Among the transmissible spongiform encephalopathies (TSEs), chronic wasting disease is unique in its high level of transmissibility and, thus, prevalence in free-ranging populations. Here we report the presence of substantial prion-converting activity in excretory tissues proximate to the saliva, urine, and feces shed by CWD-infected deer. While these results do not speak to infectivity, our previous research has shown that conventional test-negative, sPMCA-positive tissues and bodily fluids may indeed harbor infectivity (17
). The use of sPMCA as a surrogate for a bioassay to detect PrPCWD
in excretory tissues may help to explain the efficient horizontal transmission of CWD but also raises questions regarding the source and mechanism of prion shedding from glandular and mucosal tissues.
Historical cross-sectional studies of cervid herds where CWD is endemic have largely failed to demonstrate, by conventional immunohistochemistry or Western blotting, protease-resistant prions in organs involved in the production and excretion of saliva, urine, or feces (2
). These assays rely on proteolytic, heat, and/or formic acid treatments, practices which may preclude the identification of recently described protease-sensitive forms of infectious prion proteins, denoted sPrPRES
). Anecdotally, however, protease-resistant PrP has been identified in ectopic lymphoid aggregates in the kidneys of CWD-exposed deer by IHC, although no conclusions could be drawn regarding any relationship of this phenomenon with prionuria (19
). Protease-resistant PrP has also been demonstrated in the lingual epithelium of hamsters inoculated with the hyper (HY) strain of transmissible mink encephalopathy (10
) and in the salivary glands of sheep naturally exposed to scrapie (55
). Alternatively, bioassays have demonstrated infectious prions in the salivary glands of scrapie-infected goats (16
), the tongue and nasal epithelium of bovine spongiform encephalopathy (BSE)-infected cattle (3
), and the urine of animals with concurrent nephritis (18
). As sPMCA was also shown to be capable of amplifying both protease-resistant and -sensitive forms of PrPSc
), the absence of protease-resistant forms in these tissues would not preclude positive amplification, since other evidence exists for protease-sensitive infectious prions (9
). Taken together, the above-described observations raise the following interesting questions regarding the mechanisms involved in prionsialia and prionuria. (i) From where do the infectious prions in bodily fluids arise? (ii) Are the infectious prions in excreta present as traditional, protease-resistant forms at very low levels or as a more elusive protease-sensitive species? (iii) Are infectious prions transmitted in cell-free or cell-associated forms?
Excreta—urine, saliva, and feces—are made up of components from the organs and tissues responsible for their production, including aqueous, cellular, and proteinaceous constituents. We hypothesized that these organs may be involved in prion production and/or excretion, and we found a range of CWD prion amplifications from such tissues. Variation in CWD prion-amplifying activities in tissues significantly correlated with the apparent PrPCWD burden within the obex of the individual animal, while a trending relationship was observed between peripheral distribution and intensity and the source of inoculum and route of exposure (e.g., whole blood i.v. and urine and feces p.o., etc.). Perhaps with more animals per group or longer incubation times, this overall trend between inoculum and peripheral distribution may have become statistically significant.
These two findings point to an increased risk of bodily fluid infectivity with disease progression and, potentially, source of exposure. The tissue distribution variation with the route of inoculation has been described previously for both viral and bacterial infections (6
), so this finding is perhaps not surprising. Likewise, in the case of neurotropic viruses, virus levels in peripheral tissue often positively correlate with the central nervous system burden (8
). Evaluation of the prion distribution in tissues of naturally infected cervids may reveal patterns corresponding with those described in this study, i.e., widespread peripheral accumulation of prions that vary according to the central nervous system burden and, potentially, the exposure source.
The detection of amplifiable prions in peripheral excretory tissues call into question (i) whether peripheral prion amplification occurs in situ
or, alternatively, whether excretory tissues merely serve a more passive role in prion excretion and (ii) the kinetics of prion excretion, i.e., how the levels of prion excretion and infectivity in bodily fluids change over the course of the disease. It is generally accepted that TSE pathogenesis follows a pattern of centripetal spread from the periphery to the central nervous system (either solely via the peripheral nervous system or after amplification in the lymphoreticular system), followed by central amplification and centrifugal spread back to the periphery (4
). We found that some peripheral tissue levels rivaled those in the obex of the same animal, perhaps suggesting concurrent early dissemination to the central nervous system and peripheral organs. A further understanding of the kinetics of centripetal and centrifugal prion dissemination could emanate from a sequential sPMCA evaluation of the peripheral prion distribution in exposed animals.
In summary, the present study demonstrates for the first time prion-amplifying activity in organs and tissues associated with prion shedding. The ultimate source and mechanism of release into bodily fluids remain unknown. The elevated and consistent activity found in salivary gland and urinary bladder may suggest an active role in prion excretion. These findings minimally warrant additional, more detailed, and longitudinal studies of the nature and kinetics of excreted prions.