White-Tailed Deer Movements in a Chronic Wasting Disease Area in South-Central Wisconsin

Abstract: To focus white-tailed deer (Odocoileus virginianus) management within a chronic wasting disease-infected area in south-central Wisconsin, USA, we assessed deer movements and related dispersal to variation in landscape pattern, deer density, and harvest intensity. We radiocollared and monitored 165 deer between 2003 and 2005. Yearling males that dispersed (45%) had greater forest edge (i.e., fragmentation) within natal home ranges. Exploratory movements were rare for adult females. Transient and migratory movements were rare among all deer (<5%). Although yearling males have low chronic wasting disease prevalence rates, they may be infected before dispersal due to variable incubation times. Managers should increase yearling male harvest and consider removing young males in areas of higher forest edge.     

Infectious Prions in Pre-Clinical Deer and Transmission of Chronic Wasting Disease Solely by Environmental Exposure

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.     

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.Key words: scrapie, chronic wasting disease, saliva, horizontal transmission, titers     

Evaluating Spatial Overlap and Relatedness of White-tailed Deer in a Chronic Wasting Disease Management Zone

Wildlife disease transmission, at a local scale, can occur from interactions between infected and susceptible conspecifics or from a contaminated environment. Thus, the degree of spatial overlap and rate of contact among deer is likely to impact both direct and indirect transmission of infectious diseases such chronic wasting disease (CWD) or bovine tuberculosis. We identified a strong relationship between degree of spatial overlap (volume of intersection) and genetic relatedness for female white-tailed deer in Wisconsin’s area of highest CWD prevalence. We used volume of intersection as a surrogate for contact rates between deer and concluded that related deer are more likely to have contact, which may drive disease transmission dynamics. In addition, we found that age of deer influences overlap, with fawns exhibiting the highest degree of overlap with other deer. Our results further support the finding that female social groups have higher contact among related deer which can result in transmission of infectious diseases. We suggest that control of large social groups comprised of closely related deer may be an effective strategy in slowing the transmission of infectious pathogens, and CWD in particular.     

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.     

Intra-host mathematical model of chronic wasting disease dynamics in deer (Odocoileus)

Bioassays of native cervid hosts have established the presence of infectious chronic wasting disease (CWD) prions in saliva, blood, urine, and feces of clinically diseased and pre-clinical infected deer. The intra-host trafficking of prions from the time of initial infection to shedding has been less well defined. We created a discrete-time compartmentalized model to simulate the misfolding catalysis, trafficking, and shedding of infectious prions throughout the organ systems of CWD-infected cervids. Using parameter values derived from experimental infections of North American deer (Odocoileus spp.), the exponential-based model predicts prion deposition over time with: 1) nervous tissues containing the highest deposition of prions at 20 months post-infection and 2) excreted fluids containing low levels of prions throughout infection with the highest numbers of prions predicted to be shed in saliva and feces (as high as 10 lethal doses (1.34 × 10²⁹ prions) in 11–15 months). These findings are comparable to prion deposition described in literature as assayed by conventional and ultrasensitive amplification assays. The comparison of our model to published data suggests that highly sensitive assays (sPMCA, RT-QuIC, and bioassay) are appropriate for early prion detection in bodily fluids and secretions. The model provides a view of intra-host prion catalysis leading to pre-clinical shedding and provides a framework for continued development of antemortem diagnostic methods.     

Genetic assessment of environmental features that influence deer dispersal: implications for prion-infected populations

The landscape can influence host dispersal and density, which in turn, affect infectious disease transmission, spread, and persistence. Understanding how the landscape influences wildlife dispersal and pathogen epidemiology can enhance the efficacy of disease management in natural populations. We applied landscape genetics to examine relationships among landscape variables, dispersal of white-tailed deer hosts and transmission/spread of chronic wasting disease (CWD), a fatal prion encephalopathy. Our focus was on quantifying movements and population structure of host deer in infected areas as a means of predicting the spread of this pathology and promoting its adaptive management. We analyzed microsatellite genotypes of CWD-infected and uninfected deer from two disease foci (Southern Wisconsin, Northern Illinois). We quantified gene flow and population structure using F _ST, assignment tests, and spatial autocorrelation analyses. Gene flow estimates were then contrasted against a suite of landscape variables that potentially mediate deer dispersal. Forest fragmentation and grassland connectivity promoted deer movements while rivers, agricultural fields and large urbanized areas impeded movement. Landscape variables, deer dispersal, and disease transmission covaried significantly and positively in our analyses. Habitats with elevated host gene flow supported the concept of dispersal-mediated CWD transmission by reflecting a concomitant, rapid CWD expansion. Large, interrelated social groups isolated by movement barriers overlapped disease foci, suggesting that philopatry exacerbated CWD transmission. Our results promote adaptive management of CWD by predicting patterns of its spread and identifying habitats at risk for invasion. Further, our landscape genetics approach underscores the significance of topography and host behavior in wildlife disease transmission.     

In Vitro Detection of prionemia in TSE-Infected Cervids and Hamsters

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.     

Biological effects and use of PrPSc- and PrP-specific antibodies generated by immunization with purified full-length native mouse prions

The prion agent is the infectious particle causing spongiform encephalopathies in animals and humans and is thought to consist of an altered conformation (PrP(Sc)) of the normal and ubiquitous prion protein PrP(C). The interaction of the prion agent with the immune system, particularly the humoral immune response, has remained unresolved. Here we investigated the immunogenicity of full-length native and infectious prions, as well as the specific biological effects of the resulting monoclonal antibodies (MAbs) on the binding and clearance of prions in cell culture and in in vivo therapy. Immunization of prion knockout (Prnp(0/0)) mice with phosphotungstic acid-purified mouse prions resulted in PrP-specific monoclonal antibodies with binding specificities selective for PrP(Sc) or for both PrP(C) and PrP(Sc). PrP(Sc)-specific MAb W261, of the IgG1 isotype, reacted with prions from mice, sheep with scrapie, deer with chronic wasting disease (CWD), and humans with sporadic and variant Creutzfeldt-Jakob disease (CJD) in assays including a capture enzyme-linked immunosorbent assay (ELISA) system. This PrP(Sc)-specific antibody was unable to clear prions from mouse neuroblastoma cells (ScN2a) permanently infected with scrapie, whereas the high-affinity MAb W226, recognizing both isoforms, PrP(Sc) and PrP(C), did clear prions from ScN2a cells, as determined by a bioassay. However, an attempt to treat intraperitoneally prion infected mice with full-length W226 or with a recombinant variable-chain fragment (scFv) from W226 could only slightly delay the incubation time. We conclude that (i) native, full-length PrP(Sc) elicits a prion-specific antibody response in PrP knockout mice, (ii) a PrP(Sc)-specific antibody had no prion-clearing effect, and (iii) even a high-affinity MAb that clears prions in vitro (W226) may not necessarily protect against prion infection, contrary to previous reports using different antibodies.     

Aerosol Transmission of Chronic Wasting Disease in White-Tailed Deer

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.     

Prion Remains Infectious after Passage through Digestive System of American Crows (Corvus brachyrhynchos)

Avian scavengers, such as American crows (Corvus brachyrhynchos), have potential to translocate infectious agents (prions) of transmissible spongiform encephalopathy (TSE) diseases including chronic wasting disease, scrapie, and bovine spongiform encephalopathy. We inoculated mice with fecal extracts obtained from 20 American crows that were force-fed material infected with RML-strain scrapie prions. These mice all evinced severe neurological dysfunction 196–231 d postinoculation ( = 198; 95% CI: 210–216) and tested positive for prion disease. Our results suggest a large proportion of crows that consume prion-positive tissue are capable of passing infectious prions in their feces ( = 1.0; 95% CI: 0.8–1.0). Therefore, this common, migratory North American scavenger could play a role in the geographic spread of TSE diseases.     

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.     

The effect of landscape heterogeneity and host movement on a tick-borne pathogen

Landscape heterogeneity can be instrumental in determining local disease risk, pathogen persistence and spread. This is because different landscape features such as habitat type determine the abundance and spatial distributions of hosts and pathogen vectors. Therefore, disease prevalence and distribution are intrinsically linked to the hosts and vectors that utilise the different habitats. Here, we develop a simplified reaction diffusion model of the louping-ill virus and red grouse (Lagopus lagopus scoticus) system to investigate the occurrence of a tick-borne pathogen and the effect of host movement and landscape structure. Ticks (Ixodes ricinus), the virus-vector, are dispersed by a virally incompetent tick host, red deer (Cervus elephus), between different habitats, whilst the virus infects only red grouse. We investigated how deer movement between different habitats (forest and moorland) affected tick distribution and hence prevalence of infected ticks and grouse and hence, the effect of habitat size ratio and fragmentation on infection. When habitat type has a role in the survival of the pathogen vector, we demonstrated that habitat fragmentation can have a considerable effect on infection. These results highlight the importance of landscape heterogeneity and the proximity and size of adjacent habitats when predicting disease risk in a particular location. In addition, this model could be useful for other pathogen systems with generalist vectors and may inform policy on possible disease management strategies that incorporate host movements.     

Mitigation of Prion Infectivity and Conversion Capacity by a Simulated Natural Process—Repeated Cycles of Drying and Wetting

Prions enter the environment from infected hosts, bind to a wide range of soil and soil minerals, and remain highly infectious. Environmental sources of prions almost certainly contribute to the transmission of chronic wasting disease in cervids and scrapie in sheep and goats. While much is known about the introduction of prions into the environment and their interaction with soil, relatively little is known about prion degradation and inactivation by natural environmental processes. In this study, we examined the effect of repeated cycles of drying and wetting on prion fitness and determined that 10 cycles of repeated drying and wetting could reduce PrP^Sc abundance, PMCA amplification efficiency and extend the incubation period of disease. Importantly, prions bound to soil were more susceptible to inactivation by repeated cycles of drying and wetting compared to unbound prions, a result which may be due to conformational changes in soil-bound PrP^Sc or consolidation of the bonding between PrP^Sc and soil. This novel finding demonstrates that naturally-occurring environmental process can degrade prions.     

Modeling routes of chronic wasting disease transmission: environmental prion persistence promotes deer population decline and extinction

Chronic wasting disease (CWD) is a fatal disease of deer, elk, and moose transmitted through direct, animal-to-animal contact, and indirectly, via environmental contamination. Considerable attention has been paid to modeling direct transmission, but despite the fact that CWD prions can remain infectious in the environment for years, relatively little information exists about the potential effects of indirect transmission on CWD dynamics. In the present study, we use simulation models to demonstrate how indirect transmission and the duration of environmental prion persistence may affect epidemics of CWD and populations of North American deer. Existing data from Colorado, Wyoming, and Wisconsin's CWD epidemics were used to define plausible short-term outcomes and associated parameter spaces. Resulting long-term outcomes range from relatively low disease prevalence and limited host-population decline to host-population collapse and extinction. Our models suggest that disease prevalence and the severity of population decline is driven by the duration that prions remain infectious in the environment. Despite relatively low epidemic growth rates, the basic reproductive number, R(0), may be much larger than expected under the direct-transmission paradigm because the infectious period can vastly exceed the host's life span. High prion persistence is expected to lead to an increasing environmental pool of prions during the early phases (i.e. approximately during the first 50 years) of the epidemic. As a consequence, over this period of time, disease dynamics will become more heavily influenced by indirect transmission, which may explain some of the observed regional differences in age and sex-specific disease patterns. This suggests management interventions, such as culling or vaccination, will become increasingly less effective as CWD epidemics progress.     

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.     

Estimating Prion Adsorption Capacity of Soil by BioAssay of Subtracted Infectivity from Complex Solutions (BASICS)

Prions, the infectious agent of scrapie, chronic wasting disease and other transmissible spongiform encephalopathies, are misfolded proteins that are highly stable and resistant to degradation. Prions are known to associate with clay and other soil components, enhancing their persistence and surprisingly, transmissibility. Currently, few detection and quantification methods exist for prions in soil, hindering an understanding of prion persistence and infectivity in the environment. Variability in apparent infectious titers of prions when bound to soil has complicated attempts to quantify the binding capacity of soil for prion infectivity. Here, we quantify the prion adsorption capacity of whole, sandy loam soil (SLS) typically found in CWD endemic areas in Colorado; and purified montmorillonite clay (Mte), previously shown to bind prions, by BioAssay of Subtracted Infectivity in Complex Solutions (BASICS). We incubated prion positive 10% brain homogenate from terminally sick mice infected with the Rocky Mountain Lab strain of mouse-adapted prions (RML) with 10% SLS or Mte. After 24 hours samples were centrifuged five minutes at 200×g and soil-free supernatant was intracerebrally inoculated into prion susceptible indicator mice. We used the number of days post inoculation to clinical disease to calculate the infectious titer remaining in the supernatant, which we subtracted from the starting titer to determine the infectious prion binding capacity of SLS and Mte. BASICS indicated SLS bound and removed ≥ 95% of infectivity. Mte bound and removed lethal doses (99.98%) of prions from inocula, effectively preventing disease in the mice. Our data reveal significant prion-binding capacity of soil and the utility of BASICS to estimate prion loads and investigate persistence and decomposition in the environment. Additionally, since Mte successfully rescued the mice from prion disease, Mte might be used for remediation and decontamination protocols.     

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.     

Induction of PrPSc-specific systemic and mucosal immune responses in white-tailed deer with an oral vaccine for chronic wasting disease

The ongoing epidemic of chronic wasting disease (CWD) within cervid populations indicates the need for novel approaches for disease management. A vaccine that either reduces susceptibility to infection or reduces shedding of prions by infected animals, or a combination of both, could be of benefit for disease control. The development of such a vaccine is challenged by the unique nature of prion diseases and the requirement for formulation and delivery in an oral format for application in wildlife settings. To address the unique nature of prions, our group targets epitopes, termed disease specific epitopes (DSEs), whose exposure for antibody binding depends on disease-associated misfolding of PrP^C into PrP^Sc. Here, a DSE corresponding to the rigid loop (RL) region, which was immunogenic following parenteral vaccination, was translated into an oral vaccine. This vaccine consists of a replication-incompetent human adenovirus expressing a truncated rabies glycoprotein G recombinant fusion with the RL epitope (hAd5:tgG-RL). Oral immunization of white-tailed deer with hAd5:tgG-RL induced PrP^Sc-specific systemic and mucosal antibody responses with an encouraging safety profile in terms of no adverse health effects nor prolonged vector shedding. By building upon proven strategies of formulation for wildlife vaccines, these efforts generate a particular PrP^Sc-specific oral vaccine for CWD as well as providing a versatile platform, in terms of carrier protein and biological vector, for generation of other oral, peptide-based CWD vaccines.     

Prion-Seeding Activity in Cerebrospinal Fluid of Deer with Chronic Wasting Disease

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.