We transfused 4 uninfected macaques using blood from 3 macaques (CO7423, CO7422, and C16999) previously infected with vCJD (6) (Appendix). We collected 100-mL samples of blood from CO7423, at clinical onset and at terminal phase of illness, and immediately transfused the samples into 2 recipient macaques: CO1619 and 98CO19. Two years later, we transfused 2 macaques, DEIM and DFOO, with red blood cell–depleted blood prepared from whole blood of CO7422 and C16999 (Table 1). We euthanized CO1619 at 58 months posttransfusion (mpt) and euthanized 98CO19 at 104 mpt (8.7 years posttransfusion [ypt]) because of intercurrent illnesses (Table 1). DEIM developed early neurologic signs of mild ataxia and tremors at 104 mpt (8.7 ypt). Those symptoms slowly progressed to marked tremors, unsteadiness on the perch, unkept fur coat, and mild weight loss, all typical signs of vCJD in macaques. We euthanized DEIM 4 months after clinical onset. We euthanized DFOO at 120 mpt (10 ypt) when the macaque had reached the preselected experimental endpoint.

We collected blood and nasal swab specimens during and at the end of the study. We harvested brains and other tissues from each macaque. We also collected cerebrospinal fluid from DEIM and DFOO. To detect PrP^TSE, the biomarker of TSEs, we used 2 in vitro assays: real-time quaking-induced conversion (RT-QuIC) to assay nasal swab extracts, lymph nodes, and cerebrospinal fluid; and protein misfolding cyclic amplification (PMCA) to assay brain, spleen, and blood samples (8,9). All tests of tissues and fluids from CO1619 and DFOO were negative for PrP^TSE, including neuropathological examinations to show spongiform degeneration and PrP^TSE deposits in formalin-fixed paraffin-fixed brain tissue (Table 2). We included samples from vCJD-infected animals and uninfected macaques in each test as controls.

Figure 1

Figure 1. Results of real-time quaking-induced conversion testing of tissue samples from a study of abnormal prion protein in nasal swabs of macaques infected with Creutzfeldt-Jakob disease. Data based on nasal swab...

Figure 2

Figure 2. Immunohistochemistry for abnormal disease-associated prion protein (PrP^TSE) and CD21 on inguinal lymph node of macaque 98CO19 for study investigating abnormal prion protein in nasal swab specimens of macaques...

Brain homogenate from 98CO19 was negative for PrP^TSE negative using multiple biochemical detection methods, and the macaque’s blood was negative by PMCA as well (Table 2). Neuropathologic studies showed no spongiform degeneration or PrP^TSE deposits in 98CO19’s brain. However, nasal swab extracts and 2 inguinal lymph node homogenates collected at euthanasia were positive by RT-QuIC (Figure 1) when tested as 1% wt/vol tissue homogenates (4/4 positive wells) but negative in the next 10-fold dilution (0.1% wt/vol), suggesting that very small amounts of PrP^TSE were present. PMCA assays of the same 2 tissues were negative. We noted scattered clusters of PrP^TSE in sections of the lymph nodes, in the same cells staining for CD21, corresponding to follicular dendritic cells (Figure 2).

We confirmed macaque DEIM to be infected with vCJD by neuropathological and immunohistological examinations of formalin-fixed paraffin-embedded brain tissues and by Western blots of brain suspensions (Appendix Figure, panel A), RT-QuIC of cerebrospinal fluid, and PMCA of blood. PMCA first detected PrP^TSE in blood collected at 85 mpt; RT-QuIC showed PrP^TSE in nasal swab extracts 7 months later (92 mpt). PrP^TSE signals remained positive in both tissues to the end of the study (Appendix Figure). Thus, PrP^TSE appeared in those tissues several months before clinical onset, confirming the diagnostic potential of both assays with those materials.

We report the results of blood transfusions from 3 vCJD-infected macaques into 4 recipient macaques. One macaque developed clinical vCJD 9 years after transfusion, an interval consistent with a survival duration of 6.5–8.3 years seen in human recipients transfused with blood from asymptomatic vCJD-infected donors (5). One recipient macaque died early in the experiment, and 2 macaques survived ≥9 years with no clinical signs of vCJD. Macaque 98CO19 revealed PrP^TSE signals in nasal swab extracts and inguinal lymph node tissues. Researchers have used lymph node tissue previously to identify macaques incubating vCJD before clinical onset of illness (11). Our data from RT-QuIC testing showed reactivity that was weak but unequivocal and reproducible. We confirmed the presence of PrP^TSE in lymph node tissue by immunohistochemistry of the same tissue. We concluded that 98CO19 died while infected with vCJD.

Previous researchers exploring TTvCJD transfused to macaques reported a fatal neurologic syndrome, described as a myelopathy, that affected 3 of 7 macaques transfused with blood containing low infectivity (determined by low levels of peripheral PrP^TSE); the other 4 macaques remained healthy (12). The macaques with myelopathy demonstrated clinical signs that included impaired visual acuity and hind limb ataxia, but tests revealed no PrP^TSE in brain and other tissues (12). Macaque DFOO of our study showed no clinical signs associated with that described myelopathy. Macaque CO1619 died relatively early in the study—too early to know if it had been infected with vCJD or would have developed myelopathy. Our results largely agree with those of Comoy et al., who concluded that not all transfusions transmitted vCJD to macaques (12). Similarly, reports from the United Kingdom suggest that not all human recipients surviving 5 years or longer after transfusions developed TTvCJD from a donor with vCJD (13). No macaque in our smaller cohort of transfused animals showed evidence of myelopathy.

Over a 10-year period after transfusion, we collected relatively accessible biological materials, such as blood and nasal swab specimens, to test for PrP^TSE. We first detected PrP^TSE in the blood of macaque DEIM 19 months before onset of overt illness, consistent with results of our previous studies (6). Nasal swab extracts from the 2 infected macaques became positive for PrP^TSE 12 months before signs of illness for DEIM and at euthanasia for 98CO19 (98CO19’s nasal swab specimens were negative 3 months earlier). Those results support potential use of nasal swab specimens as an assay matrix to identify infected persons before clinical onset of vCJD. A caveat is that we do not know how detection of PrP^TSE might be affected by prion protein genotype. Research has demonstrated diagnostic accuracy of nasal swab testing for persons with either confirmed or presumed sporadic CJD, but the predictive value of this testing method for detecting cases before onset of neurologic illness remains uncertain (14). Acknowledging that our macaques were infected with vCJD, and not sporadic CJD, our results nonetheless suggest that testing nasal swab specimens to detect PrP^TSE may be useful in screening persons with family history of CJD, which would enable attempts at early therapeutic intervention (15).