The results of this study demonstrate a species barrier in transmission of CWD to mink. Primary oral challenge with CWD-infected elk brain did not result in clinical or pathological findings of TSE, indicating that natural interspecies transmission of CWD to mink is unlikely to occur on ranches or in wildlife. Furthermore, primary IC challenge of mink with CWD material was considerably less efficient than IC challenge with TME, as indicated by the prolonged incubation time and different lesion profiles. The lack of orally mediated disease, despite a total cumulative dose almost 200-fold greater than that given by the IC route, shows the influence of administration route on TSE pathogenesis. Species barriers in prion disease are typically defined by increased attack rate and decreased incubation time following serial IC passage of infectious material. However, IC injection, whilst useful for lesion comparison between strains or in the study of molecular pathogenesis, is an experimental technique that does not occur in nature. In the context of natural disease transmission from cervids to mustelids, and to carnivores in general, primary oral transmission is the scenario of consequence. Therefore, in this study, we defined species barriers as inefficient primary IC transmission and lack of primary oral transmission.
Differences in lesion profile were demonstrated qualitatively by TME IC recipients having more severe spongiform vacuolation and PrPd
deposition than CWD IC recipients. Quantitatively, TME recipients had significantly higher scores for both vacuolation and PrPd
deposits in all regions of the brain except for the cerebellum. Different patterns of retinal PrPd
IHC further delineated CWD from TME in mink tissue, as the CWD-infected animals had a multifocal globular signal and TME recipients had a predominantly diffuse granular signal. Significant differences in astrocyte quantification were also informative in both IC and PO recipients. Astrocyte counts were significantly higher in the hippocampus of CWD IC recipients, whereas cerebrocortical counts were significantly higher in TME IC recipients. This difference, combined with astrocyte counts that were independent of the degree of vacuolation, shows a clearly different host response for the two types of challenge inoculum. In PO recipients, astrocyte counts were evaluated as an indicator of subtle neurological change in the central nervous system. Counts were not statistically significantly different between CWD-positive PO and CWD-negative PO recipients, indicating a lack of underlying neural damage or subclinical disease that may have developed with continued observation. The rare occurrence of cerebellar lesions in both CWD and TME IC recipients is consistent with previous investigations showing minimal cerebellar involvement in mink (Hanson et al., 1971
; Marsh & Hanson, 1979
; Robinson et al., 1994
). The differences in lesion profile and extended incubation time for CWD demonstrate that CWD and TME are distinctly different diseases in the mink host.
This study complements previous ruminant-to-carnivore transmission investigations where CWD was administered to ferrets. Results in the ferrets were similar to those in mink, as primary IC administration of deer CWD caused disease (Bartz et al., 1998
), whereas primary oral challenge did not. In ferrets, serial IC passage is required before positive PrPd
IHC is demonstrable by oral challenge (Perrott et al., 2004
; Sigurdson et al., 2003
). CWD-infected tissue originated from elk in this study, and from mule deer in the ferret study. It is possible that CWD of mule deer origin may behave differently in mink tissue from that of elk origin, a hypothesis that we are currently investigating. Nevertheless, the cumulative findings demonstrate a species barrier in the development of disease in mustelid carnivores (e.g. mink and ferrets) following primary oral challenge with CWD. By extension, one may speculate that carnivores in general are resistant to consumption of CWD. Humans may also be resistant to CWD; whilst non-human primates succumb to IC CWD (Marsh et al., 2005
), epidemiological investigation has not identified a clear link between CWD and human Creutzfeldt–Jakob disease (CJD) (Belay et al., 2004
; MaWhinney et al., 2006
), and studies in humanized transgenic mice indicate CWD resistance (Kong et al., 2005
; Tamguney et al., 2006
). Continued monitoring of human disease and additional oral-transmission studies in animals are needed to confirm or refute primate and carnivore resistance to orally mediated CWD.
Host prion gene polymorphisms are associated with TSE susceptibility in some species. We examined prion genetics of both challenge material and recipient mink to identify residues that might affect interspecies transmission. Source and recipient animals were universally homozygous for methionine at codon 132/133 (elk and mink, respectively). Codon 132, the site of a methionine/leucine polymorphism in elk (O';Rourke et al., 1999
), corresponds positionally to human codon 129, where methionine homozygosity is associated with variant CJD (Zeidler et al., 1997
). The elk polymorphism segregates with disease phenotype in CWD, as leucine-homozygous elk have a prolonged incubation period and altered PrPd
migration pattern compared with methionine homozygotes (Hamir et al., 2006a
; O';Rourke et al., 2007
). Codon 96 is another site of interest, as a glycine/serine polymorphism is associated with relative CWD susceptibility in deer (O';Rourke et al., 2004
). In this study, elk and mink had conservation of methionine at codon 132 and glycine at codon 96, indicating that these residues were not limiting factors in disease transmission. Of the two CWD-positive IC recipients with disease, one was homozygous for arginine at codon 232; the other was a codon 232 arginine/tryptophan heterozygote. These two animals had similar incubation periods and lesions, thus there was no obvious effect on disease. The codon 27 polymorphism is intriguing, as it is near the cleavage site of the membrane-signalling portion of the prion protein (Prusiner, 1998
). As cytosolic accumulation of prion protein has neurotoxic effects (Ma et al., 2002
), signalling-sequence variation could influence disease pathogenesis through altered prion translocation to the cell surface. All diseased animals were homozygous at codon 27, suggesting that the polymorphism could modulate relative susceptibility; however, the small number of affected mink precludes determination of the true effect. Respective differences between mink and ferrets at codons 179 (phenylalanine/lysine) and 224 (arginine/glutamine) are associated with differential susceptibility to TME (Bartz et al., 1994
). In this study, all mink were homozygous for phenylalanine and arginine and congruous with challenge material; it is currently unknown whether these codon polymorphisms were a factor in previous CWD studies in ferrets. Overall, comparative amino acid alignment shows 23 divergent residues between cervids and mustelids that could affect transmission. Additional genetic comparison of cervid challenge material and recipient mustelids, such as by in vitro
conversion assays (Raymond et al., 2000
; Kurt et al., 2007
), is needed to delineate further possible roles of these divergent residues.
This and previous studies provide a relative comparison of mustelid susceptibility to cattle, sheep or cervid prions. Primary IC or primary oral challenge of mink with BSE results in clinicopathological abnormalities at 12 and 15 months incubation, respectively, with lesion severity that is independent of challenge route (Robinson et al., 1994
) and occurs close to the estimated 7–12 month oral incubation period for natural TME (Marsh et al., 1991
). Conversely, primary oral challenge with scrapie has not caused disease in mink, despite repeated attempts and observation up to 48 months (Marsh et al., 1991
; Marsh & Hanson, 1979
); similarly, in this study, primary oral challenge with CWD did not cause disease during 42 months incubation. CWD IC challenge resulted in minor cerebrocortical involvement, whilst the cerebral cortex is involved more extensively with scrapie or BSE IC challenge (Hanson et al., 1971
; Marsh & Hanson, 1979
; Robinson et al., 1994
). IC lesions also vary by source in the caudal brainstem, including the dorsal motor nucleus of the vagus nerve, as they are of lesser severity with scrapie or CWD, whilst severity increases with TME or BSE (Eckroade et al., 1979
; Hanson et al., 1971
; Hartsough & Burger, 1965
; Marsh & Hadlow, 1992
; Robinson et al., 1994
). IC back-passage of TME to cattle causes disease in 14.5 months, similar to TME in mink, and lesions in cattle are similar on both first and second passage (Hamir et al., 2006b
; Robinson et al., 1995
). Thus, the overall clinicopathological features do not change appreciably between mink and cattle. Cumulatively, passage of TSE between cattle and mink occurs readily, with similar lesions and incubation times, whereas passage of CWD or scrapie to mink is limited by route of administration, incubation time and appearance of lesions compared with TME. As cattle are the only ruminant without an apparent species barrier in prion transmission to and from mink, it raises the possibility that, in natural settings, previously unrecognized prion or prion-like disease in cattle may have been responsible for some cases of spongiform encephalopathy in mink.
In this study, we demonstrated a species barrier between elk CWD and mink, as shown by lack of orally mediated disease and substantive differences in lesions between CWD and TME IC recipients. Whilst CWD appears to be readily transmissible within cervid species, this study provides additional evidence that cervid prions are poorly transmissible to non-cervid hosts, and is a strong indication that mink are unlikely to be involved in natural transmission of CWD among wildlife.