Brent Race#, Kimberly Meade-White#, Richard Race and Bruce Chesebro* + Author Affiliations
In mice, prion infectivity was recently detected in fat. Since ruminant fat is consumed by humans and fed to animals, we determined infectivity titers in fat from two CWD-infected deer. Deer fat devoid of muscle contained low levels of CWD infectivity and might be a risk factor for prion infection of other species.
Here bioassays in transgenic mice expressing cervid prion protein revealed the presence of infectious prions in skeletal muscles of CWD-infected deer, demonstrating that humans consuming or handling meat from CWD-infected deer are at risk to prion exposure.
Subject: Prions in Skeletal Muscles of Deer with Chronic Wasting Disease [SCIENCE FULL TEXT]
Date: January 26, 2006 at 12:23 pm PST
Prions in Skeletal Muscles of Deer with Chronic Wasting Disease
Rachel C. Angers,1* Shawn R. Browning,1* Tanya S. Seward,2 Christina J. Sigurdson,4 Michael W. Miller,5 Edward A. Hoover,4 Glenn C. Telling1,2,3§
1Department of Microbiology, Immunology and Molecular Genetics, 2Sanders Brown Center on Aging, 3Department of Neurology, University of Kentucky, Lexington, KY 40536, USA. 4Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA. 5Colorado Division of Wildlife, Wildlife Research Center, Fort Collins, CO 80526, USA.
*These authors contributed equally to this work.
Present address: Department of Infectology, Scripps Research Institute, 5353 Parkside Drive, RF-2, Jupiter, Florida, 33458, USA.
Present address: Institute of Neuropathology, University of Zurich, Schmelzbergstrasse 12, 8091 Zurich, Switzerland.
§To whom correspondence should be addressed: E-mail: gtell2@uky.edu
Prions are transmissible proteinaceous agents of mammals that cause fatal neurodegenerative diseases of the central nervous system (CNS). The presence of infectivity in skeletal muscle of experimentally infected mice raised the possibility that dietary exposure to prions might occur through meat consumption (1). Chronic wasting disease (CWD), an enigmatic and contagious prion disease of North American cervids, is of particular concern. The emergence of CWD in an increasingly wide geographic area and the interspecies transmission of bovine spongiform encephalopathy (BSE) to humans as variant Creutzfeldt Jakob disease (vCJD) have raised concerns about zoonotic transmission of CWD.
To test whether skeletal muscle of diseased cervids contained prion infectivity, Tg(CerPrP)1536 mice (2) expressing cervid prion protein (CerPrP), were inoculated intracerebrally with extracts prepared from the semitendinosus/semimembranosus muscle group of CWD-affected mule deer or from CWD-negative deer. The availability of CNS materials also afforded direct comparisons of prion infectivity in skeletal muscle and brain. All skeletal muscle extracts from CWD-affected deer induced progressive neurological dysfunction in Tg(CerPrP)1536 mice with mean incubation times ranging between 360 and ~490 d, whereas the incubation times of prions from the CNS ranged from ~230 to 280 d (Table 1). For each inoculation group, the diagnosis of prion disease was confirmed by the presence of PrPSc in the brains of multiple infected Tg(CerPrP)1536 mice (see supporting online material for examples). In contrast, skeletal muscle and brain material from CWD-negative deer failed to induce disease in Tg(CerPrP)1536 mice (Table 1) and PrPSc was not detected in the brains of sacrificed asymptomatic mice as late as 523 d after inoculation (supporting online material).
Our results show that skeletal muscle as well as CNS tissue of deer with CWD contains infectious prions. Similar analyses of skeletal muscle BSE-affected cattle did not reveal high levels of prion infectivity (3). It will be important to assess the cellular location of PrPSc in muscle. Notably, while PrPSc has been detected in muscles of scrapie-affected sheep (4), previous studies failed to detect PrPSc by immunohistochemical analysis of skeletal muscle from deer with natural or experimental CWD (5, 6). Since the time of disease onset is inversely proportional to prion dose (7), the longer incubation times of prions from skeletal muscle extracts compared to matched brain samples indicated that prion titers were lower in muscle than in CNS where infectivity titers are known to reach high levels. Although possible effects of CWD strains or strain mixtures on these incubation times cannot be excluded, the variable 360 to ~490 d incubation times suggested a range of prion titers in skeletal muscles of CWD-affected deer. Muscle prion titers at the high end of the range produced the fastest incubation times that were ~30% longer than the incubation times of prions from the CNS of the same animal. Since all mice in each inoculation group developed disease, prion titers in muscle samples producing the longest incubation times were higher than the end point of the bioassay, defined as the infectious dose at which half the inoculated mice develop disease. Studies are in progress to accurately assess prion titers.
While the risk of exposure to CWD infectivity following consumption of prions in muscle is mitigated by relatively inefficient prion transmission via the oral route (8), these results show that semitendinosus/semimembranosus muscle, which is likely to be consumed by humans, is a significant source of prion infectivity. Humans consuming or handling meat from CWD-infected deer are therefore at risk to prion exposure.
References and Notes
1. P. J. Bosque et al., Proc. Natl. Acad. Sci. U.S.A. 99, 3812 (2002).
2. S. R. Browning et al., J. Virol. 78, 13345 (2004).
3. A. Buschmann, M. H. Groschup, J. Infect. Dis. 192, 934 (2005).
4. O. Andreoletti et al., Nat. Med. 10, 591 (2004).
5. T. R. Spraker et al., Vet. Pathol. 39, 110 (2002).
6. A. N. Hamir, J. M. Miller, R. C. Cutlip, Vet. Pathol. 41, 78 (2004).
7. S. B. Prusiner et al., Biochemistry 21, 4883 (1980).
8. M. Prinz et al., Am. J. Pathol. 162, 1103 (2003).
9. This work was supported by grants from the U.S. Public Health Service 2RO1 NS040334-04 from the National Institute of Neurological Disorders and Stroke and N01-AI-25491 from the National Institute of Allergy and Infectious Diseases.
Supporting Online Material
www.sciencemag.org/
Materials and Methods
Fig. S1
21 November 2005; accepted 13 January 2006 Published online 26 January 2006; 10.1126/science.1122864 Include this information when citing this paper.
Table 1. Incubation times following inoculation of Tg(CerPrP)1536 mice with prions from skeletal muscle and brain samples of CWD-affected deer.
Inocula Incubation time, mean d ± SEM (n/n0)*
Skeletal muscle Brain
CWD-affected deer
H92 360 ± 2 d (6/6) 283 ± 7 d (6/6)
33968 367 ± 9 d (8/8) 278 ± 11 d (6/6)
5941 427 ± 18 d (7/7)
D10 483 ± 8 d (8/8) 231 ± 17 d (7/7)
D08 492 ± 4 d (7/7)
Averages 426 d 264 d
Non-diseased deer
FPS 6.98 >523 d (0/6)
FPS 9.98 >454 d (0/7) >454 d (0/6)
None >490 d (0/6)
PBS >589 d (0/5)
*The number of mice developing prion disease divided by the original number of inoculated mice is shown in parentheses. Mice dying of intercurrent illnesses were excluded.
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Supporting Online Material for
Prions in Skeletal Muscles of Deer with Chronic Wasting Disease
Rachel C. Angers, Shawn R. Browning, Tanya S. Seward, Christina J. Sigurdson,
Michael W. Miller, Edward A. Hoover, Glenn C. Telling§
§To whom correspondence should be addressed: E-mail: gtell2@uky.edu
Published 26 January 2006 on Science Express
DOI: 10.1126/science.1122864
This PDF file includes:
Materials and Methods
Fig. S1
Supporting Online Materials
Materials and Methods
Homogenates of semitendinosus/semimembranosus muscle (10% w/v in phosphate
buffered saline) were prepared from five emaciated and somnolent mule deer, naturally
infected with CWD at the Colorado Division of Wildlife, Wildlife Research Center.
These deer were identified as D10, D08, 33968, H92, and 5941. CWD infection was
confirmed in all cases by the presence of histologic lesions in the brain including
spongiform degeneration of the perikaryon, the immunohistochemical detection of
disease-associated PrP in brain and tonsil, or by immunoblotting of protease-resistant,
disease associated PrP (CerPrPSc). Semitendinosus/semimembranosus muscle was also
obtained from two asymptomatic, mock inoculated deer, referred to as FPS 6.68 and 9.98,
that originated from a CWD non-endemic area and which were held indoors at Colorado
State University from ten days of age. These control deer were confirmed negative for
CWD by histopathological and immunohistochemical analysis of brain tissue at autopsy.
The utmost care was taken to avoid inclusion of obvious nervous tissue when muscle
biopsies were prepared and to ensure that contamination of skeletal muscle samples with
CNS tissue did not occur. Fresh, single-use instruments were used to collect each sample
biopsy and a central piece from each sample was prepared with fresh, disposable
instruments to further isolate muscle tissue for inoculum preparation. Brain samples for
transmission were prepared separately from muscle as additional insurance against cross
contamination.
1
Groups of anesthetized Tg(CerPrP)1536 mice were inoculated intracerebrally with 30 µl
of 1 % skeletal muscle or brain extracts prepared in phosphate buffered saline (PBS).
Inoculated Tg(CerPrP) mice were diagnosed with prion disease following the progressive
development of at least three neurologic symptoms including truncal ataxia, plastic tail,
loss of extensor reflex, difficultly righting, and slowed movement. The time from
inoculation to the onset of clinical signs is referred to as the incubation time.
For PrP analysis in brain extracts of Tg(CerPrP)1536 mice, 10 % homogenates prepared
in PBS were either untreated (-) or treated (+) with 40 µg/ml proteinase K (PK) for one
hour at 37oC in the presence of 2% sarkosyl. Proteins were separated by sodium dodecyl
sulfate polyacrylamide gel electrophoresis, analyzed by immunoblotting using anti PrP
monoclonal antibody 6H4 (Prionics AG, Switzerland), incubated with appropriate
secondary antibody, developed using ECL-plus detection (Amersham), and analyzed
using a FLA-5000 scanner (Fuji).
2
Fig. S1
PrP in brain extracts from representative Tg(CerPrP)1536 mice receiving muscle or CNS
tissue inocula from CWD-affected or CWD-negative deer. Extracts were either treated
(+) or untreated (-) with proteinase K (PK) as indicated. The positions of protein
molecular weight markers at 21.3, 28.7, 33.5 kDa (from bottom to top) are shown to the
left of the immunoblot.
3
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