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From: TSS ()
Subject: Transmission of New Bovine Prion to Mice
Date: July 6, 2006 at 1:18 pm PST

Transmission of

New Bovine Prion

to Mice

Thierry G.M. Baron,* Anne-Gaëlle Biacabe,*

Anna Bencsik,* and Jan P.M. Langeveld†

We previously reported that cattle were affected by a

prion disorder that differed from bovine spongiform

encephalopathy (BSE) by showing distinct molecular features

of disease-associated protease-resistant prion protein

(PrPres). We show that intracerebral injection of such

isolates into C57BL/6 mice produces a disease with preservation

of PrPres molecular features distinct from BSE.

Until recently, transmissible spongiform encephalopathy

(TSE) in cattle was believed to be caused by a single

strain of infectious agent identified at the beginning of

a foodborne epidemic of bovine spongiform encephalopathy

(BSE). Characterization of the infectious agent associated

with BSE showed unique features. These include

defined incubation periods and distribution of brain lesions

after transmission to wild-type mice, not only directly

from cattle, but also after natural or experimentally

induced cross-species transmission (1,2). The uniform features

of the disease in cattle have also been shown by

analysis of the distribution of neurodegenerative brain

lesions at different places during the BSE epidemic (3,4).

Western blot analyses of protease-resistant prion protein

(PrPres) accumulating in the brains of animals and

humans with BSE have demonstrated specific molecular

features. These include a low molecular mass of unglycosylated

PrPres with high proportions of diglycosylated

PrPres (5,6). However, recent studies reported cases of

prion abnormalities in cattle with different PrPres features

(7,8). Three cattle isolates from France have been reported,

characterized by a higher apparent molecular mass of

unglycosylated PrPres (H-type isolates) and decreased levels

of diglycosylated PrPres when compared with BSE isolates

(7). In addition. only PrPres from H-type isolates were

labeled by monoclonal antibody P4 with defined PrPres N

terminus epitope specificity, in contrast with PrPres from

BSE isolates, which suggests a different cleavage by proteinase

K of the disease-associated protein (9).

Twenty years after identification of the BSE epidemic

in cattle, the origin of the BSE agent remains controversial

(10,11). Researchers have often considered the most

likely source to be a recycled infectious agent derived

from prion-associated diseases found in other species,

such as scrapie in sheep and goats. The recent description

of unusual phenotypes of bovine prion diseases distinct

from BSE is therefore puzzling (7). This situation has

been reinforced by a second bovine amyloidotic spongiform

encephalopathy found in cattle in Italy (8). However,

whether such cases of bovine prion disorders were transmissible,

and to what extent the infectious agent caused

specific features distinct from BSE, have not been

demonstrated.

The Study

Experimental groups of 20 (4- to 6-week old) C57BL/6

female mice (Charles River, L’Arbresle, France) were

injected intracerebrally with 20 µL of 10% (weight/volume)

homogenates per mouse prepared from brain stem

samples of 3 cattle TSE isolates. Two of the isolates were

characterized, as previously described (7), by a higher

molecular mass of unglycosylated PrPres (H-type isolates)

and labeling with P4 monoclonal antibody (Table). A typical

cattle BSE isolate was also analyzed. Mice were

housed and cared for in an appropriate biohazard prevention

area (A3) according to European (directive

86/609/EEC) and French ethical committee (decree

87–848) guidelines. Mice were checked at least weekly for

neurologic clinical signs and were killed when they exhibited

signs of distress or confirmed evolution of clinical

signs. The whole brain of every second mouse was frozen

and stored at –80°C before Western blot analysis. The

other brains were fixed in 4% paraformaldehyde for other

histopathologic studies.

Frozen mouse brain tissues and fixed brain tissues were

examined by Western blot analysis and immunohistochemical

tests as previously described (12,13). PrPres extracted

from half of whole brain was detected with monoclonal

antibodies Sha31 (1:10 from TeSeE sheep/goat Western

blot, Bio-Rad, Hercules, CA, USA) (14) and (340 ng/mL)

(15). These antibodies are directed against the 144-

WEDRYYRE-151 and 88-WGQGG-92 murine amino

acid PrP sequences, respectively. Antibody 12B2, which

has an N-terminal specificity similar to that of monoclonal

antibody P4, shows poor binding to BSE-derived PrPres,

but unlike P4, binds with high affinity to prion protein

from most mammalian species, including mice and cattle.

Bound antibodies were detected by using enhanced enzymatic

chemiluminescence (Amersham, Little Chalfont,

UK) or Supersignal (Pierce, Rockford, IL, USA) and visualized

either on film (Biomax, Eastman Kodak, Rochester,

NY, USA) or directly in an image analysis system

(Versadoc, Bio-Rad). Molecular masses of PrPres glycoforms

were determined as the average of the center positions

of the bands from at least 3 repeated electrophoretic

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 7, July 2006 1125

*Agence Française de Sécurité Sanitaire des Aliments, Lyon,

France; and †Central Institute for Animal Disease Control,

Lelystad, the Netherlands

procedures, as measured by comparison with a biotinylated

marker (B2787, Sigma, Saint Louis, MO, USA)

included on each gel. Immunologic reactivities of antibodies

12B2 and Sha31 were compared in Western blots run in

parallel with the same samples with both antibodies.

After intracerebral injection of cattle brain samples into

C57BL/6 mice, disease was observed in mice with the 2 Htype

isolates, as well as with the BSE sample. Survival

periods of mice and results of PrPres detection among mice

analyzed by Western blot are shown in the Table.

Western blot analysis of PrPres from H-type–infected

mouse brains in comparison with BSE-infected mice is

shown in Figure 1. All positive mice in the same experimental

group showed the same Western blot pattern. This

pattern showed higher molecular mass PrPres glycoforms in

mice infected with H-type isolates than in mice infected

with a typical BSE agent (1.1- to 1.5-Da difference in the

unglycosylated PrPres (Figure 1A). Studies of PrPres protease

cleavage showed that only the PrPres of mice infected

with H-type isolates was recognized by antibody 12B2

(Figure 1B). This finding is in contrast to the result

obtained with monoclonal antibody Sha31 directed against

an epitope in the central region of the protein, which

showed that the 12B2 epitope was preserved in Htype–

infected mice. Thus, the molecular features of H-type

cattle isolates, which are distinct from those of the BSE

agent, were maintained after development of disease in

mice.

Histopathologic analysis showed vacuolar lesions in

the thalamus (Figure 2A) that were absent from the hypothalamus,

cochlear nucleus, and superior collicules. These

3 neuroanatomic sites were severely affected in C57BL/6

mice brain after primary passage of the BSE agent, as we

and others have reported (1). Abnormal PrP was detected

only in amyloid plaques (Figure 2B), in contrast to what

was reported after BSE transmission in C57BL/6 mice (1).

Conclusions

Our data show that the recently identified bovine Htype

isolates involve an infectious agent that can induce

development of a disease across a species barrier, while

maintaining the specific associated PrPres molecular signature.

This evidence in favor of a new bovine prion strain in

cattle suggests that BSE is not the only transmissible prion

disease in cattle. The origin of such cases has not been

determined (7). These cases suggest either the existence of

alternative origins of such diseases in cattle or phenotypic

changes of PrPres after infection with the BSE agent.

However, based on analysis of molecular features of prion

diseases in cattle, this situation is similar to that in humans

(5), in which different subtypes of sporadic Creutzfeldt-

Jakob disease agents are found.

DISPATCHES

1126 Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 7, July 2006

Figure 1. Western blot analysis of disease-associated prion protein

(PrPres) from proteinase K–treated brain homogenates of

C57BL/6 mice infected with type H (lanes 2 and 4) or bovine

spongiform encephalopathy isolates (lanes 3 and 5). PrPres of mice

infected with an experimental scrapie strain (C506M3) (6) was

used as a control (lane 1). Monoclonal antibodies used for detection

of PrPres were Sha31 in panel A and 12B2 in panel B. Lane M,

molecular mass markers: 39.8, 29, 20.1, and 14.3 kDa.

Acknowledgments

We thank Jérémy Verchère and Dominique Canal for excellent

technical assistance, Emilie Antier and Clément Lavigne for

performing animal experiments, and Karel Riepema, Esther de

Jong, and Jorg Jacobs for production and characterization of

monoclonal antibody 12B2.

This study was supported by the Agence Française de

Sécurité Sanitaire des Aliments, the Neuroprion Network of

Excellence (FOOD-CT-2004-506579) (EUROSTRAINS project),

the Dutch Ministry of Agriculture, Environmental

Management and Food (8041869000), and NeuroPrion (FOODCT-

2004-506579)(STOPPrions project).

Dr Baron is head of the Unité Agents Transmissibles Non

Conventionnels, Agence Française de Sécurité Sanitaire des

Aliments, in Lyon. His research focuses on diagnosis of prion

diseases of ruminants and characterization of the disease-associated

prion protein and infectious agents, with particular emphasis

on atypical forms of these diseases.

References

1. Fraser H, Bruce ME, Chree A, McConnell I, Wells GA. Transmission

of bovine spongiform encephalopathy and scrapie to mice. J Gen

Virol. 1992;73:1891–7.

2. Green R, Horrocks C, Wilkinson A, Hawkins SA, Ryder SJ. Primary

isolation of the bovine spongiform encephalopathy agent in mice:

agent definition based on a review of 150 transmissions. J Comp

Pathol. 2005;132:117–31.

3. Simmons MM, Harris P, Jeffrey M, Meek SC, Blamire IW, Wells GA.

BSE in Great Britain: consistency of the neurohistopathological findings

in two random annual samples of clinically suspect cases. Vet

Rec. 1996;138:175–7.

4. Orge L, Simas JP, Fernandes AC, Ramos M, Galo A. Similarity of the

lesion profile of BSE in Portuguese cattle to that described in British

cattle. Vet Rec. 2000;147:486–8.

5. Collinge J, Sidle KC, Meads J, Ironside J, Hill AF. Molecular analysis

of prion strain variation and the aetiology of ‘new variant’ CJD.

Nature. 1996;383:685–90.

6. Baron TG, Biacabe A-G. Molecular analysis of the abnormal prion

protein during coinfection of mice by bovine spongiform

encephalopathy and a scrapie agent. J Virol. 2001;75:107–14.

7. Biacabe A-G, Laplanche J-L, Baron L, Ryder SJ. Distinct molecular

phenotypes in bovine prion diseases. EMBO Rep. 2004;5:110–4.

8. 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 Creutzfeldt-

Jakob disease. Proc Natl Acad Sci U S A. 2004;101:3065–70.

9. Thuring CM, Erkens JH, Jacobs JG, Bossers JG, van Keulen LJ,

Garssen GJ, et al. Discrimination between scrapie and bovine spongiform

encephalopathy in sheep by molecular size immunoreactivity

and glycoprofile of prion protein. J Clin Microbiol. 2004;42:972–80.

10. Marsh RF. Bovine spongiform encephalopathy: a new disease of cattle?

Arch Virol Suppl. 1993;7:255–9.

11. European Commission. Opinion on: hypotheses on the origin and

transmission of BSE. Brussels: EC Health and Consumer Protection

Directorate General; 2001. p. 1–67.

Transmission of New Bovine Prion to Mice

Emerging Infectious Diseases • www.cdc.gov/eid • Vol. 12, No. 7, July 2006 1127

Figure 2. Histopathologic analysis of brain of a C57BL/6 mouse

infected with a type H isolate. A) Characteristic vacuolar lesions in

the thalamus (hematoxylin and eosin stained, scale bar = 60 µm).

B) Immunohistochemical analysis of prion protein with monoclonal

antibody 12B2 (diluted 1:200) shows the absence of granular

deposition, but the presence of plaques in the thalamus. The inset

shows that plaques are amyloids since they bind Congo red and

show birefringence in polarized light (scale bar = 60 µm, scale bar

in inset = 16 µm).

12. Baron T, Crozet C, Biacabe A-G, Philippe S, Verchère J, Bencsik A,

et al. Molecular analysis of the protease-resistant prion protein in

scrapie and bovine spongiform encephalopathy transmitted to ovine

transgenic and wild-type mice. J Virol. 2004;78:6243–51.

13. Bencsik AA, Debeer S, Baron T. An alternative pretreatment procedure

in animal transmissible spongiform encephalopathies diagnosis

using PrPsc immunohistochemistry. J Histochem Cytochem.

2005;53:1199–202.

14. Feraudet C, Morel N, Simon S, Volland H, Frobert Y, Créminon C, et

al. Screening of 145 anti-PrP monoclonal antibodies for their capacity

to inhibit PrPsc replication in infected cells. J Biol Chem.

2005;280:11247–58.

15. Yull HM, Ritchie DL, Langeveld JP, van Zijderveld FG, Bruce ME,

Ironside JW, et al. Detection of type 1 prion protein in variant

Creutzfeldt-Jakob disease. Am J Pathol. 2006;168:151–7.

Address for correspondence: Thierry G.M. Baron, Unité Agents

Transmissibles Non Conventionnels, Agence Française de Sécurité

Sanitaire des Aliments, 31 Ave Tony Garnier, 69364 Lyon CEDEX 07,

France; email: t.baron@lyon.afssa.fr

http://www.cdc.gov/ncidod/EID/vol12no07/pdfs/vol12no07.pdf

Medical Sciences
Identification of a second bovine amyloidotic spongiform encephalopathy: Molecular similarities with sporadic Creutzfeldt-Jakob disease

Cristina Casalone *, Gianluigi Zanusso , Pierluigi Acutis *, Sergio Ferrari , Lorenzo Capucci , Fabrizio Tagliavini ¶, Salvatore Monaco ||, and Maria Caramelli *
*Centro di Referenza Nazionale per le Encefalopatie Animali, Istituto Zooprofilattico Sperimentale del Piemonte, Liguria e Valle d'Aosta, Via Bologna, 148, 10195 Turin, Italy; Department of Neurological and Visual Science, Section of Clinical Neurology, Policlinico G.B. Rossi, Piazzale L.A. Scuro, 10, 37134 Verona, Italy; Istituto Zooprofilattico Sperimentale della Lombardia ed Emilia Romagna, Via Bianchi, 9, 25124 Brescia, Italy; and ¶Istituto Nazionale Neurologico "Carlo Besta," Via Celoria 11, 20133 Milan, Italy


Edited by Stanley B. Prusiner, University of California, San Francisco, CA, and approved December 23, 2003 (received for review September 9, 2003)

Transmissible spongiform encephalopathies (TSEs), or prion diseases, are mammalian neurodegenerative disorders characterized by a posttranslational conversion and brain accumulation of an insoluble, protease-resistant isoform (PrPSc) of the host-encoded cellular prion protein (PrPC). Human and animal TSE agents exist as different phenotypes that can be biochemically differentiated on the basis of the molecular mass of the protease-resistant PrPSc fragments and the degree of glycosylation. Epidemiological, molecular, and transmission studies strongly suggest that the single strain of agent responsible for bovine spongiform encephalopathy (BSE) has infected humans, causing variant Creutzfeldt-Jakob disease. The unprecedented biological properties of the BSE agent, which circumvents the so-called "species barrier" between cattle and humans and adapts to different mammalian species, has raised considerable concern for human health. To date, it is unknown whether more than one strain might be responsible for cattle TSE or whether the BSE agent undergoes phenotypic variation after natural transmission. Here we provide evidence of a second cattle TSE. The disorder was pathologically characterized by the presence of PrP-immunopositive amyloid plaques, as opposed to the lack of amyloid deposition in typical BSE cases, and by a different pattern of regional distribution and topology of brain PrPSc accumulation. In addition, Western blot analysis showed a PrPSc type with predominance of the low molecular mass glycoform and a protease-resistant fragment of lower molecular mass than BSE-PrPSc. Strikingly, the molecular signature of this previously undescribed bovine PrPSc was similar to that encountered in a distinct subtype of sporadic Creutzfeldt-Jakob disease.

--------------------------------------------------------------------------------

C.C. and G.Z. contributed equally to this work.

||To whom correspondence should be addressed.

E-mail: salvatore.monaco@mail.univr.it.
www.pnas.org/cgi/doi/10.1073/pnas.0305777101


http://www.pnas.org/cgi/content/abstract/0305777101v1

TSS




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