Squirrel monkeys were inoculated intracerebrally or orally with CWD inocula (7). We initially reported that 11/13 intracerebrally infected monkeys were euthanized at 41 months postinoculation (mpi) on average, and disease developed in 2/12 orally infected squirrel monkeys on average of 69 mpi (7). Disease developed in the 2 remaining intracerebrally infected squirrel monkeys at 61 and 75 mpi, respectively, changing the intracerebral attack rate to 100% (Figure 1, Table 1). Of the 10 remaining orally inoculated squirrel monkeys, disease developed in 9, bringing the overall oral attack rate to 92% and the average incubation period to 68 mpi (Figure 1, Table 1). Clinical signs were subtle; the most prominent finding was gradual weight loss (Table 1). A final diagnosis of CWD agent infection was made by using immunoblotting and immunohistochemical testing to determine accumulation of abnormal, disease-associated prion protein (PrPres) in brain tissue (Technical Appendix).

To compare the neuropathologic changes in intracerebrally and orally infected squirrel monkeys, we analyzed 10 brain regions for spongiform lesion severity and PrPres deposition (Figure 1, panels B, C). No statistically significant differences were noted between the 2 routes of infection (p<0.05). All squirrel monkeys studied had severe spongiform degeneration in the striatum (Figure 1, panel D) and little involvement in cerebellum and occipital lobes (Figure 1, panel E). Spongiform lesions in cortical gray matter were not consistent throughout the brain. Affected areas were commonly observed adjacent to normal regions, most frequently in the frontal, temporal, and parietal lobes (Figure 1, panel F). Except for the striatum, PrPres deposition was generally most prominent in areas that showed severe vacuolation. PrPres deposits appeared in 2 forms: dense punctate extracellular plaques (Figure 1, panel G) and less dense pericellular aggregates. The spleens of CWD agent–infected squirrel monkeys were positive for PrPres in 46% of intracerebrally infected and 60% of orally infected squirrel monkeys (Figure 1, panel H). At least 1 lymph node was positive in 30% of intracerebrally infected squirrel monkeys and in 40% of orally infected squirrel monkeys (Figure 1, panel J; Technical Appendix).

Of the squirrel monkeys under study, 3 PRNP genotypes were represented (7). In the group of orally infected squirrel monkeys, 3 had a unique heterozygous genotype that encoded either 4 or 5 octapeptide repeats. Two of these monkeys were the last orally infected monkeys to be euthanized because of clinical disease (80 and 107 mpi), and the third heterozygote was clinically normal at 108 mpi. Heterozygosity within the PRNP gene has been shown to delay or prevent prion disease (8) and may play a role in this study.

We inoculated cynomolgus macaques as another nonhuman primate model for cross-species transmission of CWD. Compared with squirrel monkeys, cynomolgus macaques are biologically closer to humans, and cynomolgus macaque PrP is more homologous to human PrP (7). Nine cynomolgus macaques were inoculated orally and 6 were inoculated intracerebrally with 1 of 3 CWD pools as described (7). Our first report included negative data from 1 cynomolgus macaque euthanized at 49 mpi (7). Since then, we have euthanized and screened 6 cynomolgus macaques for TSE (Table 2). No evidence of prion infection was detected by immunoblot and immunohistochemical methods (data not shown).

The lack of CWD transmission during >10 years suggests that a substantial species barrier exists between cervids and cynomolgus macaques. In most TSE animal models, PrPres can be detected by 1/3–1/2 of the known incubation periods. If we extrapolate this to the cynomolgus macaques in this study, negative test results at 9 years would suggest that the incubation period would be >18 years. Other prion studies of cynomolgus macaques reported clinical disease within 2–3 years after inoculation with variant Creutzfeldt-Jakob disease agents (9), 3 years after inoculation with bovine spongiform encephalopathy agents (10,11), and 5 years after inoculation with sporadic Creutzfeldt-Jakob disease agents (9,12). In contrast, our findings indicate that CWD is unlikely to develop in cynomolgus macaques.

The cause of susceptibility to CWD agents in squirrel monkeys and resistance to them in cynomolgus macaques is uncertain. Prnp/PRNP gene sequence variation has been linked to disease susceptibility (8), and differences in the PRNP genes of cynomolgus macaques and the genes of squirrel monkeys could play a major role. Comparison of PRNP sequences among cynomolgus macaques and squirrel monkeys showed differences exist at 5 codons (56, 100, 108, 159, and 182) (7). It is not clear which difference or combination of changes might confer protection to cynomolgus macaques, or if resistance is caused by other factors. Of the 5 codon differences described above, those of cynomolgus macaques and humans are identical at positions 56, 159, and 182.

Two SM-CWD brain samples were inoculated into squirrel monkeys and cynomolgus macaques to verify that SM-CWD was infectious, test for further adaptation, and to see if SM-CWD was infectious to a broader range of nonhuman primates. Two squirrel monkeys inoculated intracerebrally with SM-CWD brain homogenates (SMP2-CWD) were euthanized at 23–24 mpi (Table 1). These incubation periods decreased by >11 months compared with that of the donor squirrel monkey. Neurologic signs in the 2 SMP2-CWD were more pronounced than observed during the first passage; however, weight loss was reduced. Neuropathologic examination and Western blot for PrPres confirmed TSE in both squirrel monkeys. In contrast to SM-CWD infections, the SMP2-CWD-infected brains had spongiform lesions and PrPres deposition in the occipital lobe (Figure 2, panels A, B). Biochemical comparison of glycoform patterns among CWD, SM-CWD, and SMP2-CWD were made by using 3 different anti-PrP antibodies (L42, 6H4, and 3F4) (Technical Appendix). In all cases, SM2-CWD had a greater proportion of unglycosylated PrPres and a lower proportion of double glycosylated PrPres than did SM-CWD (Figure 2, panel C). The decreased time of manifestation of disease, differences in glycoform patterns, and distribution of PrPres in brain tissue suggested that the CWD agent was still adapting within the squirrel monkey. However, similar to CWD, SM-CWD had not caused prion disease in cynomolgus macaques by 72 mpi (Table 2).

Our studies have shown that squirrel monkeys, but not cynomolgus macaques, were susceptible to CWD. Although these nonhuman primates are not exact models of human susceptibility, they support the data from transgenic mouse studies (3–6), in vitro experiments (13), and epidemiologic evidence (14,15) that suggest humans are at a low risk of contracting CWD. Nevertheless, it remains sensible to minimize exposure to tissues potentially contaminated with the CWD agent.

Materials and methods for analysis of nonhuman primate tissues for chronic wasting disease in nonhuman primates.