Two macaques orally inoculated with L-BSE prions remained asymptomatic and healthy but were euthanized and autopsied at 75 months postinoculation. WB showed no PrP^Sc accumulation in any tissue (Table), IHC revealed no PrP^Sc accumulation, hematoxylin and eosin staining revealed no spongiform changes in brain sections, and pathologic examination revealed no abnormalities.

Figure 1

Figure 1. Sensitivity of modified protein misfolding cyclic amplification (PMCA) to detect abnormal prion protein in study of oral transmission of L-type bovine spongiform encephalopathy (L-BSE) in intracerebrally inoculated macaques to evaluate...

We next attempted to detect PrP^Sc using PMCA, performed as previously described (9), with minor modifications (Appendix). First, we evaluated the sensitivity of PMCA. Using serial amplification with 10-fold stepwise dilutions of prion-infected brain homogenates as seeds, we amplified PrP^Sc-like proteinase K (PK)–resistant prion protein (PrPres) from a 10⁻⁷ dilution of 10% brain homogenate (BH) obtained from macaque intracerebrally inoculated with L-BSE prions in the fifth amplification round (Figure 1, panel A). This method also enabled propagation of PrPres from a 10⁻⁸ dilution of BH from C-BSE–affected cattle during the second amplification round (Figure 1, panel B), suggesting PMCA’s higher efficiency and sensitivity for detecting C-BSE prions than macaque L-BSE prions.

Figure 2

Figure 2. Sensitivity of modified protein misfolding cyclic amplification (PMCA) to detect abnormal prion protein in study of oral transmission of L-type bovine spongiform encephalopathy (L-BSE) in orally inoculated macaques to evaluate...

We attempted to detect prions in the lymphoid and nervous systems, among other tissues, of the 2 orally inoculated macaques using refined PMCA (Figure 2; Appendix Figure, Table). In lymphoid tissue samples prepared using sodium phosphotungstic acid precipitation (Appendix), we amplified PrPres in the inguinal and mesenteric lymph nodes, ileum, and tonsils of both macaques (Figure 2, panels A, B), as well as in the spleen of 1 macaque (#18) and the thymus of the other (#19), in the second or third amplification round of PMCA (Figure 2, panel C). We observed no PrPres in the submandibular lymph nodes (Appendix Figure 1). Examining the central nervous system, we observed no PrPres amplification in the cerebral cortex (Figure 2, panel C), whether seeded with phosphate-buffered saline homogenates or phosphotungstic acid precipitates. The spinal cord showed no PrPres amplification upon ethanol precipitation. However, PrPres was amplified in the cervical spinal cord of macaque #19 and in the thoracic spinal cord of both macaques with phosphate-buffered saline homogenates (Figure 2, panel D). We also confirmed PrPres in the median nerve of both macaques but not in the sciatic nerve (Figure 2, panel E). We noted PrPres signals in the submandibular glands of both animals. In contrast, we found no PrPres amplification in any tissues from uninoculated control macaques.

Figure 3

Figure 3. Gel electrophoresis and Western blot testing of tissues obtained from macaques orally inoculated with L-BSE prions in study of oral transmission of L-BSE in macaques to evaluate zoonotic potential. We...

PrPres obtained from the orally inoculated macaques exhibited diverse banding patterns distinct from those generated by PMCA using L-BSE–affected cattle BH and L-BSE intracerebrally inoculated macaque BH as seeds (Figure 3, panels A–C). Of note, the lowest-molecular-weight PrPres variants from the ileum, spleen, inguinal lymph nodes, thoracic cord, submaxillary gland, and mesenteric lymph nodes of orally inoculated macaques exhibited remarkable PK resistance similarity and banding patterns indistinguishable from those of PrPres generated by PMCA with C-BSE–affected cattle BH as a seed (Figure 3, panel C, Appendix Figures 2 and 3). In contrast, the higher-molecular-weight PrPres variants from the ileum of macaque #18 exhibited a unique banding pattern distinct from those of L-BSE, C-BSE, and H-type BSE prions (Figure 3, panel C). Banding patterns and PK resistance of PrPres amplified from L-BSE–affected cattle BH and L-BSE intracerebrally inoculated macaque BH were notably similar.

This PMCA method was initially designed for the high-sensitivity detection of L-BSE intracerebrally inoculated macaque PrP^Sc but was even more efficient and sensitive in detecting bovine C-BSE PrP^Sc (Figure 1, panel B). Therefore, we believe that this method enabled the detection of both C-BSE–like PrP^Sc and potentially novel PrP^Sc variants.

We noted no detectable evidence of PrP^Sc by WB or IHC in any tissues of L-BSE orally inoculated macaques. Nevertheless, PMCA successfully amplified PrPres from lymphatic and neural tissues. The PrPres exhibited electrophoretic patterns distinct from those detected by PMCA using L-BSE–affected cattle BH as the seed (Figure 3, panel C), indicating that the PrP^Sc used as the template for PrPres amplification in orally inoculated macaques did not originate from the bovine L-BSE prions used as inoculum. Instead, PrP^Sc were newly generated by the conversion of macaque PrP^C by bovine L-BSE prions. Our results provide strong evidence that L-BSE can infect macaques via the oral route.

We found no evidence that PrP^Sc reached the brain in orally inoculated macaques; however, the macaques euthanized 6 years postinoculation might have been in the preclinical period. At low infection levels, lymph nodes play a vital role in prion spread to the central nervous system (11). Therefore, had the macaques been maintained for a longer period, they might have developed prion disease. Retrospective surveillance studies using the appendix and tonsil tissues suggested a considerable number of humans harboring vCJD in a carrier state (12). Thus, we cannot exclude that L-BSE orally inoculated macaques could similarly remain in a potentially infectious state.

The brain of L-BSE intracerebrally inoculated macaque accumulated prions with biochemical properties resembling bovine L-BSE prions (Figure 3, panel C; Appendix Figure 2); however, we observed no PrP^Sc accumulation in lymphoid tissues by WB or IHC (4). In contrast, macaques orally inoculated with C-BSE prions showed PrP^Sc accumulation in lymphoid tissues, including the spleen, tonsils, and mesenteric lymph nodes by WB and IHC (13). In our study, L-BSE orally inoculated macaques harbored C-BSE–like prions in their lymphoid and neural tissues. Interspecies transmission of L-BSE prions to ovine PrP transgenic mice can result in a shift toward C-BSE–like properties (14,15). Our data suggest that L-BSE prions may alter biophysical and biochemical properties, depending on interspecies transmission and inoculation route, acquiring traits similar to those of C-BSE prions. This transformation might result from structural changes in the L-BSE prion to C-BSE–like prions and other lymphotropic prions within lymphoid tissues or from the selective propagation of low-level lymphotropic substrains within the L-BSE prion population.

The first limitation of our study is that the oral inoculation experiment involved only 2 macaques and tissues collected at 6 years postinoculation, before disease onset. Consequently, subsequent progression of prion disease symptoms remains speculative. A larger sample size and extended observation periods are required to conclusively establish infection in orally inoculated macaques. Furthermore, we performed no bioassays for PMCA-positive samples, leaving the relationship between PMCA results and infectious titers undefined. Considering that PrPres amplifications from tissues from the orally inoculated macaque tissues required 2 rounds of PMCA, the PrP^Sc levels in positive tissues might have been extremely low and undetectable in the bioassay.

Previous studies have demonstrated that L-BSE can be orally transmitted to cattle (7) and might have caused prion disease in farm-raised minks (6), indicating that L-BSE could naturally affect various animal species. Our findings suggest that L-BSE can also be orally transmitted to macaques. Therefore, current control measures aimed at preventing primary C-BSE in cattle and humans may also need to consider the potential risk of spontaneous L-BSE transmission.