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Prion diseases are fatal neurodegenerative conditions of humans and various vertebrate species that are transmissible between individuals of the same or different species. A novel infectious moiety referred to as a prion is considered responsible for transmission of these conditions. Prion replication is believed to be the cause of the neurotoxicity that arises during prion disease pathogenesis. The prion hypothesis predicts that the transmissible prion agent consists of PrPSc, which is comprised of aggregated misfolded conformers of the normal host protein PrPC. It is important to understand the biology of transmissible prions and to identify genetic modifiers of prion-induced neurotoxicity. This information will underpin the development of therapeutic and control strategies for human and animal prion diseases. The most reliable method to detect prion infectivity is by in vivo transmission in a suitable experimental host, which to date have been mammalian species. Current prion bioassays are slow, cumbersome and relatively insensitive to low titres of prion infectivity, and do not lend themselves to rapid genetic analysis of prion disease. Here, we provide an overview of our novel studies that have led to the establishment of Drosophila melanogaster, a genetically well-defined invertebrate host, as a sensitive, versatile and economically viable animal model for the detection of mammalian prion infectivity and genetic modifiers of prion-induced toxicity.

Transmissible spongiform encephalopathy (TSE) surveillance in goats relies on tests initially approved for cattle, subsequently assessed for sheep, and approval extrapolated for use in “small ruminants.” The current EU-approved immunodetection tests employ antibodies against various epitopes of the prion protein PrPSc, which is encoded by the host PRNP gene. The caprine PRNP gene is polymorphic, mostly at codons different from the ovine PRNP. The EU goat population is much more heterogeneous than the sheep population, with more PRNP-related polymorphisms, and with marked breed-related differences. The ability of the current tests to detect disease-specific PrPSc generated against these different genetic backgrounds is currently assumed, rather than proven. We examined whether common polymorphisms within the goat PRNP gene might have any adverse effect on the relative performance of EU-approved rapid tests. The sample panel comprised goats from the UK, Cyprus, France, and Italy, with either experimental or naturally acquired scrapie at both the preclinical and/or unknown and clinical stages of disease. Test sensitivity was significantly lower and more variable when compared using samples from animals that were preclinical or of unknown status. However, all of the rapid tests included in our study were able to correctly identify all samples from animals in the clinical stages of disease, apart from samples from animals polymorphic for serine or aspartic acid at codon 146, in which the performance of the Bio-Rad tests was profoundly affected. Our data show that some polymorphisms may adversely affect one test and not another, as well as underline the dangers of extrapolating from other species.

A new alternative method for the production of biodiesel from rendered fat, including animal by‐product (ABP) Category 1 tallow, was evaluated. The method consists of a conversion phase, based on esterification and transesterification in a single step (at temperature ≥ 200°C, pressure ≥ 70 bar with a retention time ≥ 15 min), using MgO as a catalyst and in the presence of methanol (10–15%), followed by vacuum distillation (at ≥ 150°C, ≤ 10 mbar) of the end‐product, biodiesel and the co‐product, glycerine. Prions (PrPSc), which are abnormal isoforms of the prion protein, were considered by the applicant to be the most resistant hazard. In accordance with previous EFSA Opinions and current expert evaluation, a reduction in prion infectivity, or detectable PrPSc, of at least 6 log10 should be achieved for the process to be considered equivalent to the processing method laid down in the Regulation (EU) No 142/2011. Published data from an experimental replication of the conversion step of the biodiesel production process under consideration were provided, which showed an at least 6 log10 reduction in detectable PrPSc, by Western blot, in tallow that had been spiked with murine and human prion strains. In addition, it was demonstrated that the presence of methanol does not affect the recovery or detection of PrPSc from a biodiesel substrate. Based on scientific literature, the vacuum distillation step has been shown to be capable of achieving an additional 3 log10 reduction in PrPSc. Therefore, the proposed alternative method is considered to be at least equivalent to the processing method laid down in the legislation for the production of biodiesel from raw materials including Category 1 ABP. info:eu-repo/semantics/publishedVersion

Shiny Proxy files to reproduce the model outputs of the C&G opinion. To be opened using the Monte Carlo tool (risk assessment using Monte Carlo) of the EFSA’s Shiny proxy open source platform. &nbsp

Additional file 4. Sample ranking to their relative levels of N-terminal PrPres epitopes of antibodies P4 and 12B2. Figure in histogram form to compare the ranking of goat study sample series from high to low N-terminal epitope in ISS-WB and Triplex-WB.

Supplemental material, Supplemental_material for Transmissible spongiform encephalopathy in goats: is PrP rapid test sensitivity affected by genotype? by Marion M. Simmons, Leigh Thorne, Angel Ortiz-Pelaez, John Spiropoulos, Soteria Georgiadou, Penelope Papasavva-Stylianou, Olivier Andreoletti, Stephen A.C. Hawkins, Daniela Meloni and Claire Cassar in Journal of Veterinary Diagnostic Investigation

Additional file 6. Graphic comparison of the PrPres double and single triplet state in resp. CH1641 and scrapie/BSE. Figure showing difference in migration of PrPres bands in CH1641 scrapie samples, goat study sample UK-B2 and reference TSEs as obtained with mAbs Sha31 and SAF84 in Triplex-WB.

Additional file 2. Overview of the application of seven different biochemical analyses in goat and control samples. Table containing goat sample sets used in the specific type of analyses performed.

Additional file 5. Correlation between N-terminus data and molecular mass of the PrPres non-glycosylated band obtained by Triplex-WB. Figure in dotplot form showing the correspondence between total PrPres N-terminus level and molecular mass of non-glycosylated PrPres fraction.

Additional file 3. ISS-WB of goat TSE brain samples from different geographical regions with antibodies P4 and SAF84. Figure of ISS-WB with mAbs P4 and SAF84 on the set of goat study samples.

International audience; Contrasting the knowledge about prion diseases or TSEs in sheep, only a very limited number of strain typing studies are available in goats. Two cases deriving from the zoonotic bovine BSE epidemic were however detected in goats. During 2004–2012, over 70 TSE goat brain samples were collected from seven European countries and evaluated for TSE type/strain variation. A selection of these materials was chosen for in-depth analysis based on various criteria: tissue quality, genotype, broad geographical distribution, potential type variation. Of these, 37 cases were biochemically analysed (present study) and a subset of these subjected to bioassays in seven rodent models (see parallel study of Nonno et al.). Analyses of these isolates showed that in goats different PrPSc types exist, comparable to those found in sheep such as classical scrapie, CH1641-like scrapie and Nor98/atypical scrapie. However, classical scrapie isolates could be further differentiated in two molecular subtypes based on the PK susceptibility of the N-terminus and on the molecular weight of PrPres. Importantly, the two subtypes of classical scrapie showed different geographical distribution, as one subtype was only detected in goats deriving from Italy and France. These analyses also show, that none of the field samples exhibited BSE-like features and offer a comprehensive set of robust molecular PrPres properties to exclude suspicion for the presence of BSE. These are: molecular mass of the triplet bands, presence of N-terminus epitope, glycoprofile markers, a dual population marker and the structural stability of PrP-core.This work was made possible by national support and by the following European grants: Neuroprion (FP6-FOOD-506,579), GoatBSE (FOOD-CT-2006–36,353) and GOAT-TSE-FREE (EMIDA-ERA NET). We want to memorize Jorg G Jacobs, who died in 2017. Jorg developed and performed the Triplex-WB system as well as characterized antibodies like 94B4, 12B2, 9A2, 6C2, SAF84 and L42. He also was crucial in the distinction of C-, L- and H-type BSE and in 2011 he showed elegantly the allotype composition of prion material in heterozygous sheep by the use of Endo-LysC.

Additional file 1: Table S1. Primers used for the detection of mycobacteria by RT-PCR

Additional file 1: Summary of original experiment. This document gives an overview of the inoculations carried out in cattle and wild-type mice with references to the animal numbers in the original, published study [2].