Advanced survival models for risk-factor analysis in scrapie

Because of the confounding effects of long incubation duration and flock management, accurate epidemiological studies of scrapie outbreaks are difficult to carry out. In this study, 641 Manech red-faced sheep from six scrapie-affected field flocks in Pyrénées Atlantiques, France, were monitored for clinical scrapie over a 6–9 year period. Over this period, 170 scrapie clinical cases were recorded and half of the culled animals were submitted for post-mortem transmissible spongiform encephalopathy diagnosis to assess their infectious status. Collected data were analysed using a ‘mixture cure model’ approach, which allowed for the discriminating effect of PrP genotype and flock origin on incidence and incubation period. Simulations were performed to evaluate the applicability of such a statistical model to the collected data. As expected, ARR heterozygote sheep were less at risk of becoming infected than ARQ/ARQ individuals and had a greater age at clinical onset. Conversely, when compared with ARQ/ARQ, the VRQ haplotype was associated with an increased infection risk, but not a shorter incubation period. Considering the flock effect, we observed that a high incidence rate was not associated with shorter incubation periods and that the incubation period could be significantly different in flocks harbouring similar infection risks. These results strongly support the conclusion that other parameters, such as the nature of the agent or flock management, could interfere with epidemiological dynamics of the infection in scrapie-affected flocks

Simulation de l'activité corticale évoquée par les implants rétiniens artificiels

International audienc

Mammalian prion propagation in PrP transgenic Drosophila.

Mammalian prions propagate by template-directed misfolding and aggregation of normal cellular prion related protein PrPC as it converts into disease-associated conformers collectively referred to as PrPSc. Mammalian species may be permissive for prion disease because these hosts have co-evolved specific co-factors that assist PrPC conformational change and prion propagation. We have tested this hypothesis by examining whether faithful prion propagation occurs in the normally PrPC-null invertebrate host Drosophila melanogaster. Ovine PrP transgenic Drosophila exposed at the larval stage to ovine scrapie showed a progressive accumulation of transmissible prions in adult flies. Strikingly, the biological properties of distinct ovine prion strains were maintained during their propagation in Drosophila. Our observations show that the co-factors necessary for strain-specific prion propagation are not unique to mammalian species. Our studies establish Drosophila as a novel host for the study of transmissible mammalian prions.This work was supported by funds from the NC3Rs Project (Grant NC/K000462/1)

Prion protein in the cerebrospinal fluid of healthy and naturally scrapie-affected sheep

The aim of this study was to characterize the cerebrospinal fluid (CSF) prion protein (PrP) of healthy and naturally scrapie-affected sheep. The soluble form of CSF PrPC immunoblotted with an anti-octarepeat and an anti-C terminus mAb showed two isoforms of approximately 33 and 26 kDa, corresponding to the biglycosylated and unglycosylated isoforms of brain PrPC. Neither the mean concentration nor the electrophoretic profile of CSF PrP differed between healthy and scrapieaffected sheep, whereas a slightly increased resistance of CSF PrP to mild proteolysis by proteinase K was evident in the CSF of scrapie-affected sheep. No difference in susceptibility to proteolysis was observed between the two ARR and VRQ genetic variants of the purified prokaryote recombinant PrP. It was concluded that the physicochemical properties of PrPC in the CSF could be altered during scrapie and that these changes might reflect the physiopathological process of prion disease

Decoding cortical activity evoked by artificial retinal implants

International audienc

Ovine serum biomarkers of early and late phase scrapie

<p>Abstract</p> <p>Background</p> <p>Transmissible spongiform encephalopathies are fatal neurodegenerative disease occurring in animals and humans for which no <it>ante-mortem </it>diagnostic test in biological fluids is available. In such pathologies, detection of the pathological form of the prion protein (i.e., the causative factor) in blood is difficult and therefore identification of new biomarkers implicated in the pathway of prion infection is relevant.</p> <p>Methods</p> <p>In this study we used the SELDI-TOF MS technology to analyze a large number of serum samples from control sheep and animals with early phase or late phase scrapie. A few potential low molecular weight biomarkers were selected by statistical methods and, after a training analysis, a protein signature pattern, which discriminates between early phase scrapie samples and control sera was identified.</p> <p>Results</p> <p>The combination of early phase biomarkers showed a sensitivity of 87% and specificity of 90% for all studied sheep in the early stage of the disease. One of these potential biomarkers was identified and validated in a SELDI-TOF MS kinetic study of sera from Syrian hamsters infected by scrapie, by western blot analysis and ELISA quantitation.</p> <p>Conclusions</p> <p>Differential protein expression profiling allows establishing a TSE diagnostic in scrapie sheep, in the early phase of the disease. Some proteic differences observed in scrapie sheep exist in infected hamsters. Further studies are being performed to identify all the discriminant biomarkers of interest and to test our potential markers in a new cohort of animals.</p

The use of PrP transgenic Drosophila to replace and reduce vertebrate hosts in the bioassay of mammalian prion infectivity [version 1; referees: 2 approved]

Prion diseases are fatal neurodegenerative conditions of humans and vertebrate species. The transmissible prion agent is a novel infectious particle composed principally of PrPSc, an abnormal isomer of the normal host protein PrPC. The only reliable method to detect mammalian prion infectivity is by bioassay, invariably in a vertebrate host. The current prion bioassays typically involve intracerebral or peripheral inoculation of test material into the experimental host and subsequent euthanasia when clinical signs of terminal prion disease become evident. It may be months or years before the onset of clinical disease becomes evident and a pre-determined clinical end-point is reached. Consequently, bioassay of prion infectivity in vertebrate species is cumbersome, time consuming, expensive, and increasingly open to ethical debate because these animals are subjected to terminal neurodegenerative disease. Prions are a significant risk to public health through the potential for zoonotic transmission of animal prion diseases. Attention has focussed on the measurement of prion infectivity in different tissues and blood from prion-infected individuals in order to determine the distribution of infectious prions in diseased hosts. New animal models are required in order to replace or reduce, where possible, the dependency on the use of vertebrate species, including the ‘gold standard’ mouse prion bioassay, to assess prion infectivity levels. Here we highlight the development of a Drosophila-based prion bioassay, a highly sensitive and rapid invertebrate animal system that can efficiently detect mammalian prions. This novel invertebrate model system will be of considerable interest to biologists who perform prion bioassays as it will promote reduction and replacement in the number of sentient animals currently used for this purpose. This article is a composite of previous methods that provides an overview of the methodology of the model and discusses the experimental data to promote its viability for use instead of more sentient hosts

Highly Efficient Prion Transmission by Blood Transfusion

It is now clearly established that the transfusion of blood from variant CJD (v-CJD) infected individuals can transmit the disease. Since the number of asymptomatic infected donors remains unresolved, inter-individual v-CJD transmission through blood and blood derived products is a major public health concern. Current risk assessments for transmission of v-CJD by blood and blood derived products by transfusion rely on infectious titers measured in rodent models of Transmissible Spongiform Encephalopathies (TSE) using intra-cerebral (IC) inoculation of blood components. To address the biological relevance of this approach, we compared the efficiency of TSE transmission by blood and blood components when administrated either through transfusion in sheep or by intra-cerebral inoculation (IC) in transgenic mice (tg338) over-expressing ovine PrP. Transfusion of 200 µL of blood from asymptomatic infected donor sheep transmitted prion disease with 100% efficiency thereby displaying greater virulence than the transfusion of 200 mL of normal blood spiked with brain homogenate material containing 103ID50 as measured by intracerebral inoculation of tg338 mice (ID50 IC in tg338). This was consistent with a whole blood titer greater than 103.6 ID50 IC in tg338 per mL. However, when the same blood samples were assayed by IC inoculation into tg338 the infectious titers were less than 32 ID per mL. Whereas the transfusion of crude plasma to sheep transmitted the disease with limited efficacy, White Blood Cells (WBC) displayed a similar ability to whole blood to infect recipients. Strikingly, fixation of WBC with paraformaldehyde did not affect the infectivity titer as measured in tg338 but dramatically impaired disease transmission by transfusion in sheep. These results demonstrate that TSE transmission by blood transfusion can be highly efficient and that this efficiency is more dependent on the viability of transfused cells than the level of infectivity measured by IC inoculation

Screening of intact yeasts and cell extracts to reduce Scrapie prions during biotransformation of food waste

Yeasts can be used to convert organic food wastes to protein-rich animal feed in order to recapture nutrients. However, the reuse of animal-derived waste poses a risk for the transmission of infectious prions that can cause neurodegeneration and fatality in humans and animals. The aim of this study was to investigate the ability of yeasts to reduce prion activity during the biotransformation of waste substrates—thereby becoming a biosafety hurdle in such a circular food system. During pre-screening, 30 yeast isolates were spiked with Classical Scrapie prions and incubated for 72 h in casein substrate, as a waste substitute. Based on reduced Scrapie seeding activity, waste biotransformation and protease activities, intact cells and cell extracts of 10 yeasts were further tested. Prion analysis showed that five yeast species reduced Scrapie seeding activity by approximately 1 log10 or 90%. Cryptococcus laurentii showed the most potential to reduce prion activity since both intact and extracted cells reduced Scrapie by 1 log10 and achieved the highest protease activity. These results show that select forms of yeast can act as a prion hurdle during the biotransformation of waste. However, the limited ability of yeasts to reduce prion activity warrants caution as a sole barrier to transmission as higher log reductions are needed before using waste-cultured yeast in circular food systems