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Emergence of bovine spongiform encephalopathy (BSE) and the associated variant Creutzfeldt-Jakob disease (vCJD) in humans in the United Kingdom (Will et al., 1996), their spread throughout Europe, and the recent recognition of BSE in cattle in Canada and the United States has thrust the transmissible spongiform encephalopathies (TSEs) into the center of public concern about food safety, into the political and international trade arenas, and focused biomedical attention on these diseases. Although the public response to the recent recognition of a case of BSE in the United States has not caused a crisis in domestic consumption of beef, trade restrictions are imposing considerable hardship on the beef industry. If additional cases of BSE are discovered in the United States, consumer faith in the wholesomeness of beef could be challenged resulting in decreased beef consumption and major impacts to the industries.
Unique to North America is the presence of a TSE of wild ruminants (Williams and Miller, 2002). Chronic wasting disease of cervids (members of the deer family), occurs endemically in captive and free-ranging mule deer (Odocoileus hemionus), white-tailed deer (Odocoileus virginianus), and Rocky Mountain elk (Cervus elaphus) in northern Colorado, eastern Wyoming, and contiguous areas of western South Dakota and Nebraska and eastern Utah (Williams and Young, 1980, 1982; Spraker et al., 1997; Miller et al., 1998; Williams and Miller, 2002). It probably has been present in the core of this area since at least the 1960s (Gross and Miller, 2001). In February 2002, CWD was identified in white-tailed deer in southcentral Wisconsin (Joly et al., 2003); subsequent surveillance in this area indicates that CWD is endemic in this location. Concerns have been expressed by hunters, wildlife managers, physicians, and scientists about the implications of CWD for public health (Bosque, 2002; Bosque et al., 2002; Erdtmann and Sivitz, 2004); however, currently no link has been identified between CWD and any human disease (Belay et al., 2001, 2004) although this potential cannot be ruled out. Because beef and venison are significant items in the diets of many Americans it is important to determine if these foods might contain the disease associated prion.
Chronic wasting disease is horizontally transmitted among deer and elk in captivity (Williams and Young, 1992; Miller et al., 1998, 2004; Miller and Williams, 2003), and transmission among deer and elk probably occurs by direct contact and/or environmental contamination (Miller et al., 2004). Deer and elk in the CWD-endemic areas of Colorado and Wyoming share range with cattle where they may directly interact or graze on common pastures and have been since at least 1981 when the first case in a free-ranging elk was identified (Spraker et al., 1997). Thus cattle are historically and currently potentially exposed to PrPCWD although no occurrence of a bovine TSE has been identified in these areas.
Studies of cattle susceptibility to CWD are completed or are currently underway. In vitro studies of cell-free conversion of bovine PrPC by deer or elk PrPCWD showed a substantial molecular barrier between the cervids and cattle, suggesting cattle are poorly susceptible to CWD (Raymond et al., 2000). However, although in vitro and rodent laboratory models are very useful for study of the TSEs, the most reliable information and the most acceptable and understandable to the public must utilize animals of the species of interest. Therefore in 1997, we began direct studies of cattle susceptibility to CWD. Due to the very long incubation periods expected for the TSEs, these studies will not be completed until approximately 2007. Transmission of CWD to cattle was demonstrated in five of 13 head exposed by intracerebral inoculation (Hamir et al., 2001; personal communication). However, there has been no evidence of transmission of CWD to cattle in another series of experiments where cattle were exposed by more natural means either orally or by direct contact with CWD affected deer and elk (Williams, Miller, and Kreeger, unpublished data). And finally, in another study of cattle susceptibility to CWD (funded by the United States Department of Agriculture), calves were exposed to CWD agent orally and were killed sequentially to determine if there was evidence of transmission in the early post-exposure period.
Since recognition of BSE there has been concern about the possibility that the infectious agent could be present in meat. Studies including those conducted in the United Kingdom on BSE (Wells et al., 1998) and studies of scrapie in domestic sheep (Hadlow et al., 1982) have not demonstrated infectivity in muscle of naturally occurring animal TSEs. Prions have not been demonstrated in skeletal muscle of cervids with CWD or other animals with prion diseases by immunohistochemistry (Spraker et al., 2002b; Hamir et al., 2004; Williams et al., unpublished data). We were unable to detect PrPres in three skeletal muscles from mule deer and elk experimentally infected with CWD by enzyme-linked immunosorbent assay (ELISA) in a preliminary study using a small number of animals (Deslys et al., unpublished data). However, Bartz et al. (2003) were able to detect PrPres in nerve axons in the tongue of hamsters experimentally inoculated with transmissible mink encephalopathy (a TSE of mink) suggesting the possibility of anterograde transport of PrP from the brain to peripheral tissues including muscles. Recently Bosque et al. (2002), using western blotting, ELISA, and bioassay demonstrated high levels of PrPC and PrPres in skeletal muscles of mice experimentally inoculated with several strains of scrapie agent. Similarly, Thomzig et al. (2003) have shown the presence of PrPres in skeletal muscle of hamsters orally infected with scrapie. The situation in humans is similar, in that Brown et al. (1994) were not able to detect prion infectivity in muscles of humans with Creutzfeldt-Jakob disease by bioassay in primates, but PrPres was found in skeletal muscle of about a third of natural cases of sporadic CJD in humans (Glatzel et al., 2003). These observations suggest that PrPres is present in skeletal muscles of some of the TSEs and indicates a need to apply sensitive detection techniques to skeletal muscles from cervids with CWD and cattle exposed to CWD.
The stated objectives for this work were: Our objective in this study was to determine if chronic wasting disease (CWD) associated proteinase resistant prion protein (abbreviated as PrPres or PrPCWD), as a marker for infectivity, could be detected in striated muscles of deer and elk with experimental and natural CWD and in cattle experimentally exposed to CWD. Three specific objectives were proposed for this study. The first objective was to determine if cellular prion protein (the normal, nonpathogenic form of the prion protein, abbreviated as PrPC) could be detected in skeletal muscles of mule deer, white-tailed deer, elk, and cattle. The second and third objectives were similar and were to determine if PrPres could be detected in striated muscles of deer, elk, and cattle experimentally or naturally exposed to CWD.
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