Susceptibilities of nonhuman primates to chronic wasting disease

Abstract

Chronic wasting disease (CWD) is a transmissible spongiform encephalopathy, or prion disease, that affects deer, elk, and moose. Human susceptibility to CWD remains unproven despite likely exposure to CWD-infected cervids. We used 2 nonhuman primate species, cynomolgus macaques and squirrel monkeys, as human models for CWD susceptibility. CWD was inoculated into these 2 species by intracerebral and oral routes. After intracerebral inoculation of squirrel monkeys, 7 of 8 CWD isolates induced a clinical wasting syndrome within 33-53 months. The monkeys' brains showed spongiform encephalopathy and protease-resistant prion protein (PrPres) diagnostic of prion disease. After oral exposure, 2 squirrel monkeys had PrPres in brain, spleen, and lymph nodes at 69 months postinfection. In contrast, cynomolgus macaques have not shown evidence of clinical disease as of 70 months postinfection. Thus, these 2 species differed in susceptibility to CWD. Because humans are evolutionarily closer to macaques than to squirrel monkeys, they may also be resistant to CWD.

Figure 1

A) Western blot of chronic wasting disease (CWD) inocula showing protease-resistant prion protein (PrPres) in 8 CWD brain homogenate pools used for infecting nonhuman primates. Lane 1, 0.2-mg tissue equivalents of uninfected elk brain not treated with proteinase K; lanes 2–9, samples treated with proteinase K: lanes 2, 6, and 7, 0.12-mg tissue equivalents; lanes 3–5, 8, and 9, 0.67 mg tissue equivalents. PrPres was detected by using antibody L42 against PrP and enhanced chemiluminescence (GE Healthcare, Piscataway, NJ, USA). To provide optimal exposure for viewing PrP in all lanes, blot was exposed to film for 20 min. In this exposure, lanes 2, 6, 7, and 8 were exposed beyond the linear range; this blot could not be used to quantify relative PrPres levels. Values on the left are in kDa. For more accurate quantitations of PrPres, other gels with different amounts loaded were exposed for multiple times (see panel C). B) Titration of MD-3 CWD inoculum. End-point infectivity titrations were calculated for each CWD inoculum by inoculating 50 μL of serial 10-fold dilutions of each brain homogenate into transgenic mice expressing deer PrP, starting with a 1% (10^–2) brain homogenate. Shown are data for an MD-3 inoculum. As the inoculum became more dilute, the incubation period (in days) and variability within a group increased. Each open circle represents 1 mouse in which clinical CWD developed. One mouse inoculated with a 10^–6 dilution and 5 mice inoculated with a 10^–7 dilution did not become sick after 625 days (solid circles). C) Infectivity titer and PrPres levels of each CWD pool. Titers are 50% infectious dose/g of brain homogenate. Relative level (%) of PrPres in each pool was measured by Western blot with a combination of serial dilutions and sequential exposure times in the linear response range for each sample. Data obtained from these comparisons are summarized in the PrPres column. All pools were compared with the pool with the highest PrPres signal (Elk-2), which was set at 100%.

Figure 2

Western blots of squirrel monkey protease-resistant prion protein (PrPres). A) Brain homogenate from squirrel monkey 322, showing proteinase K (PK)–resistant PrPres. A downward shift of 7–9 kDa after PK digestion indicated a banding pattern typical of PrPres (lane 2). After deglycosylation with peptide-N-glycosidase F, 1 band of PrPres was present (lane 3). Lane 1, 0.5 mg of brain tissue equivalents; lane 2, 0.6 mg; lane 3, 0.4 mg. Blot was developed by using antibody 3F4 against PrP, enhanced chemiluminescence (ECL), and a 2-min exposure. B) Brains of monkeys screened for PrPres. All tissues were treated with PK, and lanes were loaded with 0.25 mg brain tissue equivalents, except for lane 5, which was loaded with 1.0 mg. All lanes contain samples of brain cortex except lanes 6 and 7, which contain thalamus. Blot was developed by using antibody 3F4, ECL, and a 30-min exposure. Lane 1, control. In lanes 2–7, PrP banding is similar among squirrel monkeys infected with different pools of chronic wasting disease (CWD) agent (Table 1). PrPres was not detected in brain of orally infected squirrel monkey 301 (lane 8) or in brain of an intracerebrally infected cynomolgus macaque (Cm) 609 (lane 9). C) Lymphatic tissues from squirrel monkey 365. For visualization of PrPres in lymph node and spleen, increased amounts of tissue were loaded, and a more sensitive detection system (femto detection; Thermo Scientific, Waltham, MA, USA) was used. Lane 1, lymph node, 0.7 mg tissue equivalents; lane 2, spleen, 1.1 mg tissue equivalents. Blot was developed by using antibody 3F4, femto-enhanced ECL, and a 1-min exposure. D) PK-treated brain and lymphatic tissues from orally infected squirrel monkeys 303 and 345. Lane 1, positive control no. 640; lane 2, negative control; lane 3, no. 345 thalamus; lane 4, no. 303 thalamus; lane 5, no. 345 spleen; lane 6, no. 345 mesenteric lymph node. Bands were visualized by using antibody 3F4 (residues 109–112) and ECL. Lanes 1–5, 10-min exposure; lane 6, overnight exposure; tissue equivalents loaded per lane: lanes 1–4, 0.25 mg; lane 5, 0.5 mg; lane 6, 1 mg. Values on the left of all blots are in kDa.

Figure 3

Immunohistochemical analysis of squirrel monkeys infected with chronic wasting disease (CWD) agent. Panels A, C, and E–M are from squirrel monkeys infected with CWD. Panels B and D are from an uninfected monkey showing no pathologic changes or positive staining for protease-resistant prion protein (PrPres). Panels A and B, cerebral cortex stained with hematoxylin and eosin; panels C and D, thalamus stained with antibody 3F4 against PrP (arrows); panels E and F, cerebellar granular cell layer and spinal cord, respectively, stained with antibody 3F4; panel G, gray matter within the internal capsule stained with antibody D13 against PrP; panel H, corpus callosum (right) stained with antibody D13 showing more intense staining than the adjacent cortex (left); panel I, frontal cortex (fc), claustrum (cl), and caudate (ca) stained with antibody 3F4 (abundant vacuoles in the putamen [arrows]); panels J–M, lymphatic tissue stained with antibody 3F4; panels J and K, PrPres staining in spleen of monkey 322; panels L and M, PrPres-positive mesenteric lymph node from orally infected monkey 301. Rectangles in panels J and L show areas enlarged in panels K and M, respectively. Antibodies D13 and 3F4 showed similar results for each monkey regarding the distribution, characteristics, and plaque size of PrPres. Scale bars: panels A–I, 50 μm; panel J, 100 μm; panels K and M, 25 μm; panel L, 250 μm.

Figure 4

Comparison of prion protein sequences from various species. The following species are shown, and GenBank accession numbers are given when available: human (M13899), cynomolgus macaque (Cyno Mac) (U08298), squirrel monkey (Sq Mk) (genotype RML-A, see Table 4), squirrel monkey from Schneider et al. (31) (AY765385), squirrel monkey from Schätzl et al. (28) (U08310), mule deer (AY330343), and elk (AF156183). Numbering is based on the human sequence. Gray boxes indicate residues different from human residues. Alignment of the sequences was conducted with MegAlign software (DNAstar/Lasergene, Madison, WI, USA).