Differentiation of Prions from L-type BSE versus Sporadic Creutzfeldt-Jakob Disease.
Subject: Creutzfeldt-Jakob disease
Bovine spongiform encephalopathy
Lemurs
Prions
Hamsters
Medical research
Medicine, Experimental
Disease transmission
Authors: Nicot, Simon
Bencsik, Anna
Morignat, Eric
Mestre-Frances, Nadine
Perret-Liaudet, Armand
Baron, Thierry
Pub Date: 12/01/2012
Publication: Name: Emerging Infectious Diseases Publisher: U.S. National Center for Infectious Diseases Audience: Academic; Professional Format: Magazine/Journal Subject: Health Copyright: COPYRIGHT 2012 U.S. National Center for Infectious Diseases ISSN: 1080-6040
Issue: Date: Dec, 2012 Source Volume: 18 Source Issue: 12
Product: Product Code: 8000200 Medical Research; 9105220 Health Research Programs; 8000240 Epilepsy & Muscle Disease R&D NAICS Code: 54171 Research and Development in the Physical, Engineering, and Life Sciences; 92312 Administration of Public Health Programs
Accession Number: 313345655
Full Text: Among transmissible spongiform encephalopathies (TSEs), the L-type bovine spongiform encephalopathy (L-BSE) in cattle requires particular attention for public health. L-BSE is transmitted more efficiently than is classical BSE among primates (7-3) as well as among transgenic mice that express human prion protein (PrP) (4,5). We recently reported that L-BSE was readily transmissible by experimental oral inoculation in a nonhuman primate species, the grey mouse lemur (Microcebus murinus) (3). These findings raise the possibility that some human Creutzfeldt-Jakob disease (CJD) cases might result from exposure to the L-BSE agent; previous studies highlighted similarities between L-BSE and some human subtypes (type 2) of sporadic CJD (sCJD) (7,6).

To examine the possible relationship between L-BSE and sCJD, we evaluated a strain-typing strategy that relies on comparative transmission characteristics in the Syrian golden hamster and in a transgenic mouse line (TgOvPrP4) expressing ovine PrP (ARQ allele). Both of these species are susceptible to L-BSE prions from cattle (7,8). The transmission of L-BSE, including after a first passage in Microcebus murinus lemurs (3), was compared with that for the MM2-cortical subtype of sCJD (9); this subtype was chosen on the basis of a study that indicated higher levels of molecular similarities of L-BSE with this sCJD subtype than with the MV2 subtype (7).

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 [micro]L, 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.sup.res]). At the terminal stage of the disease, animals were euthanized, and their brains and spleens were collected for [PrP.sup.res] analyses by Western blot and for histopathologic studies (8).

In hamsters, transmission of the MM2-cortical sCJD agent was inefficient. Clinical signs were absent up to 876 days postinoculation (dpi) (Table), and disease-associated PrP ([PrP.sup.d]) in brain samples was not detected by paraffin-embedded tissue blot (PET-blot) (Figure 1, panel A), immunohistochemical (Figure 1, panel C), or Western blot (Figure 1, panels E, F) analyses. [PrP.sup.res] was also undetectable in spleen tissues by Western blot (Table).

In contrast, the L-BSE agent passaged in a lemur was efficiently transmitted to hamsters, with a mean survival period of 529 [+ or -] 117 dpi, similar to that for L-BSE from cattle (622 [+ or -] 64 dpi) (Table). PET-blot analysis (Figure 1, panel B) showed widespread [PrP.sup.res] distribution in the brain; immunohistochemical analysis (Figure 1, panel D) showed a granular type of [PrP.sup.d] deposition that redefined the periphery of most of the blood vessels. Western blot analysis (Figure 1, panels E, F) showed [PrP.sup.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.sup.res] was characterized by low apparent molecular mass ([approximately equals] 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 efficiently transmitted, as confirmed by [PrP.sup.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/12-0342Techapp.pdf). No statistically significant differences in results were identified between the L-BSE sources (p>0.6). Mean survival period decreased to 111 [+ or -] 25 dpi at second passage in mice inoculated with the agent of MM2-cortical subtype sCJD, which differed significantly 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.sup.res] from mouse brains showed partially similar features for MM2-cortical sCJD and L-BSE, including low molecular mass ([approximately equals] 19 kDa for the unglycosylated band) (Figure 2, panel A) and similar conformational stability of [PrP.sup.d] after treatment with guanidinium hydrochloride (online Technical Appendix Figure 2). However, the proportions of diglycosylated, monoglycosylated, and unglycosylated bands of brain [PrP.sup.res] differed between sCJD and L-BSE (Figure 2, panel C); higher proportions of diglycosylated [PrP.sup.res] were found in sCJD-infected mice (mean 67% of the total signal) compared with L-BSE-infected mice ([approximately equals] 18% lower; p<0.0001). [PrP.sup.res] was readily identified in the spleens of TgOvPrP4 mice at the second passage for sCJD and L-BSE from cattle and at the first passage for L-BSE from lemur (Table). No significant differences in the proportions of [PrP.sup.res] glycoforms for sCJD-infected versus L-BSE-infected mice were observed in the spleens (Figure 2, panel D), but [PrP.sup.res] was [approximately equals] 0.5 kD higher in mice injected with sCJD (Figure 2, panel B, arrows).

[FIGURE 1 OMITTED]

[FIGURE 2 OMITTED]

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.sup.res] occurred in the frontal parts of the brain, but the intensity and appearance of [PrP.sup.res] in the cortex, thalamus, and hippocampus were distinctly different. Immunohistochemical analyses of the hippocampus showed [PrP.sup.d] deposition in the dentate gyrus in sCJD-infected 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 efficient, as previously reported for L-BSE from cattle (7,77). This result suggests that L-BSE did not undergo major modifications after this cross-species transmission and could indicate a clear biologic difference between MM2-cortical sCJD and L-BSE. We also demonstrated the efficient 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 MM2-cortical sCJD causes a much more rapidly fatal disease. Despite similar molecular features in sCJD and L-BSE, including the [PrP.sup.res] electrophoretic mobility and the conformational stability of [PrP.sup.d], sCJD and L-BSE differed in [PrP.sup.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 profiles and [PrP.sup.d] distribution, which confirms 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.

Acknowledgments

We thank the staff of the Plateforme d'Experimentation Animale of Anses-Lyon for excellent animal care; Mikael Leboidre and Jean-Michel Bridon for histotechnical assistance; Dominique Canal and Claire Aufauvre for biochemical assistance; and Francoise Didier and Nathalie Streichenberger for characterization of the human sporadic CJD case.

S.N. was supported by grants from the Agence Nationale de Securite Sanitaire and the Fondation pour la Recherche Medicale.

Mr Nicot is a PhD student at the Agence Nationale de Securite Sanitaire in Lyon. His primary research interests include characterization of the infectious agents and prion protein during intra- and interspecies transmission of animal and human prion diseases.

Author affiliations: Agence Nationale de Securite Sanitaire (Anses), Lyon, France (S. Nicot, A. Bencsik, E. Morignat, T. Baron); INSERM U710, Montpellier, France; Universite Montpellier 2, Montpellier; Ecole Pratique des Hautes Etudes, Paris, France (N. MestreFrances); and Hopitaux Civils de Lyon, Universite Lyon 1, INSERM U1028, and Centre National de la Recherche Scientifique, Lyon (A. Perret-Liaudet)

DOI: 10.3201/eid1812.120342

References

(1.) Comoy EE, Casalone C, Lescoutra-Etchegaray N, Zanusso G, Freire S, Marce D, et al. Atypical BSE (BASE) transmitted from asymptomatic aging cattle to a primate. PLoS ONE. 2008;3:e3017. http://dx.doi.org/10.1371/journal.pone.0003017

(2.) Ono F, Tase N, Kurosawa A, Hiyaoka A, Ohyama A, Tezuka Y, et al. Atypical L-type bovine spongiform encephalopathy (L-BSE) transmission to cynomolgus macaques, a non-human primate. Jpn J Infect Dis. 2011;64:81-4.

(3.) Mestre-Frances N, Nicot S, Rouland S, Biacabe AG, Quadrio I, Perret-Liaudet A, et al. Oral transmission of L-type bovine spongiform encephalopathy in primate model. Emerg Infect Dis. 2012;18:142-5. http://dx.doi.org/10.3201/eid1801.111092

(4.) Kong Q, Zheng M, Casalone C, Qing L, Huang S, Chakraborty B, et al. Evaluation of the human transmission risk of an atypical bovine spongiform encephalopathy prion strain. J Virol. 2008;82:3697-701. http://dx.doi.org/10.1128/JVI.02561-07

(5.) Beringue V, Herzog L, Reine F, Le Dur A, Casalone C, Vilotte JL, et al. Transmission of atypical bovine prions to mice transgenic for human prion protein. Emerg Infect Dis. 2008;14:1898-901. http:// dx.doi.org/10.3201/eid1412.080941

(6.) Casalone C, Zanusso G, Acutis P, Ferrari S, Capucci L, Tagliavini F, et al. Identification of a second bovine amyloidotic spongiform encephalopathy: molecular similarities with sporadic CreutzfeldtJakob disease. Proc Natl Acad Sci U S A. 2004;101:3065-70. http:// dx.doi.org/10.1073/pnas.0305777101

(7.) Nicot S, Baron T. Strain-specific barriers against bovine prions in hamsters. J Virol. 2011;85:1906-8. http://dx.doi.org/10.1128/ JVI.01872-10

(8.) Baron T, Bencsik A, Biacabe AG, Morignat E, Bessen RA. Phenotypic similarity of transmissible mink encephalopathy in cattle and L-type bovine spongiform encephalopathy in a mouse model. Emerg Infect Dis. 2007;13:1887-94. http://dx.doi.org/10.3201/ eid13112.070635

(9.) Parchi P, Giese A, Capellari S, Brown P, Schulz-Schaeffer W, Windl O, et al. Classification of sporadic Creutzfeldt-Jakob disease based on molecular and phenotypic analysis of 300 subjects. Ann Neurol. 1999;46:224-33. http://dx.doi.org/10.1002/15318249(199908)46:2<224::AID-ANA12>3.0.CO;2-W

(10.) Baron T, Bencsik A, Morignat E. Prions of ruminants show distinct splenotropisms in an ovine transgenic mouse model. PLoS ONE. 2010;5:e10310.

(11.) Shu Y, Masujin K, Okada H, Iwamaru Y, Imamura M, Matsuura Y, et al. Characterization of Syrian hamster adapted prions derived from L-type and C-type bovine spongiform encephalopathies. Prion. 2011;5:103-8. http://dx.doi.org/10.4161/pri.5.2.15847

Address for correspondence: Thierry Baron, Anses-Lyon, 31 Avenue Tony Garnier, 69364 Lyon Cedex 07, France; email: thierry.baron@anses.fr

Use of trade names is for identification only and does not imply endorsement by the Public Health Service or by the US Department of Health and Human Services.
Table. Comparison of transmission of sCJD and L-BSE in
hamsters and mice

Hosts and inoculum                       Passage

Syrian golden hamsters
  sCJD MM2-cortical                         1
  L-BSE lemur                               1
  L-BSE cattle (02-2528)                    1
TgOvPrP4 mice
  sCJD MM2-cortical                         1
  L-BSE lemur                               1
  L-BSE cattle (02-2528)                    1
  L-BSE cattle (08-0074)                    1
  sCJD MM2-cortical                         2
  L-BSE lemur                               2
  L-BSE cattle (02-2528)                    2
  L-BSE cattle (08-0074)                    2

                                          Mean
                                     survival time,
Hosts and inoculum                   dpi [+ or -] SD

Syrian golden hamsters
  sCJD MM2-cortical                  833 [+ or -] 33
  L-BSE lemur                       529 [+ or -] 177
  L-BSE cattle (02-2528)       622 [+ or -] 64 ([dagger])
TgOvPrP4 mice
  sCJD MM2-cortical                  639 [+ or -] 49
  L-BSE lemur                        509 [+ or -] 97
  L-BSE cattle (02-2528)    627 [+ or -] 74 ([double dagger])
  L-BSE cattle (08-0074)             497 [+ or -] 49
  sCJD MM2-cortical                  111 [+ or -] 25
  L-BSE lemur                        194 [+ or -] 7
  L-BSE cattle (02-2528)   202 [+ or -] 26  ([double dagger])
  L-BSE cattle (08-0074)             186 [+ or -] 37

                                          Mean
                                       [PrP.sup.d]
Hosts and inoculum                    positive/no.
                                         tested
Syrian golden hamsters
  sCJD MM2-cortical                        0/4
  L-BSE lemur                              5/5
  L-BSE cattle (02-2528)             4/5 ([dagger])
TgOvPrP4 mice
  sCJD MM2-cortical                        3/4
  L-BSE lemur                              7/7
  L-BSE cattle (02-2528)         9/10 ([double dagger])
  L-BSE cattle (08-0074)                   6/8
  sCJD MM2-cortical                       12/12
  L-BSE lemur                             12/12
  L-BSE cattle (02-2528)                  9/9J
  L-BSE cattle (08-0074)                  12/12

                                       No. spleen
                                      [PrP.sup.res]
Hosts and inoculum                    positive/no.
                                         tested
Syrian golden hamsters
  sCJD MM2-cortical                        0/4
  L-BSE lemur                              1/4
  L-BSE cattle (02-2528)                   0/5
TgOvPrP4 mice
  sCJD MM2-cortical                        0/4
  L-BSE lemur                              7/7
  L-BSE cattle (02-2528)             0/5 ([section])
  L-BSE cattle (08-0074)                   0/9
  sCJD MM2-cortical                       12/12
  L-BSE lemur                             12/12
  L-BSE cattle (02-2528)                   3/5
  L-BSE cattle (08-0074)                  9/11

* Isolate identification numbers are shown in parentheses. sCJD,
sporadic Creutzfeldt-Jakob disease; L-BSE, L-type bovine
spongiform ncephalopathy; TgOvPrP4, ovine prion protein-transgenic;
dpi, days postinoculation; PrPd, disease-associated prion protein;
PrPres, protease-resistant prion protein.

([dagger]) Data from (7).

([double dagger]) Data from (g).

([section]) Data from (70).
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