To study the possibility of cross-species transmission of CWD, two species of nonhuman primates, squirrel monkeys and cynomolgus macaques, were infected orally or intracerebrally with brain material derived from CWD affected deer or elk. At the conclusion of FY13 thirteen of thirteen intracerebrally infected squirrel monkeys had developed clinical neurological signs and were confirmed by biochemical and pathological testing of brain to have a prion disease. At the conclusion of FY14 11 of 11 orally infected squirrel monkeys also developed disease. Together these results demonstrate that CWD from elk and deer can infect and cause clinical prion disease in squirrel monkeys. Similar experiments were also done in cynomolgus macaques which are genetically closer to humans than are squirrel monkeys, and have been found to be susceptible to a variety of human prion diseases. As of July 2017, all 15 CWD-infected cynomolgus macaques had been euthanized and screened for prion disease. Based on clinical signs, histopathological examination and immunoblot screening of tissue for disease-specific PrPres, none of these cynomolgus macaques showed evidence for development of prion disease regardless of inoculation route. The lack of susceptibility of cynomolgus macaques to CWD was in marked contrast to the high susceptibility of squirrel monkeys to these same CWD isolates. Thus, there appeared to be strong species-specific differences in susceptibility of non-human primates to CWD. Based on the closer genetic relationship between humans and cynomolgus macaques, these data suggest that humans may also be resistant to CWD infection. To attempt to detect adaptation of CWD prions to primates after passage in squirrel monkeys, in FY08 two cynomolgus macaques and two squirrel monkeys were inoculated with brain homogenates derived from a CWD-infected squirrel monkey. During FY10 both squirrel monkeys inoculated with squirrel monkey-adapted CWD developed disease. The incubation period from this inocula was much faster and clinical signs were slightly different suggesting adaptation to a new host or a higher titer of inocula. The successful passage of CWD derived from a squirrel monkey into additional squirrel monkeys confirmed the presence of prion infectivity. This confirmation was important since the same squirrel monkey CWD inocula did not cause disease in transgenic mice expressing human or deer prion protein and has not caused disease in any of the cynomolgus macaques still under study. During FY13 one of the cynomolgus macaques was euthanized due to chronic weight loss and liver failure, and this monkey was tested negative for TSE disease, indicating that squirrel monkey-adapted CWD may still be unable to infect other species of non-human primates. In FY17 the remaining monkey in this study was euthanized and also tested negative for prion disease by all criteria listed above. In addition to testing infected squirrel monkey brain we are also evaluating blood from infected squirrel monkeys for the presence of prion infectivity. None of the blood product recipient monkeys have shown any signs of disease as of July 2017. The lack of transmission to the humanized mice is encouraging data that supports the conclusion that CWD will likely not infect humans. During FY15 we started a study to directly test the transmission of cervid derived CWD into tg66 mice which express human PrP. By July, 2017, these mice did not develop clinical signs of prion disease and prion disease pathology was not seen in the mice euthanized so far. These will continue to be monitored for prion disease for approximately 600-700 days post-infection.