Norwegian moose CWD induces clinical disease and neuroinvasion in gene-targeted mice expressing cervid S138N prion protein.

Chronic wasting disease (CWD) is a prion disease affecting deer, elk and moose in North America and reindeer, moose and red deer in Northern Europe. Pathogenesis is driven by the accumulation of PrPSc, a pathological form of the host's cellular prion protein (PrPC), in the brain. CWD is contagious among North American cervids and Norwegian reindeer, with prions commonly found in lymphatic tissue. In Nordic moose and red deer CWD appears exclusively in older animals, and prions are confined to the CNS and undetectable in lymphatic tissues, indicating a sporadic origin. We aimed to determine transmissibility, neuroinvasion and lymphotropism of Nordic CWD isolates using gene-targeted mice expressing either wild-type (138SS/226QQ) or S138N (138NN/226QQ) deer PrP. When challenged with North American CWD strains, mice expressing S138N PrP did not develop clinical disease but harbored prion seeding activity in brain and spleen. Here, we infected these models intracerebrally or intraperitoneally with Norwegian moose, red deer and reindeer CWD isolates. The moose isolate was the first CWD type to cause full-blown disease in the 138NN/226QQ model in the first passage, with 100% attack rate and shortened survival times upon second passage. Furthermore, we detected prion seeding activity or PrPSc in brains and spinal cords, but not spleens, of 138NN/226QQ mice inoculated intraperitoneally with the moose isolate, providing evidence of prion neuroinvasion. We also demonstrate, for the first time, that transmissibility of the red deer CWD isolate was restricted to transgenic mice overexpressing elk PrPC (138SS/226EE), identical to the PrP primary structure of the inoculum. Our findings highlight that susceptibility to clinical disease is determined by the conformational compatibility between prion inoculum and host PrP primary structure. Our study indicates that neuroinvasion of Norwegian moose prions can occur without, or only very limited, replication in the spleen, an unprecedented finding for CWD.

Extraneural infection route restricts prion conformational variability and attenuates the impact of quaternary structure on infectivity.

Prions can exist as different strains that consist of conformational variants of the misfolded, pathogenic prion protein isoform PrPSc. Defined by stably transmissible biological and biochemical properties, strains have been identified in a spectrum of prion diseases, including chronic wasting disease (CWD) of wild and farmed cervids. CWD is highly contagious and spreads via direct and indirect transmission involving extraneural sites of infection, peripheral replication and neuroinvasion of prions. Here, we investigated the impact of infection route on CWD prion conformational selection and propagation. We used gene-targeted mouse models expressing deer PrP for intracerebral or intraperitoneal inoculation with fractionated or unfractionated brain homogenates from white-tailed deer, harboring CWD strains Wisc-1 or 116AG. Upon intracerebral inoculation, Wisc-1 and 116AG-inoculated mice differed in conformational stability of PrPSc. In brains of mice infected intraperitoneally with either inoculum, PrPSc propagated with identical conformational stability and fewer PrPSc deposits in most brain regions than intracerebrally inoculated animals. For either inoculum, PrPSc conformational stability in brain and spinal cord was similar upon intracerebral infection but significantly higher in spinal cords of intraperitoneally infected animals. Inoculation with fractionated brain homogenates resulted in lower variance of survival times upon intraperitoneal compared to intracerebral infection. In summary, we demonstrate that extraneural infection mitigates the impact of PrPSc quaternary structure on infection and reduces conformational variability of PrPSc propagated in the brain. These findings provide new insights into the evolution of stable CWD strains in natural, extraneural transmissions.

Lack of Transmission of Chronic Wasting Disease Prions to Human Cerebral Organoids.

Chronic wasting disease (CWD) is a cervid prion disease with unknown zoonotic potential that might pose a risk to humans who are exposed. To assess the potential of CWD to infect human neural tissue, we used human cerebral organoids with 2 different prion genotypes, 1 of which has previously been associated with susceptibility to zoonotic prion disease. We exposed organoids from both genotypes to high concentrations of CWD inocula from 3 different sources for 7 days, then screened for infection periodically for up to 180 days. No de novo CWD propagation or deposition of protease-resistant forms of human prions was evident in CWD-exposed organoids. Some persistence of the original inoculum was detected, which was equivalent in prion gene knockout organoids and thus not attributable to human prion propagation. Overall, the unsuccessful propagation of CWD in cerebral organoids supports a strong species barrier to transmission of CWD prions to humans.

A review of chronic wasting disease (CWD) spread, surveillance, and control in the United States captive cervid industry.

Chronic wasting disease (CWD) is a fatal prion disease of the family Cervidae that circulates in both wild and captive cervid populations. This disease threatens the health and economic viability of the captive cervid industry, which raises cervids in contained spaces for purposes such as hunting and breeding. Given the high transmissibility and long incubation period of CWD, the introduction and propagation of the infectious prion protein within and between captive cervid farms could be devastating to individual facilities and to the industry as a whole. Despite this risk, there does not yet exist a literature review that summarizes the scientific knowledge, to date, about CWD spread, surveillance, or control measures. Our review, which focused on peer reviewed, primary research conducted in the United States, sought to address this need by searching Google Scholar, Scopus, and Web of Science with a five-term keyword string containing terms related to the (1) location, (2) species affected, (3) disease, (4) captive cervid industry, and (5) topic of focus. Between the three databases, there were 190 articles that were selected for further examination. Those articles were then read to determine if they were about CWD spread, surveillance, and/or control in captive cervid facilities. The 22 articles that met these inclusion criteria were evaluated in detail and discussed, with recommendations for future collaborative work between captive cervid owners, government agencies, and researchers. This work will help to address, inform, and mitigate the rising problem of CWD spread and establishment.

Prion forensics: a multidisciplinary approach to investigate CWD at an illegal deer carcass disposal site.

Infectious prions are resistant to degradation and remain infectious in the environment for several years. Chronic wasting disease (CWD) has been detected in cervids inhabiting North America, the Nordic countries, and South Korea. CWD-prion spread is partially attributed to carcass transport and disposal. We employed a forensic approach to investigate an illegal carcass dump site connected with a CWD-positive herd. We integrated anatomic, genetic, and prion amplification methods to discover CWD-positive remains from six white-tailed deer (Odocoileus virginianus) and, using microsatellite markers, confirmed a portion originated from the CWD-infected herd. This approach provides a foundation for future studies of carcass prion transmission risk.

Oral vaccination as a potential strategy to manage chronic wasting disease in wild cervid populations.

Prion diseases are a novel class of infectious disease based in the misfolding of the cellular prion protein (PrPC) into a pathological, self-propagating isoform (PrPSc). These fatal, untreatable neurodegenerative disorders affect a variety of species causing scrapie in sheep and goats, bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease (CWD) in cervids, and Creutzfeldt-Jacob disease (CJD) in humans. Of the animal prion diseases, CWD is currently regarded as the most significant threat due its ongoing geographical spread, environmental persistence, uptake into plants, unpredictable evolution, and emerging evidence of zoonotic potential. The extensive efforts to manage CWD have been largely ineffective, highlighting the need for new disease management tools, including vaccines. Development of an effective CWD vaccine is challenged by the unique biology of these diseases, including the necessity, and associated dangers, of overcoming immune tolerance, as well the logistical challenges of vaccinating wild animals. Despite these obstacles, there has been encouraging progress towards the identification of safe, protective antigens as well as effective strategies of formulation and delivery that would enable oral delivery to wild cervids. In this review we highlight recent strategies for antigen selection and optimization, as well as considerations of various platforms for oral delivery, that will enable researchers to accelerate the rate at which candidate CWD vaccines are developed and evaluated.

Detection of Chronic Wasting Disease Prions in Fetal Tissues of Free-Ranging White-Tailed Deer.

The transmission of chronic wasting disease (CWD) has largely been attributed to contact with infectious prions shed in excretions (saliva, urine, feces, blood) by direct animal-to-animal exposure or indirect contact with the environment. Less-well studied has been the role that mother-to-offspring transmission may play in the facile transmission of CWD, and whether mother-to-offspring transmission before birth may contribute to the extensive spread of CWD. We thereby focused on a population of free-ranging white-tailed deer from West Virginia, USA, in which CWD has been detected. Fetal tissues, ranging from 113 to 158 days of gestation, were harvested from the uteri of CWD+ dams in the asymptomatic phase of infection. Using serial protein misfolding amplification (sPMCA), we detected evidence of prion seeds in 7 of 14 fetuses (50%) from 7 of 9 pregnancies (78%), with the earliest detection at 113 gestational days. This is the first report of CWD detection in free ranging white-tailed deer fetal tissues. Further investigation within cervid populations across North America will help define the role and impact of mother-to-offspring vertical transmission of CWD.

Chronic wasting disease: a cervid prion infection looming to spillover.

The spread of chronic wasting disease (CWD) during the last six decades has resulted in cervid populations of North America where CWD has become enzootic. This insidious disease has also been reported in wild and captive cervids from other continents, threatening ecosystems, livestock and public health. These CWD "hot zones" are particularly complex given the interplay between cervid PRNP genetics, the infection biology, the strain diversity of infectious prions and the long-term environmental persistence of infectivity, which hinder eradication efforts. Here, we review different aspects of CWD including transmission mechanisms, pathogenesis, epidemiology and assessment of interspecies infection. Further understanding of these aspects could help identify "control points" that could help reduce exposure for humans and livestock and decrease CWD spread between cervids.

Evaluation of Winter Ticks (Dermacentor albipictus) Collected from North American Elk (Cervus canadensis) in an Area of Chronic Wasting Disease Endemicity for Evidence of PrPCWD Amplification Using Real-Time Quaking-Induced Conversion Assay.

Chronic wasting disease (CWD) is a progressive and fatal spongiform encephalopathy of deer and elk species, caused by a misfolded variant of the normal prion protein. Horizontal transmission of the misfolded CWD prion between animals is thought to occur through shedding in saliva and other forms of excreta. The role of blood in CWD transmission is less clear, though infectivity has been demonstrated in various blood fractions. Blood-feeding insects, including ticks, are known vectors for a range of bacterial and viral infections in animals and humans, though to date, there has been no evidence for their involvement in prion disease transmission. In the present study, we evaluated winter ticks (Dermacentor albipictus) collected from 136 North American elk (Cervus canadensis) in an area where CWD is endemic for evidence of CWD prion amplification using the real-time quaking-induced conversion assay (RT-QuIC). Although 30 elk were found to be CWD positive (22%) postmortem, amplifiable prions were found in just a single tick collected from an elk in advanced stages of CWD infection, with some evidence for prions in ticks collected from elk in mid-stage infection. These findings suggest that further investigation of ticks as reservoirs for prion disease may be warranted. IMPORTANCE This study reports the first finding of detectable levels of prions linked to chronic wasting disease in a tick collected from a clinically infected elk. Using the real-time quaking-induced conversion assay (RT-QuIC), "suspect" samples were also identified; these suspect ticks were more likely to have been collected from CWD-positive elk, though suspect amplification was also observed in ticks collected from CWD-negative elk. Observed levels were at the lower end of our detection limits, though our findings suggest that additional research evaluating ticks collected from animals in late-stage disease may be warranted to further evaluate the role of ticks as potential vectors of chronic wasting disease.

Chronic Wasting Disease Transmission Risk Assessment for Farmed Cervids in Minnesota and Wisconsin.

CWD (chronic wasting disease) has emerged as one of the most important diseases of cervids and continues to adversely affect farmed and wild cervid populations, despite control and preventive measures. This study aims to use the current scientific understanding of CWD transmission and knowledge of farmed cervid operations to conduct a qualitative risk assessment for CWD transmission to cervid farms and, applying this risk assessment, systematically describe the CWD transmission risks experienced by CWD-positive farmed cervid operations in Minnesota and Wisconsin. A systematic review of literature related to CWD transmission informed our criteria to stratify CWD transmission risks to cervid operations into high-risk low uncertainty, moderate-risk high uncertainty, and negligible-risk low uncertainty categories. Case data from 34 CWD-positive farmed cervid operations in Minnesota and Wisconsin from 2002 to January 2019 were categorized by transmission risks exposure and evaluated for trends. The majority of case farms recorded high transmission risks (56%), which were likely sources of CWD, but many (44%) had only moderate or negligible transmission risks, including most of the herds (62%) detected since 2012. The presence of CWD-positive cervid farms with only moderate or low CWD transmission risks necessitates further investigation of these risks to inform effective control measures.

Adaptive selection of a prion strain conformer corresponding to established North American CWD during propagation of novel emergent Norwegian strains in mice expressing elk or deer prion protein.

Prions are infectious proteins causing fatal, transmissible neurodegenerative diseases of animals and humans. Replication involves template-directed refolding of host encoded prion protein, PrPC, by its infectious conformation, PrPSc. Following its discovery in captive Colorado deer in 1967, uncontrollable contagious transmission of chronic wasting disease (CWD) led to an expanded geographic range in increasing numbers of free-ranging and captive North American (NA) cervids. Some five decades later, detection of PrPSc in free-ranging Norwegian (NO) reindeer and moose marked the first indication of CWD in Europe. To assess the properties of these emergent NO prions and compare them with NA CWD we used transgenic (Tg) and gene targeted (Gt) mice expressing PrP with glutamine (Q) or glutamate (E) at residue 226, a variation in wild type cervid PrP which influences prion strain selection in NA deer and elk. Transmissions of NO moose and reindeer prions to Tg and Gt mice recapitulated the characteristic features of CWD in natural hosts, revealing novel prion strains with disease kinetics, neuropathological profiles, and capacities to infect lymphoid tissues and cultured cells that were distinct from those causing NA CWD. In support of strain variation, PrPSc conformers comprising emergent NO moose and reindeer CWD were subject to selective effects imposed by variation at residue 226 that were different from those controlling established NA CWD. Transmission of particular NO moose CWD prions in mice expressing E at 226 resulted in selection of a kinetically optimized conformer, subsequent transmission of which revealed properties consistent with NA CWD. These findings illustrate the potential for adaptive selection of strain conformers with improved fitness during propagation of unstable NO prions. Their potential for contagious transmission has implications for risk analyses and management of emergent European CWD. Finally, we found that Gt mice expressing physiologically controlled PrP levels recapitulated the lymphotropic properties of naturally occurring CWD strains resulting in improved susceptibilities to emergent NO reindeer prions compared with over-expressing Tg counterparts. These findings underscore the refined advantages of Gt models for exploring the mechanisms and impacts of strain selection in peripheral compartments during natural prion transmission.

New and distinct chronic wasting disease strains associated with cervid polymorphism at codon 116 of the Prnp gene.

Chronic wasting disease (CWD) is a prion disease affecting cervids. Polymorphisms in the prion protein gene can result in extended survival of CWD-infected animals. However, the impact of polymorphisms on cellular prion protein (PrPC) and prion properties is less understood. Previously, we characterized the effects of a polymorphism at codon 116 (A>G) of the white-tailed deer (WTD) prion protein and determined that it destabilizes PrPC structure. Comparing CWD isolates from WTD expressing homozygous wild-type (116AA) or heterozygous (116AG) PrP, we found that 116AG-prions were conformationally less stable, more sensitive to proteases, with lower seeding activity in cell-free conversion and reduced infectivity. Here, we aimed to understand CWD strain emergence and adaptation. We show that the WTD-116AG isolate contains two different prion strains, distinguished by their host range, biochemical properties, and pathogenesis from WTD-116AA prions (Wisc-1). Serial passages of WTD-116AG prions in tg(CerPrP)1536+/+ mice overexpressing wild-type deer-PrPC revealed two populations of mice with short and long incubation periods, respectively, and remarkably prolonged clinical phase upon inoculation with WTD-116AG prions. Inoculation of serially diluted brain homogenates confirmed the presence of two strains in the 116AG isolate with distinct pathology in the brain. Interestingly, deglycosylation revealed proteinase K-resistant fragments with different electrophoretic mobility in both tg(CerPrP)1536+/+ mice and Syrian golden hamsters infected with WTD-116AG. Infection of tg60 mice expressing deer S96-PrP with 116AG, but not Wisc-1 prions induced clinical disease. On the contrary, bank voles resisted 116AG prions, but not Wisc-1 infection. Our data indicate that two strains co-existed in the WTD-116AG isolate, expanding the variety of CWD prion strains. We argue that the 116AG isolate does not contain Wisc-1 prions, indicating that the presence of 116G-PrPC diverted 116A-PrPC from adopting a Wisc-1 structure. This can have important implications for their possible distinct capacities to cross species barriers into both cervids and non-cervids.

Second passage of chronic wasting disease of mule deer to sheep by intracranial inoculation compared to classical scrapie.

The origin of chronic wasting disease (CWD) in cervids is unclear. One hypothesis suggests that CWD originated from scrapie in sheep. We compared the disease phenotype of sheep-adapted CWD to classical scrapie in sheep. We inoculated sheep intracranially with brain homogenate from first-passage mule deer CWD in sheep (sCWDmd). The attack rate in second-passage sheep was 100% (12 of 12). Sheep had prominent lymphoid accumulations of PrPSc reminiscent of classical scrapie. The pattern and distribution of PrPSc in the brains of sheep with CWDmd was similar to scrapie strain 13-7 but different from scrapie strain x124. The western blot glycoprofiles of sCWDmd were indistinguishable from scrapie strain 13-7; however, independent of sheep genotype, glycoprofiles of sCWDmd were different than x124. When sheep genotypes were evaluated individually, there was considerable overlap in the glycoprofiles that precluded significant discrimination between sheep CWD and scrapie strains. Our data suggest that the phenotype of CWD in sheep is indistinguishable from some strains of scrapie in sheep. Given our results, current detection techniques would be unlikely to distinguish CWD in sheep from scrapie in sheep if cross-species transmission occurred naturally. It is unknown if sheep are naturally vulnerable to CWD; however, the susceptibility of sheep after intracranial inoculation and lymphoid accumulation indicates that the species barrier is not absolute.

Exposure Risk of Chronic Wasting Disease in Humans.

The majority of human prion diseases are sporadic, but acquired disease can occur, as seen with variant Creutzfeldt-Jakob disease (vCJD) following consumption of bovine spongiform encephalopathy (BSE). With increasing rates of cervid chronic wasting disease (CWD), there is concern that a new form of human prion disease may arise. Currently, there is no evidence of transmission of CWD to humans, suggesting the presence of a strong species barrier; however, in vitro and in vivo studies on the zoonotic potential of CWD have yielded mixed results. The emergence of different CWD strains is also concerning, as different strains can have different abilities to cross species barriers. Given that venison consumption is common in areas where CWD rates are on the rise, increased rates of human exposure are inevitable. If CWD was to infect humans, it is unclear how it would present clinically; in vCJD, it was strain-typing of vCJD prions that proved the causal link to BSE. Therefore, the best way to screen for CWD in humans is to have thorough strain-typing of harvested cervids and human CJD cases so that we will be in a position to detect atypical strains or strain shifts within the human CJD population.

Experimental oral transmission of chronic wasting disease to sika deer (Cervus nippon).

Chronic wasting disease (CWD) affects a broad array of cervid species and continues to be detected in an expanding geographic range. Initially introduced into the Republic of Korea through the importation of CWD-infected elk (Cervus canadensis), additional cases of CWD were subsequently detected in farmed Korean elk and sika deer (Cervus nippon). Wild and farmed sika deer are found in many regions of Asia, North America, and Europe, although natural transmission to this species has not been detected outside of the Republic of Korea. In this study, the oral transmission of CWD to sika deer was investigated using material from CWD-affected elk. Pathological prion (PrPCWD) immunoreactivity was detected in oropharyngeal lymphoid tissues of one sika deer at 3.9 months post-inoculation (mpi) and was more widely distributed in a second sika deer examined at 10.9 mpi. The remaining four sika deer progressed to clinical disease between 21 and 24 mpi. Analysis of PrPCWD tissue distribution in clinical sika deer revealed widespread deposition in central and peripheral nervous systems, lymphoreticular tissues, and the gastrointestinal tract. Prion protein gene (PRNP) sequences of these sika deer were identical and consistent with those reported in natural sika deer populations. These findings demonstrate the efficient oral transmission of CWD from elk to sika deer.

Studies in bank voles reveal strain differences between chronic wasting disease prions from Norway and North America.

Chronic wasting disease (CWD) is a relentless epidemic disorder caused by infectious prions that threatens the survival of cervid populations and raises increasing public health concerns in North America. In Europe, CWD was detected for the first time in wild Norwegian reindeer (Rangifer tarandus) and moose (Alces alces) in 2016. In this study, we aimed at comparing the strain properties of CWD prions derived from different cervid species in Norway and North America. Using a classical strain typing approach involving transmission and adaptation to bank voles (Myodes glareolus), we found that prions causing CWD in Norway induced incubation times, neuropathology, regional deposition of misfolded prion protein aggregates in the brain, and size of their protease-resistant core, different from those that characterize North American CWD. These findings show that CWD prion strains affecting Norwegian cervids are distinct from those found in North America, implying that the highly contagious North American CWD prions are not the proximate cause of the newly discovered Norwegian CWD cases. In addition, Norwegian CWD isolates showed an unexpected strain variability, with reindeer and moose being caused by different CWD strains. Our findings shed light on the origin of emergent European CWD, have significant implications for understanding the nature and the ecology of CWD in Europe, and highlight the need to assess the zoonotic potential of the new CWD strains detected in Europe.

Successful transmission of the chronic wasting disease (CWD) agent to white-tailed deer by intravenous blood transfusion.

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy (TSEs) that affects free-ranging and captive cervid species. The infectious agent of CWD may be transmitted from ingestion of prions shed in bodily fluids (e.g. feces, urine, saliva, placenta tissue) of infected animals, contaminated pastures, and/or decomposing carcasses from dead animals. Studies have also demonstrated prion infectivity in whole blood or blood fractions of CWD infected animals. To determine if CWD-infected blood contained sufficient levels of prion infectivity to cause disease, recipient deer were inoculated intravenously (IV) with blood derived from a CWD-infected white-tailed deer. We found that the CWD agent can be successfully transmitted to white-tailed deer by a single intravenous blood transfusion. The incubation period was associated with recipient prion protein genotype at codon 96 with the GG96 recipient incubating for 25.6 months and the GS96 recipient incubating for 43.6 months. This study complements and supports an earlier finding that CWD can be transmitted to deer by intravenous blood transfusion from white-tailed deer with CWD.

Hunting strategies to increase detection of chronic wasting disease in cervids.

The successful mitigation of emerging wildlife diseases may involve controversial host culling. For livestock, 'preemptive host culling' is an accepted practice involving the removal of herds with known contact to infected populations. When applied to wildlife, this proactive approach comes in conflict with biodiversity conservation goals. Here, we present an alternative approach of 'proactive hunting surveillance' with the aim of early disease detection that simultaneously avoids undesirable population decline by targeting demographic groups with (1) a higher likelihood of being infected and (2) a lower reproductive value. We applied this harvesting principle to populations of reindeer to substantiate freedom of chronic wasting disease (CWD) infection. Proactive hunting surveillance reached 99% probability of freedom from infection (<4 reindeer infected) within 3-5 years, in comparison to ~10 years using ordinary harvest surveillance. However, implementation uncertainties linked to social issues appear challenging also with this kind of host culling.

Failure To Detect Prion Infectivity in Ticks following Prion-Infected Blood Meal.

Chronic wasting disease (CWD) is an emerging and fatal contagious prion disease that affects cervids, including mule deer, white-tailed deer, black-tailed deer, red deer reindeer, elk, and moose. CWD prions are widely distributed throughout the bodies of CWD-infected animals and are found in the nervous system, lymphoid tissues, muscle, blood, urine, feces, and antler velvet. The mechanism of CWD transmission in natural settings is unknown. Potential mechanisms of transmission include horizontal, maternal, or environmental routes. Due to the presence of prions in the blood of CWD-infected animals, the potential exists for invertebrates that feed on mammalian blood to contribute to the transmission of CWD. The geographic range of the Rocky Mountain Wood tick, Dermancentor andersoni, overlaps with CWD throughout the northwest United States and southwest Canada, raising the possibility that D. andersoni parasitization of cervids may be involved in CWD transmission. We investigated this possibility by examining the blood meal of D. andersoni that fed upon prion-infected hamsters for the presence of prion infectivity by animal bioassay. None of the hamsters inoculated with a D. andersoni blood meal that had been ingested from prion-infected hamsters developed clinical signs of prion disease or had evidence for a subclinical prion infection. Overall, the data do not demonstrate a role for D. andersoni in the transmission of prion disease.IMPORTANCE Chronic wasting disease (CWD) is an emerging prion disease that affects cervids, including mule deer, white-tailed deer, black-tailed deer, red deer reindeer, elk, and moose. The mechanism of CWD transmission in unknown. Due to the presence of prions in the blood of CWD-infected animals, it is possible for invertebrates that feed on cervid blood to contribute to the transmission of CWD. We examined the blood meal of D. andersoni, a tick with a similar geographic range as cervids, that fed upon prion-infected hamsters for the presence of prion infectivity by animal bioassay. None of the D. andersoni blood meals that had been ingested from prion-infected hamsters yielded evidence of prion infection. Overall, the data do not support a role of D. andersoni in the transmission of prion disease.

Very low oral exposure to prions of brain or saliva origin can transmit chronic wasting disease.

The minimum infectious dose required to induce CWD infection in cervids remains unknown, as does whether peripherally shed prions and/or multiple low dose exposures are important factors in CWD transmission. With the goal of better understand CWD infection in nature, we studied oral exposures of deer to very low doses of CWD prions and also examined whether the frequency of exposure or prion source may influence infection and pathogenesis. We orally inoculated white-tailed deer with either single or multiple divided doses of prions of brain or saliva origin and monitored infection by serial longitudinal tissue biopsies spanning over two years. We report that oral exposure to as little as 300 nanograms (ng) of CWD-positive brain or to saliva containing seeding activity equivalent to 300 ng of CWD-positive brain, were sufficient to transmit CWD disease. This was true whether the inoculum was administered as a single bolus or divided as three weekly 100 ng exposures. However, when the 300 ng total dose was apportioned as 10, 30 ng doses delivered over 12 weeks, no infection occurred. While low-dose exposures to prions of brain or saliva origin prolonged the time from inoculation to first detection of infection, once infection was established, we observed no differences in disease pathogenesis. These studies suggest that the CWD minimum infectious dose approximates 100 to 300 ng CWD-positive brain (or saliva equivalent), and that CWD infection appears to conform more with a threshold than a cumulative dose dynamic.