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Short Communication |
1 Robert Koch-Institut (P24 – Transmissible Spongiform Encephalopathies), Nordufer 20, 13353 Berlin, Germany
2 Department of Cell Biology and Neurosciences, Istituto Superiore di Sanità, Viale Regina Elena 299, 00161 Rome, Italy
3 7815 Exeter Road, Bethesda, MD 20814, USA
Correspondence
Michael Beekes
BeekesM{at}rki.de
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ABSTRACT |
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These authors contributed equally to this work. 
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reported the detection of disease-associated prion protein (hereafter referred to as PrPTSE; Brown & Cervenakova, 2005), the biochemical marker for infectious agents causing transmissible spongiform encephalopathies (TSEs), in muscles from sheep infected experimentally with scrapie by the intracerebral or oral route and from sheep with natural scrapie. Although dietary exposure to ovine meat products contaminated with the scrapie agent is currently considered non-hazardous to humans, the report of Andréoletti et al. (2004) has gained considerable attention because it calls for a review of whether muscle of sheep should be considered as a potential source of infectious material for humans, especially in the case of spread of bovine spongiform encephalopathy (BSE) to sheep. Independently of the route of infection, PrPTSE is also deposited in muscle fibres of hamsters perorally (Thomzig et al., 2004a), intraperitoneally or intracerebrally (A. Thomzig, unpublished data) challenged with 263K scrapie agent, providing further evidence that scrapie-infected hamsters are relevant model animals mimicking key features of the spread of infection through the body in ovine TSEs (McBride et al., 2001; van Keulen et al., 2000, 2002).
Here, we report on PrPTSE deposition in muscle tissue of hamsters and mice infected experimentally with BSE or variant Creutzfeldt–Jakob disease (vCJD) agents. We have examined muscles from terminally ill rodents inoculated intracerebrally with hamster-adapted BSE (isolate BSE-H; Thomzig et al., 2004b), mouse-adapted BSE (isolate 6PB1; Maignien et al., 1999) or mouse-adapted vCJD (Cervenakova et al., 2003) for the presence of PrPTSE. The recipients inoculated with these TSE agents [50 or 30 µl 1 % (w/v) brain homogenate from terminally diseased hamsters or mice, respectively] showed incubation times of 287±28, 142±9 and 140±11 days until terminal disease (expressed as means±SD), respectively. Western blot and immunohistochemical detection of PrPTSE were performed as described previously (Thomzig et al., 2004a), with the exception that, in murine samples, we used the monoclonal anti-PrP antibody ICSM-18 (D-Gen Ltd). The specificity of this antibody for the detection of PrP has been demonstrated previously (White et al., 2003). Our study revealed widespread accumulation of PrPTSE in muscle tissue from animals in all three experimental models (Table 1).
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) and in muscle specimens from BSE- or vCJD-infected mice corresponding to 15–40 mg tissue (Fig. 1b) reached levels similar to or higher than those observed in the positive-control samples, i.e. in muscle specimens from uninfected animals that were spiked with 5x10–6 g BSE-infected hamster brain or 5x10–5 g BSE- (not shown) or vCJD-infected mouse brain.
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) and sheep with scrapie (Andréoletti et al., 2004): in the lingual muscle, PrPTSE was found in individual muscle fibres predominantly in the region of the fibre surface (Fig. 1c
), but also scattered within myocytes (Fig. 1d).
Muscle extracts from terminally ill C57Bl mice infected intracerebrally with the 6PB1 BSE agent showed substantial PrPTSE deposition in many of the skeletal muscles examined (Fig. 1b, lanes 1–3). We also observed similar deposits of PrPTSE in muscles of C57Bl mice inoculated with the mouse-adapted vCJD agent (Fig. 1b, lanes 4–6). Positive muscle specimens from BSE- or vCJD-infected mice showed an approximately 500- to 1000-fold lower concentration of PrPTSE than in the brain, similar to what has been reported previously for scrapie-infected mice (Bosque et al., 2002).
All of this special interest in muscle involvement derives from the fact that meat, and particularly beef, is a staple of the human diet and would thus constitute a public-health risk if it were found to be infectious in cattle or small ruminants like sheep and goats (European Food Safety Authority, 2005) with TSEs. Studies to investigate the involvement of muscle in the pathogenesis of TSEs date back to a report on scrapie infectivity in the muscle of an affected goat in 1962 (Pattison & Millson, 1962) that remained, for decades, the sole successful transmission attempt. However, increasingly sensitive methodologies developed within the past few years have documented the presence of PrPTSE in rodents infected experimentally with various strains of TSE and, as noted above, in both natural and experimental scrapie infections of sheep (Table 2). Although the detection of PrPTSE cannot be equated directly to the presence of infectious TSE agent, a consistent association between PrPTSE and infectivity was found when these two parameters were assayed in muscles of scrapie-infected hamsters and mice (Bosque et al., 2002; Thomzig et al., 2004a).
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). However, at each time point, only approximately 0·5 g muscle was bioassayed, representing a minute sample of the >500 kg muscle from the slaughtered cattle, so that any lack of homogeneity in the distribution of infectivity could easily have led to a negative result. Moreover, a statistical analysis of the sampling shows that, even with a homogeneous distribution, there was only a 50 % chance of detecting one infectious dose. This caveat to pronouncing BSE muscle to be non-infectious was fully appreciated by the authors of the study, but may not have been widely understood by its interpreters.
It must be borne in mind that the data of this study have been obtained from intracerebrally infected rodents, whereas natural transmission of scrapie and BSE probably occurs via the oral route. However, the relevance of the reported findings is corroborated by the detection of PrPTSE in muscles of sheep and hamsters after both oral and intracerebral infection with scrapie (Andréoletti et al., 2004; Thomzig et al., 2004a; A. Thomzig, unpublished data for intracerebrally infected hamsters). If rodents challenged intracerebrally with BSE or vCJD agents mirror the pathophysiology of muscle targeting in ovine BSE and human vCJD to a similar extent, this would point to muscles as reservoirs for infectivity in these diseases. Thus, our findings emphasize the need for further assessment of the risks for public health that may result from prions in skeletal muscle (Bosque et al., 2002), either by oral exposure to BSE in ruminant meat products or by surgical exposure to human vCJD.
This is the first report showing deposition of BSE- and vCJD-associated PrPTSE in muscle tissue. Kinetic studies in intracerebrally and perorally challenged animals will be necessary to further reveal whether BSE- and vCJD-related agents target muscle tissue prior to the onset of clinical symptoms after either highly invasive inoculation or alimentary infection.
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ACKNOWLEDGEMENTS |
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Received 22 June 2005;
accepted 12 September 2005.
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