Administration of L-type Bovine Spongiform Encephalopathy to Macaques to Evaluate Zoonotic Potential

Volume 31, Number 5—May 2025

Dispatch

Administration of L-type Bovine Spongiform Encephalopathy to Macaques to Evaluate Zoonotic Potential

Morikazu Imamura1Comments to Author , Ken’ichi Hagiwara, Minoru Tobiume, Minako Ohno, Hiromi Iguchi, Hanae Takatsuki, Tsuyoshi Mori, Ryuichiro Atarashi, Hiroaki Shibata, and Fumiko Ono1 Author affiliation: University of Miyazaki, Miyazaki, Japan (M. Imamura, M. Ohno, H. Iguchi, H. Takatsuki, T. Mori, R. Atarashi); National Institute of Infectious Diseases, Tokyo, Japan (K. Hagiwara, M. Tobiume); The Corporation for Production and Research of Laboratory Primates, Tsukuba, Japan (H. Shibata); Okayama University of Science, Imabari, Japan (F. Ono) Suggested citation for this article

Abstract

We administered L-type bovine spongiform encephalopathy prions to macaques to determine their potential for transmission to humans. After 75 months, no clinical symptoms appeared, and prions were undetectable in any tissue by Western blot or immunohistochemistry. Protein misfolding cyclic amplification, however, revealed prions in the nerve and lymphoid tissues.

Worldwide emergence of classical bovine spongiform encephalopathy (C-BSE) is associated with variant Creutzfeldt-Jakob disease in humans (1). Two other naturally occurring BSE variants have been identified, L-type (L-BSE) and H-type. Studies using transgenic mice expressing human normal prion protein (PrPC) (2) and primates (3–5) have demonstrated that L-BSE is more virulent than C-BSE. Although L-BSE is orally transmissible to minks (6), cattle (7), and mouse lemurs (5), transmissibility to cynomolgus macaques, a suitable model for investigating human susceptibility to prions, remains unclear. We orally inoculated cynomolgus macaques with L-BSE prions and explored the presence of abnormal prion proteins (PrPSc) in tissues using protein misfolding cyclic amplification (PMCA) along with Western blot (WB) and immunohistochemistry (IHC). PMCA markedly accelerates prion replication in vitro, and its products retain the biochemical properties and transmissibility of seed prion strains (8).

The Study

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

We next attempted to detect PrPSc 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 PrPSc-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 😎, suggesting PMCA’s higher efficiency and sensitivity for detecting C-BSE prions than macaque L-BSE prions.

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, 😎, 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.

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 PrPSc but was even more efficient and sensitive in detecting bovine C-BSE PrPSc (Figure 1, panel 😎. Therefore, we believe that this method enabled the detection of both C-BSE–like PrPSc and potentially novel PrPSc variants.

Conclusion

We noted no detectable evidence of PrPSc 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 PrPSc used as the template for PrPres amplification in orally inoculated macaques did not originate from the bovine L-BSE prions used as inoculum. Instead, PrPSc were newly generated by the conversion of macaque PrPC 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 PrPSc 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 PrPSc accumulation in lymphoid tissues by WB or IHC (4). In contrast, macaques orally inoculated with C-BSE prions showed PrPSc 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 PrPSc 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.

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Dr. Imamura is an associate professor in the Division of Microbiology, Department of Infectious Diseases, Faculty of Medicine, University of Miyazaki, Miyazaki, Miyazaki, Japan. His research interests are focused on elucidating the mechanisms underlying prion formation.

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Acknowledgment This study was supported by the Health Labor Sciences Research Grant (H29-Shokuhin-Ippan-004, 20KA1003, and 23KA1004).

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References…snip…end

https://wwwnc.cdc.gov/eid/article/31/5/24-1257_article

***> Our results provide strong evidence that L-BSE can infect macaques via the oral route.

Wednesday, May 24, 2023

***> WAHIS, WOAH, OIE, United States of America Bovine spongiform encephalopathy Immediate notification

https://wahis.woah.org/#/in-review/5067

https://woahoie.blogspot.com/2023/05/wahis-woah-oie-united-states-of-america.html

https://prpsc.proboards.com/thread/125/wahis-woah-oie-immediate-notification

SATURDAY, MAY 20, 2023

***> Tennessee State Veterinarian Alerts Cattle Owners to Disease Detection Mad Cow atypical L-Type BSE

https://bse-atypical.blogspot.com/2023/05/tennessee-state-veterinarian-alerts.html

https://prpsc.proboards.com/thread/123/tennessee-veterinarian-alerts-cattle-confirmed

USDA Announces Atypical Bovine Spongiform Encephalopathy Detection Published: May 19, 2023 

The U.S. Department of Agriculture (USDA) is announcing an atypical case of Bovine Spongiform Encephalopathy (BSE), a neurologic disease of cattle, in an approximately five-year-old or older beef cow at a slaughter plant in South Carolina. This animal never entered slaughter channels and at no time presented a risk to the food supply or to human health in the United States. Given the United States’ negligible risk status for BSE, we do not expect any trade impacts as a result of this finding.

USDA Animal and Plant Health Inspection Service’s (APHIS) National Veterinary Services Laboratories (NVSL) confirmed that this cow was positive for atypical L-type BSE. The animal was tested as part of APHIS’s routine surveillance of cattle that are deemed unsuitable for slaughter. The radio frequency identification tag present on the animal is associated with a herd in Tennessee. APHIS and veterinary officials in South Carolina and Tennessee are gathering more information during this ongoing investigation.

Atypical BSE generally occurs in older cattle and seems to arise rarely and spontaneously in all cattle populations.

This is the nation’s 7th detection of BSE. Of the six previous U.S. cases, the first, in 2003, was a case of classical BSE in a cow imported from Canada; the rest have been atypical (H- or L-type) BSE.

The World Organization for Animal Health (WOAH) recognizes the United States as negligible risk for BSE. As noted in the WOAH guidelines for determining this status, atypical BSE cases do not impact official BSE risk status recognition as this form of the disease is believed to occur spontaneously in all cattle populations at a very low rate. Therefore, this finding of an atypical case will not change the negligible risk status of the United States, and should not lead to any trade issues.

The United States has a longstanding system of interlocking safeguards against BSE that protects public and animal health in the United States, the most important of which is the removal of specified risk materials - or the parts of an animal that would contain BSE should an animal have the disease - from all animals presented for slaughter. The second safeguard is a strong feed ban that protects cattle from the disease. Another important component of our system - which led to this detection - is our ongoing BSE surveillance program that allows USDA to detect the disease if it exists at very low levels in the U.S. cattle population.

More information about this disease is available in the BSE factsheet.

http://web.archive.org/web/20230522150242/https://www.aphis.usda.gov/aphis/newsroom/stakeholder-info/sa_by_date/sa-2023/bse

2 weeks before the announcement of this recent mad cow case in the USA, i submitted this to the APHIS et al;

***> APPRX. 2 weeks before the recent mad cow case was confirmed in the USA, in Tennessee, atypical L-Type BSE, I submitted this to the APHIS et al;

Document APHIS-2023-0027-0001 BSE Singeltary Comment Submission May 2, 2023

''said 'burden' cost, will be a heavy burden to bear, if we fail with Bovine Spongiform Encephalopathy BSE TSE Prion disease, that is why this information collection is so critical''...

https://www.regulations.gov/comment/APHIS-2023-0027-0002

https://downloads.regulations.gov/APHIS-2023-0027-0002/attachment_1.pdf

TUESDAY, APRIL 15, 2025

Cases of Creutzfeldt-Jakob disease in young individuals: open questions regarding aetiology

Recently, two young individuals, aged 15 and 21, were diagnosed with sporadic Creutzfeldt-Jakob disease (sCJD) in Canada and the United States, respectively (D'Arcy et al., 2019; Ahn et al., 2024).

https://www.frontiersin.org/journals/cellular-neuroscience/articles/10.3389/fncel.2025.1571662/full

https://creutzfeldt-jakob-disease.blogspot.com/2025/04/cases-of-creutzfeldt-jakob-disease-in.html

SUNDAY, MARCH 23, 2025

Creutzfeldt Jakob Disease TSE Prion Increasing 2025 Update

https://creutzfeldt-jakob-disease.blogspot.com/2025/03/creutzfeldt-jakob-disease-tse-prion.html

https://creutzfeldt-jakob-disease.blogspot.com/2024/12/creutzfeldt-jacob-disease-cjd-bse-cwd.html

Neuropsychiatric symptoms in sporadic Creutzfeldt-Jakob disease

Jennifer Zitser, Sven Forner, Katherine Wong, Jin Chengshi, John Neuhaus, Jennifer Martindale, Ben J Raudabaugh, Kendra Benisano, Kelly Goodman-O’Leary, Stacy Metcalf ...

Brain, awaf077, https://doi.org/10.1093/brain/awaf077 Published: 28 March 2025

Although no neuropsychiatric symptom is pathognomonic for sCJD, certain symptoms might help differentiate sCJD from some other neurodegenerative diseases. Our findings support the inclusion of behavioral symptoms in sCJD diagnostic criteria.

https://academic.oup.com/brain/advance-article-abstract/doi/10.1093/brain/awaf077/8098176?login=false

https://creutzfeldt-jakob-disease.blogspot.com/2025/03/neuropsychiatric-symptoms-in-sporadic.html

Terry S. Singeltary Sr.