Figure 1.
Recovery of PrPTSE after extraction from muscle tissue.
Western blot detection of PrP27-30, the Proteinase K-resistant core of PrPTSE, 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; PrPTSE extract from 100 mg of skeletal muscle (M. glutaeobiceps) from an uninfected control WTD (blotted also in lane 9 of figure 2; Fa-WTD 10) spiked before extraction with 1×10−5 g of CWD brain tissue from a WTD in the clinical stage of disease.
Figure 2.
Detection and semi-quantitative assessment of PrPTSE in skeletal muscles of CWD-infected WTD.
Western blot detection of PrP27-30, the Proteinase K-resistant core of PrPTSE, after labelling with anti-PrP monoclonal antibody ICSM-18. Lanes 1–3, PrPTSE extracts from 100 mg of skeletal muscle from an uninfected control WTD (blotted also in lane 9) spiked before extraction with 1×10−4 g (lane 1), 5×10−5 g (lane 2), and 1×10−5 g of CWD brain tissue from a WTD in the clinical stage of disease. Lanes 4–8, PrPTSE extracts from 100 mg samples of the following muscles from CWD-infected WTD: M. glutaeobiceps (farmed animal Fa-WTD 8, lane 4), M. semimembranosus/tendinosus (farmed animal Fa-WTD 1, lane 5), M. semimembranosus/tendinosus (hunter-kill animal FR-WTD 1, lane 6), M. semimembranosus/tendinosus (hunter-kill animal FR-WTD 2, lane 7), M. semimembranosus/tendinosus (farmed animal Fa-WTD 2, lane 8). Lane 9, extract from 100 mg sample of M. glutaeobiceps from an uninfected control WTD (Fa-WTD 10).
Table 1.
Presence and seeding actvity of PrPTSE in skeletal muscles from CWD-infected white-tailed deer.
Figure 3.
PrPTSE-associated seeding activity in muscles from CWD-infected WTD.
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 PrPTSE-extract from M. semimembranosus/tendinosus of a CWD-infected WTD (Fa-WTD 1, B). In (C) PMCA was seeded with M. semimembranosus/tendinosus from Fa-WTD 2 that had been tested positive for PrPTSE in brain tissue while no PrPTSE could be detected by Western blotting in muscle extracts of this animal (the negative result with this muscle is shown in figure 2, 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.
Figure 4.
Intramuscular location of PrPTSE in skeletal muscle tissue of CWD-infected WTD.
(A) Tissue-blot from M. glutaeobiceps of CWD-infected WTD (FR-WTD 2). Distinct granular endoneural PrPTSE accumulation in muscle-associated nerve fascicles visualised by immunolabelling with the monoclonal anti-PrP antibody P4. No PrPTSE 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 M. glutaeobiceps 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.