Highly efficient amplification of chronic wasting disease agent by protein misfolding cyclic amplification with beads (PMCAb)

Protein misfolding cyclic amplification (PMCA) has emerged as an important technique for detecting low levels of pathogenic prion protein in biological samples. The method exploits the ability of the pathogenic prion protein to convert the normal prion protein to a proteinase K-resistant conformation. Inclusion of Teflon® beads in the PMCA reaction (PMCAb) has been previously shown to increase the sensitivity and robustness of detection for the 263 K and SSLOW strains of hamster-adapted prions. Here, we demonstrate that PMCAb with saponin dramatically increases the sensitivity of detection for chronic wasting disease (CWD) agent without compromising the specificity of the assay (i.e., no false positive results). Addition of Teflon® beads increased the robustness of the PMCA reaction, resulting in a decrease in the variability of PMCA results. Three rounds of serial PMCAb allowed detection of CWD agent from a 6.7×10⁻¹³ dilution of 10% brain homogenate (1.3 fg of source brain). Titration of the same brain homogenate in transgenic mice expressing cervid prion protein (Tg(CerPrP)1536^+/− mice) allowed detection of CWD agent from the 10⁻⁶ dilution of 10% brain homogenate. PMCAb is, thus, more sensitive than bioassay in transgenic mice by a factor exceeding 10⁵. Additionally, we are able to amplify CWD agent from brain tissue and lymph nodes of CWD-positive white-tailed deer having Prnp alleles associated with reduced disease susceptibility.     

Detection of Sub-Clinical CWD Infection in Conventional Test-Negative Deer Long after Oral Exposure to Urine and Feces from CWD+ Deer

Background

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.

Objectives

We sought to investigate whether conventional test-negative deer, previously exposed orally to urine and feces from CWD+ sources, may be harboring low level CWD infection not evident in the 19 month observation period. We further attempted to determine the peripheral PrP^CWD distribution in these animals.

Methods

Various neural and lymphoid tissues from conventional test-negative deer were reanalyzed for CWD prions by sPMCA and cervid transgenic mouse bioassay in parallel with appropriate tissue-matched positive and negative controls.

Results

PrP^CWD was detected in the tissues of orally exposed deer by both sPMCA and Tg[CerPrP] mouse bioassay; each assay revealed very low levels of CWD prions previously undetectable by western blot, ELISA, or IHC. Serial PMCA analysis of individual tissues identified that obex alone was positive in 4 of 5 urine/feces exposed deer. PrP^CWD was amplified from both lymphoid and neural tissues of positive control deer but not from identical tissues of negative control deer.

Discussion

Detection of subclinical infection in deer orally exposed to urine and feces (1) suggests that a prolonged subclinical state can exist, necessitating observation periods in excess of two years to detect CWD infection, and (2) illustrates the sensitive and specific application of sPMCA in the diagnosis of low-level prion infection. Based on these results, it is possible that low doses of prions, e.g. following oral exposure to urine and saliva of CWD-infected deer, bypass significant amplification in the LRS, perhaps utilizing a neural conduit between the alimentary tract and CNS, as has been demonstrated in some other prion diseases.     

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.     

Assessing the Susceptibility of Transgenic Mice Overexpressing Deer Prion Protein to Bovine Spongiform Encephalopathy

Several transgenic mouse models have been developed which facilitate the transmission of chronic wasting disease (CWD) of cervids and allow prion strain discrimination. The present study was designed to assess the susceptibility of the prototypic mouse line, Tg(CerPrP)1536^+/−, to bovine spongiform encephalopathy (BSE) prions, which have the ability to overcome species barriers. Tg(CerPrP)1536^+/− mice challenged with red deer-adapted BSE resulted in 90% to 100% attack rates, and BSE from cattle failed to transmit, indicating agent adaptation in the deer.     

Efficient in vitro amplification of chronic wasting disease PrPRES

Chronic wasting disease (CWD) of cervids is associated with conversion of the normal cervid prion protein, PrP(C), to a protease-resistant conformer, PrP(CWD). Here we report the use of both nondenaturing amplification and protein-misfolding cyclic amplification (PMCA) to amplify PrP(CWD) in vitro. Normal brains from deer, transgenic mice expressing cervid PrP(C) [Tg(cerPrP)1536 mice], and ferrets supported amplification. PMCA using normal Tg(cerPrP)1536 brains as the PrP(C) substrate produced >6.5 x 10(9)-fold amplification after six rounds. Highly efficient in vitro amplification of PrP(CWD) is a significant step toward detection of PrP(CWD) in the body fluids or excreta of CWD-susceptible species.     

Chronic wasting disease prion infection of differentiated neurospheres

A possible strategy to develop more diverse cell culture systems permissive to infection with naturally occurring prions is to exploit culture of neurospheres from transgenic mice expressing the normal prion protein (PrP) of the native host species. Accordingly, we developed differentiated neurosphere cultures from the cervid PrP-expressing mice to investigate whether this in vitro system would support replication of non-adapted cervid-origin chronic wasting disease (CWD) prions. Here we report the successful amplification of disease-associated PrP in differentiated neurosphere cultures within 3 weeks after exposure to CWD prions from both white-tailed deer or elk. This neurosphere culture system provides a new in vitro tool that can be used to assess non-adapted CWD prion propagation and transmission.     

Identification of two prion protein regions that modify scrapie incubation time

A series of prion transmission experiments was performed in transgenic (Tg) mice expressing either wild-type, chimeric, or truncated prion protein (PrP) molecules. Following inoculation with Rocky Mountain Laboratory (RML) murine prions, scrapie incubation times for Tg(MoPrP)4053, Tg(MHM2)294/Prnp(0/0), and Tg(MoPrP, Delta23-88)9949/Prnp(0/0) mice were approximately 50, 120, and 160 days, respectively. Similar scrapie incubation times were obtained after inoculation of these lines of Tg mice with either MHM2(MHM2(RML)) or MoPrP(Delta23-88)(RML) prions, excluding the possibility that sequence-dependent transmission barriers could account for the observed differences. Tg(MHM2)294/Prnp(0/0) mice displayed prolonged scrapie incubation times with four different strains of murine prions. These data provide evidence that the N terminus of MoPrP and the chimeric region of MHM2 PrP (residues 108 through 111) both influence the inherent efficiency of prion propagation.     

Prion protein of 106 residues creates an artifical transmission barrier for prion replication in transgenic mice

A redacted prion protein (PrP) of 106 amino acids with two large deletions was expressed in transgenic (Tg) mice deficient for wild-type (wt) PrP (Prnp0/0) and supported prion propagation. RML prions containing full-length PrP(Sc)produced disease in Tg(PrP106)Prnp0/0 mice after approximately 300 days, while transmission of RML106 prions containing PrP(Sc)106 created disease in Tg(PrP106) Prnp0/0 mice after only approximately 66 days on repeated passage. This artificial transmission barrier for the passage of RML prions was diminished by the coexpression of wt MoPrPc in Tg(PrP106)Prnp+/0 mice that developed scrapie in approximately 165 days, suggesting that wt MoPrP acts in trans to accelerate replication of RML106 prions. Purified PrP(Sc)106 was protease resistant, formed filaments, and was insoluble in nondenaturing detergents. The unique features of RML106 prions offer insights into the mechanism of prion replication, and the small size of PrP(Sc)106 should facilitate structural analysis.     

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 of elk and deer prions to transgenic mice

Chronic wasting disease (CWD) is a fatal prion disease in deer and elk. Unique among the prion diseases, it is transmitted among captive and free-ranging animals. To facilitate studies of the biology of CWD prions, we generated five lines of transgenic (Tg) mice expressing prion protein (PrP) from Rocky Mountain elk (Cervus elaphus nelsoni), denoted Tg(ElkPrP), and two lines of Tg mice expressing PrP common to white-tailed deer (Odocoileus virginianus) and mule deer (Odocoileus hemionus), denoted Tg(DePrP). None of the Tg(ElkPrP) or Tg(DePrP) mice exhibited spontaneous neurologic dysfunction at more than 600 days of age. Brain samples from CWD-positive elk, white-tailed deer, and mule deer produced disease in Tg(ElkPrP) mice between 180 and 200 days after inoculation and in Tg(DePrP) mice between 300 and 400 days. One of eight cervid brain inocula transmitted disease to Tg(MoPrP)4053 mice overexpressing wild-type mouse PrP-A in approximately 540 days. Neuropathologic analysis revealed abundant PrP amyloid plaques in the brains of ill mice. Brain homogenates from symptomatic Tg(ElkPrP) mice produced disease in 120 to 190 days in Tg(ElkPrP) mice. In contrast to the Tg(ElkPrP) and Tg(DePrP) mice, Tg mice overexpressing human, bovine, or ovine PrP did not develop prion disease after inoculation with CWD prions from among nine different isolates after >500 days. These findings suggest that CWD prions from elk, mule deer, and white-tailed deer can be readily transmitted among these three cervid species.     

Detection of chronic wasting disease prions in salivary, urinary, and intestinal tissues of deer: potential mechanisms of prion shedding and transmission

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.     

Mother to Offspring Transmission of Chronic Wasting Disease in Reeves’ Muntjac Deer

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.     

Strain-Specific Role of RNAs in Prion Replication

Several lines of evidence suggest that various cofactors may be required for prion replication. PrP binds to polyanions, and RNAs were shown to promote the conversion of PrP(C) into PrP(Sc) in vitro. In the present study, we investigated strain-specific differences in RNA requirement during in vitro conversion and the potential role of RNA as a strain-specifying component of infectious prions. We found that RNase treatment impairs PrP(Sc)-converting activity of 9 murine prion strains by protein misfolding cyclic amplification (PMCA) in a strain-specific fashion. While the addition of RNA restored PMCA conversion efficiency, the effect of synthetic polynucleotides or DNA was strain dependent, showing a different promiscuity of prion strains in cofactor utilization. The biological properties of RML propagated by PMCA under RNA-depleted conditions were compared to those of brain-derived and PMCA material generated in the presence of RNA. Inoculation of RNA-depleted RML in Tga20 mice resulted in an increased incidence of a distinctive disease phenotype characterized by forelimb paresis. However, this abnormal phenotype was not conserved in wild-type mice or upon secondary transmission. Immunohistochemical and cell panel assay analyses of mouse brains did not reveal significant differences between mice injected with the different RML inocula. We conclude that replication under RNA-depleted conditions did not modify RML prion strain properties. Our study cannot, however, exclude small variations of RML properties that would explain the abnormal clinical phenotype observed. We hypothesize that RNA molecules may act as catalysts of prion replication and that variable capacities of distinct prion strains to utilize different cofactors may explain strain-specific dependency upon RNA.     

Transgenic Mouse Bioassay: Evidence That Rabbits Are Susceptible to a Variety of Prion Isolates

Interspecies transmission of prions is a well-established phenomenon, both experimentally and under field conditions. Upon passage through new hosts, prion strains have proven their capacity to change their properties and this is a source of strain diversity which needs to be considered when assessing the potential risks associated with consumption of prion contaminated protein sources. Rabbits were considered for decades to be a prion resistant species until proven otherwise recently. To determine the extent of rabbit susceptibility to prions and to assess the effects of passage of different prion strains through this species a transgenic mouse model overexpressing rabbit PrP^C was developed (TgRab). Intracerebral challenges with prion strains originating from a variety of species including field isolates (ovine SSBP/1 scrapie, Nor98- scrapie; cattle BSE, BSE-L and cervid CWD), experimental murine strains (ME7 and RML) and experimentally obtained ruminant (sheepBSE) and rabbit (de novo NZW) strains were performed. On first passage TgRab were susceptible to the majority of prions (Cattle BSE, SheepBSE, BSE-L, de novo NZW, ME7 and RML) tested with the exception of SSBP/1 scrapie, CWD and Nor98 scrapie. Furthermore, TgRab were capable of propagating strain-specific features such as differences in incubation periods, histological brain lesions, abnormal prion (PrP^d) deposition profiles and proteinase-K (PK) resistant western blotting band patterns. Our results confirm previous studies proving that rabbits are not resistant to prion infection and show for the first time that rabbits are susceptible to PrP^d originating in a number of other species. This should be taken into account when choosing protein sources to feed rabbits.     

Propagation of prion strains through specific conformers of the prion protein.

Two prion strains with identical incubation periods in mice exhibited distinct incubation periods and different neuropathological profiles upon serial transmission to transgenic mice expressing chimeric Syrian hamster/mouse (MH2M) prion protein (PrP) genes [Tg(MH2M) mice] and subsequent transmission to Syrian hamsters. After transmission to Syrian hamsters, the Me7 strain was indistinguishable from the previously established Syrian hamster strain Sc237, despite having been derived from an independent ancestral source. This apparent convergence suggests that prion diversity may be limited. The Me7 mouse strain could also be transmitted directly to Syrian hamsters, but when derived in this way, its properties were distinct from those of Me7 passaged through Tg(MH2M) mice. The Me7 strain did not appear permanently altered in either case, since the original incubation period could be restored by effectively reversing the series of passages. Prion diversity enciphered in the conformation of the scrapie isoform of PrP (PrP(Sc)) (G. C. Telling et al., Science 274:2079-2082, 1996) seems to be limited by the sequence of the PrP substrates serially converted into PrP(Sc), while prions are propagated through interactions between the cellular and scrapie isoforms of PrP.     

Crossing the species barrier by PrP(Sc) replication in vitro generates unique infectious prions

Prions are unconventional infectious agents composed exclusively of misfolded prion protein (PrP(Sc)), which transmits the disease by propagating its abnormal conformation to the cellular prion protein (PrP(C)). A key characteristic of prions is their species barrier, by which prions from one species can only infect a limited number of other species. Here, we report the generation of infectious prions by interspecies transmission of PrP(Sc) misfolding by in vitro PMCA amplification. Hamster PrP(C) misfolded by mixing with mouse PrP(Sc) generated unique prions that were infectious to wild-type hamsters, and similar results were obtained in the opposite direction. Successive rounds of PMCA amplification result in adaptation of the in vitro-produced prions, in a process reminiscent of strain stabilization observed upon serial passage in vivo. Our results indicate that PMCA is a valuable tool for the investigation of cross-species transmission and suggest that species barrier and strain generation are determined by the propagation of PrP misfolding.     

Salivary prions in sheep and deer

Scrapie of sheep and chronic wasting disease (CWD) of cervids are transmissible prion diseases. Milk and placenta have been identified as sources of scrapie prions but do not explain horizontal transmission. In contrast, CWD prions have been reported in saliva, urine and feces, which are thought to be responsible for horizontal transmission. While the titers of CWD prions have been measured in feces, levels in saliva or urine are unknown. Because sheep produce ~17 L/day of saliva, and scrapie prions are present in tongue and salivary glands of infected sheep, we asked if scrapie prions are shed in saliva. We inoculated transgenic (Tg) mice expressing ovine prion protein, Tg(OvPrP) mice, with saliva from seven Cheviot sheep with scrapie. Six of seven samples transmitted prions to Tg(OvPrP) mice with titers of -0.5 to 1.7 log ID₅₀ U/ml. Similarly, inoculation of saliva samples from two mule deer with CWD transmitted prions to Tg(ElkPrP) mice with titers of -1.1 to -0.4 log ID₅₀ U/ml. Assuming similar shedding kinetics for salivary prions as those for fecal prions of deer, we estimated the secreted salivary prion dose over a 10-mo period to be as high as 8.4 log ID₅₀ units for sheep and 7.0 log ID₅₀ units for deer. These estimates are similar to 7.9 log ID₅₀ units of fecal CWD prions for deer. Because saliva is mostly swallowed, salivary prions may reinfect tissues of the gastrointestinal tract and contribute to fecal prion shedding. Salivary prions shed into the environment provide an additional mechanism for horizontal prion transmission.     

Strain specific resistance to murine scrapie associated with a naturally occurring human prion protein polymorphism at residue 171

Transmissible spongiform encephalopathies (TSE) or prion diseases are neurodegenerative disorders associated with conversion of normal host prion protein (PrP) to a misfolded, protease-resistant form (PrPres). Genetic variations of prion protein in humans and animals can alter susceptibility to both familial and infectious prion diseases. The N171S PrP polymorphism is found mainly in humans of African descent, but its low incidence has precluded study of its possible influence on prion disease. Similar to previous experiments of others, for laboratory studies we created a transgenic model expressing the mouse PrP homolog, PrP-170S, of human PrP-171S. Since PrP polymorphisms can vary in their effects on different TSE diseases, we tested these mice with four different strains of mouse-adapted scrapie. Whereas 22L and ME7 scrapie strains induced typical clinical disease, neuropathology and accumulation of PrPres in all transgenic mice at 99-128 average days post-inoculation, strains RML and 79A produced clinical disease and PrPres formation in only a small subset of mice at very late times. When mice expressing both PrP-170S and PrP-170N were inoculated with RML scrapie, dominant-negative inhibition of disease did not occur, possibly because interaction of strain RML with PrP-170S was minimal. Surprisingly, in vitro PrP conversion using protein misfolding cyclic amplification (PMCA), did not reproduce the in vivo findings, suggesting that the resistance noted in live mice might be due to factors or conditions not present in vitro. These findings suggest that in vivo conversion of PrP-170S by RML and 79A scrapie strains was slow and inefficient. PrP-170S mice may be an example of the conformational selection model where the structure of some prion strains does not favor interactions with PrP molecules expressing certain polymorphisms.     

Detection of protease-resistant cervid prion protein in water from a CWD-endemic area

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. CWD-positive animals may contribute to environmental prion load via decomposing carcasses and biological materials including saliva, blood, urine and feces. 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 10⁷ 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.