Chronic wasting disease prions in elk antler velvet

Abstract

Chronic wasting disease (CWD) is a contagious, fatal prion disease of deer and elk that continues to emerge in new locations. To explore the means by which prions are transmitted with high efficiency among cervids, we examined prion infectivity in the apical skin layer covering the growing antler (antler velvet) by using CWD-susceptible transgenic mice and protein misfolding cyclic amplification. Our finding of prions in antler velvet of CWD-affected elk suggests that this tissue may play a role in disease transmission among cervids. Humans who consume antler velvet as a nutritional supplement are at risk for exposure to prions. The fact that CWD prion incubation times in transgenic mice expressing elk prion protein are consistently more rapid raises the possibility that residue 226, the sole primary structural difference between deer and elk prion protein, may be a major determinant of CWD pathogenesis.

Figure 1

Levels of transgene expression in transgenic (Tg) mice expressing deer or elk cellular prion protein (PrP^C). Representative Western blot analysis of PrP^C expression from different total protein loads in brain extracts from Tg mice Tg(CerPrP)1536^+/–, Tg(CerPrP-E226)5029^+/–, and Tg(CerPrP-E226)5037^+/– compared with wild type and Prnp^0/0 mice (knock-out mice for PrP gene).

Figure 2

Accumulation of PrP^Sc (disease-associated form of prion protein) in diseased transgenic (Tg) mice. Tg(CerPrP)1536^+/– and Tg(CerPrPE226)5037^+/– mice inoculated with phosphate-buffered saline (PBS), elk brain (B), or antler velvet (A) were treated with or without proteinase K (PK). Membranes were probed with monoclonal antibody 6H4. Molecular weights indicated are 37, 29, and 20 kD.

Figure 3

Distribution of PrP^Sc (disease-associated form of prion protein) in brains of diseased mice. Histoblots of mice inoculated with 01-0306 brain or antler velvet material were treated with proteinase K and probed with monoclonal antibody 6H4. Tg, transgenic.

Figure 4

PrP^Sc (disease-associated form of prion protein)–specific immunohistochemistry in the brains of diseased mice. Transgenic (Tg) mice Tg(CerPrP)1536^+/– inoculated with brain (A) and antler velvet (B) preparations from elk 01-0306 exhibit florid PrP^Sc-reactive plaques in the cerebral cortex at the level of the thalamus but retain integrity of cerebellar granular cells (C and D). Tg(CerPrP-E226)5037^+/– mice inoculated with brain (E) and antler velvet (F) preparations from elk 01-0306 display small plaques and diffuse granular staining in the cerebral cortex, PrP^Sc deposition, and marked cerebellar neuronal loss (G and H).

Figure 5

Quantification of chronic wasting disease prions. Diseased transgenic (Tg) mice Tg(CerPrP)1536^+/– inoculated with dilutions of brain homogenate are indicated by filled symbols Asymptomatic mice are indicated by open circles; time at which asymptomatic aged mice were either euthanized or died of illnesses unrelated to prion disease is shown. Error bars indicate SEM. n/n⁰ refers to the number of mice developing prion disease divided by the number of inoculated mice. Also shown are the mean incubation times of diseased Tg(CerPrP)1536^+/– mice inoculated with antler velvet preparations (filled squares).

Figure 6

Detection of CerPrP^Sc (disease-associated form of cervid prion protein [PrP]) in brain and antler velvet from chronic wasting disease (CWD)–affected elk after serial protein misfolding cyclic amplification (PMCA). Western blots demonstrate amplification of protease-resistant prion protein (PrP) after serial PMCA when seeded with brain or velvet antler material from CWD-affected elk. Brain samples: lane 1, Tg(CerPrP)1536^+/– brain material not treated with proteinase K (PK); lane 2, Tg(CerPrP)1536^+/– brain material used as a negative control seed for the PMCA reactions; lanes 3–9, 10^–2 to 10^–8 dilutions of 10% elk brain homogenate. Antler velvet samples: lane 1, Tg(CerPrP)1536^+/– brain material not treated with PK; lane 2, Tg(CerPrP)1536^+/– brain material used as a negative control seed for the PMCA reactions; lanes 3–9, replicate 10^–2 dilutions of 10% antler velvet homogenate. Samples were either treated or not treated with PK as indicated. Membranes were probed with monoclonal antibody 6H4. Tg, transgenic.