Validation of a real-time quaking-induced conversion (RT-QuIC) assay protocol to detect chronic wasting disease using rectal mucosa of naturally infected, pre-clinical white-tailed deer (Odocoileus virginianus).

Chronic wasting disease (CWD) is a fatal prion disease of cervids spreading across North America. More effective mitigation efforts may require expansion of the available toolkit to include new methods that provide earlier antemortem detection, higher throughput, and less expense than current immunohistochemistry (IHC) methods. The rectal mucosa near the rectoanal junction is a site of early accumulation of CWD prions and is safely sampled in living animals by pinch biopsy. A fluorescence-based, 96-well format, protein-aggregation assay-the real-time quaking-induced conversion (RT-QuIC) assay-is capable of ultra-sensitive detection of CWD prions. Notably, the recombinant protein substrate is crucial to the assay's performance and is now commercially available. In this blinded independent study, the preclinical diagnostic performance of a standardized RT-QuIC protocol using a commercially sourced substrate (MNPROtein) and a laboratory-produced substrate was studied using mock biopsy samples of the rectal mucosa from 284 white-tailed deer (Odocoileus virginianus). The samples were from a frozen archive of intact rectoanal junctions collected at depopulations of farmed herds positive for CWD in the United States. All deer were pre-clinical at the time of depopulation and infection status was established from the regulatory record, which evaluated the medial retropharyngeal lymph nodes (MRPLNs) and obex by CWD-IHC. A pre-analytic sample precipitation step was found to enhance the protocol's detection limit. Performance metrics were influenced by the choice of RT-QuIC diagnostic cut points (minimum number of positive wells and assay time) and by deer attributes (preclinical infection stage and prion protein genotype). The peak overall diagnostic sensitivities of the protocol were similar for both substrates (MNPROtein, 76.8%; laboratory-produced, 73.2%), though each was achieved at different cut points. Preclinical infection stage and prion protein genotype at codon 96 (G = glycine, S = serine) were primary predictors of sensitivity. The diagnostic sensitivities in late preclinical infections (CWD-IHC positive MPRLNs and obex) were similar, ranging from 96% in GG96 deer to 80% in xS96 deer (x = G or S). In early preclinical infections (CWD-IHC positive MRPLNs only), the diagnostic sensitivity was 64-71% in GG96 deer but only 25% in xS96 deer. These results demonstrate that this standardized RT-QuIC protocol for rectal biopsy samples using a commercial source of substrate produced stratified diagnostic sensitivities similar to or greater than those reported for CWD-IHC but in less than 30 hours of assay time and in a 96-well format. Notably, the RT-QuIC protocol used herein represents a standardization of protocols from several previous studies. Alignment of the sensitivities across these studies suggests the diagnostic performance of the assay is robust given quality reagents, optimized diagnostic criteria, and experienced staff.

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.

Nasal bots carry relevant titers of CWD prions in naturally infected white-tailed deer.

Chronic wasting disease (CWD) is a prion disease affecting farmed and free-ranging cervids. CWD is rapidly expanding across North America and its mechanisms of transmission are not completely understood. Considering that cervids are commonly afflicted by nasal bot flies, we tested the potential of these parasites to transmit CWD. Parasites collected from naturally infected white-tailed deer were evaluated for their prion content using the protein misfolding cyclic amplification (PMCA) technology and bioassays. Here, we describe PMCA seeding activity in nasal bot larvae collected from naturally infected, nonclinical deer. These parasites efficiently infect CWD-susceptible mice in ways suggestive of high infectivity titers. To further mimic environmental transmission, bot larvae homogenates were mixed with soils, and plants were grown on them. We show that both soils and plants exposed to CWD-infected bot homogenates displayed seeding activity by PMCA. This is the first report describing prion infectivity in a naturally occurring deer parasite. Our data also demonstrate that CWD prions contained in nasal bots interact with environmental components and may be relevant for disease transmission.

Neonatal intensive care nurses' accounts of care for mothers/families with substance-exposed pregnancies: A critical discourse analysis.

AIMS: To explore the effects of power dynamics and hospital organizational structure upon neonatal intensive care nurses' experiences caring for infants and families from a substance-exposed pregnancy (SEP). DESIGN: This secondary data analysis further investigated the results of a primary study after the original analysis suggested differences in work environments may impact relationship-building opportunities between nurses and mothers/families. Critical discourse analysis served as both the theoretical lens and analytic technique. METHOD: Nine (9) nurses from the southeast region of the United States (U.S.) were interviewed in 2019. Fifty-one (51) stories of caregiving experiences were analysed with a focus on narratives related to organizational structure and care delivery. RESULTS: Study findings revealed nurses experienced challenges providing high-quality, family-centered care for patients in the neonatal intensive care unit (NICU) affected by substances during pregnancy. Nurses described the central challenge of workload, exacerbated by power imbalances and structural constraints within the hospital's organizational structure. Findings suggest workload issues may endorse stigma by inhibiting opportunities to build relationships. Nurses report manageable workloads can support healthcare teams and recipients of care. CONCLUSION: The study suggests power imbalances between nurses, families and adjacent healthcare professionals can inhibit the delivery of high-quality care. Supporting healthcare teams and recipients of care while centering the role of organizational structure is critical. Questions emerged about workload demands impacting the potential production of stigma in clinical environments. IMPACT: This study examines the intersection of nurses' care experiences and hospital organizational structure. It identifies how the unique needs of caring for infants and families from a SEP increase the complexity of power imbalances and organizational constraints to further increase workload demands. Findings have implications for global healthcare organization leaders who build and maintain the structural integrity of clinical environments and nurse leaders who advocate and guide clinical teams to provide high-quality care in stressful healthcare environments. REPORTING METHOD: EQUATOR guidelines were followed, using the COREQ checklist. PATIENT OR PUBLIC CONTRIBUTION: NICU nurses were interviewed about their care-provision experiences. Interviews were analysed in the primary study and the current analysis of secondary data.

Dynamics of CWD prion detection in feces and blood from naturally infected white-tailed deer.

Chronic wasting disease (CWD) is a prion disease affecting cervids. Confirmatory testing of CWD is currently performed postmortem in obex and lymphoid tissues. Extensive evidence demonstrates the presence of infectious prions in feces of CWD-infected deer using in vitro prion-amplification techniques and bioassays. In experimental conditions, this has been achieved as soon as 6-month post-inoculation, suggesting this sample type is a candidate for antemortem diagnosis. In the present study, we optimized the detection of CWD-prions in fecal samples from naturally infected, pre-clinical white-tailed deer by comparing protocols aiming to concentrate CWD-prions with direct spiking of the sample into the PMCA reactions. Results of this screening were compared with similar analyses made in blood. Our data shows that CWD-prion detection in feces using PMCA is best in the absence of sample pre-treatments. We performed a screening of 169 fecal samples, detecting CWD-prions with diagnostic sensitivity and specificity of 54.81% and 98.46%, respectively. In addition, the PMCA seeding activity of 76 fecal samples was compared with that on blood of matched deer. Our findings, demonstrate that CWD-prions in feces and blood are increased at late pre-clinical stages, exhibiting similar detection in both sample types (> 90% sensitivity) when PrP96GG animals are tested. Our findings contribute to understand prion distribution across different biological samples and polymorphic variants in white-tailed deer. This information is also relevant for the current efforts to identify platforms to diagnose CWD.

Tonsil biopsy to detect chronic wasting disease in white-tailed deer (Odocoileus virginianus) by immunohistochemistry.

Chronic wasting disease (CWD) continues to spread in wild and farmed cervid populations. Early antemortem CWD testing of farmed cervids is of considerable interest to producers and regulatory agencies as a tool to combat this spread. The tissues accessible for antemortem sampling are limited and include biopsy of the tonsil and recto-anal mucosa-associated lymphoid tissue (RAMALT). The sensitivity to detect CWD by immunohistochemistry (IHC)-the regulatory gold standard-using biopsy samples of RAMALT from naturally infected white-tailed deer (WTD) has been determined by several studies. However, similar information is lacking for tonsil biopsy. In this study, two-bite tonsil biopsies from 79 naturally infected farmed WTD were used to determine the diagnostic sensitivity of tonsil IHC compared to the official CWD status based on results from the medial retropharyngeal lymph nodes and obex. IHC detection of CWD by tonsil biopsy was compared to the result and follicle metrics from the contralateral whole tonsil. The sensitivity of two-bite tonsil biopsy for detecting CWD by IHC was 72% overall. When the stage of infection was considered, the sensitivity was 92% for deer in late preclinical infection but only 55% for early preclinical infection. For deer with early preclinical infection, the sensitivity for deer homozygous for the prion protein gene (PRNP) coding for glycine at codon 96 (GG) was 66% but only 30% when heterozygous for the serine substitution (GS). The results indicate that the sensitivity of two-bite tonsil biopsy in WTD, and consequently its potential utility as an antemortem diagnostic, is limited during early infection, especially in WTD heterozygous for the serine substitution at PRNP codon 96.

Disclosure and comfort during genetic counseling sessions with LGBTQ+ patients: An updated assessment.

Disclosure is the act of sharing a stigmatized identity, and members of the LGBTQ+ community make decisions related to disclosure multiple times throughout their life. Disclosure in medical settings can impact perceptions of care and outcomes for LGBTQ+ patients; however, little is understood about the process of decision-making regarding disclosure in the genetic counseling setting. As such, this study aimed to explore LGBTQ+ experiences in genetic counseling sessions and their disclosure behaviors. Fifty-five LGBTQ+ individuals who attended a genetic counseling session and 91 genetic counselors completed online surveys. The patient survey assessed for disclosure behaviors, experiences of discrimination, and comfort in genetic counseling sessions. The counselor survey evaluated comfort with the LGBTQ+ population in a counseling setting, whether counselors facilitate disclosure in sessions, and whether counseling is tailored for the LGBTQ+ population. Eighty-two percent of genetic counselors "rarely" or "never" ask about sexual orientation, and 69% "rarely" or "never" ask about gender identity. The majority of patients indicated they were not asked about their sexual orientation (87%) or gender identity (80%). Some patients reported experiencing discrimination or homo/transphobia in their genetic counseling sessions, with 6.12% of LGBQ+ patients experiencing discrimination and 24.1% of trans+ patients reporting discrimination. Over half of genetic counselors reported receiving training in LGBTQ+ healthcare and the majority reported comfort with providing care to LGBTQ+ patients. However, discrepancies between patient-reported experiences and genetic counselors' descriptions of their care for the LGBTQ+ population warrant further research and suggest additional training or changes in practice may be necessary.

Canine detection of chronic wasting disease (CWD) in laboratory and field settings.

Chronic wasting disease (CWD) is a fatal transmissible spongiform encephalopathy that affects both free-ranging and farmed cervid species, including mule deer, white-tailed deer, and elk (Odocoileus hemionus, Odocoileus virginianus, and Cervus canadensis). Due to the long incubation period and variability of clinical signs, CWD can expand and spread to new areas before they reach diagnostically detectable levels. Antemortem testing methods currently available can be difficult to obtain and to be applied to the large numbers required for adequate surveillance. However, key volatile biomarkers could be harnessed for non-invasive antemortem surveillance. Detection dogs are the most effective tool currently available for volatile detection; dogs can effectively complete wildlife surveys at rates surpassing that of humans. This study is the first to demonstrate that trained detection dogs can be used as an antemortem test for CWD. First, we trained three dogs to differentiate between CWD-positive and CWD-negative white-tailed deer faeces in a laboratory setting. Dogs spent significantly more time at the positive sample than the negative samples, suggesting that they differentiated between the positive and negative volatile signatures. We then trained the same dogs to search for CWD-positive faecal samples in a more naturalistic field setting. In the field, dogs found 8/11 CWD-positive samples and had an average false detection rate of 13%. These results suggest that dogs can be trained to differentiate CWD-positive faeces from CWD-negative faeces in both laboratory and field settings. Future studies will compare canine accuracy to other antemortem methods, as well as improved canine training methods.

Detection of chronic wasting disease in feces and recto-anal mucosal associated lymphoid tissues with RT-QuIC in a naturally infected farmed white-tailed deer herd.

Chronic wasting disease (CWD) is an infectious prion disease affecting the cervids, including white-tailed deer (WTD) (Odocoileus virginianus). CWD is typically diagnosed postmortem in farmed cervids by immunohistochemistry (IHC). Amplification-based detection methods are newer generation tests currently being evaluated to improve the detection of prion disease. In addition to improving sensitivity, antemortem detection by amplification assays is a focus for improving disease control and management. In this study, we evaluate the use of real-time quaking-induced conversion (RT-QuIC) to detect CWD in fecal and recto-anal mucosal-associated lymphoid tissue (RAMALT) samples from naturally infected farmed WTD herds at postmortem. We successfully detected the presence of CWD prions in WTD RAMALT with a specificity of 100% and a sensitivity of 85.7% (n = 71) and in feces with a specificity of 100% and a sensitivity of 60% (n = 69), utilizing RT-QuIC on samples collected postmortem. Seeding activity detected in RAMALT (15.3 ± 4.2%, n = 18) was much stronger than in feces (44.4 ± 4.2%, n = 15), as measured by cycle threshold (Ct) and rise in relative fluorescence in samples collected from the same WTD. Prion detection in the RAMALT (94.7%) and feces (70.5%) was highest when both obex and retropharyngeal lymph nodes (RPLNs) were positive for CWD via IHC. In the study group, we were also able to test prion protein gene variants and associated disease susceptibility. A majority of tested WTD were the CWD genotype (96 GG) and also harbored the highest percentage of positive animals (43.7%). The second highest population of WTD was the genotype 96 GS and had a CWD positivity rate of 37.5%. Each of these groups showed no difference in RAMALT or fecal detection of CWD.

Genotype by environment interactions for chronic wasting disease in farmed US white-tailed deer.

Despite implementation of enhanced management practices, chronic wasting disease in US white-tailed deer (Odocoileus virginianus) continues to expand geographically. Herein, we perform the largest genome-wide association analysis to date for chronic wasting disease (n = 412 chronic wasting disease-positive; n = 758 chronic wasting disease-nondetect) using a custom Affymetrix Axiom single-nucleotide polymorphism array (n = 121,010 single-nucleotide polymorphisms), and confirm that differential susceptibility to chronic wasting disease is a highly heritable (h2= 0.611 ± 0.056) polygenic trait in farmed US white-tailed deer, but with greater trait complexity than previously appreciated. We also confirm PRNP codon 96 (G96S) as having the largest-effects on risk (P ≤ 3.19E-08; phenotypic variance explained ≥ 0.025) across 3 US regions (Northeast, Midwest, South). However, 20 chronic wasting disease-positive white-tailed deer possessing codon 96SS genotypes were also observed, including one that was lymph node and obex positive. Beyond PRNP, we also detected 23 significant single-nucleotide polymorphisms (P-value ≤ 5E-05) implicating ≥24 positional candidate genes; many of which have been directly implicated in Parkinson's, Alzheimer's and prion diseases. Genotype-by-environment interaction genome-wide association analysis revealed a single-nucleotide polymorphism in the lysosomal enzyme gene ARSB as having the most significant regional heterogeneity of effects on chronic wasting disease (P ≤ 3.20E-06); with increasing copy number of the minor allele increasing susceptibility to chronic wasting disease in the Northeast and Midwest; but with opposite effects in the South. In addition to ARSB, 38 significant genotype-by-environment single-nucleotide polymorphisms (P-value ≤ 5E-05) were also detected, thereby implicating ≥ 36 positional candidate genes; the majority of which have also been associated with aspects of Parkinson's, Alzheimer's, and prion diseases.

Exploration of the Care Giving Dynamic Between NICU Nurses and Mothers with a Substance-Exposed Pregnancy.

PURPOSE: To explore the caregiving dynamic between NICU nurses and mothers with a substance-exposed pregnancy (SEP) by examining how nurses view these mothers compared to mothers without an SEP. DESIGN: A qualitative design using interviews with NICU nurses who care for infants and families with an SEP. SAMPLE: The sample (n = 9) was all female, with an average of approximately 10 years of nursing experience in the NICU, with two-thirds having achieved a Bachelor of Science in Nursing (BSN) or higher. MAIN OUTCOME VARIABLE: Perceptions of nurses working with families affected by an SEP. RESULTS: Stigmatized views and trust issues shaped nurses' views of mothers with an SEP as different from other mothers. Empathy levels toward mothers differed related to nurses' views of substance use as either a moral decision or chronic disease. These viewpoints shaped the nurse's judgment of the mother's potential to provide a safe environment and independently care for the infant at home. Nurses described personal distress when caring for these families.

Detection of two dissimilar chronic wasting disease isolates in two captive Rocky Mountain elk (Cervus canadensis) herds.

Chronic wasting disease (CWD) continues to spread in both wild and captive cervid herds in North America and has now been identified in wild reindeer and moose in Norway, Finland and Sweden. There is limited knowledge about the variety and characteristics of isolates or strains of CWD that exist in the landscape and their implications on wild and captive cervid herds. In this study, we evaluated brain samples from two captive elk herds that had differing prevalence, history and timelines of CWD incidence. Site 1 had a 16-year history of CWD with a consistently low prevalence between 5% and 10%. Twelve of fourteen naïve animals placed on the site remained CWD negative after 5 years of residence. Site 2 herd had a nearly 40-year known history of CWD with long-term environmental accrual of prion leading to nearly 100% of naïve animals developing clinical CWD within two to 12 years. Obex samples of several elk from each site were compared for CWD prion strain deposition, genotype in prion protein gene codon 132, and conformational stability of CWD prions. CWD prions in the obex from site 2 had a lower conformational stability than those from site 1, which was independent of prnp genotype at codon 132. These findings suggest the existence of different CWD isolates between the two sites and suggest potential differential disease attack rates for different CWD strains.

Detection of CWD prions in naturally infected white-tailed deer fetuses and gestational tissues by PMCA.

Chronic wasting disease (CWD) is a prevalent prion disease affecting cervids. CWD is thought to be transmitted through direct animal contact or by indirect exposure to contaminated environmental fomites. Other mechanisms of propagation such as vertical and maternal transmissions have also been suggested using naturally and experimentally infected animals. Here, we describe the detection of CWD prions in naturally-infected, farmed white-tailed deer (WTD) fetal tissues using the Protein Misfolding Cyclic Amplification (PMCA) technique. Prion seeding activity was identified in a variety of gestational and fetal tissues. Future studies should demonstrate if prions present in fetuses are at sufficient quantities to cause CWD after birth. This data confirms previous findings in other animal species and furthers vertical transmission as a relevant mechanism of CWD dissemination.

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.

Chronic wasting disease (CWD) prion detection in blood from pre-symptomatic white-tailed deer harboring PRNP polymorphic variants.

Chronic wasting disease (CWD) is a prionopathy affecting wild and farmed cervids. This disease is endemic in North America and has been recently identified in Europe. Ante-mortem CWD tests of pre-clinical cervids may be an important tool in helping control the spread of this disease. Unfortunately, current CWD diagnostic methods are not suitable for non-tissue type samples. We reported that CWD prions can be detected in blood of pre-clinical CWD-infected white-tailed deer (WTD) with high sensitivity and specificity using the Protein Misfolding Cyclic Amplification (PMCA) assay. However, that report only included animals homozygous for codon 96G, the most common polymorphic version of the prion protein within this animal species. Here, we report CWD prion detection using blood of naturally infected WTD coding one or two copies of the PrP-96S polymorphic variant. Our results, from a blinded screening, show 100% specificity and ~ 58% sensitivity for animals harboring one 96S codon, regardless of their stage within the pre-clinical phase. Detection efficiency for PrP-96S homozygous animals was substantially lower, suggesting that this allele affect peripheral prion replication/tropism. These results provide additional information on the influence of codon 96 polymorphisms and the ability of PMCA to detect CWD in the blood of pre-clinical WTD.

The Role of Bias in Knowledge Translation: Lessons Learned from a Dissemination of Best Practices in Maternal Opioid Use.

INTRODUCTION: Increases in opioid use during pregnancy fueled concerns among reproductive health advocates, policy makers, and providers in North Carolina, United States. A stakeholder group designed a set of knowledge translation (KT) interventions to increase the use of evidence-based practices across relevant health care and social service arenas. Efforts to decrease stigma was included as a best practice. Understanding the process and the contexts in which KT intervention activities occur can help increase their effectiveness. Toward that end, this study examined how stigma was addressed and how audiences responded to evidence-based messages. METHODS: Data were collected over a seven-year period and included observations of KT activities (conferences, workshops, and community meetings), focus groups and interviews conducted with providers, and a review of publicly available documents, including KT intervention materials. Clarke's situational mapping was initially applied, and stigma emerged as a critical contextual element. Data were then analyzed to identify how stigma was addressed and how evidence-based messages were received. RESULTS: Despite direct stigma-reduction messages, biases against maternal drug use and ambivalence toward evidence-based recommendations of harm reduction principles were found in audience responses. Findings also revealed tensions around the interpretation of knowledge and the appropriate implementation of best practices between "experts" and among practitioners. DISCUSSION: Stigma and professional biases may serve as significant barriers to KT activities. Results suggest that even under the best of circumstances, evidence-based practices for highly stigmatized and controversial issues are difficult to disseminate and may require unique approaches.

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.

Accurate Genomic Predictions for Chronic Wasting Disease in U.S. White-Tailed Deer.

The geographic expansion of chronic wasting disease (CWD) in U.S. white-tailed deer (Odocoileus virginianus) has been largely unabated by best management practices, diagnostic surveillance, and depopulation of positive herds. Using a custom Affymetrix Axiom single nucleotide polymorphism (SNP) array, we demonstrate that both differential susceptibility to CWD, and natural variation in disease progression, are moderately to highly heritable ([Formula: see text] among farmed U.S. white-tailed deer, and that loci other than PRNP are involved. Genome-wide association analyses using 123,987 quality filtered SNPs for a geographically diverse cohort of 807 farmed U.S. white-tailed deer (n = 284 CWD positive; n = 523 CWD non-detect) confirmed the prion gene (PRNP; G96S) as a large-effect risk locus (P-value < 6.3E-11), as evidenced by the estimated proportion of phenotypic variance explained (PVE ≥ 0.05), but also demonstrated that more phenotypic variance was collectively explained by loci other than PRNP Genomic best linear unbiased prediction (GBLUP; n = 123,987 SNPs) with k-fold cross validation (k = 3; k = 5) and random sampling (n = 50 iterations) for the same cohort of 807 farmed U.S. white-tailed deer produced mean genomic prediction accuracies ≥ 0.81; thereby providing the necessary foundation for exploring a genomically-estimated CWD eradication program.

In Vitro detection of Chronic Wasting Disease (CWD) prions in semen and reproductive tissues of white tailed deer bucks (Odocoileus virginianus).

Chronic Wasting Disease (CWD) is a prion disease affecting several cervid species. Among them, white-tailed deer (WTD) are of relevance due to their value in farming and game hunting. The exact events involved in CWD transmission in captive and wild animals are still unclear. An unexplored mechanism of CWD spread involves transmissions through germplasm, such as semen. Surprisingly, the presence and load of CWD prions in semen and male sexual tissues from WTD has not been explored. Here, we described the detection of CWD prions in semen and sexual tissues of WTD bucks utilizing the Protein Misfolding Cyclic Amplification (PMCA) technology. Samples were obtained post-mortem from farmed pre-clinical, CWD positive WTD bucks possessing polymorphisms at position 96 of the PRNP gene. Our results show that overall CWD detection in these samples had a sensitivity of 59.3%, with a specificity of 97.2%. The data indicate that the presence of CWD prions in male sexual organs and fluids is prevalent in late stage, pre-clinical, CWD-infected WTD (80%-100% of the animals depending on the sample type analyzed). Our findings reveal the presence of CWD prions in semen and sexual tissues of prion infected WTD bucks. Future studies will be necessary to determine whether sexual contact and/or artificial inseminations are plausible means of CWD transmission in susceptible animal species.

Estimating relative CWD susceptibility and disease progression in farmed white-tailed deer with rare PRNP alleles.

Chronic wasting disease is a prion disease affecting both free-ranging and farmed cervids in North America and Scandinavia. A range of cervid species have been found to be susceptible, each with variations in the gene for the normal prion protein, PRNP, reportedly influencing both disease susceptibility and progression in the respective hosts. Despite the finding of several different PRNP alleles in white-tailed deer, the majority of past research has focused on two of the more common alleles identified-the 96G and 96S alleles. In the present study, we evaluate both infection status and disease stage in nearly 2100 farmed deer depopulated in the United States and Canada, including 714 CWD-positive deer and correlate our findings with PRNP genotype, including the more rare 95H, 116G, and 226K alleles. We found significant differences in either likelihood of being found infected or disease stage (and in many cases both) at the time of depopulation in all genotypes present, relative to the most common 96GG genotype. Despite high prevalence in many of the herds examined, infection was not found in several of the reported genotypes. These findings suggest that additional research is necessary to more properly define the role that these genotypes may play in managing CWD in both farmed and free-ranging white-tailed deer, with consideration for factors including relative fitness levels, incubation periods, and the kinetics of shedding in animals with these rare genotypes.