Chronic wasting disease prions in elk antler velvet.

Chronic wasting disease (CWD) of deer, elk, and moose is the only recognized prion disease of wild animals. To date, 15 US states and 2 Canada provinces have reported CWD in wild and/or farm-raised cervids. Outbreaks have also occurred in South Korea as a result of importation of subclinically infected animals (1,2). The unparalleled efficiency of prion transmission in cervids by a largely undefined mechanism, combined with high deer densities in certain areas of North America, complicates strategies for controlling CWD as it continues to emerge in new locations.

Growing antlers of male cervids are covered by a highly innervated and vascularized apical skin layer, referred to as velvet, which is shed after an increase in testosterone and ossification of antlers. Our study objective was to assess whether velvet from CWD-infected elk contains prion infectivity. Our rationale was 2-fold. First, the annual shedding of this material raises the possibility that it may play a role in CWD transmission. Second, although the most likely means of human exposure to CWD prions is consumption of contaminated venison (3), the substantial market for velvet in traditional Asian medicine also warrants concern.

We used CWD-susceptible transgenic (Tg) mice as a sensitive means to detect prions in antler velvet. Bioassays in Tg mice expressing deer prion protein (PrP) (4) and newly created Tg mice expressing elk PrP, demonstrated low levels of CWD prions in antler velvet. We also show that the associated protease-resistant PrP could be amplified in vitro (for detection by Western blot) by protein misfolding cyclic amplification (PMCA). Finally, comparative CWD transmissions in Tg mice indicated that the glutamine (Q) to glutamic acid (E) variation at residue 226, which is the sole primary structural difference between deer and elk PrP, may be a major determinant of CWD pathogenesis in these 2 species.

Materials and Methods

Production of Transgenic Mice

[Tg(CerPrP)1536.sup.+/-] mice expressing deer PrP have been described previously (4). To generate Tg(CerPrPE226) mice expressing the elk PrP coding sequence, codon 226 of the deer PrP gene (PRNP) (GenBank accession no. AF009180) was mutated from Q to E by site-directed mutagenesis (Quick Change; Stratagene, La Jolla, CA, USA).

The resulting expression cassette, CerPrP-E226, highlights the single amino acid difference between deer and elk PrP at this position (5). The coding sequence was inserted into the MoPrP.Xho expression vector, and the purified transgene was microinjected into pronuclei of fertilized FVB/ [Prnp.sup.0/0] oocytes. Transgenic founders were identified by PCR screening of genomic DNA. Three Tg(CerPrP-E226) founders (Tg5029, Tg5034, Tg5037) were mated to FVB/ [Prnp.sup.0/0] mice to produce hemizygous transgenic lines. Because of equivalent levels of CerPrP-E226 expression, studies were not duplicated in the [Tg5034.sup.+/-] and Tg5037+/lines. Estimates of the levels of PrP expression in 3 different [Tg5037.sup.+/-] mice and 5 different [Tg5029.sup.+/-] mice were accomplished by immuno-dot blotting and Western blotting using monoclonal antibody (MAb) 6H4 (Prionics, Schlieren, Switzerland).

Preparation of Prion Inocula

Antler velvet and matching brain samples were obtained from 4 elk from Canada that were naturally affected with CWD. Elk 01-0306 had severe CWD-specific neuropathologic changes in the obex and neurologic signs indicative of CWD; elk 02-0306 had moderate neuropathologic changes in the obex and was clinically normal. Although information about the clinical status of elk 03-0306 and 04-0306 was not available, these elk had mild and severe neuropathologic changes, respectively, in the obex. Brain samples were also obtained from CWD-affected mule deer D10 and D92 at the Colorado Division of Wildlife, Wildlife Research Center, and from CWD-affected elk 7378 and 99W12389 at the Wyoming Game and Fish Department's Sybille Wildlife Research Unit. Homogenates of brain and antler velvet (10% wt/vol) were prepared in sterile phosphatebuffered saline (PBS) lacking [Ca.sup.2+] and [Mg.sup.2+] ions.

Measurement of Incubation Times

Groups of 5-week-old Tg mice were given general anesthesia and inoculated with 30 [micro]L of brain or antler velvet homogenate through a 27-gauge needle inserted into the right parietal lobe of the brain. Mice were observed 3 times a week for clinical signs indicative of prion infection, e.g., ataxia, weight loss, hyperactivity, flattened posture, absent extensor reflex, or kyphosis. The incubation period is the time between inoculation and the first day on which subsequently progressive clinical signs were identified. For endpoint titration, groups of 8 Tg mice were inoculated with [10.sup.-1] to [10.sup.-10] dilutions of a 10% brain homogenate of D92 prepared in PBS lacking [Ca.sup.2+] and [Mg.sup.2+] ions.

Western Blotting

Protein content in 10% brain homogenates was determined by bicinchoninic acid assay. Total protein (50 [micro]g) was then digested with 40 [micro]g/mL proteinase K (PK) (Roche, Mannheim, Germany) in the presence of 2% sarkosyl for 1 h at 37[degrees]C. Digestion was terminated with phenylmethylsulfonyl fluoride at a final concentration of 5 [micro]M. Proteins were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride Immobilon-FLmembranes (Millipore, Billerica, MA, USA), which were immunoprobed with MAb 6H4 followed by horseradish peroxidase-conjugated antimouse secondary antibody. Proteins were visualized by using ECL Plus (GE Healthcare, Piscataway, NJ, USA) and an FLA-5000 scanner (Fujifilm Life Science, Woodbridge, CT, USA).

Histoblotting

Coronal cryostat sections (10 [micro]m) were transferred to nitrocellulose and probed with MAb 6H4 after PK digestion as described previously (6). Images were photographed with a NikonDMX 1200F (Tokyo, Japan) digital camera in conjunction with Metamorph software (Molecular Devices, Sunnyvale, CA, USA).

Immunohistochemical Analysis

Sections (8 [micro]m) of formalin-fixed, paraffin-embedded mouse brains on positively charged microscope slides were deparaffinized and subjected to immunohistochemical analysis to deter the disease-associated form of PrP ([PrP.sup.Sc]) as described previously (7). [PrP.sup.Sc] was detected with MAb 6H4 after hydrolytic autoclaving for 15 min in 10 mmol/L HCl. Biotinylated secondary antibody in conjunction with 3,3'-diaminobenzidine was used to visualize [PrP.sup.Sc].

PMCA

Healthy [Tg(CerPrP)1536.sup.+/-] mice were perfused with PBS/5 mM EDTA. Brain homogenates (10% wt/vol) were prepared in conversion buffer consisting of PBS containing 150 mmol/L NaCl, 1.0% Triton X-100, and Roche's Complete Protease Inhibitor Cocktail. Samples were clarified by centrifugation at 500 rpm for 60 s. Each PMCA cycle consisted of 30 min incubation at 37[degrees]C followed by a 20 s sonication pulse at setting 7 using a Misonix 3000 Sonicator (Misonix, Farmingdale, NY, USA). After 96 cycles, 6 [micro]L of the 60-[micro]L reaction was diluted into 54 [micro]L of fresh [Tg(CerPrP)1536.sup.+/-] substrate for a subsequent round of PMCA. Amplification products were digested with 50 [micro]g/mL PK at 50[degrees]C for 75 min.

Results

CWD Prions in Elk Antler Velvet

Mean incubation periods for mice inoculated with CWD prions from elk brains were more uniform (225-335 days) than were those for mice inoculated with CWD prions from antler velvet. Not all [Tg(CerPrP)1536.sup.+/-] mice inoculated with antler velvet developed disease; incubation times for those that did were relatively long and highly variable (Table). Clinical signs did develop in [Tg(CerPrP)1536.sup.+/-] mice challenged with antler velvet from elk 01-0306 and 03-0306; mean incubation periods were [approximately equal to]440 d and [approximately equal to]460 d and attack rates were 75% and 66%, respectively. [Tg(CerPrP)1536.sup.+/-] mice inoculated with antler velvet from elk 02-0306 and 04-0306 remained healthy until the mice were euthanized at [approximately equal to]600 d postinoculation.

We also tested the susceptibility of Tg mice expressing elk PrP. Mean incubation times after inoculation of [Tg(CerPrP-E226)5037.sup.+/-] mice with CWD prions from brains of elk 01-0306 and 04-0306 were 174 [+ or -] 7 d and 224 [+ or -] 6 d, respectively (Table). Of the [Tg(CerPrPE226)5037.sup.+/-] mice inoculated with antler velvet from elk 01-0306, 29% died of prion disease in <400 d, and antler velvet from elk 04-0306 failed to induce disease (Table); this finding confirmed the antler velvet transmission results in [Tg(CerPrP)1536.sup.+/-] mice.

[Tg(CerPrP-E226)5037.sup.+/-] mice express PrP at [approximately equal to]5-fold the level of PrP in the brains of wild type mice, similar to transgene expression levels in [Tg(CerPrP)1536.sup.+/-] mice; the level of expression in Tg(CerPrP-E226)5029+/- mice was approximately equal to that in wild type mice (Figure 1). Induction of disease by CWD prions in brain from diseased elk and deer was consistently and significantly more rapid in [Tg(CerPrP-E226)5037.sup.+/-] than in [Tg(CerPrP)1536.sup.+/-]mice (Table). Mean incubation times of the D92 isolate were equivalent in [Tg(CerPrP)1536.sup.+/-] and [Tg(CerPrPE226)5029.sup.+/-] mice (Table), which have a 5-fold difference in transgene expression levels (Figure 1).

[PrP.sup.Sc] Accumulation and Neuropathologic

Changes in Diseased Tg Mice

Diagnoses for all [Tg(CerPrP)1536.sup.+/-] and Tg(CerPrPE226) 5037+/- mice were confirmed by the presence or absence of protease-resistant [PrP.sup.Sc] in brains, according to Western blotting (Figure 2) and histoblotting (Figure 3) and finding of disease-specific neuropathologic changes (Figure 4). [PrP.sup.Sc] immunostaining in histoblots of diseased [Tg(CerPrP)1536.sup.+/-] and [Tg(CerPrP-E226)5037.sup.+/-] mice was punctate (Figure 3). Although cortical florid plaques were observed in the brains of diseased [Tg(CerPrP)1536.sup.+/-]mice (Figure 4, panels A and B), [PrP.sup.Sc] accumulation in [Tg(CerPrP-E226)5037.sup.+/-] mice was more diffuse and granular. The extensive loss of cerebellar granular cells and accompanying [PrP.sup.Sc] deposition that characterized disease in [Tg(CerPrP-E226)5037.sup.+/-] mice (Figure 4, panels G and H) was not noted for [Tg(CerPrP)1536.sup.+/-] mice (Figure 4, panels C and D).

Estimates of CWD Prion Titers in Antler Velvet

A brainstem preparation from CWD-affected mule deer D92, which contained high levels of [PrP.sup.Sc] according to Western blot (data not shown), was selected for endpoint titration of CWD prions in [Tg(CerPrP)1536.sup.+/-] mice. Disease developed in all mice inoculated with the 3 lowest dilutions; mean incubation periods ranged from 268 to 390 d (Figure 5). Disease did not develop in any of the mice inoculated with the [10.sup.-4] dilution, but disease did develop after 471 d in 1 mouse from the [10.sup.-5] dilution group. The remaining mice in the [10.sup.-5] dilution group and all mice inoculated with higher dilutions remained free of disease and were euthanized after 560-645 d. The disease status of all mice was confirmed by Western blotting (data not shown). We estimated the titer of CWD prions in D92 brain tissue to be 6 log i.c. [ID.sub.50]/g (i.c. [ID.sub.50] refers to the dose of CWD prions that produces infection in 50% of the intracerebrally inoculated Tg mice) (8).

[FIGURE 1 OMITTED]

The inefficient transmission of prions from antler velvet samples (Table) is consistent with low levels of CWD prions. Because the incubation times of CWD prions in antler velvet from elk 01-0306 and 03-0306 were outside the linear range of dose and incubation time (Figure 5), we were unable to assign a definitive titer. The infection attack rates <100% suggested that CWD prion titers in these 1% inocula were close to, or at, the end point of the bioassay ([less than or equal to]3.5 log i.c.[ID.sub.50] units).

Amplification of [PrP.sup.Sc] in Antler Velvet Preparations

When Western blot, ELISA, and immunohistochemical analyses failed to detect [PrP.sup.Sc] in antler velvet samples from 14 CWD-affected elk, including the 4 samples analyzed by bioassay (data not shown), we attempted to amplify [PrP.sup.Sc] in antler velvet samples by using serial PMCA (Figure 6). Whereas [PrP.sup.Sc] in brain homogenates of elk 01-0306, 03-0306, and 04-0306 was efficiently amplified, even at high dilution, after 1 or 2 rounds of PMCA, [PrP.sup.Sc] in antler velvet homogenates of elk 01-0306 and 03-0306 was not amplified until either round 3 or 4. [PrP.sup.Sc] was not amplified in antler velvet from elk 04-0306 or after PMCA of negative-control preparations. [PrP.sup.Sc] amplification correlated with the transmission efficiency of CWD prions in antler velvet. The antler velvet sample from elk 01-0306, which produced detectable amplification products after 3 rounds of PMCA, caused disease in [Tg(CerPrP)1536.sup.+/-]mice (mean incubation time 442 d, attack rate 75%) and in [Tg(CerPrP-E226)5037.sup.+/-] mice. The velvet sample from elk 03-0306, which produced detectable amplification products after 4 rounds of PMCA, and resulted in a 463-d mean incubation time and a 66% attack rate. In contrast, the antler velvet sample from elk 04-0306, which failed to amplify after 4 rounds of PMCA, also failed to transmit prions to either Tg mouse line.

[FIGURE 2 OMITTED]

Discussion

The transmission of CWD prions in antler velvet from 2 naturally affected elk to mice in 2 Tg models demonstrates that this tissue contains low, but detectable, amounts of CWD prions. In addition, serial PMCA amplified otherwise undetectable levels of [PrP.sup.Sc] in antler velvet.

We characterized CWD prion infectivity by end-point titration. The [approximately equal to]6 log i.c.[ID.sub.50]/g CWD prion titer estimated by this method contrasts with [approximately equal to]9 log i.c.[ID.sub.50]/g titers of mouse-adapted scrapie prions in rodent brains (9) and [approximately equal to]7-7.7 log i.c.[ID.sub.50]/g titers of BSE prions estimated by bioassay in transgenic mice (10,11). The linear relationship between dose and incubation time (12) provides an opportunity to estimate the level of prions in materials containing an unknown amount of infectivity. The attack rates of <100% after inoculation with antler velvet preparations from elk 01-0306 and 03-0306 and the failure to transmit disease from the remaining antler velvet samples suggest that CWD prion titers are close to, or at, the end point of the [Tg(CerPrP)1536.sup.+/-] bioassay. Although we are aware of the limitations of comparing levels of prions in tissues from different CWD-affected cervids, we estimated the end point of the CWD prion titration using D92 to be <3.5 log i.c.[ID.sub.50] units. Other factors could also influence levels of infectivity in the 4 tested samples, e.g., the portion of the antler processed and the age of the antler when harvested. Histologic evaluation indicated that the velvet samples used in these transmission studies came from elk antlers in the early stages of seasonal growth (data not shown). Whether CWD prion titers in antler velvet vary according to the state of antler growth remains to be determined. Whether prion infectivity is derived from nervous system tissue, blood (13), or another component of velvet, is also unclear.

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Implications for Horizontal CWD Transmission and Human Exposure

Our studies indicate that antler velvet represents a previously unrecognized source of CWD prions in the environment. Whereas oral transmission of rodent-adapted scrapie prions is known to be [approximately equal to]5 orders of magnitude less efficient than transmission by intracerebral inoculation (14,15), the relative efficiency of oral CWD prion transmission is unknown. Multiple exposures to low levels of CWD prions in the environment (16,17), as well as increased infectivity when prions are bound to soil minerals (18), are factors that may influence transmission.

The appearance of variant Creutzfeldt-Jakob disease in humans exposed to bovine spongiform encephalopathy (BSE) (19,20) and the demonstration of CWD prions in muscle (3) placed the human species barrier to CWD prions at the forefront of public health concerns. Our studies indicate that antler velvet represents an additional source for human exposure to CWD prions. Widely used in traditional Asian medicine to treat a variety of ailments including impotence, arthritis, and high blood pressure, antler velvet can be readily purchased in caplet form and its usage has increased worldwide.

Fortunately, to date there is no epidemiologic evidence that rates of CJD in the CWD-endemic region (Colorado, USA) have increased (21,22). Also reassuring is the inefficient in vitro conversion of human PrP to protease-resistant PrP by CWD (23). Two studies have shown that CWD prions failed to induce disease in Tg mice expressing human PrP (24,25). However, the failure of BSE to be transmitted to Tg mice expressing human prion protein (HuPrP) was cited as early evidence of a BSE transmission barrier in humans (26); subsequent studies demonstrated a strong effect of the codon 129 polymorphism on transmissibility of BSE prions (27). To date, only mice expressing HuPrP with methionine at 129 have been challenged with CWD. In support of the argument that humans might be susceptible to CWD, intracerebral inoculation of squirrel monkeys produced disease after >30 months (28). Prion strain properties are also critical when considering the potential for interspecies transmission. The existence of multiple CWD strains has been suggested by several studies (4,25,29,30), but strain isolation and host range characterization have not been reported. Finally, it is worth considering that if CWD were to cross the species barrier into humans, this transmission source might not be recognized if the disease profile overlapped with one of the forms of sporadic CJD reported in North America.

Possible Role for Residue 226 in CWD Pathogenesis

Previous studies that demonstrated more rapid CWD prion incubation times in Tg mice expressing elk PrP (24,29) than in [Tg(CerPrP)1536.sup.+/-] mice (4) raised the possibility that the single amino acid difference at residue 226 between elk and deer PrP (5) may influence CWD pathogenesis (29). However, when the transmission characteristics of CWD isolates were directly compared in Tg mice expressing differing levels of deer or elk PrP, Tamguney et al. concluded that CWD incubation times were related solely to the level of PrP transgene expression (25). We compared CWD transmission in [Tg(CerPrP-E226)5037.sup.+/-] and [Tg(CerPrP)1536.sup.+/-] mice, which express PrP at levels [approximately equal to]5-fold higher than PrP levels in wild type mouse brain (Figure 1A), and found that CWD transmission was consistently and substantially more rapid in [Tg(CerPrP-E226)5037.sup.+/-] mice. Our results appear compatible with more efficient CWD prion propagation by elk cellular prion protein ([CerPrP.sup.C]) containing E at residue 226 than by deer [CerPrP.sup.C] containing Q at this position. Consistent with this interpretation, despite 5-fold lower levels of transgene expression in Tg(CerPrP-E226)5029+/- mice than in [Tg(CerPrP)1536.sup.+/-] mice, mean incubation times of the D92 isolate were equivalent in these 2 lines (Table). Nonetheless, undetected differences in [CerPrP.sup.C] expression, for example in particular cell types, might result in more rapid disease and/or altered pathologic changes. The generation of transgenic mice expressing elk and deer coding sequences using gene replacement strategies would seem to be an excellent approach for resolving this issue.

[FIGURE 6 OMITTED]

The different responses to CWD in Tg mice also appear to recapitulate aspects of CWD pathogenesis in the natural hosts. Previous limited comparative transmission studies indicated that CWD developed [approximately equal to]25% more rapidly in orally challenged elk than deer (31). Although plaques were not detected in brains of CWD-affected elk, florid plaques have been observed in the brains of diseased deer (32,33). Similar differences in pathologic changes were observed in [Tg(CerPrP-E226)5037.sup.+/-] and [Tg(CerPrP)1536.sup.+/-] mice (Figure 4). Structural analyses suggest that residue 226 is located within a region of [PrP.sup.C] proposed to interact with a factor (34), possibly equivalent to the postulated protein X (35). Although mutation of the equivalent residue from Q to lysine (K) in epitope-tagged mouse PrP had no effect on [PrP.sup.Sc] formation in transfected chronically infected ScN2A cells, the effects of the Q-to-E substitution were not assessed (36).

DOI: 10.3201/eid1505.081458

Acknowledgments

We thank Dongyue Zhuang for excellent technical assistance. This work was supported by grants 2RO1NS040334-04 from the National Institute of Neurological Disorders and Stroke, N01 AI-25491 and 1 P01 AI077774-01 from the National Institute of Allergy and Infectious Diseases, and Specific Cooperative Agreement 58-5348-6-131 from the US Department of Agriculture, Agricultural Research Service. R.C.A. was supported by the T32 DA022738 Training Program in Therapeutic Strategies for Neurodegeneration.

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Rachel C. Angers, [1] Tanya S. Seward, Dana Napier, Michael Green, Edward Hoover, Terry Spraker, Katherine O'Rourke, Aru Balachandran, and Glenn C. Telling

[1] Current affiliation: Medical Research Council Laboratory of Molecular Biology, Cambridge, UK.

Author affiliations: University of Kentucky Medical Center, Lexington, Kentucky, USA (R.C. Angers, T.S. Seward, D. Napier, M. Green, G.C. Telling); Colorado State University, Fort Collins, Colorado, USA (E. Hoover, T. Spraker); US Department of Agriculture, Pullman, Washington, USA (K. O'Rourke); and Canadian Food Inspection Agency, Ottawa, Ontario, Canada (A. Balachandran)

At the time of this study, Dr Angers was pursuing a PhD in the Department of Microbiology, Immunology and Molecular Biology at the Sanders Brown Center on Aging, University of Kentucky College of Medicine, under the supervision of Dr Telling. Her research focused on the generation and characterization of transgenic mouse models to study mechanisms of CWD pathogenesis.

Address for correspondence: Glenn C. Telling, Microbiology, Immunology and Molecular Genetics, University of Kentucky Medical Center, 800 Rose St, Lexington, KY 40536, USA; email: [e-mail]

Table. Transmission of chronic wasting disease prions to
transgenic mice *

                     Mice expressing deer [PrP.sup.c]

                        Tg(CerPrP)[1536.sup.+/-]
Isolate from
animal no.      Antler velvet                Brain

01-0306         442 [+ or -] 16 (6/8)        322 [+ or -] 9 (8/8)
02-0306         >594 (0/5)                   225 [+ or -] 3 (7/7)
03-0306         463 [+ or -] 23 (2/3)        335 [+ or -] 5 (7/7)
                  ([section])
04-0306         >601 (0/6)                   281 [+ or -] 5 (7/7)
7378                                         235 [+ or -] 2 (8/8)
99W12389                                     230 [+ or -] 9 (8/8)
D10                                          225 [+ or -] 1 (8/8)
D92                                          268 [+ or -] 15 (7/7)
PBS                                          >510 (0/6)
None

                       Mice expressing elk [PrP.sup.c]

                        Tg(CerPrP-E226)[5037.sup.+/-]
Isolate from
animal no.      Antler velvet                Brain

01-0306         305 [+ or -] 86 (2/7)        174 [+ or -] 7 (8/8)
                  ([double dagger])
02-0306
03-0306
04-0306         >505 (0/5)                   224 [+ or -] 6 (7/7)
7378                                         177 [+ or -] 15 (7/7)
99W12389                                     158 [+ or -] 13 (7/7)
D10                                          201 [+ or -] 8 (8/8)
D92                                          201 [+ or -] 15 (8/8)
PBS
None                                         >592 (0/4) ([paragraph])

                       Mice expressing elk [PrP.sup.c]

                        Tg(CerPrP-E226)[5029.sup.+/-]
Isolate from
animal no.      Brain                        % Decrease ([dagger])

01-0306                                      46 (<0.0001)
02-0306
03-0306
04-0306                                      20 (<0.0001)
7378                                         25 (0.0061)
99W12389                                     31 (0.0004)
D10                                          11 (0.013)
D92             263 [+ or -] 10 (7/7)        25 (0.0061)
PBS
None

* Incubation times are given as mean [+ or -] SEM days. Values in
parentheses are no. diseased mice/no. inoculated mice. [PrP.sup.c],
cellular prion protein; Tg, transgenic; PBS, phosphate-buffered
saline.

([dagger]) Values represent the percent decrease in mean incubation
time of isolates in Tg(CerPrP-E226)[5037.sup.+/-] mice compared with
Tg(CerPrP)[1536.sup.+/-] mice. p values, calculated by the Student
t-test, are shown in parentheses.

([double dagger]) The remaining 5 of 7 inoculated mice remained free
of disease until 525 d postinoculation, at which time they were
euthanized.

([section]) Mice inoculated with the antler velvet preparation from
elk 03-0306 had an unusually high rate of intercurrent illnesses,
resulting in early euthanasia of 5 inoculated mice.

([paragraph])Four uninoculated Tg(CerPrP E2226) [5037.sup.+/-] mice
were euthanized at 437 d.