Presence and Seeding Activity of Pathological Prion Protein (PrPTSE) in Skeletal Muscles of White-Tailed Deer Infected with Chronic Wasting Disease

Chronic wasting disease (CWD) is a contagious, rapidly spreading transmissible spongiform encephalopathy (TSE), or prion disease, occurring in cervids such as white tailed-deer (WTD), mule deer or elk in North America. Despite efficient horizontal transmission of CWD among cervids natural transmission of the disease to other species has not yet been observed. Here, we report for the first time a direct biochemical demonstration of pathological prion protein PrPTSE and of PrPTSE-associated seeding activity, the static and dynamic biochemical markers for biological prion infectivity, respectively, in skeletal muscles of CWD-infected cervids, i. e. WTD for which no clinical signs of CWD had been recognized. The presence of PrPTSE was detected by Western- and postfixed frozen tissue blotting, while the seeding activity of PrPTSE was revealed by protein misfolding cyclic amplification (PMCA). Semi-quantitative Western blotting indicated that the concentration of PrPTSE in skeletal muscles of CWD-infected WTD was approximately 2000-10000 -fold lower than in brain tissue. Tissue-blot-analyses revealed that PrPTSE was located in muscle-associated nerve fascicles but not, in detectable amounts, in myocytes. The presence and seeding activity of PrPTSE in skeletal muscle from CWD-infected cervids suggests prevention of such tissue in the human diet as a precautionary measure for food safety, pending on further clarification of whether CWD may be transmissible to humans

Intramuscular location of PrP^TSE in skeletal muscle tissue of CWD-infected WTD.

<p>(A) Tissue-blot from <i>M. glutaeobiceps</i> of CWD-infected WTD (FR-WTD 2). Distinct granular endoneural PrP^TSE accumulation in muscle-associated nerve fascicles visualised by immunolabelling with the monoclonal anti-PrP antibody P4. No PrP^TSE accumulation could be detected in muscle fibres. (B) H&E stained slice from the same tissue block as in (A) showing nerves embedded in the muscle tissue. (C) Control tissue-blot of <i>M. glutaeobiceps</i> from uninfected WTD (Fa-WTD 10). (D) H&E stained slice from the same tissue block as in (C). Arrows indicate muscle-associated nerve fascicles in A–D. Bars = 2 mm.</p

PrP^TSE-associated seeding activity in muscles from CWD-infected WTD.

<p>Western blot detection of Proteinase K-resistant prion protein (PrPres) after PMCA. For PMCA normal hamster brain homogenate was seeded with 10 µl of a 10% (w/v) CWD brain homogenate from an infected WTD (A), or with PrP^TSE-extract from <i>M. semimembranosus/tendinosus</i> of a CWD-infected WTD (Fa-WTD 1, B). In (C) PMCA was seeded with <i>M. semimembranosus/tendinosus</i> from Fa-WTD 2 that had been tested positive for PrP^TSE in brain tissue while no PrP^TSE could be detected by Western blotting in muscle extracts of this animal (the negative result with this muscle is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018345#pone-0018345-g002" target="_blank">figure 2</a>, lane 8). (D) Unseeded control in which PMCA was performed with normal hamster brain homogenate only. Lanes 1–7 represent Western blot findings after 1, 2, 3, 4, 5, 6 and 7 rounds of PMCA. Lane 8, Proteinase K-digested brain homogenate from a hamster in the clinical stage of scrapie representing 1×10⁻⁷ g of hamster brain tissue (loaded as an internal blotting control). M: molecular mass marker. Anti-PrP monoclonal antibody 3F4 was used as primary antibody for the detection of PrPres.</p

Presence and seeding actvity of PrP^TSE in skeletal muscles from CWD-infected white-tailed deer.

<p><b>Footnote to </b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018345#pone-0018345-t001" target="_blank"><b>Table 1</b></a><b>.</b> Explanation of abbreviations: n.a. – no tissue available for testing; n.e. – not examined; SA – PMCA testing for seeding activity of PrP^TSE; TB – Tissue blot testing for PrP^TSE; WB – Western blot testing for PrP^TSE. Explanation of symbols:</p>†<p>Western blot testing of WTD brain tissue and/or lymph nodes for PrP^TSE was performed by the responsible authorities in Canada or the USA in the context of CWD surveillance.</p

Recovery of PrP^TSE after extraction from muscle tissue.

<p>Western blot detection of PrP27-30, the Proteinase K-resistant core of PrP^TSE, after labelling with anti-PrP monoclonal antibody ICSM-18. Lanes 1–4; Proteinase K-digested brain homogenates from WTD in the clinical stage of CWD representing 5×10⁻⁵ g (lane 1), 1×10⁻⁵ g (lane 2), 5×10⁻⁶ g (lane 3) and 1×10⁻⁶ g (lane 4) of CWD brain tissue. Lane 5; PrP^TSE extract from 100 mg of skeletal muscle (<i>M. glutaeobiceps</i>) from an uninfected control WTD (blotted also in lane 9 of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018345#pone-0018345-g002" target="_blank">figure 2</a>; Fa-WTD 10) spiked before extraction with 1×10⁻⁵ g of CWD brain tissue from a WTD in the clinical stage of disease.</p