01-2009
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Chronic wasting disease (CWD) is a contagious, fatal prion disease of deer and elk that continues to emerge in new locations. To explore the means by which prions are transmitted with high efficiency among cervids, we examined prion infectivity in the apical skin layer covering the growing antler (antler velvet) by using CWD-susceptible transgenic mice and protein misfolding cyclic amplification. Our finding of prions in antler velvet of CWD-affected elk suggests that this tissue may play a role in disease transmission among cervids. Humans who consume antler velvet as a nutritional supplement are at risk for exposure to prions. The fact that CWD prion incubation times in transgenic mice expressing elk prion protein are consistently more rapid raises the possibility that residue 226, the sole primary structural difference between deer and elk prion protein, may be a major determinant of CWD pathogenesis.
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Infectious prions in pre-clinical deer and transmission of chronic wasting disease solely by environmental exposure.
PLoS ONE
PUBLISHED: 03-06-2009
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Key to understanding the epidemiology and pathogenesis of prion diseases, including chronic wasting disease (CWD) of cervids, is determining the mode of transmission from one individual to another. We have previously reported that saliva and blood from CWD-infected deer contain sufficient infectious prions to transmit disease upon passage into naïve deer. Here we again use bioassays in deer to show that blood and saliva of pre-symptomatic deer contain infectious prions capable of infecting naïve deer and that naïve deer exposed only to environmental fomites from the suites of CWD-infected deer acquired CWD infection after a period of 15 months post initial exposure. These results help to further explain the basis for the facile transmission of CWD, highlight the complexities associated with CWD transmission among cervids in their natural environment, emphasize the potential utility of blood-based testing to detect pre-clinical CWD infection, and could augur similar transmission dynamics in other prion infections.
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Detection of CWD prions in urine and saliva of deer by transgenic mouse bioassay.
PLoS ONE
PUBLISHED: 02-03-2009
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Chronic wasting disease (CWD) is a prion disease affecting captive and free-ranging cervids (e.g. deer, elk, and moose). The mechanisms of CWD transmission are poorly understood, though bodily fluids are thought to play an important role. Here we report the presence of infectious prions in the urine and saliva of deer with chronic wasting disease (CWD). Prion infectivity was detected by bioassay of concentrated, dialyzed urine and saliva in transgenic mice expressing the cervid PrP gene (Tg[CerPrP] mice). In addition, PrP(CWD) was detected in pooled and concentrated urine by protein misfolding cyclic amplification (PMCA). The concentration of abnormal prion protein in bodily fluids was very low, as indicated by: undetectable PrP(CWD) levels by traditional assays (western blot, ELISA) and prolonged incubation periods and incomplete TSE attack rates in inoculated Tg(CerPrP) mice (373(+/-)3 days in 2 of 9 urine-inoculated mice and 342(+/-)109 days in 8 of 9 saliva-inoculated mice). These findings help extend our understanding of CWD prion shedding and transmission and portend the detection of infectious prions in body fluids in other prion infections.
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Trans-species amplification of PrP(CWD) and correlation with rigid loop 170N.
Virology
PUBLISHED: 01-24-2009
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Chronic wasting disease (CWD) is an efficiently transmitted spongiform encephalopathy of cervids. Whether CWD could represent a threat to non-cervid species remains speculative. Here we show that brain homogenates from several CWD-susceptible non-cervid species, such as ferrets and hamsters, support amplification of PrP(CWD) by sPMCA, whereas brain homogenates from CWD-resistant species, such as laboratory mice and transgenic mice expressing human PrP(C) [Tg(HuPrP) mice], do not. We also investigated whether several North American species that share the environment with cervids would support amplification of PrP(CWD) by sPMCA. Three native rodent species, including voles and field mice, supported PrP(CWD) amplification, whereas other species (e.g. prairie dog, coyote) did not. Analysis of PrP sequences suggests that an ability to support amplification of PrP(CWD) in trans-species sPMCA is correlated with the presence of asparagine at position 170 of the substrate species PrP. Serial PMCA may offer insights into species barriers to transmission of CWD.
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Susceptibility of domestic cats to chronic wasting disease.
J. Virol.
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Domestic and nondomestic cats have been shown to be susceptible to feline spongiform encephalopathy (FSE), almost certainly caused by consumption of bovine spongiform encephalopathy (BSE)-contaminated meat. Because domestic and free-ranging nondomestic felids scavenge cervid carcasses, including those in areas affected by chronic wasting disease (CWD), we evaluated the susceptibility of the domestic cat (Felis catus) to CWD infection experimentally. Cohorts of 5 cats each were inoculated intracerebrally (i.c.) or orally (p.o.) with CWD-infected deer brain. At 40 and 42 months postinoculation, two i.c.-inoculated cats developed signs consistent with prion disease, including a stilted gait, weight loss, anorexia, polydipsia, patterned motor behaviors, head and tail tremors, and ataxia, and the cats progressed to terminal disease within 5 months. Brains from these two cats were pooled and inoculated into cohorts of cats by the i.c., p.o., and intraperitoneal and subcutaneous (i.p./s.c.) routes. Upon subpassage, feline CWD was transmitted to all i.c.-inoculated cats with a decreased incubation period of 23 to 27 months. Feline-adapted CWD (Fel(CWD)) was demonstrated in the brains of all of the affected cats by Western blotting and immunohistochemical analysis. Magnetic resonance imaging revealed abnormalities in clinically ill cats, which included multifocal T2 fluid attenuated inversion recovery (FLAIR) signal hyperintensities, ventricular size increases, prominent sulci, and white matter tract cavitation. Currently, 3 of 4 i.p./s.c.- and 2 of 4 p.o. secondary passage-inoculated cats have developed abnormal behavior patterns consistent with the early stage of feline CWD. These results demonstrate that CWD can be transmitted and adapted to the domestic cat, thus raising the issue of potential cervid-to-feline transmission in nature.
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Aerosol transmission of chronic wasting disease in white-tailed deer.
J. Virol.
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While the facile transmission of chronic wasting disease (CWD) remains incompletely elucidated, studies in rodents suggest that exposure of the respiratory mucosa may be an efficient pathway. The present study was designed to address this question in the native cervid host. Here, we demonstrate aerosol transmission of CWD to deer with a prion dose >20-fold lower than that used in previous oral inoculations. Inhalation of prions may facilitate transmission of CWD and, perhaps, other prion infections.
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Mucosal transmission and pathogenesis of chronic wasting disease in ferrets.
J. Gen. Virol.
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Chronic wasting disease (CWD) of cervids is almost certainly transmitted by mucosal contact with the causative prion, whether by direct (animal-to-animal) or indirect (environmental) means. Yet the sites and mechanisms of prion entry remain to be further understood. This study sought to extend this understanding by demonstrating that ferrets exposed to CWD via several mucosal routes developed infection, CWD prion protein (PrP(CWD)) amplification in lymphoid tissues, neural invasion and florid transmissible spongiform encephalopathy lesions resembling those in native cervid hosts. The ferrets developed extensive PrP(CWD) accumulation in the nervous system, retina and olfactory epithelium, with lesser deposition in tongue, muscle, salivary gland and the vomeronasal organ. PrP(CWD) accumulation in mucosal sites, including upper respiratory tract epithelium, olfactory epithelium and intestinal Peyers patches, make the shedding of prions by infected ferrets plausible. It was also observed that regionally targeted exposure of the nasopharyngeal mucosa resulted in an increased attack rate when compared with oral exposure. The latter finding suggests that nasal exposure enhances permissiveness to CWD infection. The ferret model has further potential for investigation of portals for initiation of CWD infection.
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Sensitivity of protein misfolding cyclic amplification versus immunohistochemistry in ante-mortem detection of chronic wasting disease.
J. Gen. Virol.
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As the only prion disease affecting free-ranging animals, ante-mortem identification of affected cervids has become paramount in understanding chronic wasting disease (CWD) pathogenesis, prevalence and control of horizontal or vertical transmission. To seek maximal sensitivity in ante-mortem detection of CWD infection, this study used paired tonsil biopsy samples collected at various time points from 48 CWD-exposed cervids to compare blinded serial protein misfolding cyclic amplification (sPMCA) with the assay long considered the gold standard for CWD detection, immunohistochemistry (IHC). sPMCA-negative controls (34?% of the samples evaluated) included tissues from mock-inoculated animals and unspiked negative controls, all of which tested negative throughout the course of the study. It was found that sPMCA on tonsil biopsies detected CWD infection significantly earlier (2.78 months, 95?% confidence interval 2.40-3.15) than conventional IHC. Interestingly, a correlation was observed between early detection by sPMCA and host PRNP genotype. These findings demonstrate that in vitro-amplification assays provide enhanced sensitivity and advanced detection of CWD infection in the peripheral tissues of cervids, with a potential role for spike or substrate genotype in sPMCA amplification efficiency.
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