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.

Identification of a novel risk factor for chronic wasting disease (CWD) in elk: S100G single nucleotide polymorphism (SNP) of the prion protein gene (PRNP).

Prion diseases are fatal and malignant infectious encephalopathies induced by the pathogenic form of prion protein (PrPSc) originating from benign prion protein (PrPC). A previous study reported that the M132L single nucleotide polymorphism (SNP) of the prion protein gene (PRNP) is associated with susceptibility to chronic wasting disease (CWD) in elk. However, a recent meta-analysis integrated previous studies that did not find an association between the M132L SNP and susceptibility to CWD. Thus, there is controversy about the effect of M132L SNP on susceptibility to CWD. In the present study, we investigated novel risk factors for CWD in elk. We investigated genetic polymorphisms of the PRNP gene by amplicon sequencing and compared genotype, allele, and haplotype frequencies between CWD-positive and CWD-negative elk. In addition, we performed a linkage disequilibrium (LD) analysis by the Haploview version 4.2 program. Furthermore, we evaluated the 3D structure and electrostatic potential of elk prion protein (PrP) according to the S100G SNP using AlphaFold and the Swiss-PdbViewer 4.1 program. Finally, we analyzed the free energy change of elk PrP according to the S100G SNP using I-mutant 3.0 and CUPSAT. We identified 23 novel SNP of the elk PRNP gene in 248 elk. We found a strong association between PRNP SNP and susceptibility to CWD in elk. Among those SNP, S100G is the only non-synonymous SNP. We identified that S100G is predicted to change the electrostatic potential and free energy of elk PrP. To the best of our knowledge, this was the first report of a novel risk factor, the S100G SNP, for 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.

Longitudinal detection of prion shedding in nasal secretions of CWD-infected white-tailed deer.

Chronic wasting disease (CWD) is an emergent prion disease spreading in cervid populations in North America, South Korea and Scandinavia. Rapid detection of CWD prions shed by live animals using minimally invasive methods remains an important need. Previous studies in deer, elk and hamsters have demonstrated prion replication in the nasal olfactory mucosa, yet the temporal profile of CWD prion shedding in nasal secretions has not been well characterized. Here we report nasal prion shedding in 18 deer orally exposed to low doses of CWD prions and monitored longitudinally by several parameters. Serially collected nasal swabs were assayed for CWD prion seeding activity using iron oxide magnetic extraction and real-time quaking-induced conversion (IOME RT-QuIC). These findings were correlated with the results from longitudinal tonsil biopsies, terminal tissues and PRNP genotype. We detected nasal prion shedding 3-16 months after the first positive tonsil biopsy in ten of the 18 deer; detectable shedding persisted thereafter in nine of the ten animals. Surprisingly, nasal swabs were negative in eight deer, even though all were CWD-infected as determined by tonsil biopsies and terminal tissue assays. Nasal shedding was detected more often in deer that were homozygous for glycine at codon 96, and those that were near or demonstrating symptoms of clinical disease shed earlier and more frequently, irrespective of prion exposure dose. The results of this study demonstrate nasal shedding of CWD prions that can be detected using minimally invasive nasal swab sampling and RT-QuIC analysis.

Diagnostic testing of chronic wasting disease in white-tailed deer (Odocoileus virginianus) by RT-QuIC using multiple tissues.

Chronic wasting disease (CWD) is a fatal prion disease affecting cervids (deer, elk, moose). Current methods to monitor individual disease state include highly invasive antemortem rectal biopsy or postmortem brain biopsy. Efficient, sensitive, and selective antemortem and postmortem testing of populations would increase knowledge of the dynamics of CWD epizootics as well as provide a means to track CWD progression into previously unaffected areas. Here, we analyzed the presence of CWD prions in skin samples from two easily accessed locations (ear and belly) from 30 deceased white-tailed deer (Odocoileus viginianus). The skin samples were enzymatically digested and analyzed by real-time quaking-induced conversion (RT-QuIC). The diagnostic sensitivity of the ear and belly skin samples were both 95%, and the diagnostic specificity of the ear and belly skin were both 100%. Additionally, the location of the skin biopsy on the ear does not affect specificity or sensitivity. These results demonstrate the efficacy of CWD diagnosis with skin biopsies using RT-QuIC. This method could be useful for large scale antemortem population testing.

CAUSE OF DEATH, PATHOLOGY, AND CHRONIC WASTING DISEASE STATUS OF WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS) MORTALITIES IN WISCONSIN, USA.

White-tailed deer (WTD; Odocoileus virginianus) are a critical species for ecosystem function and wildlife management. As such, studies of cause-specific mortality among WTD have long been used to understand population dynamics. However, detailed pathological information is rarely documented for free-ranging WTD, especially in regions with a high prevalence of chronic wasting disease (CWD). This leaves a significant gap in understanding how CWD is associated with disease processes or comorbidities that may subsequently alter broader population dynamics. We investigated unknown mortalities among collared WTD in southwestern Wisconsin, USA, an area of high CWD prevalence. We tested for associations between CWD and other disease processes and used a network approach to test for co-occurring disease processes. Predation and infectious disease were leading suspected causes of death, with high prevalence of CWD (42.4%; of 245 evaluated) and pneumonia (51.2%; of 168 evaluated) in our sample. CWD prevalence increased with age, before decreasing among older individuals, with more older females than males in our sample. Females were more likely to be CWD positive, and although this was not statistically significant when accounting for age, females were significantly more likely to die with end-stage CWD than males and may consequently be an underrecognized source of CWD transmission. Presence of CWD was associated with emaciation, atrophy of marrow fat and hematopoietic cells, and ectoparasitism (lice and ticks). Occurrences of severe infectious disease processes clustered together (e.g., pneumonia, CWD), as compared to noninfectious or low-severity processes (e.g., sarcocystosis), although pneumonia cases were not fully explained by CWD status. With the prevalence of CWD increasing across North America, our results highlight the critical importance of understanding the potential role of CWD in favoring or maintaining disease processes of importance for deer population health and dynamics.

First report of a strong association between genetic polymorphisms of the prion protein gene (PRNP) and susceptibility to chronic wasting disease in sika deer (Cervus nippon).

Prion diseases are incurable neurodegenerative disorders caused by proteinase K-resistant prion protein (PrPSc ) derived from normal prion protein (PrPC ) encoded by the prion protein gene (PRNP). Although the cervid PRNP gene plays a pivotal role in the pathological mechanism of chronic wasting disease (CWD), there is no existing association analysis between susceptibility to CWD and genetic polymorphisms of the PRNP gene in sika deer. We investigated genetic polymorphisms of the PRNP gene using amplicon sequencing in sika deer. In addition, to identify a genetic susceptibility factor, we compared the genotype, allele and haplotype frequencies of the PRNP gene between CWD-positive and CWD-negative sika deer. Furthermore, to assess the effect of the genetic polymorphisms on sika deer prion protein (PrP), we performed in silico analysis using PolyPhen-2, PROVEAN and AMYCO. Finally, we analysed the tertiary structure and electrostatic potential of sika deer PrP based on single nucleotide polymorphisms (SNPs) using the SWISS-MODEL and Swiss-PdbViewer programs. We found a total of 24 SNPs of the PRNP gene, including 22 novel SNPs (10 synonymous SNPs and 12 nonsynonymous SNPs), in sika deer. Among the nonsynonymous SNPs, we found a strong association of susceptibility to CWD with c.56G > A (Ser19Asn). In addition, we found that c.56G > A (Ser19Asn), c.296A > T (His99Leu) and c.560T > A (Val187Asp) were predicted to have damaging effects on sika deer PrP. Furthermore, we observed significant alterations in the electrostatic potential of sika deer PrP by genetic polymorphisms of the 187Asp allele. To the best of our knowledge, this was the first association study between genetic polymorphisms of the PRNP gene and susceptibility to CWD in sika deer.

Vivir con dolor crónico desde la experiencia de adultos mayores con enfermedades crónico degenerativas / Living with cronic pain from the experience of older adults with chronic degenerative diseases

INTRODUCCIÓN: Ser adulto mayor conlleva a cambios físicos, psicológicos y sociales que se ven aún más afectados por comorbilidades como las enfermedades crónicas y el dolor. El propósito es interpretar las experiencias de vivir con dolor de los adultos mayores con enfermedades crónico-degenerativas. METODOLOGÍA: Diseño cualitativo de tipo fenomenológico, recolectado mediante un muestreo intencional a través de una entrevista semi estructurada, en la cual se seleccionó a la población adulta mayor con una patología crónico-degenerativa que haya experimentado dolor crónico; se analizaron los datos con el proceso cognitivo de Janice Morse, hasta llegar a la saturación de la información. Participaron 8 personas entre los 66 y 72 años, habiendo siete mujeres y un hombre. RESULTADOS: Se encontraron cuatro categorías: 1. El desgaste de vivir con dolor; 2. Cotidianidad del vivir con dolor; 3. Alternativas para aliviar el dolor, y 4. Afectación en el entorno social. CONCLUSIÓN: Ser adulto mayor trae muchos cambios en todas las esferas de la vida, sin embargo, el padecer una enfermedad crónica acelera toda esta transición, viéndose afectados la salud mental, físico y social, adaptando esta situación a su cotidianidad, buscando a su vez medios alternos que mitiguen o disminuya el dolor.

INTRODUCTION: Being an older adult leads to physical, psychological, and social changes that are further affected by comorbidities such as chronic diseases and pain. The purpose is to interpret the experiences of living in pain of older adults with chronic-degenerative diseases. METHOD: Qualitative phenomenological research, recollected by an intentional sampling through a semi-structured interview in which was selected the adult population with a chronic-degenerative pathology that have experienced an state of chronic pain. The data was analyzed with the cognitive process of Janice Morse and reached to a saturation of 8 participants. There were 8 participants between the ages of 66 and 72, with seven women and one man. RESULTS: Four categories were found: 1. Wearing of living with pain; 2. Daily Living with Pain; 3. Alternatives for Pain Relief, and 4. Social Affectation. CONCLUSION: Being an older adult brings many changes in all spheres of life, however, suffering from a chronic disease accelerates this whole transition, being affected mental, physical and social health, adapting this situation to its daily life, seeking in turn alternate means to mitigate or decrease pain.

[Effects of SGI-1027 on Formation and Elimination of PrP

Recently, SGI-1027, a well-known inhibitor of DNA-methyl transferases (DNMTs), was reported to effectively reduce formation of pathogenic PrP^(Sc) in prion-infected cells. Herein, we confirm the elimination of PrP^(Sc) in chronic wasting disease (CWD) prion-infected neurons by SGI-1027, and pinpoint the binding region of human prion protein to SGI-1027. SGI-1027 is broadly functional against various prion disease types, including human prions. Previously, the inhibitory effects of SGI-1027 on DNMT function is well tested in various cell culture models. While neither treatment with a DNMTs enhancer S-adenosyl-L-methionine (SAM), nor with their inhibitor, 5-azacytidine, prevented PrP^(Sc) propagation, SGI-1027 did. Our study suggest that the anti-prion effects of SGI-1027 are a result of its direct interaction with PrP^(C), which effectively interferes with the pathogenic conformational change of PrP^(C) to PrP^(Sc). We conclude that SGI-1027 driven suppression of pathogenic PrP^(Sc) is independent of DNMT.

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.

EFFECT OF ORAL COPPER SUPPLEMENTATION ON SUSCEPTIBILITY IN WHITE-TAILED DEER (ODOCOILEUS VIRGINIANUS) TO CHRONIC WASTING DISEASE.

Chronic wasting disease (CWD) is an infectious disease, but reported associations suggest several metals-especially copper (Cu) and manganese-potentially play a role in this and other prion diseases. To assess the utility of dietary Cu supplementation in protecting white-tailed deer (Odocoileus virginianus) from CWD, we compared incidence and disease course among individuals naturally exposed to CWD while being maintained on sustained-release Cu boluses or unsupplemented (control). Oral Cu supplementation increased liver tissue Cu concentrations compared to controls but did not affect susceptibility to CWD or survival after natural exposure in the captive white-tailed deer we studied. Over the 27 mo study, 89% (8/9) of the Cu-supplemented deer and 86% (6/7) of control deer became CWD-infected. Survival to 27 mo postexposure did not differ between Cu-supplemented and control deer: model-averaged survival probabilities to 27 mo were 0.45-0.47 for all combinations of Cu treatment and PRNP gene haplotype presence. The PRNP gene haplotype influenced the probability of deer remaining biopsy negative for at least 17 mo but did not affect overall susceptibility.

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.

Cellulose ether treatment in vivo generates chronic wasting disease prions with reduced protease resistance and delayed disease progression.

Chronic wasting disease (CWD) is a prion disease of free-ranging and farmed cervids that is highly contagious because of extensive prion shedding and prion persistence in the environment. Previously, cellulose ether compounds (CEs) have been shown to significantly extend the survival of mice inoculated with mouse-adapted prion strains. In this study, we used CEs, TC-5RW, and 60SH-50, in vitro and in vivo to assess their efficacy to interfere with CWD prion propagation. In vitro, CEs inhibited CWD prion amplification in a dose-dependent manner. Transgenic mice over-expressing elk PrPC (tgElk) were injected subcutaneously with a single dose of either of the CEs, followed by intracerebral inoculation with different CWD isolates from white tailed deer, mule deer, or elk. All treated groups showed a prolonged survival of up to more than 30 % when compared to the control group regardless of the CWD isolate used for infection. The extended survival in the treated groups correlated with reduced proteinase K resistance of prions. Remarkably, passage of brain homogenates from treated or untreated animals in tgElk mice resulted in a prolonged life span of mice inoculated with homogenates from CE-treated mice (of + 17%) even in the absence of further treatment. Besides the delayed disease onset upon passage in TgElk mice, the reduced proteinase K resistance was maintained but less pronounced. Therefore, these compounds can be very useful in limiting the spread of CWD in captive and wild-ranging cervids.

Chronic wasting disease associated with prion protein gene (PRNP) variation in Norwegian wild reindeer (Rangifer tarandus).

The emergence of CWD in Europe in 2016 and the first natural infection in wild reindeer warranted disease management. This led to the testing of 2424 hunted or culled reindeer during 2016-2018, from the infected subpopulation in the Nordfjella mountain range in Southern Norway. To identify any association between PRNP variation and CWD susceptibility, we characterized the open reading frame of the PRNP gene in 19 CWD positive reindeer and in 101 age category- and sex-matched CWD negative controls. Seven variant positions were identified: 6 single nucleotide variants (SNVs) and a 24 base pair (bp) deletion located between nucleotide position 238 and 272, encoding four instead of five octapeptide repeats. With a single exception, all variant positions but one were predicted to be non-synonymous. The synonymous SNV and the deletion are novel in reindeer. Various combinations of the non-synonymous variant positions resulted in the identification of five PRNP alleles (A-E) that structured into 14 genotypes. We identified an increased CWD risk in reindeer carrying two copies of the most common allele, A, coding for serine in position 225 (Ser225) and in those carrying allele A together with the 24 bp deletion.

Melatonin attenuates inflammation-related venous endothelial cells apoptosis through modulating the MST1-MIEF1 pathway.

Chronic venous disease (CVD) is a prevalent and potentially debilitating condition that affects millions of individuals. An excessive endothelial inflammatory response is reportedly involved in the development of CVD. In this study, we explored the effect and mechanism of melatonin on venous endothelial damage induced by tumor necrosis factor α (TNF-α). Our data demonstrated that inflammation injury triggered mitochondrial dysfunction, activated reactive oxygen species-related oxidative damage, inhibited mitochondrial potential and ultimately initiated caspase-involved cellular death. Interestingly, melatonin preserved inflammation-attacked mitochondrial performance and thus increased cell survival under TNF-α. Cellular experiments illustrated that inflammation injury promoted the levels of mammalian sterile 20-like kinase 1 (MST1) and mitochondrial elongation factor 1 (MIEF1); active MST1-MIEF1 pathway disturbed mitochondria-related energy production, leading to mitochondria-induced cell damage. Interestingly, melatonin effectively suppressed MST1-MIEF1 axis and thus improved cell survival ratio under TNF-α-mediated inflammation injury. Reactivation of MST1-MIEF1 pathway attenuated melatonin-related endothelial protective actions. Herein, our results illuminate that melatonin is an effective approach to attenuate inflammation-related venous endothelial cell damage through handling the MST1-MIEF1 signaling pathway.

FUS-ALS presenting with myoclonic jerks in a 17-year-old man.

Fused in sarcoma-related amyotrophic lateral sclerosis (FUS-ALS) accounts for 4% of all familial motor neurone disease, but has a much higher incidence in juvenile ALS. We present a case of a 17-year-old British man with rapidly progressive bulbar and respiratory failure. On examination he had weak periocular muscles, neck flexion weakness, and a wasted, fasciculating and weak tongue. There were no sensory, cerebellar, or extrapyramidal features but he had frequent myoclonic jerks of the limbs. Routine bloods were normal and an MRI of the neuroaxis as well as CT chest, abdomen and pelvis were unremarkable. NCS/EMG was consistent with anterior horn cell disorder and EEG showed multiple paroxysmal generalized spike-wave discharges. DNA sequencing demonstrated that he was heterozygous for the c.1483C>T pathogenic nonsense mutation in exon 14 of the FUS gene, consistent with ALS6. This is the first reported case of FUS-ALS presenting with prominent myoclonus.

Accelerated onset of chronic wasting disease in elk (Cervus canadensis) vaccinated with a PrPSc-specific vaccine and housed in a prion contaminated environment.

Chronic wasting disease (CWD) is a fatal prion disease affecting multiple cervid species. Effective management tools for this disease, particularly in free-ranging populations, are currently limited. We evaluated a novel CWD vaccine in elk (Cervus canadensis) naturally exposed to CWD through a prion-contaminated environment. The vaccine targets a YYR disease-specific epitope to induce antibody responses specific to the misfolded (PrPSc) conformation. Female elk calves (n = 41) were captured from western Wyoming and transported to the Thorne-Williams Wildlife Research Center where CWD has been documented since 1979. Elk were held in contaminated pens for 14 to 20 days before being alternately assigned to either a vaccine (n = 21) or control group (n = 20). Vaccinated animals initially received two vaccinations approximately 42 days apart and annual vaccinations thereafter. Vaccination induced elevated YYR-specific antibody titers in all animals. Elk were genotyped for the prion protein gene at codon 132, monitored for clinical signs of CWD through daily observation, for disease status through periodic biopsy of rrectoanal mucosa-associated lympoid tissue (RAMALT), and monitored for YYR-specific serum antibody titres. Mean survival of vaccinated elk with the 132MM genotype (n = 15) was significantly shorter (800 days) than unvaccinated elk (n = 13) of the same genotype (1062 days; p = 0.003). Mean days until positive RAMALT biopsy for 132MM vaccinated elk (6 7 8) were significantly shorter than unvaccinated 132MM elk (990; p = 0.012). There was, however, no significant difference in survival between vaccinated (n = 4) and control (n = 5) elk with the 132ML genotype (p = 0.35) or in timing of positive RAMALT biopsies of 132ML elk (p = 0.66). There was no strong (p = 0.17) correlation between YYR-specific antibody titers and survival time. Determining the mechanism by which this vaccine accelerates onset of CWD will be important to direct further CWD vaccine research.

A Bioluminescent Cell Assay to Quantify Prion Protein Dimerization.

The prion protein (PrP) is a cell surface protein that in disease misfolds and becomes infectious causing Creutzfeldt-Jakob disease in humans, scrapie in sheep, and chronic wasting disease in deer and elk. Little is known regarding the dimerization of PrP and its role in disease. We developed a bioluminescent prion assay (BPA) to quantify PrP dimerization by bimolecular complementation of split Gaussia luciferase (GLuc) halves that are each fused to PrP. Fusion constructs between PrP and N- and C-terminal GLuc halves were expressed on the surface of RK13 cells (RK13-DC cells) and dimerized to yield a bioluminescent signal that was decreased in the presence of eight different antibodies to PrP. Dimerization of PrP was independent of divalent cations and was induced under stress. Challenge of RK13-DC cells with seven different prion strains did not lead to detectable infection but was measurable by bioluminescence. Finally, we used BPA to screen a compound library for compounds inhibiting PrP dimerization. One of the most potent compounds to inhibit PrP dimerization was JTC-801, which also inhibited prion replication in RML-infected ScN2a and SMB cells with an EC50 of 370 nM and 220 nM, respectively. We show here that BPA is a versatile tool to study prion biology and to identify anti-prion compounds.

Prion protein modulates iron transport in the anterior segment: Implications for ocular iron homeostasis and prion transmission.

Iron is an essential biometal in the aqueous humor, the principal source of nutrients for the avascular cornea and the lens. Here, we explored whether the ciliary body (CB), the source of aqueous humor, transports iron, and if the prion protein (PrPC) facilitates this process as in the outer retina. Using a combination of human, bovine, and mouse eyes as models, we report the expression of iron export proteins ferroportin and ceruloplasmin, and major iron uptake and storage proteins transferrin, transferrin receptor, and ferritin in the ciliary epithelium, indicating active exchange of iron at this site. Ferroportin and transferrin receptor are also expressed in the corneal endothelium. However, the relative expression of iron export and uptake proteins suggests export from the ciliary epithelium and import by corneal endothelium. In addition, abundant expression of PrPC, a ferrireductase that facilitates iron transport, is noted in pigmented and non-pigmented epithelium of the CB, posterior pigmented epithelium of the iris, corneal endothelium and epithelium, and lens epithelium. Notably, majority of PrPC in the ciliary epithelium is cleaved at the ß-site as in retinal pigment epithelial cells, suggesting a role in iron transport. Most of the PrPC in the cornea, however, is full-length, and susceptible to aggregation by intracerebrally inoculated PrP-scrapie, an infectious conformation of PrPC responsible for human and animal prion disorders. Soluble PrPC is present in the aqueous and vitreous humor, a provocative observation with significant implications. Together, these observations suggest independent cycling of iron in the anterior segment, and a prominent role of PrPC in this process. Aggregation of PrPC in the cornea of PrP-scrapie-infected animals raises the alarming possibility of transmission of animal prions through corneal abrasions.

EVALUATION OF A TEST AND CULL STRATEGY FOR REDUCING PREVALENCE OF CHRONIC WASTING DISEASE IN MULE DEER ( ODOCOILEUS HEMIONUS).

We evaluated a test and cull strategy for lowering chronic wasting disease (CWD) prevalence in a naturally-infected, free-ranging mule deer ( Odocoileus hemionus) herd wintering in the town of Estes Park, Colorado, US and in nearby Rocky Mountain National Park. We tested 48-68% of the estimated number of adult (≥1 yr old) deer annually for 5 yr via tonsil biopsy immunohistochemistry (IHC), collecting 1,251 samples from >700 individuals and removing IHC-positive deer. Among males, CWD prevalence during the last 3 yr of selective culling was lower (one-sided Fisher's exact test P=0.014) than in the period prior. In contrast, CWD prevalence among females before culling and after culling were equivalent ( P=0.777). Relatively higher annual testing of males (mean 77%) compared to females (mean 51%) might have contributed to differences seen in responses to management. A more intensive and sustained effort or modified spatial approach might have reduced prevalence more consistently in both sexes. Limitations of this technique in wider management application include cost and labor as well as property access and animal tolerance to repeated capture. However, elements of this approach could potentially be used to augment harvest-based disease management.