PLoS ONE. 2009; 4(6): e5916. Published online 2009 June 16. doi: 10.1371/journal.pone.0005916. PMCID: PMC2691594
Copyright Mathiason et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Infectious Prions in Pre-Clinical Deer and Transmission of Chronic Wasting Disease Solely by Environmental Exposure
Candace K. Mathiason,1 Sheila A. Hays,1 Jenny Powers,2 Jeanette Hayes-Klug,1 Julia Langenberg,3 Sallie J. Dahmes,4 David A. Osborn,5 Karl V. Miller,5 Robert J. Warren,5 Gary L. Mason,1 and Edward A. Hoover1* 1Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, Colorado, United States of America 2National Park Service, Fort Collins, Colorado, United States of America 3Wisconsin Department of Natural Resources, Madison, Wisconsin, United States of America 4WASCO Inc., Monroe, Georgia, United States of America 5Warnell School of Forestry and Natural Resources, University of Georgia, Athens, Georgia, United States of America Per Westermark, Editor Uppsala University, Sweden * E-mail: Edward.Hoover@ColoState.edu
Conceived and designed the experiments: CKM EAH. Performed the experiments: CKM SAH JGP JHK. Analyzed the data: CKM GLM. Contributed reagents/materials/analysis tools: JL SJD DAO KVM RJW. Wrote the paper: CKM. Paper editing: EAH JGP JL. Received March 6, 2009; Accepted May 9, 2009.
Key to understanding the epidemiology and pathogenesis of prion diseases, including chronic wasting disease (CWD) of cervids, is determining the mode of transmission from one individual to another. We have previously reported that saliva and blood from CWD-infected deer contain sufficient infectious prions to transmit disease upon passage into naïve deer. Here we again use bioassays in deer to show that blood and saliva of pre-symptomatic deer contain infectious prions capable of infecting naïve deer and that naïve deer exposed only to environmental fomites from the suites of CWD-infected deer acquired CWD infection after a period of 15 months post initial exposure. These results help to further explain the basis for the facile transmission of CWD, highlight the complexities associated with CWD transmission among cervids in their natural environment, emphasize the potential utility of blood-based testing to detect pre-clinical CWD infection, and could augur similar transmission dynamics in other prion infections.
Time interval to detection of CWD infection by tonsil biopsy The high transmission rate of CWD among cervids in their natural environment sets CWD apart from other prion diseases. The results of this study help provide a plausible basis for this facile transmission and extend our earlier findings  (Fig. 4) in demonstrating infectious prions in blood and saliva of pre-clinical CWD+ donors. The time from exposure to first detection of PrPCWD by tonsil biopsy was variable-as short as 6 months but as long as 18 months. We assume that the time until appearance of PrPCWD in tonsil is an underestimate due to the inherent variability in prion deposition kinetics  and the logistical limitations of tonsil biopsies, which require general anesthesia. The incubation periods prior to clinical CWD in our study were similar to those observed previously in experimental and naturally acquired infections , , . While we can not exclude horizontal transmission from the first positive deer in each cohort, the timeframe for detection in the remaining deer (3 months) is less than half that which we have historically observed in deer inoculated orally with a brain homogenate from terminal CWD-infected deer , suggesting much earlier exposure to infectious prions.
Infectious prions in saliva of CWD+ deer
The presence of infectious CWD prions in saliva suggests the potential for disease transmission via grooming interactions, shared water sources and communal feeding grounds-especially in high density cervid populations, such as those that exist for some cervid species during the breeding season, at baiting sites, in captivity, and in low predation situations. Other investigators have detected the presence of the aberrant misfolded prion protein (PrPRES) in alimentary tract tissue, and have suggested saliva as a possible vehicle for prion transmission , , , . While the volume of saliva used in this study was large, the results nevertheless provide evidence to support the above premise. Salivary dissemination of prions may not be limited to CWD. Prions associated with transmissible mink encephalopathy (TME) have been detected in the submandibular salivary gland of mink  and TME protease-resistant prion protein has been detected in the lamina propria of the oral cavity, taste buds and squamous epithelium of the tongue, and the vomeronasal organ and olfactory mucosa of infected hamsters . Hamster-adapted scrapie agent has been found in the tongue and taste buds of prion-infected hamsters . Vascellari et al reported the presence of the pathological prion protein in both major and minor salivary glands of naturally and experimentally infected sheep , and we have made similar observations in the olfactory mucosa of ferrets experimentally infected with CWD  and in the taste buds of deer (Haley, NJ, personal communication). The exact source of prions shed in saliva remains speculative; possible sources include centrifugal/retrograde passage from nerve fiber terminations in the oral-nasal mucosa, or from lymphoid cells emanating from infected tonsilar or other alimentary lymphoid tissues.
Infectious prions in blood of CWD+ deer
Blood-borne transmission of TSEs has long been feared, and the identification of a prion pathogen associated with blood-borne transmission has been pursued with disparate results , , . Here we report the induction of CWD infection by a single blood transfusion from each of two pre-clinical CWD+ blood donors. This result is consistent with previous findings in substantiating the transmission of infectious prions by the blood of asymptomatic animal ,  and human -, ,  donors, thus providing support for a subclinical hematogenous carrier state in TSE infections.
Direct detection of blood-borne PrPRES has been difficult. Saa et al were the first to use protein misfolding cyclic amplification assay (PMCA) ,  to detect protease-resistant prion protein in the blood of asymptomatic scrapie-infected hamsters . More recently, Thorne et al reported PMCA amplification of PrPSC from the blood of scrapie-infected sheep . Continued efforts toward the development of sensitive, noninvasive, diagnostic tools are paramount. We are presently re-examining by serial PMCA the tissues of exposed but conventional PrPCWD test negative animals that may harbor infectious prions not manifested in the observation periods used in our CWD studies.
Hunter and colleagues ,  provided the first evidence for blood-borne TSE transmission for bovine spongiform encephalopathy (BSE) and scrapie by transfusion of whole blood ,  and buffy-coat white blood cells  from infected donor sheep to naïve sheep. Sparse but compelling evidence has accumulated for blood transmission of variant Creutzfeldt-Jakob Disease (vCJD) -,  and PrPRES has been found in peripheral organs of some sporadic CJD patients , raising the possibility that peripheral distribution of PrPRES is not limited to vCJD. In an ongoing study of sixty-six individuals who received blood products from asymptomatic blood donors who later developed vCJD , three of the 66 blood transfusion recipients developed vCJD 6.5 to 8.5 years after receiving blood , ,  and a fourth blood recipient died of causes unrelated to vCJD five years after receiving the blood donation. Upon autopsy of this individual, PrPRES was detected in lymphoid tissue but not brain, thus providing presumptive evidence for a case of subclinical infection . Our findings with CWD further support the tenet that blood products from subclinical prion-infected individuals may transmit disease.
Additional cases of subclinical human prion disease may exist. While in vitro conversion studies have indicated an inefficient conversion of human PrP into a protease-resistant form ,  and no evidence exists of CWD transmission to non-cervid species cohabitating with or on CWD contaminated environments , -, it is reasonable to surmise that cross-species transmission of prions may require extenuating circumstances, i.e. origin of specific strains , , prolonged incubation time , and permissive genotypes . At least two studies provide information bearing on these concerns. The first study, an ongoing longitudinal study to closely monitor 81 Americans who inadvertently consumed, or were exposed to, CWD+ venison at an upstate New York sportsman's feast, will conduct health evaluations of these individuals over the next six years . The second, a retrospective study using western blot analysis of human tonsil and appendix samples collected in the United Kingdom (UK) to investigate possible exposure to the BSE agent, reported the detection of abnormal prion protein in three of 12,674 samples . Mathematical modeling based on the results of this study predicts a minimum estimate of 3000 BSE infected people in the UK between 10-30 years of age. If this model is accurate, it predicts that 93% of these individuals could develop long-term subclinical infection .
Environmental sources of CWD infection
Previous studies have confirmed direct animal-to-animal contact-horizontal transmission-as an efficient mode for prion disease transmission , . Moreover, Miller and colleagues , ,  have provided substantial evidence for environmental contamination as a source of CWD infection. Our bioassay study inocula doses (50 ml saliva/deer), while efficient in establishing the infectious nature of saliva, are likely unrealistic doses to be acquired in a natural setting. To emulate a more feasible natural environment-associated dose, while negating direct animal-to-animal contact, we exposed naïve deer to repeated exposures to fomites from the suites of CWD-infected deer. The study design was meant to mirror the daily habits and movements of a deer in its natural setting in which it may return to an area contaminated with small amounts of infectious prions over time. Here we provide the first report that under controlled indoor conditions CWD-naïve deer can acquire infection by exposure to fomites from the environment of CWD-infected deer, supporting the findings of Miller et al in the natural environment , , , in demonstrating that there are sufficient infectious prions in bedding and water to transmit CWD. Efficient transmission, as evidenced by tonsillar lymphoid PrPCWD detection, was seen in as little as 15 months post initial exposure. These results are also consistent with the findings of Georgsson  and Miller  as part of their attempts to decontaminate areas heavily contaminated with scrapie and CWD. Animals reintroduced to these areas after decontamination developed clinical signs of prion disease within two years. The presence of infectious CWD prions in the environment therefore strongly suggests that natural prion infection occurs by routes additional to direct animal-to-animal contact. Based on the present and our previous findings , we speculate that saliva may harbor the greatest concentration of CWD prions available for horizontal transmission and environmental contamination, but recognize that other routes of excretion at lower concentration and greater volume still remain plausible.
Lack of detectable infectious prions in the urine and feces of CWD+ deer
Previous studies have postulated that environmental contamination by excreta from infected cervids seems the most plausible explanation for the dissemination of CWD , yet at 19 months pi we were not able to detect PrPCWD in the three deer inoculated with urine and feces. Our earlier report  indicated that 2 of 2 deer expressing the prnp gene G/S polymorphism at codon 96 remained negative 19 mo. pi. In the present study all three deer inoculated with urine and feces expressed the G/G polymorphism at prnp codon 96, which is associated with susceptibility to CWD infection . We report no detection of PrPCWD in the obex or lymphoid tissues of deer with either G/G or G/S polymorphisms at 19 mo pi. Although both of our bioassay studies in deer have failed to transmit CWD infection by oral exposure to urine and feces from CWD-infected deer, these results must still be interpreted with caution in light of ongoing PMCA and cervid transgenic mouse intracerebral bioassay studies which suggest that very low concentrations of prions may be present in urine and feces of CWD+ cervids -. Perhaps an incubation time longer than 19 months is necessary for a detectable accumulation of lymphoid PrPCWD, or a larger dose of inoculum by the oral route is necessary for efficient passage of prions across the alimentary mucosa.
In summary, the results reported here reconfirm that blood and saliva are sources of infectious CWD prions, consistent with previous findings , and further support a mechanism for efficient CWD transmission in nature. We also show that infectious prions shed into the environment by CWD+ deer are sufficient to transmit the disease to naïve deer in the absence of direct animal-to-animal contact. These observations reinforce the exposure risk associated with body fluids, excreta, and all tissues from CWD+ cervids and suggest that similar dynamics may exist in other prion infections.
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