Chronic Wasting Disease Infection Research Articles

Assessing freedom from chronic wasting disease in semi-domesticated reindeer in Norway and Sweden

Establishing freedom from disease is a key component of surveillance and may have direct consequences for trade and economy. Transboundary populations pose challenges in terms of variable legislation, efforts, and data availability between countries, often limiting surveillance efficiency. Chronic wasting disease (CWD) is a contagious prion disease of cervids. The long incubation period and slow initial epidemic growth make it notoriously difficult to detect CWD in the early phase of an epidemic. The recent emergence of CWD in wild reindeer in Norway poses a threat to approximately 250,000 semi-domesticated reindeer in Norway and 250,000 in Sweden, including transboundary populations. Here, we provide a first analysis of surveillance data (2016–2022) from all reindeer districts in Norway and Sweden to determine the probability of freedom from CWD infection. During the six years, 6017 semi-domesticated reindeer were tested in Sweden and 51,974 in Norway. Most samples came from healthy slaughtered animals (low risk). Reindeer use large and remote areas and (high risk) samples from fallen stock and animals with clinical signs were difficult to obtain. A scenario tree model was run for seven different set of values for the input parameters (design prevalence within and between districts, probability of introduction, and relative risks) to determine the effect on surveillance sensitivity. At the national level, the mean probability of disease freedom was 59.0 % in Sweden and 87.0 % in Norway by 2021. The most marked effect on sensitivity was varying the design prevalence both within and between districts. Uncertainty about relative risk ratios affected sensitivity for Sweden more than for Norway, due to the higher proportion of animals in the high-risk group in the former (13.8 % vs. 2.1 %, respectively). A probability of disease freedom of 90 % or higher was reached in 8.2 % of the 49 districts in Sweden and 43.5 % of the 46 districts in Norway for a design prevalence of 0.5 %. The probability of freedom remained below 60 % in 29 districts (59.2 %) in Sweden and 10 districts (21.7 %) in Norway. At the national level, only Norway had a sufficiently large number of samples to reach a probability of more than 95 % of disease freedom within a period of 10 years. Our cross-border assessment forms an important knowledge base for designing future surveillance efforts depending on the spatial pattern of prevalence of CWD and risk of spread.

Prospective fecal microbiomic biomarkers for chronic wasting disease.

Chronic wasting disease (CWD) is a naturally occurring prion disease in cervids that has been rapidly proliferating in the United States. Here, we investigated a potential link between CWD infection and gut microbiome by analyzing 50 fecal samples obtained from CWD-positive animals of different sexes from various regions in the USA compared to 50 CWD-negative controls using high throughput sequencing of the 16S ribosomal RNA and targeted metabolomics. Our analysis reveals promising trends in the gut microbiota that could potentially be CWD-dependent, including several bacterial taxa at each rank level, as well as taxa pairs, that can differentiate between CWD-negative and CWD-positive deer. Through machine-learning, these taxa and taxa pairs at each rank level could facilitate identification of around 70% of both the CWD-negative and the CWD-positive samples. Our results provide a potential tool for diagnostics and surveillance of CWD in the wild, as well as conceptual advances in our understanding of the disease.IMPORTANCEThis is a comprehensive study that tests the connection between the composition of the gut microbiome in deer in response to chronic wasting disease (CWD). We analyzed 50 fecal samples obtained from CWD-positive animals compared to 50 CWD-negative controls to identify CWD-dependent changes in the gut microbiome, matched with the analysis of fecal metabolites. Our results show promising trends suggesting that fecal microbial composition can directly correspond to CWD disease status. These results point to the microbial composition of the feces as a potential tool for diagnostics and surveillance of CWD in the wild, including non-invasive CWD detection in asymptomatic deer and deer habitats, and enable conceptual advances in our understanding of the disease.

Herd-level risk factors associated with chronic wasting disease-positive herd status in Minnesota, Pennsylvania, and Wisconsin cervid herds.

The objective of this study was to evaluate risks related to introduction of Chronic Wasting Disease (CWD) to farmed cervid herds in Minnesota (MN), Pennsylvania (PA), and Wisconsin (WI). This was the first study to evaluate risk factors related to multiple pathways of CWD transmission to farmed cervid herds. Participating herds in this case-control study included 22 case and 49 control herds identified through participation in the respective mandatory State CWD herd program. Data was voluntarily collected from white-tailed deer (WTD) study herds using a questionnaire and state animal health agency databases. Univariable analyses identified associations between CWD-positive herds and variables representing different transmission pathways, including direct contact with infected farmed cervids (imported from a herd that later tested positive for CWD) odds ratio (OR):7.16, 95 % confidence intervals (CI):1.64-31.21 and indirect contact with infected wild cervids (access of domestic cats to pens or feed storage area) OR:4.07, 95 % CI:1.35-12.26, observed evidence of mammalian scavengers inside or outside of fenceline in the previous 12 months OR:6.55, 95 % CI:1.37-31.32, ≤5km distance to nearest detected CWD-positive wild cervid OR:3.08, 95 % CI:1.01-9.39, forested area crosses the perimeter fenceline OR:3.54, 95 % CI:1.13-11.11, ≤0.3m distance of water source to fenceline OR:4.71, 95 % CI:1.60-13.83, and water source shared with wild cervids (running or standing water) OR:4.17, 95 % CI:1.34-12.92. Three variables from univariable analyses that represented different biological transmission pathways were placed in a Firth's penalized maximum likelihood multivariable logistic regression to evaluate associations between transmission pathway and CWD herd infection status. For the issue of low sample size and overfitting, 95 % CIs for estimated coefficients for the three variables were computed via bootstrapping of 10,000 independent bootstrap samples. The three biological variables were significantly associated with herd CWD infection status: imported cervids from a herd that later tested positive for CWD (OR:5.63; 95 % CI:1.1-28.2), ≤0.3m distance of cervid water source to perimeter fenceline (OR:4.83; 95 % CI:1.5-16.1), and ≤5km distance to nearest detected CWD-positive wild cervid (OR:4.10; 95 % CI:1.1-15.2). The risk factors associated with CWD herd status identified in this study indicated the importance of transmission through direct contact pathways with infected cervid herds (introduction of cervids from herds later identified as CWD-infected) and indirect contact pathways with infected wild cervids that could be related to other animals through the perimeter fence. Further studies are needed to confirm and clarify understanding of indirect pathways to allow development of improved biosecurity practices to prevent CWD introduction to cervid herds.

Movement of Chronic Wasting Disease Prions in Prairie, Boreal and Alpine Soils.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy negatively impacting cervids on three continents. Soil can serve as a reservoir for horizontal transmission of CWD by interaction with the infectious prion protein (PrPCWD) shed by diseased individuals and from infected carcasses. We investigated the pathways for PrPCWD migration in soil profiles using lab-scale soil columns, comparing PrPCWD migration through pure soil minerals (quartz, illite and montmorillonite), and diverse soils from boreal (Luvisol, Brunisol) and prairie (Chernozem) regions. We analyzed the leachate of the soil columns by immunoblot and protein misfolding cyclic amplification (PMCA) and detected PrP in the leachates of columns composed of quartz, illite, Luvisol and Brunisol. Animal bioassay confirmed the presence of CWD infectivity in the leachates from quartz, illite and Luvisol columns. Leachates from columns with montmorillonite and prairie Chernozems did not contain PrP detectable by immunoblotting or PMCA; bioassay confirmed that the Chernozemic leachate was not infectious. Analysis of the solid phase of the columns confirmed the migration of PrP to lower layers in the illite column, while the strongest signal in the montmorillonite column remained close to the surface. Montmorillonite, the prevalent clay mineral in prairie soils, has the strongest prion binding ability; by contrast, illite, the main clay mineral in northern boreal and tundra soils, does not bind prions significantly. This suggests that in soils of North American CWD-endemic regions (Chernozems), PrPCWD would remain on the soil surface due to avid binding to montmorillonite. In boreal Luvisols and mountain Brunisols, prions that pass through the leaf litter will continue to move through the soil mineral horizon, becoming less bioavailable. In light-textured soils where quartz is a dominant mineral, the majority of the infectious prions will move through the soil profile. Local soil properties may consequently determine the efficiency of environmental transmission of CWD.

Second passage experiments of chronic wasting disease in transgenic mice overexpressing human prion protein

Chronic wasting disease (CWD) is a prion disease of cervids including deer, elk, reindeer, and moose. Human consumption of cervids is common, therefore assessing the risk potential of CWD transmission to humans is critical. In a previous study, we tested CWD transmission via intracerebral inoculation into transgenic mice (tg66 and tgRM) that over-expressed human prion protein. Mice screened by traditional prion detection assays were negative. However, in a group of 88 mice screened by the ultrasensitive RT-QuIC assay, we identified 4 tg66 mice that produced inconsistent positive RT-QuIC reactions. These data could be false positive reactions, residual input inoculum or indicative of subclinical infections suggestive of cross species transmission of CWD to humans. Additional experiments were required to understand the nature of the prion seeding activity in this model. In this manuscript, second passage experiments using brains from mice with weak prion seeding activity showed they were not infectious to additional recipient tg66 mice. Clearance experiments showed that input CWD prion seeding activity was eliminated by 180 days in tg66 mice and PrPKO mice, which are unable to replicate prion protein, indicating that the weak positive levels of seeding activity detected at later time points was not likely residual inoculum. The failure of CWD prions to cause disease in tg66 after two sequential passages suggested that a strong species barrier prevented CWD infection of mice expressing human prion protein.

Susceptibility of Beavers to Chronic Wasting Disease.

Simple SummaryChronic wasting disease is increasing across the landscape, and this is threatening other wildlife species in addition to cervids. Our objective was to evaluate the possibility that chronic wasting disease could transmit to beavers. Our results indicate that beavers are susceptible to multiple types of prion diseases, including chronic wasting disease.Chronic wasting disease (CWD) is a contagious, fatal, neurodegenerative prion disease of cervids. The expanding geographical range and rising prevalence of CWD are increasing the risk of pathogen transfer and spillover of CWD to non-cervid sympatric species. As beavers have close contact with environmental and food sources of CWD infectivity, we hypothesized that they may be susceptible to CWD prions. We evaluated the susceptibility of beavers to prion diseases by challenging transgenic mice expressing beaver prion protein (tgBeaver) with five strains of CWD, four isolates of rodent-adapted prions and one strain of Creutzfeldt–Jakob disease. All CWD strains transmitted to the tgBeaver mice, with attack rates highest from moose CWD and the 116AG and H95+ strains of deer CWD. Mouse-, rat-, and especially hamster-adapted prions were also transmitted with complete attack rates and short incubation periods. We conclude that the beaver prion protein is an excellent substrate for sustaining prion replication and that beavers are at risk for CWD pathogen transfer and spillover.

Large animal models for chronic wasting disease.

Chronic wasting disease (CWD) is a fatal neurodegenerative prion disease of cervid species including deer, elk, moose and reindeer. The disease has shown both geographic and species expansion since its discovery in the late 1960’s and is now recognized in captive and free-ranging cervid populations in North America, Asia and Europe. The facile transmission of CWD is unique among prion diseases and has resulted in growing concern for cervid populations and human public health. The development of native cervid host models with longitudinal monitoring has revealed new insights about CWD pathogenesis and transmission dynamics. More than 20 years of experimental studies conducted in these models, using biologically relevant routes of infection, have led to better understanding of many aspect of CWD infections. This review addresses some of these insights, including: (i) the temporal intra-host trafficking of CWD prions in tissues and bodily fluids, (ii) the presence of infectivity shed in bodily excretions that may help explain the facile transmission of CWD, (iii) mother-to-offspring CWD transmission, (iv) the influence of some Prnp polymorphisms on CWD susceptibility, and (vi) continued development of vaccine strategies to mitigate CWD.

Examination of the interaction between age-specific predation and chronic disease in the Greater Yellowstone Ecosystem.

Predators may create healthier prey populations by selectively removing diseased individuals. Predators typically prefer some ages of prey over others, which may, or may not, align with those prey ages that are most likely to be diseased. The interaction of age-specific infection and predation has not been previously explored and likely has sizable effects on disease dynamics. We hypothesize that predator cleansing effects will be greater when the disease and predation occur in the same prey age groups. We examine the predator cleansing effect using a model where both vulnerability to predators and pathogen prevalence vary with age. We tailor this model to chronic wasting disease (CWD) in mule deer and elk populations in the Greater Yellowstone Ecosystem, with empirical data from Yellowstone grey wolves and cougars. Model results suggest that under moderate, yet realistic, predation pressure from cougars and wolves independently, predators may decrease CWD outbreak size substantially and delay the accumulation of symptomatic deer and elk. The magnitude of this effect is driven by the ability of predators to selectively remove late-stage CWD infections that are likely the most responsible for transmission, but this may not be the age class they typically select. Thus, predators that select for infected young adults over uninfected juveniles have a stronger cleansing effect, and these effects are strengthened when transmission rates increase with increasing prey morbidity. There are also trade-offs from a management perspective-that is, increasing predator kill rates can result in opposing forces on prey abundance and CWD prevalence. Our modelling exploration shows that predators have the potential to reduce prevalence in prey populations when prey age and disease severity are considered, yet the strength of this effect is influenced by predators' selection for demography or body condition. Current CWD management focuses on increasing cervid hunting as the primary management tool, and our results suggest predators may also be a useful tool under certain conditions, but not necessarily without additional impacts on host abundance and demography. Protected areas with predator populations will play a large role in informing the debate over predator impacts on disease.

Pathogen‐mediated selection and management implications for white‐tailed deer exposed to chronic wasting disease

Abstract Pathogens can cause host extinction, affect population dynamics and influence natural selection. Host susceptibility to pathogens can vary by species, demographics and genetics which affect epizootic and population dynamics, ultimately determining population trends and evolution. Chronic wasting disease (CWD), a fatal neuro‐degenerative prion disease of cervids, has varying host susceptibility conferred by polymorphisms of the prion protein gene (PRNP) at codon 96 for white‐tailed deer Odocoileus virginianus. Deer with the homozygous Glycine allele (96GG) are most susceptible and a single Serine allele (96GS/96SS) reduces the risk of infection and mortality. We developed epizootiological models that demonstrate CWD infection and disease‐associated mortality were higher for the more susceptible (96GG) genotype; and, infection was higher for males than females. We used population models to evaluate future shifts in genotype frequencies under alternative harvest and infection rate scenarios. Genetic shifts towards less susceptible genotypes were predicted as CWD prevalence increased during the course of an outbreak. This further increased CWD prevalence, and likely environmental contamination from prion shedding, due to longer incubation periods. Alternative harvest management strategies directly influenced CWD prevalence and spread, the rate of genetic selection and deer population growth. Synthesis and applications. We show that chronic wasting disease (CWD) transmission varied by sex, age class and PRNP genotype, and that CWD disease‐associated mortality varied by genotype. Together, these forces lead to CWD‐mediated genetic selection for a white‐tailed deer population. We predict that genetic selection pressure increases when hunter harvest pressure is lowered, and conversely, increasing hunter harvest can reduce genetic selection rates of antlered deer. Our results support the control of CWD prevalence by aggressive harvest of adult males, because they have the highest infection rates. Our results have strong implications for evolution, disease ecology, geographical spread, disease mitigation and cervid population management.

Neural transcriptomic signature of chronic wasting disease in white-tailed deer

BackgroundThe increasing prevalence and expanding geographical range of the chronic wasting disease (CWD) panzootic in cervids is threatening human, animal, environmental and economic health. The pathogenesis of CWD in cervids is, however, not well understood. We used RNA sequencing (RNA-seq) to compare the brain transcriptome from white-tailed deer (WTD; Odocoileus virginianus) clinically affected with CWD (n = 3) to WTD that tested negative (n = 8) for CWD. In addition, one preclinical CWD+ brain sample was analyzed by RNA-seq.ResultsWe found 255 genes that were significantly deregulated by CWD, 197 of which were upregulated. There was a high degree of overlap in differentially expressed genes (DEGs) identified when using either/both the reference genome assembly of WTD for mapping sequenced reads to or the better characterized genome assembly of a closely related model species, Bos taurus. Quantitative PCR of a subset of the DEGs confirmed the RNA-seq data. Gene ontology term enrichment analysis found a majority of genes involved in immune activation, consistent with the neuroinflammatory pathogenesis of prion diseases. A metagenomic analysis of the RNA-seq data was conducted to look for the presence of spiroplasma and other bacteria in CWD infected deer brain tissue.ConclusionsThe gene expression changes identified highlight the role of innate immunity in prion infection, potential disease associated biomarkers and potential targets for therapeutic agents. An association between CWD and spiroplasma infection was not found.

Evaluation of Real-Time Quaking-Induced Conversion, ELISA, and Immunohistochemistry for Chronic Wasting Disease Diagnosis.

Chronic wasting disease (CWD) is a transmissible prion disorder, primarily affecting free-ranging and captive cervids in North America (United States and Canada), South Korea, and Europe (Finland, Norway, and Sweden). Current diagnostic methods used in the United States for detection of CWD in hunter harvested deer involve demonstration of the causal misfolded prion protein (PrPCWD) in the obex or retropharyngeal lymph nodes (RLNs) using an antigen detection ELISA as a screening tool, followed by a confirmation by the gold standard method, immunohistochemistry (IHC). Real-time quaking-induced conversion (RT-QuIC) assay is a newer approach that amplifies misfolded CWD prions in vitro and has facilitated CWD prion detection in a variety of tissues, body fluids, and excreta. The current study was undertaken to compare ELISA, IHC, and RT-QuIC on RLNs (n = 1,300 animals) from white-tailed deer (WTD) in Michigan. In addition, prescapular, prefemoral and popliteal lymph nodes collected from a small subset (n = 7) of animals were tested. Lastly, the location of the positive samples within Michigan was documented and the percentage of CWD positive RLNs was calculated by sex and age. ELISA and RT-QuIC detected PrPCWD in 184 and 178 out of 1,300 RLNs, respectively. Of the 184 ELISA positive samples, 176 were also IHC positive for CWD. There were seven discordant results when comparing IHC and ELISA. RT-QuIC revealed that six of the seven samples matched the IHC outcomes. One RLN was negative by IHC, but positive by ELISA and RT-QuIC. RT-QuIC, IHC, and ELISA also detected PrPCWD in prescapular, prefemoral and popliteal lymph nodes. CWD infection heterogeneities were observed in different age and sex groups, with young males having higher CWD prevalence. All, except one, CWD positive RLNs analyzed were from ten Counties geographically located in the West Michigan region of the Lower Peninsula. Taken together, we show evidence that the RT-QuIC assay is comparable to ELISA and IHC and could be helpful for routine CWD detection in surveillance programs. RT-QuIC also demonstrated that CWD prions are distributed across lymph nodes in a variety of anatomic locations. A multi-laboratory validation on blinded sample panels is underway and is likely to help to provide insight into the variability (lab-to-lab), analytical sensitivity, and specificity of gold standard diagnostics vs. RT-QuIC assay.

The impact of maternal infection with chronic wasting disease on fetal characteristics in wild white-tailed deer (Odocoileus virginianus) in Illinois, USA

Chronic wasting disease (CWD) is a fatal neurodegenerative disease of cervids caused by a misfolded and infectious protein, called a prion. Infection with CWD is characterized by an asymptomatic period, followed by a decline that inevitably leads to death. The disease is transmitted mainly through direct contact between infected and uninfected animals, but vertical transmission has been documented in muntjac deer ( Muntiacus reevesi ), along with the presence of infectious prions in the reproductive tissues of elk ( Cervus canadensis ) and white-tailed deer ( Odocoileus virginianus ). Therefore, questions have been raised about the effects of CWD on the health and characteristics of the offspring gestated by infected animals, and about how vertical transmission may complicate disease management efforts and drive reduction of herd fitness. To begin finding quantitative answers to these questions, this study used multilevel Bayesian linear regression to model the influence of maternal infection with CWD, along with several other environmental and biological variables, on fetal weight, sex, and length using data collected on white-tailed deer as part of the CWD management program in Illinois for the time period between fiscal years 2003 and 2020. The regression analyses found no statistically significant relationship between maternal CWD status and fetal length or the fraction of males in a litter but did find a significant relationship between maternal infection and fetal weight. Fetuses of CWD positive deer weighed 1% less than fetuses from deer in which CWD was not detected. Given that 88% of the fetuses in this data were in the second trimester of gestation, the impact of CWD on fetuses through the entire course of gestation remains to be evaluated. • Fetal weights in the second trimester were 1% lower in CWD positive female deer • Fetal length showed no significant relationship with maternal CWD infection • Fetal sex ratios were 1:1 regardless of maternal CWD infection status

Inferring Chronic Wasting Disease Incidence from Prevalence Data.

Incidence of chronic wasting disease infection showed strong, positive correlation (r≥0.944) with apparent prevalence among female and male mule deer (Odocoileus hemionus) in seven herds previously studied in Colorado and Wyoming, US. With attention to monitoring method consistency and context, inferring that observed prevalence trends reflect underlying epidemic dynamics in mule deer herds appears justifiable.

Detection of chronic wasting disease in mule and white-tailed deer by RT-QuIC analysis of outer ear

Efforts to contain the spread of chronic wasting disease (CWD), a fatal, contagious prion disease of cervids, would be aided by the availability of additional diagnostic tools. RT-QuIC assays allow ultrasensitive detection of prion seeds in a wide variety of cervid tissues, fluids and excreta. The best documented antemortem diagnostic test involving RT-QuIC analysis targets lymphoid tissue in rectal biopsies. Here we have tested a more easily accessed specimen, ear pinna punches, using an improved RT-QuIC assay involving iron oxide magnetic extraction to detect CWD infections in asymptomatic mule and white-tailed deer. Comparison of multiple parts of the ear pinna indicated that a central punch spanning the auricular nerve provided the most consistent detection of CWD infection. When compared to results obtained from gold-standard retropharyngeal lymph node specimens, our RT-QuIC analyses of ear samples provided apparent diagnostic sensitivity (81%) and specificity (91%) that rivaled, or improved upon, those observed in previous analyses of rectal biopsies using RT-QuIC. These results provide evidence that RT-QuIC analysis of ear pinna punches may be a useful approach to detecting CWD infections in cervids.

An Ex Vivo Brain Slice Culture Model of Chronic Wasting Disease: Implications for Disease Pathogenesis and Therapeutic Development

Chronic wasting disease (CWD) is a rapidly spreading prion disease of cervids, yet antemortem diagnosis, treatment, and control remain elusive. We recently developed an organotypic slice culture assay for sensitive detection of scrapie prions using ultrasensitive prion seeding. However, this model was not established for CWD prions due to their strong transmission barrier from deer (Odocoileus spp) to standard laboratory mice (Mus musculus). Therefore, we developed and characterized the ex vivo brain slice culture model for CWD, using a transgenic mouse model (Tg12) that expresses the elk (Cervus canadensis) prion protein gene (PRNP). We tested for CWD infectivity in cultured slices using sensitive seeding assays such as real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA). Slice cultures from Tg12, but not from prnp−/− mice, tested positive for CWD. Slice-generated CWD prions transmitted efficiently to Tg12 mice. Furthermore, we determined the activity of anti-prion compounds and optimized a screening protocol for the infectivity of biological samples in this CWD slice culture model. Our results demonstrate that this integrated brain slice model of CWD enables the study of pathogenic mechanisms with translational implications for controlling CWD.

Long-Term Incubation PrPCWD with Soils Affects Prion Recovery but Not Infectivity.

Chronic wasting disease (CWD) is a contagious prion disease of cervids. The infectious agent is shed from animals at the preclinical and clinical stages of disease where it persists in the environment as a reservoir of CWD infectivity. In this study, we demonstrate that long-term incubation of CWD prions (generated from tg-mice infected with deer or elk prions) with illite, montmorillonite (Mte) and whole soils results in decreased recovery of PrPCWD, suggesting that binding becomes more avid and irreversible with time. This continual decline of immunoblot PrPCWD detection did not correlate with prion infectivity levels. Bioassay showed no significant differences in incubation periods between mice inoculated with 1% CWD brain homogenate (BH) and with the CWD-BH pre-incubated with quartz or Luvisolic Ae horizon for 1 or 30 weeks. After 55 weeks incubation with Chernozem and Luvisol, bound PrPCWD was not detectable by immunoblotting but remained infectious. This study shows that although recovery of PrPCWD bound to soil minerals and whole soils with time become more difficult, prion infectivity is not significantly altered. Detection of prions in soil is, therefore, not only affected by soil type but also by length of time of the prion–soil interaction.

Progression of chronic wasting disease in white-tailed deer analyzed by serial biopsy RT-QuIC and immunohistochemistry.

Chronic wasting disease (CWD) continues to spread or be recognized in the United States, Canada, and Europe. CWD is diagnosed by demonstration of the causative misfolded prion protein (PrPCWD) in either brain or lymphoid tissue using immunodetection methods, with immunohistochemistry (IHC) recognized as the gold standard. In recent years, in vitro amplification assays have been developed that can detect CWD prion seeding activity in tissues, excreta, and body fluids of affected cervids. These methods potentially offer earlier and more facile detection of CWD, both pre- and post-mortem. Here we provide a longitudinal profile of CWD infection progression, as assessed by both real-time quaking-induced conversion (RT-QuIC) and IHC on serial biopsies of mucosal lymphoid tissues of white-tailed deer orally exposed to low doses of CWD prions. We report that detection of CWD infection by RT-QuIC preceded that by IHC in both tonsil and recto-anal lymphoid tissue (RAMALT) in 14 of 19 deer (74%). Of the 322 biopsy samples collected in post-exposure longitudinal monitoring, positive RT-QuIC results were obtained for 146 samples, 91 of which (62%) were concurrently also IHC-positive. The lower frequency of IHC positivity was manifest most in the earlier post-exposure periods and in biopsies in which lymphoid follicles were not detected. For all deer in which RT-QuIC seeding activity was detected in a tonsil or RAMALT biopsy, PrPCWD was subsequently or concurrently detected by IHC. Overall, this study (a) provides a longitudinal profile of CWD infection in deer after low yet infectious oral prion exposure; (b) illustrates the value of RT-QuIC for sensitive detection of CWD; and (c) demonstrates an ultimate high degree of correlation between RT-QuIC and IHC positivity as CWD infection progresses.

Association of chronic wasting disease susceptibility with prion protein variation in white-tailed deer (Odocoileus virginianus)

ABSTRACT Chronic wasting disease (CWD) is caused by prions, infectious proteinaceous particles, PrPCWD. We sequenced the PRNP gene of 2,899 white-tailed deer (WTD) from Illinois and southern Wisconsin, finding 38 haplotypes. Haplotypes A, B, D, E, G and 10 others encoded Q95G96S100N103A123Q226, designated ‘PrP variant A.’ Haplotype C and five other haplotypes encoded PrP ‘variant C’ (Q95S96S100N103A123Q226). Haplotype F and three other haplotypes encoded PrP ‘variant F’ (H95G96S100N103A123Q226). The association of CWD with encoded PrP variants was examined in 2,537 tested WTD from counties with CWD. Relative to PrP variant A, CWD susceptibility was lower in deer with PrP variant C (OR = 0.26, p < 0.001), and even lower in deer with PrP variant F (OR = 0.10, p < 0.0001). Susceptibility to CWD was highest in deer with both chromosomes encoding PrP variant A, lower with one copy encoding PrP variant A (OR = 0.25, p < 0.0001) and lowest in deer without PrP variant A (OR = 0.07, p < 0.0001). There appeared to be incomplete dominance for haplotypes encoding PrP variant C in reducing CWD susceptibility. Deer with both chromosomes encoding PrP variant F (FF) or one encoding PrP variant C and the other F (CF) were all CWD negative. Our results suggest that an increased population frequency of PrP variants C or F and a reduced frequency of PrP variant A may reduce the risk of CWD infection. Understanding the population and geographic distribution of PRNP polymorphisms may be a useful tool in CWD management.

Can genetic assignment tests provide insight on the influence of captive egression on the epizootiology of chronic wasting disease?

Identifying the sources of ongoing and novel disease outbreaks is critical for understanding the diffusion of epizootic diseases. Identifying infection sources is difficult when few physical differences separate individuals with different origins. Genetic assignment procedures show great promise for assessing transmission dynamics in such situations. Here, we use genetic assignment tests to determine the source of chronic wasting disease infections in free‐ranging white‐tailed deer (Odocoileus virginianus) populations. Natural dispersal is thought to facilitate the geographic diffusion of chronic wasting disease, but egression from captive cervid populations represents an alternative source of infection that is difficult to detect due to physical similarities with wild deer. Simulated reference populations were created based on allele frequencies from 1,912 empirical microsatellite genotypes collected in four sampling subregions and five captive facilities. These reference populations were used to assess the likelihood of ancestry and assignment of 1,861 free‐ranging deer (1,834 noninfected and 27 infected) and 51 captive individuals to captive or wild populations. The ancestry (Q) and assignment scores (A) for free‐ranging deer to wild populations were high (average Q wild = 0.913 and average A wild = 0.951, respectively), but varied among subregions (Q wild = 0.800–0.947, A wild = 0.857–0.976). These findings suggest that captive egression and admixture are rare, but risk may not be spatially uniform. Ancestry and assignment scores for two free‐ranging deer with chronic wasting disease sampled in an area where chronic wasting disease was previously unobserved in free‐ranging herds indicated a higher likelihood of assignment and proportion of ancestry attributable to captive populations. While we cannot directly assign these individuals to infected facilities, these findings suggest that rare egression events may influence the epizootiology of chronic wasting disease in free‐ranging populations. Continued disease surveillance and genetic analyses may further elucidate the relative disease risk attributable to captive and wild sources.

Inactivation of chronic wasting disease prions using sodium hypochlorite.

Chronic wasting disease (CWD) is a fatal prion disease that can infect deer, elk and moose. CWD has now been detected in 26 states of the USA, 3 Canadian provinces, South Korea, Norway, Sweden and Finland. CWD continues to spread from endemic areas, and new foci of infections are frequently detected. As increasing numbers of cervids become infected, the likelihood for human exposure increases. To date, no cases of CWD infection in humans have been confirmed, but experience with the BSE zoonosis in the United Kingdom suggests exposure to CWD should be minimized. Specifically, hunters, meat processors and others in contact with tissues from potentially CWD-infected cervids need a practical method to decontaminate knives, saws and other equipment. Prions are notoriously difficult to inactivate, and most effective methods require chemicals or sterilization processes that are either dangerous, caustic, expensive or not readily available. Although corrosive, sodium hypochlorite (bleach) is widely available and affordable and has been shown to inactivate prion agents including those that cause scrapie, bovine spongiform encephalopathy and Creutzfeldt-Jakob disease. In the current study, we confirm that bleach is an effective disinfectant for CWD prions and establish minimum times and bleach concentrations to eliminate prion seeding activity from stainless steel and infected brain homogenate solutions. We found that a five-minute treatment with a 40% dilution of household bleach was effective at inactivating CWD seeding activity from stainless-steel wires and CWD-infected brain homogenates. However, bleach was not able to inactivate CWD seeding activity from solid tissues in our studies.