A natural case of chronic wasting disease in a free-ranging moose (Alces alces shirasi)

Chronic wasting disease (CWD) was diagnosed in a free-ranging moose (Alces alces shirasi) killed by a hunter in Jackson County, Colorado, USA, in September 2005. The diagnosis was based upon immunohistochemistry (IHC) demonstrating the presence of accumulations of CWD-associated prion protein (PrP(CWD)) in tissue sections of medulla oblongata at the level of the obex (dorsal motor nucleus of the vagus) and in retropharyngeal lymph node (RPLN); additional testing by IHC revealed deposits of PrP(CWD) in multiple sections of medulla oblongata and cervical spinal cord as well as palatine tonsil and submandibular lymph node tissues. Western blot confirmed the presence of PrP(CWD) in RPLN and tonsil tissue. The PrP(CWD) also was detected via enzyme-linked immunosorbent assay of RPLN tissue. Spongiform encephalopathy was observed in sections of the brainstem and cervical spinal cord, although no clinical signs were noted by the hunter who killed the animal. The affected moose was homozygous for methionine at codon 209 of the prion protein coding region. In October 2006, two additional free-ranging moose were diagnosed with CWD. Epidemiology and implications of CWD in moose remain to be determined.     

Species barriers for chronic wasting disease by in vitro conversion of prion protein

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy that can affect North American cervids (deer, elk, and moose). Using a novel in vitro conversion system based on incubation of prions with normal brain homogenates, we now report that PrP^CWD of elk can readily induce the conversion of normal cervid PrP (PrP^C) molecules to a protease-resistant form, but is less efficient in converting the PrP^C of other species, such as human, bovine, hamster, and mouse. However, when substrate brain homogenates are partially denatured by acidic conditions (pH 3.5), PrP^CWD-induced conversion can be greatly enhanced in all species. Our results demonstrate that PrP^C from cervids (including moose) can be efficiently converted to a protease-resistant form by incubation with elk CWD prions, presumably due to sequence and structural similarities between these species. Moreover, partial denaturation of substrate PrP^C can apparently overcome the structural barriers between more distant species.     

Adaptive selection of a prion strain conformer corresponding to established North American CWD during propagation of novel emergent Norwegian strains in mice expressing elk or deer prion protein

Prions are infectious proteins causing fatal, transmissible neurodegenerative diseases of animals and humans. Replication involves template-directed refolding of host encoded prion protein, PrP^C, by its infectious conformation, PrP^Sc. Following its discovery in captive Colorado deer in 1967, uncontrollable contagious transmission of chronic wasting disease (CWD) led to an expanded geographic range in increasing numbers of free-ranging and captive North American (NA) cervids. Some five decades later, detection of PrP^Sc in free-ranging Norwegian (NO) reindeer and moose marked the first indication of CWD in Europe. To assess the properties of these emergent NO prions and compare them with NA CWD we used transgenic (Tg) and gene targeted (Gt) mice expressing PrP with glutamine (Q) or glutamate (E) at residue 226, a variation in wild type cervid PrP which influences prion strain selection in NA deer and elk. Transmissions of NO moose and reindeer prions to Tg and Gt mice recapitulated the characteristic features of CWD in natural hosts, revealing novel prion strains with disease kinetics, neuropathological profiles, and capacities to infect lymphoid tissues and cultured cells that were distinct from those causing NA CWD. In support of strain variation, PrP^Sc conformers comprising emergent NO moose and reindeer CWD were subject to selective effects imposed by variation at residue 226 that were different from those controlling established NA CWD. Transmission of particular NO moose CWD prions in mice expressing E at 226 resulted in selection of a kinetically optimized conformer, subsequent transmission of which revealed properties consistent with NA CWD. These findings illustrate the potential for adaptive selection of strain conformers with improved fitness during propagation of unstable NO prions. Their potential for contagious transmission has implications for risk analyses and management of emergent European CWD. Finally, we found that Gt mice expressing physiologically controlled PrP levels recapitulated the lymphotropic properties of naturally occurring CWD strains resulting in improved susceptibilities to emergent NO reindeer prions compared with over-expressing Tg counterparts. These findings underscore the refined advantages of Gt models for exploring the mechanisms and impacts of strain selection in peripheral compartments during natural prion transmission.     

Applicability of current bovine spongiform encephalopathy (BSE) diagnostic procedures for chronic wasting disease (CWD)

Chronic wasting disease (CWD) in cervids is one of the transmissible spongiform encephalopathies ; however, its risk to humans is still obscure. An increase in number of diseased deer in North America has raised concerns regarding the CWD risk to humans. We demonstrated that the con-firmatory procedures and the commercial diagnostic kits for bovine spongiform encephalopathy (BSE) can be adopted for the diagnosis of CWD. No CWD case was confirmed in the surveillance of 558 cervids that were examined between 2003 and 2006 in Japan.     

Molecular characterization of the Rocky Mountain elk (Cervus elaphus nelsoni) PRNP putative promoter

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) affecting deer (Odocoileus spp.), moose (Alces alces), and Rocky Mountain elk (Cervus elaphus nelsoni). Leucine homozygosity at elk PRNP codon 132 has been associated with reduced CWD susceptibility. However, naturally acquired CWD has been detected in elk possessing the 132 Leu/Leu genotype. Recent human and bovine studies indicate that PRNP regulatory polymorphisms may also influence TSE occurrence. Therefore, we generated sequences for the elk PRNP putative promoter (2.2 kb), exon 1 (predicted; 54 bp), intron 1 (predicted; 193 bp), and exon 3 (771 bp). Promoter prediction analysis using CpGProD yielded a single elk PRNP promoter that was homologous to regions of known promoter activity in cow and sheep. Molecular interrogation of the elk PRNP putative promoter revealed 32 diallelic single-nucleotide polymorphisms (SNPs). No variation was detected within the predicted exon 1 or intron 1 sequences. Evaluation of elk PRNP exon 3 revealed 3 SNPs (63Y, 312R, 394W-->Met/Leu). Bayesian haplotype reconstruction resulted in 3 elk PRNP haplotypes, with complete linkage disequilibrium observed between all PRNP putative promoter SNPs and codon 132. The results of this study provide the initial genomic foundation for future comparative and haplotype-based elk PRNP studies.     

The role of genetics in chronic wasting disease of North American cervids

Chronic wasting disease (CWD) is a major concern for the management of North American cervid populations. This fatal prion disease has led to declines in populations which have high CWD prevalence and areas with both high and low infection rates have experienced economic losses in wildlife recreation and fears of potential spill-over into livestock or humans. Research from human and veterinary medicine has established that the prion protein gene (Prnp) encodes the protein responsible for transmissible spongiform encephalopathies (TSEs). Polymorphisms in the Prnp gene can lead to different prion forms that moderate individual susceptibility to and progression of TSE infection. Prnp genes have been sequenced in a number of cervid species including those currently infected by CWD (elk, mule deer, white-tailed deer, moose) and those for which susceptibility is not yet determined (caribou, fallow deer, sika deer). Over thousands of sequences examined, the Prnp gene is remarkably conserved within the family Cervidae; only 16 amino acid polymorphisms have been reported within the 256 amino acid open reading frame in the third exon of the Prnp gene. Some of these polymorphisms have been associated with lower rates of CWD infection and slower progression of clinical CWD. Here we review the body of research on Prnp genetics of North American cervids. Specifically, we focus on known polymorphisms in the Prnp gene, observed genotypic differences in CWD infection rates and clinical progression, mechanisms for genetic TSE resistance related to both the cervid host and the prion agent and potential for natural selection for CWD-resistance. We also identify gaps in our knowledge that require future research.     

Ante-mortem detection of chronic wasting disease in recto-anal mucosa-associated lymphoid tissues from elk (Cervus elaphus nelsoni) using real-time quaking-induced conversion (RT-QuIC) assay: A blinded collaborative study

Prion diseases are transmissible spongiform encephalopathies (TSEs) characterized by fatal, progressive neurologic diseases with prolonged incubation periods and an accumulation of infectious misfolded prion proteins. Antemortem diagnosis is often difficult due to a long asymptomatic incubation period, differences in the pathogenesis of different prions, and the presence of very low levels of infectious prion in easily accessible samples. Chronic wasting disease (CWD) is a TSE affecting both wild and captive populations of cervids, including mule deer, white-tailed deer, elk, moose, muntjac, and most recently, wild reindeer. This study represents a well-controlled evaluation of a newly developed real-time quaking-induced conversion (RT-QuIC) assay as a potential CWD diagnostic screening test using rectal biopsy sections from a depopulated elk herd. We evaluated 69 blinded samples of recto-anal mucosa-associated lymphoid tissue (RAMALT) obtained from USDA Veterinary Services. The results were later un-blinded and statistically compared to immunohistochemical (IHC) results from the USDA National Veterinary Services Laboratories (NVSL) for RAMALT, obex, and medial retropharyngeal lymph node (MRPLN). Comparison of RAMALT RT-QuIC assay results with the IHC results of RAMALT revealed 92% relative sensitivity (95% confidence limits: 61.52–99.8%) and 95% relative specificity (95% confidence limits: 85.13–99%). Collectively, our results show a potential utility of the RT-QuIC assay to advance the development of a rapid, sensitive, and specific prion diagnostic assay for CWD prions.     

Comparison of abnormal prion protein glycoform patterns from transmissible spongiform encephalopathy agent-infected deer,elk, sheep,and cattle

Analysis of abnormal prion protein glycoform patterns from chronic wasting disease (CWD)-affected deer and elk, scrapie-affected sheep and cattle, and cattle with bovine spongiform encephalopathy failed to identify patterns capable of reliably distinguishing these transmissible spongiform encephalopathy diseases. However, PrP-res patterns sometimes differed among individual animals, suggesting infection by different or multiple CWD strains in some species.     

High prevalence of prion protein genotype associated with resistance to chronic wasting disease in one Alberta woodland caribou population

Chronic wasting disease (CWD) is a prion disease found in deer, elk and moose in North America and since recently, wild reindeer in Norway. Caribou are at-risk to encounter CWD in areas such as Alberta, Canada, where the disease spreads toward caribou habitats. CWD susceptibility is modulated by species-specific polymorphisms in the prion protein gene (Prnp). We sequenced Prnp of woodland caribou from 9 Albertan populations. In one population (Chinchaga) a significantly higher frequency of the 138N allele linked to reduced CWD susceptibility was observed. These data are relevant for developing CWD management strategies including conservation of threatened caribou populations.     

Chronic wasting disease: Fingerprinting the culprit in risk assessments

Transmissible spongiform encephalopathies (prion diseases) in animals may be associated with a zoonotic risk potential for humans as shown by the occurrence of variant Creutzfeldt-Jakob disease in the wake of the bovine spongiform encephalopathy epidemic. Thus, the increasing exposure of humans in North America to cervid prions of chronic wasting disease (CWD) in elk and deer has prompted comprehensive risk assessments. The susceptibility of humans to CWD infections is currently under investigation in different studies using macaques as primate models. The necessity for such studies was recently reinforced when disease-associated prion protein and its seeding activity were detected in muscles of clinically inconspicuous CWD-infected white-tailed deer (WTD). Increasing evidence points to the existence of different CWD strains and CWD prions may also change or newly emerge over time. Therefore, CWD isolates examined in macaques should be characterized as precisely as possible for their molecular identity. On this basis other CWD field samples collected in the past, present or future could be systematically compared with macaque-tested inocula in order to assess whether they are covered by the ongoing risk assessments in primates. CWD typing by Fourier transform-infrared spectroscopy of pathological prion protein may provide a method of choice for this purpose.     

Early detection of chronic wasting disease prions in urine of pre-symptomatic deer by real-time quaking-induced conversion assay

Chronic wasting disease (CWD) is a prion disease of captive and free-ranging deer (Odocoileus spp), elk (Cervus elaphus nelsonii) and moose (Alces alces shirasi). Unlike in most other prion diseases, in CWD prions are shed in urine and feces, which most likely contributes to the horizontal transmission within and between cervid species. To date, CWD ante-mortem diagnosis is only possible by immunohistochemical detection of protease resistant prion protein (PrP^Sc) in tonsil or recto-anal mucosa-associated lymphoid tissue (RAMALT) biopsies, which requires anesthesia of animals. We report on detection of CWD prions in urine collected from pre-symptomatic deer and in fecal extracts by using real time quaking-induced conversion (RT-QuIC). This assay can be useful for non-invasive pre-symptomatic diagnosis and surveillance of CWD.     

Interspecies transmission of chronic wasting disease prions to squirrel monkeys (Saimiri sciureus)

Chronic wasting disease (CWD) is an emerging prion disease of deer and elk. The risk of CWD transmission to humans following exposure to CWD-infected tissues is unknown. To assess the susceptibility of nonhuman primates to CWD, two squirrel monkeys were inoculated with brain tissue from a CWD-infected mule deer. The CWD-inoculated squirrel monkeys developed a progressive neurodegenerative disease and were euthanized at 31 and 34 months postinfection. Brain tissue from the CWD-infected squirrel monkeys contained the abnormal isoform of the prion protein, PrP-res, and displayed spongiform degeneration. This is the first reported transmission of CWD to primates.     

Identification of the prion-related protein gene (PRNT) sequences in various species of the Cervidae family

Molecular Biology Reports - Chronic wasting disease (CWD) is caused by abnormal deleterious prion protein (PrPSc), and transmissible spongiform encephalopathy occurs in the Cervidae family. In...     

Evidence of a molecular barrier limiting susceptibility of humans, cattle and sheep to chronic wasting disease

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSE) of deer and elk, and little is known about its transmissibility to other species. An important factor controlling interspecies TSE susceptibility is prion protein (PrP) homology between the source and recipient species/genotypes. Furthermore, the efficiency with which the protease-resistant PrP (PrP-res) of one species induces the in vitro conversion of the normal PrP (PrP-sen) of another species to the protease-resistant state correlates with the cross-species transmissibility of TSE agents. Here we show that the CWD-associated PrP-res (PrP(CWD)) of cervids readily induces the conversion of recombinant cervid PrP-sen molecules to the protease-resistant state in accordance with the known transmissibility of CWD between cervids. In contrast, PrP(CWD)-induced conversions of human and bovine PrP-sen were much less efficient, and conversion of ovine PrP-sen was intermediate. These results demonstrate a barrier at the molecular level that should limit the susceptibility of these non-cervid species to CWD.     

Detection of PrPCWD in feces from naturally exposed Rocky Mountain elk (Cervus elaphus nelsoni) using protein misfolding cyclic amplification

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy affecting captive and free-ranging cervids. Currently, tests for CWD in live animals involve relatively invasive procedures to collect lymphoid tissue biopsies and examine them for CWD-associated, protease-resistant cervid prion protein (PrP(CWD)) detected by immunohistochemistry (IHC). We adapted an ultrasensitive prion detection system, protein misfolding cyclic amplification (PMCA), to detect PrP(CWD) in Rocky Mountain elk (Cervus elaphus nelsoni) feces. Our PMCA reproducibly detected a 1.2 × 10(7) dilution of PrP(CWD) (a 10% infected brain homogenate diluted 1.2 × 10(6)-fold into 10% fecal homogenates), equivalent to approximately 100 pg of PrP(CWD)/g of feces. We developed a semiquantitative scoring system based on the first PMCA round at which PrP(CWD) was detected and fit a nonlinear regression curve to our serial dilutions to correlate PMCA scores with known PrP(CWD) concentrations. We used this PMCA scoring system to detect PrP(CWD) and estimate its concentration in feces from free-ranging elk from Rocky Mountain National Park, Colorado. We compared our results to PrP(CWD) IHC of rectoanal mucosa-associated lymphoid tissue and obex from the same animals. The PMCA successfully detected PrP(CWD) in feces from elk that were positive by IHC, with estimated prion loads from 100 to 5,000 pg PrP(CWD)/g of feces. These data show for the first time PrP(CWD) in feces from naturally exposed free-ranging elk and demonstrate the potential of PMCA as a new, noninvasive CWD diagnostic tool to complement IHC.     

Inhibition of protease-resistant prion protein formation in a transformed deer cell line infected with chronic wasting disease

Chronic wasting disease (CWD) is an emerging transmissible spongiform encephalopathy (prion disease) of North American cervids, i.e., mule deer, white-tailed deer, and elk (wapiti). To facilitate in vitro studies of CWD, we have developed a transformed deer cell line that is persistently infected with CWD. Primary cultures derived from uninfected mule deer brain tissue were transformed by transfection with a plasmid containing the simian virus 40 genome. A transformed cell line (MDB) was exposed to microsomes prepared from the brainstem of a CWD-affected mule deer. CWD-associated, protease-resistant prion protein (PrP(CWD)) was used as an indicator of CWD infection. Although no PrP(CWD) was detected in any of these cultures after two passes, dilution cloning of cells yielded one PrP(CWD)-positive clone out of 51. This clone, designated MDB(CWD), has maintained stable PrP(CWD) production through 32 serial passes thus far. A second round of dilution cloning yielded 20 PrP(CWD)-positive subclones out of 30, one of which was designated MDB(CWD2). The MDB(CWD2) cell line was positive for fibronectin and negative for microtubule-associated protein 2 (a neuronal marker) and glial fibrillary acidic protein (an activated astrocyte marker), consistent with derivation from brain fibroblasts (e.g., meningeal fibroblasts). Two inhibitors of rodent scrapie protease-resistant PrP accumulation, pentosan polysulfate and a porphyrin compound, indium (III) meso-tetra(4-sulfonatophenyl)porphine chloride, potently blocked PrP(CWD) accumulation in MDB(CWD) cells. This demonstrates the utility of these cells in a rapid in vitro screening assay for PrP(CWD) inhibitors and suggests that these compounds have potential to be active against CWD in vivo.     

Experimental oral transmission of chronic wasting disease to reindeer (Rangifer tarandus tarandus)

Chronic wasting disease (CWD), a transmissible spongiform encephalopathy of cervids, remains prevalent in North American elk, white-tailed deer and mule deer. A natural case of CWD in reindeer (Rangifer tarandus tarandus) has not been reported despite potential habitat overlap with CWD-infected deer or elk herds. This study investigates the experimental transmission of CWD from elk or white-tailed deer to reindeer by the oral route of inoculation. Ante-mortem testing of the three reindeer exposed to CWD from white-tailed deer identified the accumulation of pathological PrP (PrP(CWD)) in the recto-anal mucosa associated lymphoid tissue (RAMALT) of two reindeer at 13.4 months post-inoculation. Terminal CWD occurred in the two RAMALT-positive reindeer at 18.5 and 20 months post-inoculation while one other reindeer in the white-tailed deer CWD inoculum group and none of the 3 reindeer exposed to elk CWD developed disease. Tissue distribution analysis of PrP(CWD) in CWD-affected reindeer revealed widespread deposition in central and peripheral nervous systems, lymphoreticular tissues, the gastrointestinal tract, neuroendocrine tissues and cardiac muscle. Analysis of prion protein gene (PRNP) sequences in the 6 reindeer identified polymorphisms at residues 2 (V/M), 129 (G/S), 138 (S/N) and 169 (V/M). These findings demonstrate that (i) a sub-population of reindeer are susceptible to CWD by oral inoculation implicating the potential for transmission to other Rangifer species, and (ii) certain reindeer PRNP polymorphisms may be protective against CWD infection.     

Alteration of the chronic wasting disease species barrier by in vitro prion amplification

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.     

Transmission and adaptation of chronic wasting disease to hamsters and transgenic mice: evidence for strains

In vitro screening using the cell-free prion protein conversion system indicated that certain rodents may be susceptible to chronic wasting disease (CWD). Therefore, CWD isolates from mule deer, white-tailed deer, and elk were inoculated intracerebrally into various rodent species to assess the rodents' susceptibility and to develop new rodent models of CWD. The species inoculated were Syrian golden, Djungarian, Chinese, Siberian, and Armenian hamsters, transgenic mice expressing the Syrian golden hamster prion protein, and RML Swiss and C57BL10 wild-type mice. The transgenic mice and the Syrian golden, Chinese, Siberian, and Armenian hamsters had limited susceptibility to certain of the CWD inocula, as evidenced by incomplete attack rates and long incubation periods. For serial passages of CWD isolates in Syrian golden hamsters, incubation periods rapidly stabilized, with isolates having either short (85 to 89 days) or long (408 to 544 days) mean incubation periods and distinct neuropathological patterns. In contrast, wild-type mouse strains and Djungarian hamsters were not susceptible to CWD. These results show that CWD can be transmitted and adapted to some species of rodents and suggest that the cervid-derived CWD inocula may have contained or diverged into at least two distinct transmissible spongiform encephalopathy strains.     

The ecology of chronic wasting disease in wildlife

Prions are misfolded infectious proteins responsible for a group of fatal neurodegenerative diseases termed transmissible spongiform encephalopathy or prion diseases. Chronic Wasting Disease (CWD) is the prion disease with the highest spillover potential, affecting at least seven Cervidae (deer) species. The zoonotic potential of CWD is inconclusive and cannot be ruled out. A risk of infection for other domestic and wildlife species is also plausible. Here, we review the current status of the knowledge with respect to CWD ecology in wildlife. Our current understanding of the geographic distribution of CWD lacks spatial and temporal detail, does not consider the biogeography of infectious diseases, and is largely biased by sampling based on hunters' cooperation and funding available for each region. Limitations of the methods used for data collection suggest that the extent and prevalence of CWD in wildlife is underestimated. If the zoonotic potential of CWD is confirmed in the short term, as suggested by recent results obtained in experimental animal models, there will be limited accurate epidemiological data to inform public health. Research gaps in CWD prion ecology include the need to identify specific biological characteristics of potential CWD reservoir species that better explain susceptibility to spillover, landscape and climate configurations that are suitable for CWD transmission, and the magnitude of sampling bias in our current understanding of CWD distribution and risk. Addressing these research gaps will help anticipate novel areas and species where CWD spillover is expected, which will inform control strategies. From an ecological perspective, control strategies could include assessing restoration of natural predators of CWD reservoirs, ultrasensitive CWD detection in biotic and abiotic reservoirs, and deer density and landscape modification to reduce CWD spread and prevalence.