Differentiation of Prions from L-type BSE versus Sporadic Creutzfeldt-Jakob Disease

We compared transmission characteristics for prions from L-type bovine spongiform encephalopathy and MM2-cortical sporadic Creutzfeldt-Jakob disease in the Syrian golden hamster and an ovine prion protein–transgenic mouse line and isolated distinct prion strains. Our findings suggest the absence of a causal relationship between these diseases, but further investigation is warranted.


The Study
The TSE brain inocula used in this study, conducted during November 2010-December 2011, were derived from 2 natural L-BSE isolates from France (02-2528 and 08-0074); a lemur injected intracerebrally (i.c.) with the 02-2528 L-BSE cattle isolate (3); and a human patient with MM2-cortical sCJD. Consent was obtained for using tissues from the human patient in research, including genetic analyses. Animal experiments were performed in the biohazard prevention area (A3) of the Anses-Lyon animal facilities, in accordance with the guidelines of the French Ethical Committee (decree 87-848) and European Community Directive 86/609/EEC.
Six-week-old TgOvPrP4 mice and 4-week-old Syrian golden hamsters were injected i.c. with 20 and 30 μL, respectively, of 10% (wt/vol) brain homogenates in 5% sterile glucose. Serial passages were performed in TgOvPrP4 mice by i.c. inoculation of 1% (wt/vol) homogenates from mice positive for protease-resistant PrP (PrP res ). At the terminal stage of the disease, animals were euthanized, and their brains and spleens were collected for PrP res analyses by Western blot and for histopathologic studies (8).
In hamsters, transmission of the MM2-cortical sCJD agent was ineffi cient. Clinical signs were absent up to 876 days postinoculation (dpi) ( Table), and disease-associated PrP (PrP d ) in brain samples was not detected by paraffi nembedded tissue blot (PET-blot) ( Figure 1, panel A), immunohistochemical ( Figure 1, panel C), or Western blot ( Figure 1, panels E, F) analyses. PrP res was also undetectable in spleen tissues by Western blot (Table).
In contrast, the L-BSE agent passaged in a lemur was effi ciently transmitted to hamsters, with a mean survival period of 529 ± 117 dpi, similar to that for L-BSE from cattle (622 ± 64 dpi) (Table). PET-blot analysis ( Figure  1, panel B) showed widespread PrP res distribution in the brain; immunohistochemical analysis ( Figure 1, panel D) showed a granular type of PrP d deposition that redefi ned the periphery of most of the blood vessels. Western blot analysis ( Figure 1, panels E, F) showed PrP res in the brains of hamsters inoculated with L-BSE from cattle and lemur and in 1/4 spleens of hamsters injected with L-BSE passaged in lemur (Table). Brain PrP res was characterized by low apparent molecular mass (≈19 kDa for the unglycosylated band) associated with a lack of reactivity toward the N terminal 12B2 antibody, in contrast to that for the control animal with scrapie ( Figure 1, panels E, F). In TgOvPrP4 mice, all TSEs were effi ciently transmitted, as confi rmed by PrP d accumulation in the mouse brains (Table). After serial passages in additional TgOvPrP4 mice, the survival periods in each experiment became considerably shorter (Table; online Technical Appendix Figure 1, wwwnc.cdc.gov/EID/pdfs/12-0342-Techapp.pdf). No statistically signifi cant differences in results were identifi ed between the L-BSE sources (p>0.6). Mean survival period decreased to 111 ± 25 dpi at second passage in mice inoculated with the agent of MM2-cortical subtype sCJD, which differed signifi cantly from that of mice inoculated with L-BSE (p<0.0001). A third passage of both cattle L-BSE and human sCJD did not reduce the survival periods in TgOvPrP4 mice (data not shown).
Western blot analyses of PrP res from mouse brains showed partially similar features for MM2-cortical sCJD and L-BSE, including low molecular mass (≈19 kDa for the unglycosylated band) (Figure 2, panel A) and similar conformational stability of PrP d after treatment with guanidinium hydrochloride (online Technical Appendix Figure 2). However, the proportions of diglycosylated, monoglycosylated, and unglycosylated bands of brain PrP res differed between sCJD and L-BSE ( Figure 2 (7). ‡Data from (8). §Data from (10).
from cattle and at the fi rst passage for L-BSE from lemur (Table). No signifi cant differences in the proportions of PrP res glycoforms for sCJD-infected versus L-BSEinfected mice were observed in the spleens (Figure 2, panel D), but PrP res was ≈0.5 kD higher in mice injected with sCJD ( Figure 2, panel B, arrows). Histopathologic analysis showed severe vacuolar lesions in TgOvPrP4 mice infected at second passage with sCJD and lemur-passaged L-BSE (online Technical Appendix Figure 3). However, in sCJD-infected mice, vacuolar lesions were mostly observed in the anterior parts of the brain (except the parietal cortex), whereas in mice infected with lemur-passaged L-BSE, the lesions were more widely distributed, involving the colliculi and the hypothalamus. In mice infected with sCJD and lemurpassaged L-BSE, PET-blot analyses showed that most of the PrP res occurred in the frontal parts of the brain, but the intensity and appearance of PrP res in the cortex, thalamus, and hippocampus were distinctly different. Immunohistochemical analyses of the hippocampus showed PrP d deposition in the dentate gyrus in sCJDinfected mice, in contrast to a lack of deposition in lemurpassaged L-BSE-infected mice.

Conclusions
We report the isolation of 2 prion strains derived from L-BSE and MM2-cortical sCJD after transmission in Syrian hamsters and ovine PrP-transgenic mice. In hamsters, we did not transmit any disease with sCJD, but the transmission of L-BSE from lemur was effi cient, as previously reported for L-BSE from cattle (7,11). This result suggests that L-BSE did not undergo major modifi cations after this cross-species transmission and could indicate a clear biologic difference between MM2cortical sCJD and L-BSE. We also demonstrated the effi cient transmission of both L-BSE and MM2-cortical sCJD in TgOvPrP4 mice, which enabled us to compare these diseases in a single model. Unexpectedly, during serial passages, we observed that the agent of MM2cortical sCJD causes a much more rapidly fatal disease. Despite similar molecular features in sCJD and L-BSE, including the PrP res electrophoretic mobility and the conformational stability of PrP d , sCJD and L-BSE differed in PrP res glycosylation for the mouse brains and gel migrations for the mouse spleens. Mice infected with MM2-cortical sCJD versus those infected with L-BSE also showed distinct lesion profi les and PrP d distribution, which confi rms clear biologic differences between these diseases. Although only 1 case of sCJD of a unique molecular subtype was examined in our study, our observations do not support the hypothesis of a causal relationship between L-BSE and this human sCJD subtype. Our study thus encourages further investigations using the proposed bioassay approach for a more complete evaluation of possible relationships between L-BSE and human prion diseases.