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Tang Y.,Animal Health and Veterinary Laboratories Agency | Gielbert A.,Animal Health and Veterinary Laboratories Agency | Jacobs J.G.,Central Veterinary Institute of Wageningen UR | Baron T.,Agence Francaise de Securite Sanitaire des Aliments Lyon | And 4 more authors.
Journal of Immunological Methods | Year: 2012

Prion diseases or transmissible spongiform encephalopathies (TSEs) in small ruminants are presented in many forms: classical scrapie, Nor98/atypical scrapie, CH1641 scrapie and bovine spongiform encephalopathy (BSE). We previously described a multiplex immunofluorometric assay (mIFMA), based on a bead array flow cytometry technology, which provided, in a single assay, discrimination between BSE (in cattle and sheep) and classical scrapie (Tang et al., 2010). In this study, we extended the mlFMA to differentiate classical scrapie, atypical scrapie, BSE (experimentally infected sheep and naturally infected cattle) and CH1641 (both experimental and natural CH1641-like infections in sheep). Three capture antibodies were used, two distinct PrP N-terminus specific antibodies 12B2 and 9A2, and a PrP core specific antibody 94B4. All three antibodies were shown to bind classical scrapie PrP res strongly, whereas in Nor98/atypical scrapie PrP res only 12B2 and 9A2 binding was observed. PrP res binding of 12B2 was low for both BSE and CH1641, as expected.Furthermore, analysis of serially diluted samples indicated that the assay provided a similar level of sensitivity for atypical scrapie as that found using a well established commercial test. Unexpectedly, 9A2 binding to CH1641 PrP res was reduced by 2.1 fold both for experimental CH1641 and CH1641-like scrapie when compared with BSE, suggesting that major cleavage of the N-terminus occurs further towards the C-terminus in CH1641 than in BSE. The ratios of 12B2/94B4 and 9A2/94B4 were similar between experimental CH1641 and CH1641-like cases, although two CH1641-like subjects displayed slightly elevated ratios of both 12B2/94B4 and 9A2/94B4. To verify this finding for PrP res, mass spectrometry based quantification was used to determine the absolute abundance of the peptides associated with all three antibody binding regions. There was a 2.2 fold reduction of peptides containing the 9A2 epitope for experimental CH1641 PrP res in comparison to BSE PrP res. Observation of reduced PrP res may serve as a new marker for CH1641. This mIFMA may thus provide the basis for simplified TSE diagnosis with capability for simultaneous screening and differential diagnosis. © 2012.

Baron T.,Agence Francaise de Securite Sanitaire des Aliments Lyon | Vulin J.,Agence Francaise de Securite Sanitaire des Aliments Lyon | Biacabe A.,Agence Francaise de Securite Sanitaire des Aliments Lyon | Lakhdar L.,Agence Francaise de Securite Sanitaire des Aliments Lyon | And 3 more authors.
PLoS ONE | Year: 2011

Background: Two distinct forms of atypical spongiform encephalopathies (H-BSE and L-BSE) have recently been identified in cattle. Transmission studies in several wild-type or transgenic mouse models showed that these forms were associated with two distinct major strains of infectious agents, which also differed from the unique strain that had been isolated from cases of classical BSE during the food-borne epizootic disease. Methodology/Principal Findings: H-BSE was monitored during three serial passages in C57BL/6 mice. On second passage, most of the inoculated mice showed molecular features of the abnormal prion protein (PrPd) and brain lesions similar to those observed at first passage, but clearly distinct from those of classical BSE in this mouse model. These features were similarly maintained during a third passage. However, on second passage, some of the mice exhibited distinctly different molecular and lesion characteristics, reminiscent of classical BSE in C57Bl/6 mice. These similarities were confirmed on third passage from such mice, for which the same survival time was also observed as with classical BSE adapted to C57Bl/6 mice. Lymphotropism was rarely detected in mice with H-BSE features. In contrast, PrPd was detectable, on third passage, in the spleens of most mice exhibiting classical BSE features, the pattern being indistinguishable from that found in C57Bl/6 mice infected with classical BSE. Conclusion/Significance: Our data demonstrate the emergence of a prion strain with features similar to classical BSE during serial passages of H-BSE in wild-type mice. Such findings might help to explain the origin of the classical BSE epizootic disease, which could have originated from a putatively sporadic form of BSE.©2011 Baron et al.

Nicot S.,Agence Francaise de Securite Sanitaire des Aliments Lyon | Baron T.G.M.,Agence Francaise de Securite Sanitaire des Aliments Lyon
Journal of General Virology | Year: 2010

The cerebral prion protein (PrP) isolated in the absence of proteinase K digestion, from ruminants prion sources transmitted to ovine transgenic mice, was studied by Western blot analysis. A C2 PrP fragment, showing strain-specific cleavages, similar to those observed after proteinase K or thermolysin digestion, accumulated in the brain. 'CH1641-like' scrapie was characterized by the unique accumulation of a more C-terminally cleaved PrP fragment (CTF14). A similar, protease-resistant, PrP product was observed after proteinase K or thermolysin digestion. Whereas classical BSE appeared highly resistant to thermolysin digestion, CH1641 and 'CH1641-like' natural isolates did not show any remarkable feature regarding resistance to thermolysin. Thus, the molecular strain-specific features in the brain of transmissible spongiform encephalopathy infected mice essentially reflect the PrP proteolytic processing occurring in vivo. © 2010 SGM.

Baron T.,Agence Francaise de Securite Sanitaire des Aliments Lyon | Bencsik A.,Agence Francaise de Securite Sanitaire des Aliments Lyon | Morignat E.,Agence Francaise de Securite Sanitaire des Aliments Lyon
PLoS ONE | Year: 2010

Background: Transmissible agents involved in prion diseases differ in their capacities to target different regions of the central nervous system and lymphoid tissues, which are also host-dependent. Methodology/Principal Findings:Protease-resistant prion protein (PrP res) was analysed by Western blot in the spleen of transgenic mice (TgOvPrP4) that express the ovine prion protein under the control of the neuron-specific enolase promoter, after infection by intra-cerebral route with a variety of transmissible spongiform encephalopathies (TSEs) from cattle and small ruminants. Splenic PrP res was consistently detected in classical BSE and in most natural scrapie sources, the electrophoretic pattern showing similar features to that of cerebral PrP res. However splenic PrP res was not detected in L-type BSE and TME-in-cattle, or in the CH1641 experimental scrapie isolate, indicating that some TSE strains showed reduced splenotropism in the ovine transgenic mice. In contrast with CH1641, PrP res was also consistently detected in the spleen of mice infected with six natural "CH1641-like" scrapie isolates, but then showed clearly different molecular features from those identified in the brains (unglycosylated PrP res at ~ 18 kDa with removal of the 12B2 epitope) of ovine transgenic mice or of sheep. These features included different cleavage of the main PrP res cleavage product (unglycosylated PrP res at ~ 19 kDa with preservation of the 12B2 epitope) and absence of the additional C-terminally cleaved PrP res product (unglycosylated form at,14 kDa) that was detected in the brain. Conclusion/Significance: Studies in a transgenic mouse model expressing the sheep prion protein revealed different capacities of ruminant prions to propagate in the spleen. They showed unexpected features in "CH1641-like" ovine scrapie suggesting that such isolates contain mixed conformers with distinct capacities to propagate in the brain or lymphoid tissues of these mice. © 2010 Baron et al.

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