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Sergio Migliore,
Elena Esposito,
Laura Pirisinu, Stefano Marcon,
Michele Di Bari,
Claudia D'Agostino,
Barbara Chiappini,
Michela Conte,
Erminia Sezzi,
Luigi De Grossi,
Umberto Agrimi,
Gabriele Vaccari,
Romolo Nonno
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ABSTRACT: Procedures for discriminating scrapie from bovine spongiform encephalopathy (BSE) in sheep are relevant to ascertain whether BSE has entered the sheep population. This study was aimed at investigating whether the suitability of an official EU discriminative method is affected by the sheep PrP genotype and route of infection.
Journal of General Virology 02/2012; 93(Pt 2):450-5. · 3.36 Impact Factor
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Laura Pirisinu,
Michele Di Bari, Stefano Marcon,
Gabriele Vaccari,
Claudia D'Agostino,
Paola Fazzi,
Elena Esposito,
Roberta Galeno,
Jan Langeveld,
Umberto Agrimi,
Romolo Nonno
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ABSTRACT: Although proteinacious in nature, prions exist as strains with specific self-perpetuating biological properties. Prion strains are thought to be associated with different conformers of PrP(Sc), a disease-associated isoform of the host-encoded cellular protein (PrP(C)). Molecular strain typing approaches have been developed which rely on the characterization of protease-resistant PrP(Sc). However, PrP(Sc) is composed not only of protease-resistant but also of protease-sensitive isoforms. The aim of this work was to develop a protocol for the molecular characterization of both, protease-resistant and protease-sensitive PrP(Sc) aggregates. We first set up experimental conditions which allowed the most advantageous separation of PrP(C) and PrP(Sc) by means of differential centrifugation. The conformational solubility and stability assay (CSSA) was then developed by measuring PrP(Sc) solubility as a function of increased exposure to GdnHCl. Brain homogenates from voles infected with human and sheep prion isolates were analysed by CSSA and showed strain-specific conformational stabilities, with mean [GdnHCl](1/2) values ranging from 1.6 M for MM2 sCJD to 2.1 for scrapie and to 2.8 M for MM1/MV1 sCJD and E200K gCJD. Interestingly, the rank order of [GdnHCl](1/2) values observed in the human and sheep isolates used as inocula closely matched those found following transmission in voles, being MM1 sCJD the most resistant (3.3 M), followed by sheep scrapie (2.2 M) and by MM2 sCJD (1.6 M). In order to test the ability of CSSA to characterise protease-sensitive PrP(Sc), we analysed sheep isolates of Nor98 and compared them to classical scrapie isolates. In Nor98, insoluble PrP(Sc) aggregates were mainly protease-sensitive and showed a conformational stability much lower than in classical scrapie. Our results show that CSSA is able to reveal strain-specified PrP(Sc) conformational stabilities of protease-resistant and protease-sensitive PrP(Sc) and that it is a valuable tool for strain typing in natural hosts, such as humans and sheep.
PLoS ONE 01/2010; 5(9):e12723. · 4.09 Impact Factor
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Gabriele Vaccari,
Claudia D'Agostino,
Romolo Nonno,
Francesca Rosone,
Michela Conte,
Michele Angelo Di Bari,
Barbara Chiappini,
Elena Esposito,
Luigi De Grossi,
Francesco Giordani, Stefano Marcon,
Luisella Morelli,
Renata Borroni,
Umberto Agrimi
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ABSTRACT: The susceptibility of sheep to classical scrapie and bovine spongiform encephalopathy (BSE) is mainly influenced by prion protein (PrP) polymorphisms A136V, R154H, and Q171R, with the ARR allele associated with significantly decreased susceptibility. Here we report the protective effect of the amino acid substitution M137T, I142K, or N176K on the ARQ allele in sheep experimentally challenged with either scrapie or BSE. Such observations suggest the existence of additional PrP alleles that significantly decrease the susceptibility of sheep to transmissible spongiform encephalopathies, which may have important implications for disease eradication strategies.
Journal of Virology 08/2007; 81(13):7306-9. · 5.40 Impact Factor
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Marta Vascellari,
Romolo Nonno,
Franco Mutinelli,
Michela Bigolaro,
Michele Angelo Di Bari,
Erica Melchiotti, Stefano Marcon,
Claudia D'Agostino,
Gabriele Vaccari,
Michela Conte,
Luigi De Grossi,
Francesca Rosone,
Francesco Giordani,
Umberto Agrimi
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ABSTRACT: The salivary glands of scrapie-affected sheep and healthy controls were investigated for the presence of the pathological prion protein (PrP(Sc)). PrP(Sc) was detected in major (parotid and mandibular) and minor (buccal, labial, and palatine) salivary glands of naturally and experimentally infected sheep. Using Western blotting, the PrP(Sc) concentration in glands was estimated to be 0.02 to 0.005% of that in brain. Immunohistochemistry revealed intracellular depositions of PrP(Sc) in ductal and acinar epithelia and occasional labeling in the lumina of salivary ducts. The presence of PrP(Sc) in salivary glands highlights the possible role of saliva in the horizontal transmission of scrapie.
Journal of Virology 06/2007; 81(9):4872-6. · 5.40 Impact Factor
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Gabriele Vaccari,
Michele A Di Bari,
Luisella Morelli,
Romolo Nonno,
Barbara Chiappini,
Giovanni Antonucci, Stefano Marcon,
Elena Esposito,
Paola Fazzi,
Nadia Palazzini,
Pasquale Troiano,
Antonio Petrella,
Giovanni Di Guardo,
Umberto Agrimi
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ABSTRACT: The association between PrP gene variations and scrapie susceptibility was studied in a single herd of Ionica breed goats. The entire herd comprised 100 animals, 11 of which were clinically affected and showed pathological prion protein (PrP(Sc)) deposition in both their central nervous system (CNS) and lymphoreticular system (LRS). Among asymptomatic goats, nine harboured PrP(Sc) in both CNS and LRS, 19 showed PrP(Sc) only at the LRS level and 61 animals had no PrP(Sc) deposition. Genetic analysis of the PrP gene coding sequence revealed the presence of several polymorphisms, namely G37V, T110P, H143R, R154H, Q222K and P240S. Silent polymorphisms were also found at codons 42, 138, 219 and 232. The effect of PrP polymorphism on scrapie susceptibility was assessed by comparing the genotype distribution at each locus among animals with different pathogenetic and clinical disease stages. Significant differences in the distribution of genotypes were observed for codons 154 and 222, with polymorphism at codon 154 modulating susceptibility to scrapie and lysine at codon 222 being associated with scrapie resistance. The allelic variant encoding lysine at position 222 could be a valuable candidate to select in the framework of appropriate breeding programmes for scrapie resistance in goats.
Journal of General Virology 06/2006; 87(Pt 5):1395-402. · 3.36 Impact Factor
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ABSTRACT: Concerns have been raised about the possibility that the bovine spongiform encephalopathy (BSE) agent could have been transmitted to sheep populations via contaminated feedstuffs. The objective of our study was to investigate the suitability of molecular strain typing methods as a surveillance tool for studying scrapie strain variations and for differentiating PrP(Sc) from sheep scrapie, BSE, and sheep BSE. We studied 38 Italian sheep scrapie cases from 13 outbreaks, along with a British scrapie case, an experimental ovine BSE, and 3 BSE cases, by analyzing the glycoform patterns and the apparent molecular masses of the nonglycosylated forms of semipurified, proteinase-treated PrP(Sc). Both criteria were able to clearly differentiate sheep scrapie from BSE and ovine experimental BSE. PrP(Sc) from BSE and sheep BSE showed a higher glycoform ratio and a lower molecular mass of the nonglycosylated form compared to scrapie PrP(Sc). Scrapie cases displayed homogeneous PrP(Sc) features regardless of breed, flock, and geographic origin. The glycoform patterns observed varied with the antibody used, but either a monoclonal antibody (MAb) (F99/97.6.1) or a polyclonal antibody (P7-7) was able to distinguish scrapie from BSE PrP(Sc). While more extensive surveys are needed to further corroborate these findings, our results suggest that large-scale molecular screening of sheep populations for BSE surveillance may be eventually possible.
Journal of Clinical Microbiology 10/2003; 41(9):4127-33. · 4.15 Impact Factor
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ABSTRACT: Gli autori descrivono i quadri istopatologici e l’immunolocalizzazione della GFAP e della PrP<sub>sc</sub> in 25 aree del SNC di 8 ovini con scrapie naturale, provenienti da due allevamenti della Sardegna. Il quadro clinico e lesivo sostanzialmente omogeneo portano a supporre una identità del ceppo, o dei ceppi conoivoli nei due focolai di malattia.
