Chronic Wasting Disease: State of the Science.

Chronic wasting disease (CWD) is a prion disease affecting cervid species, both free-ranging and captive populations. As the geographic range continues to expand and disease prevalence continues to increase, CWD will have an impact on cervid populations, local economies, and ecosystem health. Mitigation of this "wicked" disease will require input from many different stakeholders including hunters, landowners, research biologists, wildlife managers, and others, working together. The NC1209 (North American interdisciplinary chronic wasting disease research consortium) is composed of scientists from different disciplines involved with investigating and managing CWD. Leveraging this broad breadth of expertise, the Consortium has created a state-of-the-science review of five key aspects of CWD, including current diagnostic capabilities for detecting prions, requirements for validating these diagnostics, the role of environmental transmission in CWD dynamics, and potential zoonotic risks associated with CWD. The goal of this review is to increase stakeholders', managers', and decision-makers' understanding of this disease informed by current scientific knowledge.

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.

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.

PMCA screening of retropharyngeal lymph nodes in white-tailed deer and comparisons with ELISA and IHC.

Chronic wasting disease (CWD) is a prion disease affecting cervids. CWD diagnosis is conducted through enzyme-linked immunosorbent assay (ELISA) and immunohistochemistry (IHC) in retropharyngeal lymph nodes. Unfortunately, these techniques have limited sensitivity against the biomarker (CWD-prions). Two in vitro prion amplification techniques, real-time quaking-induced conversion (RT-QuIC) and protein misfolding cyclic amplification (PMCA), have shown promise in detecting CWD-prions in tissues and bodily fluids. Recent studies have demonstrated that RT-QuIC yields similar results compared to ELISA and IHC. Here, we analyzed 1003 retropharyngeal lymph nodes (RPLNs) from Texas white-tailed deer. PMCA detected CWD at a higher rate compared to ELISA/IHC, identified different prion strains, and revealed the presence of CWD-prions in places with no previous history. These findings suggest that PMCA exhibits greater sensitivity than current standard techniques and could be valuable for rapid and strain-specific CWD detection.

Identification of chronic wasting disease prions in decaying tongue tissues from exhumed white-tailed deer.

Chronic wasting disease (CWD) prions cause fatal neuropathies in farmed and free-ranging cervids. The deposition of prions in natural and humanmade environmental components has been implicated as a major mechanism mediating CWD spread in wild and captive populations. Prions can be deposited in the environment through excreta, tissues, and carcasses from pre-clinical and clinical animals. Furthermore, burial of CWD-positive animals may reduce but not completely mitigate prion spread from carcasses into the surrounding environment. Here, we analyzed exhumed, decaying deer carcasses for the presence of CWD prions. By analyzing tongue tissues through the protein misfolding cyclic amplification (PMCA) technique, we were able to identify seven out of 95 exhumed white-tailed deer carcasses as CWD prions carriers. Confirmatory analyses were performed using the real-time quaking-induced conversion (RT-QuIC) technique. In addition, we evaluated the potential contamination of the pens that housed these animals by swabbing feeders and waterers. PMCA analyses of swabs confirmed CWD contamination on farming equipment. This work demonstrates the usefulness of PMCA to detect CWD prions in a variety of contexts, including exhumed/decaying tissues. In addition, this is the first report demonstrating swabbing coupled with PMCA as a method for the detection of prion seeding activity on naturally exposed surfaces. Considering that this study was focused on a single site, further studies should confirm whether prion amplification assays are useful to identify CWD prions not only in animals but also in the environment that contains them. IMPORTANCE Environmental contamination is thought to be a major player in the spread of chronic wasting disease (CWD), a fatal prion disease affecting a wide variety of cervid species. At present, there are no officially approved methods allowing for the detection of prion infectivity in environmental components. Importantly, animal as well as anthropogenic activities are thought to contribute to prion environmental contamination. Here, we detected CWD prions in exhumed white-tailed deer carcasses by using the protein misfolding cyclic amplification (PMCA) assay. In addition, we identified CWD prions in feeders used within the infected facility. These results highlight the potential role of PMCA in identifying prion infectivity in a variety of scenarios, ranging from decaying tissues to farming equipment.

Blocking ATP-releasing channels prevents high extracellular ATP levels and airway hyperreactivity in an asthmatic mouse model.

Allergic asthma is a chronic airway inflammatory response to different triggers like inhaled allergens. Excessive ATP in fluids from patients with asthma is considered an inflammatory signal and an important autocrine/paracrine modulator of airway physiology. Here, we investigated the deleterious effect of increased extracellular ATP (eATP) concentration on the mucociliary clearance (MCC) effectiveness and determined the role of ATP releasing channels during airway inflammation in an ovalbumin (OVA)-sensitized mouse model. Our allergic mouse model exhibited high levels of eATP measured in the tracheal fluid with a luciferin-luciferase assay and reduced MCC velocity determined by microspheres tracking in the trachea ex vivo. Addition of ATP had a dual effect on MCC, where lower ATP concentration (µM) increased microspheres velocity, whereas higher concentration (mM) transiently stopped microspheres movement. Also, an augmented ethidium bromide uptake by the allergic tracheal airway epithelium suggests an increase in ATP release channel functionality during inflammatory conditions. The use of carbenoxolone, a nonspecific inhibitor of connexin and pannexin1 channels reduced the eATP concentration in the allergic mouse tracheal fluid and dye uptake by the airway epithelium, providing evidence that these ATP release channels are facilitating the net flux of ATP to the lumen during airway inflammation. However, only the specific inhibition of pannexin1 with 10Panx peptide significantly reduced eATP in bronchoalveolar lavage and decreased airway hyperresponsiveness in OVA-allergic mouse model. These data provide evidence that blocking eATP may be a pharmacological alternative to be explored in rescue therapy during episodes of airflow restriction in patients with asthma.