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

Cristina Casalone

Gianluigi Zanusso

Pierluigi Acutis

Sergio Ferrari

^{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}

^{To whom correspondence should be addressed. E-mail:}ti.rvinu.liam@ocanom.erotavlas.

^{C.C. and G.Z. contributed equally to this work.}

Edited by Stanley B. Prusiner, University of California, San Francisco, CA, and approved December 23, 2003

Received 2003 Sep 9

Abstract

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 (PrP^{) of the host-encoded cellular prion protein (PrP}^{). 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 PrP}^{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 PrP}^{accumulation. In addition, Western blot analysis showed a PrP}^{type with predominance of the low molecular mass glycoform and a protease-resistant fragment of lower molecular mass than BSE-PrP}^{. Strikingly, the molecular signature of this previously undescribed bovine PrP}^{was similar to that encountered in a distinct subtype of sporadic Creutzfeldt-Jakob disease.}

Abstract

The transmissible spongiform encephalopathies (TSEs), or prion diseases (1), encompass a group of progressive neurodegenerative disorders, including Creutzfeldt-Jakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE) (1-4). These disorders are characterized by brain deposition of an insoluble, protease-resistant isoform of the host-encoded cellular prion protein (PrP^{), named PrP}⁽1, 4, 5) In different TSE phenotypes, PrP^{exhibits disease-specific properties, including distinctive cleavage sites after proteolytic treatment, ratio of glycoforms, and deposition patterns, all features useful in providing a means of strain identification (}6-10).

Although not contagious, TSEs are potentially infective, and in humans may present as sporadic, inherited, and acquired diseases. Human-to-human transmission of TSE is well documented and has occurred either through oral or mucocutaneous route of infection, as in kuru (11), or after medical and surgical procedures, as in iatrogenic CJD (12). Recently, animal-to-human transmission has also occurred. Epidemiological (13), experimental transmission (14), and biochemical PrP^{typing (}8) have provided strong evidence that the single prion strain responsible for BSE has infected humans, causing variant CJD (vCJD), in addition to several animal species. In BSE and BSE-related disorders, including vCJD, the molecular typing of disease-associated PrP^{shows identical PrP fragment sizes and predominance of the high molecular mass glycoform both in natural hosts and in experimentally inoculated animals. To date, at variance with CJD in humans and scrapie in sheep, only a single strain and a single PrP}^{type have been detected in BSE.}

The spreading of the BSE agent across mammalian species barriers has aroused considerable concern for the following reasons: (i) the possible existence of new or previously unrecognized cattle TSE strains, potentially pathogenic for humans; and (ii) the occurrence of phenotypic variation of the BSE strain, with propagation of a new agent encoding distinctive molecular and biological properties.

In Italy, an active surveillance system on BSE in cattle was started in January 2001, and by August 2003 a total of 103 BSE cases had been diagnosed of 1,638,275 statutory tested brainstem samples. Confirmatory positive results have been obtained in all cases by immunohistochemical and Western immunoblot demonstration of disease-specific protease-resistant PrP^.

To assess molecular and neuropathological characteristics in Italian BSE cases, we have over the last few months collected whole brains of eight Italian cattle that were PrP^{-positive in Western immunoblots. In two cattle, older than other affected bovines, the PrP}^{glycotype was clearly different from the BSE-associated PrP}^{molecule, and widespread PrP-amyloid plaques were seen in supratentorial brain regions. Unlike typical BSE, the brainstem was less involved and no PrP deposition was detected in the dorsal nucleus of the vagus nerve. Given the biochemical and pathological similarities with sporadic CJD (sCJD) cases linked to type-2 PrP}⁽9) and methionine/valine (M/V) polymorphism at codon 129 in the prion protein gene (PRNP), these findings have prompted ongoing strain typing in inbred mice. Although the present findings dictate caution, here we show that a PrP^{type associated with sCJD and the previously undescribed bovine PrP}^{show convergent molecular signatures.}

The presence of amyloid plaques was assessed after thioflavin-S staining, PrP immunohistochemistry, and ultrastructural examination. Codon 70 in control cattle and other affected animals was CAG/CAG, encoding Q/Q.

Acknowledgments

We are grateful to Giuseppe Ru (Centro di Referenza Nazionale per le Encefalopatie Animali, Istituto Zooprofilattico Sperimentale di Torino) for the provision of surveillance data. We also thank Diana Bazan for preparing material for transmission electron microscopy, and Ines Crescio, Cristiano Corona, Cristiano Longo, Michele Fiorini, Alessia Farinazzo, and Matteo Gelati for technical assistance. This work was supported by a grant from the Italian Ministry of Health (IZS PLV 004/01 to M.C. and S.M.), a grant from Fondazione Cariverona (2002-Malattie neurodegenerative to S.M.), and in part by the Italian Ministry of Health (RF 2001.96 to F.T.).

Acknowledgments

Notes

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: TSE, transmissible spongiform encephalopathy, BSE, bovine spongiform encephalopathy; CJD, Creutzfeldt-Jakob disease; vCJD, variant CJD; sCJD, sporadic CJD; PrP, prion protein, PrPSc pathological PrP; BASE, bovine amyloidotic spongiform encephalopathy.

Notes

This paper was submitted directly (Track II) to the PNAS office.

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