Aerosol Transmission of Chronic Wasting Disease in White-Tailed Deer

While the facile transmission of chronic wasting disease (CWD) remains incompletely elucidated, studies in rodents suggest that exposure of the respiratory mucosa may be an efficient pathway. The present study was designed to address this question in the native cervid host. Here, we demonstrate aerosol transmission of CWD to deer with a prion dose >20-fold lower than that used in previous oral inoculations. Inhalation of prions may facilitate transmission of CWD and, perhaps, other prion infections.     

Resistance to chronic wasting disease in transgenic mice expressing a naturally occurring allelic variant of deer prion protein

Prion protein (PrP) is a required factor for susceptibility to transmissible spongiform encephalopathy or prion diseases. In transgenic mice, expression of prion protein (PrP) from another species often confers susceptibility to prion disease from that donor species. For example, expression of deer or elk PrP in transgenic mice has induced susceptibility to chronic wasting disease (CWD), the prion disease of cervids. In the current experiments, transgenic mice expressing two naturally occurring allelic variants of deer PrP with either glycine (G) or serine (S) at residue 96 were found to differ in susceptibility to CWD infection. G96 mice were highly susceptible to infection, and disease appeared starting as early as 160 days postinfection. In contrast, S96 mice showed no evidence of disease or generation of disease-associated protease-resistant PrP (PrPres) over a 600-day period. At the time of clinical disease, G96 mice showed typical vacuolar pathology and deposition of PrPres in many brain regions, and in some individuals, extensive neuronal loss and apoptosis were noted in the hippocampus and cerebellum. Extraneural accumulation of PrPres was also noted in spleen and intestinal tissue of clinically ill G96 mice. These results demonstrate the importance of deer PrP polymorphisms in susceptibility to CWD infection. Furthermore, this deer PrP transgenic model is the first to demonstrate extraneural accumulation of PrPres in spleen and intestinal tissue and thus may prove useful in studies of CWD pathogenesis and transmission by oral or other natural routes of infection.     

Mother to Offspring Transmission of Chronic Wasting Disease in Reeves’ Muntjac Deer

The horizontal transmission of prion diseases has been well characterized in bovine spongiform encephalopathy (BSE), chronic wasting disease (CWD) of deer and elk and scrapie of sheep, and has been regarded as the primary mode of transmission. Few studies have monitored the possibility of vertical transmission occurring within an infected mother during pregnancy. To study the potential for and pathway of vertical transmission of CWD in the native cervid species, we used a small cervid model–the polyestrous breeding, indoor maintainable, Reeves’ muntjac deer–and determined that the susceptibility and pathogenesis of CWD in these deer reproduce that in native mule and white-tailed deer. Moreover, we demonstrate here that CWD prions are transmitted from doe to fawn. Maternal CWD infection also appears to result in lower percentage of live birth offspring. In addition, evolving evidence from protein misfolding cyclic amplification (PMCA) assays on fetal tissues suggest that covert prion infection occurs in utero. Overall, our findings demonstrate that transmission of prions from mother to offspring can occur, and may be underestimated for all prion diseases.     

Experimental oral transmission of chronic wasting disease to reindeer (Rangifer tarandus tarandus)

Chronic wasting disease (CWD), a transmissible spongiform encephalopathy of cervids, remains prevalent in North American elk, white-tailed deer and mule deer. A natural case of CWD in reindeer (Rangifer tarandus tarandus) has not been reported despite potential habitat overlap with CWD-infected deer or elk herds. This study investigates the experimental transmission of CWD from elk or white-tailed deer to reindeer by the oral route of inoculation. Ante-mortem testing of the three reindeer exposed to CWD from white-tailed deer identified the accumulation of pathological PrP (PrP(CWD)) in the recto-anal mucosa associated lymphoid tissue (RAMALT) of two reindeer at 13.4 months post-inoculation. Terminal CWD occurred in the two RAMALT-positive reindeer at 18.5 and 20 months post-inoculation while one other reindeer in the white-tailed deer CWD inoculum group and none of the 3 reindeer exposed to elk CWD developed disease. Tissue distribution analysis of PrP(CWD) in CWD-affected reindeer revealed widespread deposition in central and peripheral nervous systems, lymphoreticular tissues, the gastrointestinal tract, neuroendocrine tissues and cardiac muscle. Analysis of prion protein gene (PRNP) sequences in the 6 reindeer identified polymorphisms at residues 2 (V/M), 129 (G/S), 138 (S/N) and 169 (V/M). These findings demonstrate that (i) a sub-population of reindeer are susceptible to CWD by oral inoculation implicating the potential for transmission to other Rangifer species, and (ii) certain reindeer PRNP polymorphisms may be protective against CWD infection.     

Interspecies transmission of chronic wasting disease prions to squirrel monkeys (Saimiri sciureus)

Chronic wasting disease (CWD) is an emerging prion disease of deer and elk. The risk of CWD transmission to humans following exposure to CWD-infected tissues is unknown. To assess the susceptibility of nonhuman primates to CWD, two squirrel monkeys were inoculated with brain tissue from a CWD-infected mule deer. The CWD-inoculated squirrel monkeys developed a progressive neurodegenerative disease and were euthanized at 31 and 34 months postinfection. Brain tissue from the CWD-infected squirrel monkeys contained the abnormal isoform of the prion protein, PrP-res, and displayed spongiform degeneration. This is the first reported transmission of CWD to primates.     

Transmission of chronic wasting disease identifies a prion strain causing cachexia and heart infection in hamsters

Chronic wasting disease (CWD) is an emerging prion disease of free-ranging and captive cervids in North America. In this study we established a rodent model for CWD in Syrian golden hamsters that resemble key features of the disease in cervids including cachexia and infection of cardiac muscle. Following one to three serial passages of CWD from white-tailed deer into transgenic mice expressing the hamster prion protein gene, CWD was subsequently passaged into Syrian golden hamsters. In one passage line there were preclinical changes in locomotor activity and a loss of body mass prior to onset of subtle neurological symptoms around 340 days. The clinical symptoms included a prominent wasting disease, similar to cachexia, with a prolonged duration. Other features of CWD in hamsters that were similar to cervid CWD included the brain distribution of the disease-specific isoform of the prion protein, PrP(Sc), prion infection of the central and peripheral neuroendocrine system, and PrP(Sc) deposition in cardiac muscle. There was also prominent PrP(Sc) deposition in the nasal mucosa on the edge of the olfactory sensory epithelium with the lumen of the nasal airway that could have implications for CWD shedding into nasal secretions and disease transmission. Since the mechanism of wasting disease in prion diseases is unknown this hamster CWD model could provide a means to investigate the physiological basis of cachexia, which we propose is due to a prion-induced endocrinopathy. This prion disease phenotype has not been described in hamsters and we designate it as the 'wasting' or WST strain of hamster CWD.     

Early and Non-Invasive Detection of Chronic Wasting Disease Prions in Elk Feces by Real-Time Quaking Induced Conversion

Chronic wasting disease (CWD) is a fatal prion disease of wild and captive cervids in North America. Prions are infectious agents composed of a misfolded version of a host-encoded protein, termed PrP^Sc. Infected cervids excrete and secrete prions, contributing to lateral transmission. Geographical distribution is expanding and case numbers in wild cervids are increasing. Recently, the first European cases of CWD have been reported in a wild reindeer and two moose from Norway. Therefore, methods to detect the infection early in the incubation time using easily available samples are desirable to facilitate effective disease management. We have adapted the real-time quaking induced conversion (RT-QuIC) assay, a sensitive in vitro prion amplification method, for pre-clinical detection of prion seeding activity in elk feces. Testing fecal samples from orally inoculated elk taken at various time points post infection revealed early shedding and detectable prion seeding activity throughout the disease course. Early shedding was also found in two elk encoding a PrP genotype associated with reduced susceptibility for CWD. In summary, we suggest that detection of CWD prions in feces by RT-QuIC may become a useful tool to support CWD surveillance in wild and captive cervids. The finding of early shedding independent of the elk’s prion protein genotype raises the question whether prolonged survival is beneficial, considering accumulation of environmental prions and its contribution to CWD transmission upon extended duration of shedding.     

Cross-species transmission of CWD prions

Prions cause fatal neurodegenerative diseases in humans and animals and can be transmitted zoonotically. Chronic wasting disease (CWD) is a highly transmissible prion disease of wild deer and elk that affects cervids over extensive regions of the United States and Canada. The risk of cross-species CWD transmission has been experimentally evaluated in a wide array of mammals, including non-human primates and mouse models expressing human cellular prion protein. Here we review the determinants of cross-species CWD transmission, and propose a model that may explain a structural barrier for CWD transmission to humans.     

The ecology of chronic wasting disease in wildlife

Prions are misfolded infectious proteins responsible for a group of fatal neurodegenerative diseases termed transmissible spongiform encephalopathy or prion diseases. Chronic Wasting Disease (CWD) is the prion disease with the highest spillover potential, affecting at least seven Cervidae (deer) species. The zoonotic potential of CWD is inconclusive and cannot be ruled out. A risk of infection for other domestic and wildlife species is also plausible. Here, we review the current status of the knowledge with respect to CWD ecology in wildlife. Our current understanding of the geographic distribution of CWD lacks spatial and temporal detail, does not consider the biogeography of infectious diseases, and is largely biased by sampling based on hunters' cooperation and funding available for each region. Limitations of the methods used for data collection suggest that the extent and prevalence of CWD in wildlife is underestimated. If the zoonotic potential of CWD is confirmed in the short term, as suggested by recent results obtained in experimental animal models, there will be limited accurate epidemiological data to inform public health. Research gaps in CWD prion ecology include the need to identify specific biological characteristics of potential CWD reservoir species that better explain susceptibility to spillover, landscape and climate configurations that are suitable for CWD transmission, and the magnitude of sampling bias in our current understanding of CWD distribution and risk. Addressing these research gaps will help anticipate novel areas and species where CWD spillover is expected, which will inform control strategies. From an ecological perspective, control strategies could include assessing restoration of natural predators of CWD reservoirs, ultrasensitive CWD detection in biotic and abiotic reservoirs, and deer density and landscape modification to reduce CWD spread and prevalence.     

Chronic wasting disease: Fingerprinting the culprit in risk assessments

Transmissible spongiform encephalopathies (prion diseases) in animals may be associated with a zoonotic risk potential for humans as shown by the occurrence of variant Creutzfeldt-Jakob disease in the wake of the bovine spongiform encephalopathy epidemic. Thus, the increasing exposure of humans in North America to cervid prions of chronic wasting disease (CWD) in elk and deer has prompted comprehensive risk assessments. The susceptibility of humans to CWD infections is currently under investigation in different studies using macaques as primate models. The necessity for such studies was recently reinforced when disease-associated prion protein and its seeding activity were detected in muscles of clinically inconspicuous CWD-infected white-tailed deer (WTD). Increasing evidence points to the existence of different CWD strains and CWD prions may also change or newly emerge over time. Therefore, CWD isolates examined in macaques should be characterized as precisely as possible for their molecular identity. On this basis other CWD field samples collected in the past, present or future could be systematically compared with macaque-tested inocula in order to assess whether they are covered by the ongoing risk assessments in primates. CWD typing by Fourier transform-infrared spectroscopy of pathological prion protein may provide a method of choice for this purpose.     

Prion protein gene sequence and chronic wasting disease susceptibility in white-tailed deer (Odocoileus virginianus)

ABSTRACT

The sequence of the prion protein gene (PRNP) affects susceptibility to spongiform encephalopathies, or prion diseases in many species. In white-tailed deer, both coding and non-coding single nucleotide polymorphisms have been identified in this gene that correlate to chronic wasting disease (CWD) susceptibility. Previous studies examined individual nucleotide or amino acid mutations; here we examine all nucleotide polymorphisms and their combined effects on CWD. A 626 bp region of PRNP was examined from 703 free-ranging white-tailed deer. Deer were sampled between 2002 and 2010 by hunter harvest or government culling in Illinois and Wisconsin. Fourteen variable nucleotide positions were identified (4 new and 10 previously reported). We identified 68 diplotypes comprised of 24 predicted haplotypes, with the most common diplotype occurring in 123 individuals. Diplotypes that were found exclusively among positive or negative animals were rare, each occurring in less than 1% of the deer studied. Only one haplotype (C, odds ratio 0.240) and 2 diplotypes (AC and BC, odds ratios of 0.161 and 0.108 respectively) has significant associations with CWD resistance. Each contains mutations (one synonymous nucleotide 555C/T and one nonsynonymous nucleotide 286G/A) at positions reported to be significantly associated with reduced CWD susceptibility. Results suggest that deer populations with higher frequencies of haplotype C or diplotypes AC and BC might have a reduced risk for CWD infection – while populations with lower frequencies may have higher risk for infection. Understanding the genetic basis of CWD has improved our ability to assess herd susceptibility and direct management efforts within CWD infected areas.     

Rapid prion neuroinvasion following tongue infection

Food-borne transmission of prions can lead to infection of the gastrointestinal tract and neuroinvasion via the splanchnic and vagus nerves. Here we report that the transmission of transmissible mink encephalopathy (TME) is 100,000-fold more efficient by inoculation of prions into the tongues of hamsters than by oral ingestion. The incubation period following TME agent (hereinafter referred to as TME) inoculation into the lingual muscles was the shortest among the five nonneuronal routes of inoculation, including another intramuscular route. Deposition of the abnormal isoform of the prion protein, PrP(Sc), was first detected in the tongue and submandibular lymph node at 1 to 2 weeks following inoculation of the tongue with TME. PrP(Sc) deposits in the tongue were associated with individual axons, and the initial appearance of TME in the brain stem was found in the hypoglossal nucleus at 2 weeks postinfection. At later time points, PrP(Sc) was localized to brain cell groups that directly project to the hypoglossal nucleus, indicating the transneuronal spread of TME. TME PrP(Sc) entry into the brain stem preceded PrP(Sc) detection in the rostral cervical spinal cord. These results demonstrate that TME can replicate in both the tongue and regional lymph nodes but indicate that the faster route of brain invasion is via retrograde axonal transport within the hypoglossal nerve to the hypoglossal nucleus. Topical application of TME to a superficial wound on the surface of the tongue resulted in a higher incidence of disease and a shorter incubation period than with oral TME ingestion. Therefore, abrasions of the tongue in livestock and humans may predispose a host to oral prion infection of the tongue-associated cranial nerves. In a related study, PrP(Sc) was detected in tongues following the intracerebral inoculation of six hamster-adapted prion strains, which demonstrates that prions can also travel from the brain to the tongue in the anterograde direction along the tongue-associated cranial nerves. These findings suggest that food products containing ruminant or cervid tongue may be a potential source of prion infection for humans.     

Modeling routes of chronic wasting disease transmission: environmental prion persistence promotes deer population decline and extinction

Chronic wasting disease (CWD) is a fatal disease of deer, elk, and moose transmitted through direct, animal-to-animal contact, and indirectly, via environmental contamination. Considerable attention has been paid to modeling direct transmission, but despite the fact that CWD prions can remain infectious in the environment for years, relatively little information exists about the potential effects of indirect transmission on CWD dynamics. In the present study, we use simulation models to demonstrate how indirect transmission and the duration of environmental prion persistence may affect epidemics of CWD and populations of North American deer. Existing data from Colorado, Wyoming, and Wisconsin's CWD epidemics were used to define plausible short-term outcomes and associated parameter spaces. Resulting long-term outcomes range from relatively low disease prevalence and limited host-population decline to host-population collapse and extinction. Our models suggest that disease prevalence and the severity of population decline is driven by the duration that prions remain infectious in the environment. Despite relatively low epidemic growth rates, the basic reproductive number, R(0), may be much larger than expected under the direct-transmission paradigm because the infectious period can vastly exceed the host's life span. High prion persistence is expected to lead to an increasing environmental pool of prions during the early phases (i.e. approximately during the first 50 years) of the epidemic. As a consequence, over this period of time, disease dynamics will become more heavily influenced by indirect transmission, which may explain some of the observed regional differences in age and sex-specific disease patterns. This suggests management interventions, such as culling or vaccination, will become increasingly less effective as CWD epidemics progress.     

Genetic Predictions of Prion Disease Susceptibility in Carnivore Species Based on Variability of the Prion Gene Coding Region

Mammalian species vary widely in their apparent susceptibility to prion diseases. For example, several felid species developed prion disease (feline spongiform encephalopathy or FSE) during the bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom, whereas no canine BSE cases were detected. Whether either of these or other groups of carnivore species can contract other prion diseases (e.g. chronic wasting disease or CWD) remains an open question. Variation in the host-encoded prion protein (PrP^C) largely explains observed disease susceptibility patterns within ruminant species, and may explain interspecies differences in susceptibility as well. We sequenced and compared the open reading frame of the PRNP gene encoding PrP^C protein from 609 animal samples comprising 29 species from 22 genera of the Order Carnivora; amongst these samples were 15 FSE cases. Our analysis revealed that FSE cases did not encode an identifiable disease-associated PrP polymorphism. However, all canid PrPs contained aspartic acid or glutamic acid at codon 163 which we propose provides a genetic basis for observed susceptibility differences between canids and felids. Among other carnivores studied, wolverine (Gulo gulo) and pine marten (Martes martes) were the only non-canid species to also express PrP-Asp163, which may impact on their prion diseases susceptibility. Populations of black bear (Ursus americanus) and mountain lion (Puma concolor) from Colorado showed little genetic variation in the PrP protein and no variants likely to be highly resistant to prions in general, suggesting that strain differences between BSE and CWD prions also may contribute to the limited apparent host range of the latter.     

Generation of a new form of human PrP(Sc) in vitro by interspecies transmission from cervid prions

Prion diseases are infectious neurodegenerative disorders that affect humans and animals and that result from the conversion of normal prion protein (PrP(C)) into the misfolded prion protein (PrP(Sc)). Chronic wasting disease (CWD) is a prion disorder of increasing prevalence within the United States that affects a large population of wild and captive deer and elk. Determining the risk of transmission of CWD to humans is of utmost importance, considering that people can be infected by animal prions, resulting in new fatal diseases. To study the possibility that human PrP(C) can be converted into the misfolded form by CWD PrP(Sc), we performed experiments using the protein misfolding cyclic amplification technique, which mimics in vitro the process of prion replication. Our results show that cervid PrP(Sc) can induce the conversion of human PrP(C) but only after the CWD prion strain has been stabilized by successive passages in vitro or in vivo. Interestingly, the newly generated human PrP(Sc) exhibits a distinct biochemical pattern that differs from that of any of the currently known forms of human PrP(Sc). Our results also have profound implications for understanding the mechanisms of the prion species barrier and indicate that the transmission barrier is a dynamic process that depends on the strain and moreover the degree of adaptation of the strain. If our findings are corroborated by infectivity assays, they will imply that CWD prions have the potential to infect humans and that this ability progressively increases with CWD spreading.     

Prion transmission

Prion diseases range from being highly infectious, for example scrapie and CWD which show facile transmission between susceptible individuals, to showing negligible horizontal transmission, such as BSE and CJD which are spread via food or iatrogenically respectively. Scrapie and CWD display considerable in vivo dissemination, with PrP^Sc and infectivity being found in a range of peripheral tissues. This in vivo dissemination appears to facilitate the recently reported excretion of prion through multiple routes such as from skin, faeces, urine, milk, nasal secretions, saliva and placenta. Furthermore, excreted scrapie and CWD agent is detected within environmental samples such as water and on the surfaces of inanimate objects. The cycle of ‘uptake of prion from the environment - widespread in vivo prion dissemination - prion excretion - prion persistence in the environment’ is likely to explain the facile transmission and maintenance of these diseases within wild and farmed populations over many years.     

CWD prions remain infectious after passage through the digestive system of coyotes (Canis latrans)

ABSTRACT

Chronic wasting disease (CWD) is a geographically expanding prion disease of wild and captive cervids in North America. Disease can be transmitted directly, animal to animal, or indirectly via the environment. CWD contamination can occur residually in the environment via soil, water, and forage following deposition of bodily fluids such as urine, saliva, and feces, or by the decomposition of carcasses. Recent work has indicated that plants may even take up prions into the stems and leaves. When a carcass or gut pile is present in the environment, a large number of avian and mammalian species visit and consume the carrion. Additionally, predators like coyotes, likely select for disease-compromised cervids. Natural cross-species CWD transmission has not been documented, however, passage of infectious prion material has been observed in the feces of crows. In this study we evaluated the ability of CWD-infected brain material to pass through the gastrointestinal tract of coyotes (Canis latrans) following oral ingestion, and be infectious in a cervidized transgenic mouse model. Results from this study indicate that coyotes can pass infectious prions via their feces for at least 3 days post ingestion, demonstrating that mammalian scavengers could contribute to the translocation and contamination of CWD in the environment.     

Transmission and adaptation of chronic wasting disease to hamsters and transgenic mice: evidence for strains

In vitro screening using the cell-free prion protein conversion system indicated that certain rodents may be susceptible to chronic wasting disease (CWD). Therefore, CWD isolates from mule deer, white-tailed deer, and elk were inoculated intracerebrally into various rodent species to assess the rodents' susceptibility and to develop new rodent models of CWD. The species inoculated were Syrian golden, Djungarian, Chinese, Siberian, and Armenian hamsters, transgenic mice expressing the Syrian golden hamster prion protein, and RML Swiss and C57BL10 wild-type mice. The transgenic mice and the Syrian golden, Chinese, Siberian, and Armenian hamsters had limited susceptibility to certain of the CWD inocula, as evidenced by incomplete attack rates and long incubation periods. For serial passages of CWD isolates in Syrian golden hamsters, incubation periods rapidly stabilized, with isolates having either short (85 to 89 days) or long (408 to 544 days) mean incubation periods and distinct neuropathological patterns. In contrast, wild-type mouse strains and Djungarian hamsters were not susceptible to CWD. These results show that CWD can be transmitted and adapted to some species of rodents and suggest that the cervid-derived CWD inocula may have contained or diverged into at least two distinct transmissible spongiform encephalopathy strains.     

In vivo comparison of chronic wasting disease infectivity from deer with variation at prion protein residue 96

Chronic wasting disease (CWD) is a prion disease of cervids that causes neurodegeneration and death. Susceptibility to prion infections, including CWD, can be dependent on the amino acid sequence of the host prion protein (PrP). Here, CWD agent obtained from a deer expressing the 96SS genotype, associated with partial resistance to CWD, was used to infect transgenic (tg) mice expressing either 96GG or 96SS deer PrP. Transgenic mice expressing 96GG deer PrP succumbed to this agent, but tg mice expressing 96SS deer PrP did not. Additional studies using inocula from 96GG deer showed no transmission to 96SS PrP mice and delayed disease in 96GS mice. Thus, 96S PrP played an inhibitory role in disease progression in tg mice.     

Prion transmission: Prion excretion and occurrence in the environment

Prion diseases range from being highly infectious, for example scrapie and CWD, which show facile transmission between susceptible individuals, to showing negligible horizontal transmission, such as BSE and CJD, which are spread via food or iatrogenically, respectively. Scrapie and CWD display considerable in vivo dissemination, with PrP^Sc and infectivity being found in a range of peripheral tissues. This in vivo dissemination appears to facilitate the recently reported excretion of prion through multiple routes such as from skin, feces, urine, milk, nasal secretions, saliva and placenta. Furthermore, excreted scrapie and CWD agent is detected within environmental samples such as water and on the surfaces of inanimate objects. The cycle of “uptake of prion from the environment—widespread in vivo prion dissemination—prion excretion—prion persistence in the environment” is likely to explain the facile transmission and maintenance of these diseases within wild and farmed populations over many years.Key words: prion, PrP, excretion, secretion, transmission