Classical Bovine Spongiform Encephalopathy Research Articles

Lack of prion transmission barrier in human PrP transgenic Drosophila

While animal prion diseases are a threat to human health, their zoonotic potential is generally inefficient because of interspecies prion transmission barriers. New animal models are required to provide an understanding of these prion transmission barriers and to assess the zoonotic potential of animal prion diseases. To address this goal, we generated Drosophila transgenic for human or non-human primate PrP and determined their susceptibility to known pathogenic prion diseases, namely vCJD and classical BSE, and that with unknown pathogenic potential, namely CWD. Adult Drosophila transgenic for M129 or V129 human PrP, or non-human primate PrP developed a neurotoxic phenotype and showed an accelerated loss of survival after exposure to vCJD, classical BSE, or CWD prions at the larval stage. vCJD prion strain identity was retained after passage in both M129 and V129 human PrP Drosophila. All of the primate PrP fly lines accumulated prion seeding activity and concomitantly developed a neurotoxic phenotype, generally including accelerated loss of survival, after exposure to CWD prions derived from different cervid species, including North American white-tailed deer and muntjac, and European reindeer and moose. These novel studies show that primate PrP transgenic Drosophila lack known prion transmission barriers since, in mammalian hosts, V129 human PrP is associated with severe resistance to classical BSE prions, while both human and cynomolgus macaque PrP are associated with resistance to CWD prions. Significantly, our data suggest that interspecies differences in the amino acid sequence of PrP may not be a principal determinant of the prion transmission barrier.

Classical BSE dismissed as the cause of CWD in Norwegian red deer despite strain similarities between both prion agents

The first case of CWD in a Norwegian red deer was detected by a routine ELISA test and confirmed by western blotting and immunohistochemistry in the brain stem of the animal. Two different western blotting tests were conducted independently in two different laboratories, showing that the red deer glycoprofile was different from the Norwegian CWD reindeer and CWD moose and from North American CWD. The isolate showed nevertheless features similar to the classical BSE (BSE-C) strain. Furthermore, BSE-C could not be excluded based on the PrPSc immunohistochemistry staining in the brainstem and the absence of detectable PrPSc in the lymphoid tissues. Because of the known ability of BSE-C to cross species barriers as well as its zoonotic potential, the CWD red deer isolate was submitted to the EURL Strain Typing Expert Group (STEG) as a BSE-C suspect for further investigation. In addition, different strain typing in vivo and in vitro strategies aiming at identifying the BSE-C strain in the red deer isolate were performed independently in three research groups and BSE-C was not found in it. These results suggest that the Norwegian CWD red deer case was infected with a previously unknown CWD type and further investigation is needed to determine the characteristics of this potential new CWD strain.

Assessment of the Zoonotic Potential of Atypical Scrapie Prions in Humanized Mice Reveals Rare Phenotypic Convergence but Not Identity With Sporadic Creutzfeldt-Jakob Disease Prions.

Atypical/Nor98 scrapie (AS) is an idiopathic infectious prion disease affecting sheep and goats. Recent findings suggest that zoonotic prions from classical bovine spongiform encephalopathy (C-BSE) may copropagate with atypical/Nor98 prions in AS sheep brains. Investigating the risk AS poses to humans is crucial. To assess the risk of sheep/goat-to-human transmission of AS, we serially inoculated brain tissue from field and laboratory isolates into transgenic mice overexpressing human prion protein (Met129 allele). We studied clinical outcomes as well as presence of prions in brains and spleens. No transmission occurred on the primary passage, with no clinical disease or pathological prion protein in brains and spleens. On subsequent passages, 1 isolate gradually adapted, manifesting as prions with a phenotype resembling those causing MM1-type sporadic Creutzfeldt-Jakob disease in humans. However, further characterization using in vivo and in vitro techniques confirmed both prion agents as different strains, revealing a case of phenotypic convergence. Importantly, no C-BSE prions emerged in these mice, especially in the spleen, which is more permissive than the brain for C-BSE cross-species transmission. The results obtained suggest a low zoonotic potential for AS. Rare adaptation may allow the emergence of prions phenotypically resembling those spontaneously forming in humans.

Experimental transmission of ovine atypical scrapie to cattle

Classical bovine spongiform encephalopathy (BSE) in cattle was caused by the recycling and feeding of meat and bone meal contaminated with a transmissible spongiform encephalopathy (TSE) agent but its origin remains unknown. This study aimed to determine whether atypical scrapie could cause disease in cattle and to compare it with other known TSEs in cattle. Two groups of calves (five and two) were intracerebrally inoculated with atypical scrapie brain homogenate from two sheep with atypical scrapie. Controls were five calves intracerebrally inoculated with saline solution and one non-inoculated animal. Cattle were clinically monitored until clinical end-stage or at least 96 months post-inoculation (mpi). After euthanasia, tissues were collected for TSE diagnosis and potential transgenic mouse bioassay. One animal was culled with BSE-like clinical signs at 48 mpi. The other cattle either developed intercurrent diseases leading to cull or remained clinical unremarkable at study endpoint, including control cattle. None of the animals tested positive for TSEs by Western immunoblot and immunohistochemistry. Bioassay of brain samples from the clinical suspect in Ov-Tg338 and Bov-Tg110 mice was also negative. By contrast, protein misfolding cyclic amplification detected prions in the examined brains from atypical scrapie-challenged cattle, which had a classical BSE-like phenotype. This study demonstrates for the first time that a TSE agent with BSE-like properties can be amplified in cattle inoculated with atypical scrapie brain homogenate.

Detection of classical BSE prions in asymptomatic cows after inoculation with atypical/Nor98 scrapie

The emergence of bovine spongiform encephalopathy (BSE) prions from atypical scrapie has been recently observed upon experimental transmission to rodent and swine models. This study aimed to assess whether the inoculation of atypical scrapie could induce BSE-like disease in cattle. Four calves were intracerebrally challenged with atypical scrapie. Animals were euthanized without clinical signs of prion disease and tested negative for PrPSc accumulation by immunohistochemistry and western blotting. However, an emergence of BSE-like prion seeding activity was detected during in vitro propagation of brain samples from the inoculated animals. These findings suggest that atypical scrapie may represent a potential source of BSE infection in cattle.

Conventional and State-of-the-Art Detection Methods of Bovine Spongiform Encephalopathy (BSE).

Bovine spongiform encephalopathy (BSE) is a fatal neurodegenerative disease that belongs to a group of diseases known as transmissible spongiform encephalopathies (TSEs). It is believed that the infectious agent responsible for prion diseases is abnormally folded prion protein (PrPSc), which derives from a normal cellular protein (PrPC), which is a cell surface glycoprotein predominantly expressed in neurons. There are three different types of BSE, the classical BSE (C-type) strain and two atypical strains (H-type and L-type). BSE is primarily a disease of cattle; however, sheep and goats also can be infected with BSE strains and develop a disease clinically and pathogenically indistinguishable from scrapie. Therefore, TSE cases in cattle and small ruminants require discriminatory testing to determine whether the TSE is BSE or scrapie and to discriminate classical BSE from the atypical H- or L-type strains. Many methods have been developed for the detection of BSE and have been reported in numerous studies. Detection of BSE is mainly based on the identification of characteristic lesions or detection of the PrPSc in the brain, often by use of their partial proteinase K resistance properties. The objective of this paper was to summarize the currently available methods, highlight their diagnostic performance, and emphasize the advantages and drawbacks of the application of individual tests.

Discrimination of Classical and Atypical BSE by a Distinct Immunohistochemical PrPSc Profile

Bovine spongiform encephalopathy (BSE) belongs to the group of transmissible spongiform encephalopathies and is associated with the accumulation of a pathological isoform of the host-encoded glycoprotein, designated prion protein (PrPSc). Classical BSE (C-type) and two atypical BSE forms (L- and H-type) are known, and can be discriminated by biochemical characteristics. The goal of our study was to identify type-specific PrPSc profiles by using Immunohistochemistry. In our study, brain samples from 21 cattle, intracerebrally inoculated with C-, H-, and L-type BSE, were used. In addition, the corresponding samples from three orally C-type BSE infected animals were also included. From all animals, a lesion and PrPSc-profiles of six brain regions were determined. The lesion profile and the neuroanatomical distribution of PrPSc was highly consistent between the groups, but the immunohistochemical analysis revealed a distinct PrPSc profile for the different BSE-types, which included both the topographic and cellular pattern of PrPSc. This qualitative and quantitative analysis of PrPSc affected structures sheds new light into the pathogenesis of the different BSE types. Furthermore, immunohistochemical characterization is supported as an additional diagnostic tool in BSE surveillance programs, especially when only formalin-fixed tissue samples are available.

Experimental infection of bovine spongi-form encephalopathy in guinea pigs: clinical symptoms, pathohistology and immunohistochemistry

Prion diseases are infectious neurodegenerative diseases affecting humans and animals that are invariably fatal and have no currently available prevention or treatment methods. These diseases are also known as transmissible spongiform encephalopathies (TSE), characterized by similar neurological disorders, vacuolization of the neuroparenchyma, accumulation of the pathogenic isoform of the prion protein in it, the absence of an immunological response to the pathogen, and the inevitable death. The most famous of them is the classic bovine spongiform encephalopathy, because. has unprecedented social and economic consequences. C-BSE of cattle is also dangerous for humans, causing one of the forms of Creutzfeldt-Jakob disease, leading to a gradual progression of irreversible C-BSE lesions, leading to the death of a sick person. Laboratory animal models make a huge contribution to the study of the biological properties of prions and the diseases they cause. Thus, mouse models of prion infections have played a major role in deciphering fundamental questions of prion biology and assessing the potential transmissibility of neurodegenerative diseases. However, despite the economic acceptability and other positive properties of this model, it has a number of disadvantages. It produces low-volume tissue samples, and it is very difficult to obtain the required volume of serum and cerebrospinal fluid for many studies. Therefore, the search for and study of the disease in new models of laboratory animals sensitive to bovine C-BSE is of great scientific importance in the study of prion diseases. This work shows that during experimental intracerebral infection of guinea pigs with the causative agent of classical bovine spongiform encephalopathy, they are 100% sensitive to the pathogen. The clinical picture of the disease, developing severe pathomorphological changes in all parts of the brain with vacuolization of neurons and neropil and massive deposition of the prion protein PrPSc are described. Guinea pigs can be a highly sensitive object for laboratory study of the causative agent of bovine spongiform encephalopathy.

Low frequency of protective variants at regulatory region of PRNP gene indicating the genetically high risk of BSE in Ethiopian Bos indicus and Bos taurus africanus

Susceptibility to classical bovine spongiform encephalopathy (BSE) has been linked to 23 bp indel in promoter and 12 bp indel in the first intron of cattle prion protein gene. This study aimed to investigate 23/12 bp indel polymorphisms in the polymorphisms in cattle prion protein (PRNP) gene to reveal the risk of BSE in Ethiopian cattle. Also, frequency of each polymorphism was compared to the other Bos taurus and Bos indicus breeds. According to results, the insertion variant was detected at a low frequency in all of the study populations at both loci. The 23 bp insertion allele in Fogera breed was relatively lower than Borona and Arsi and the same allele at the same locus in Afar breed was higher than the rest of the breeds (0.16). Due to high linkage disequilibrium (LD) of the deletion allele in Bos taurus, the frequencies of deletion allele at 23 bp (0.84) and 12 bp (0.86) loci in Afar breed were relatively closer than the rest of the breeds. In addition, DD/DD was found as the highly frequent diplotype in all of the breeds. The low frequency of insertion alleles at 23 and 12 bp indel sites demonstrate that Ethiopian cattle have a genetically high risk for BSE.

Have the Biological Properties of Classical BSE Changed After the Implementation of the Reinforced Feed Ban in 1996 in the UK?

Have the Biological Properties of Classical BSE Changed After the Implementation of the Reinforced Feed Ban in 1996 in the UK?

Prion Infectivity and PrPBSE in the Peripheral and Central Nervous System of Cattle 8 Months Post Oral BSE Challenge.

After oral exposure of cattle with classical bovine spongiform encephalopathy (C-BSE), the infectious agent ascends from the gut to the central nervous system (CNS) primarily via the autonomic nervous system. However, the timeline of this progression has thus far remained widely undetermined. Previous studies were focused on later time points after oral exposure of animals that were already 4 to 6 months old when challenged. In contrast, in this present study, we have orally inoculated 4 to 6 weeks old unweaned calves with high doses of BSE to identify any possible BSE infectivity and/or PrPBSE in peripheral nervous tissues during the first eight months post-inoculation (mpi). For the detection of BSE infectivity, we used a bovine PrP transgenic mouse bioassay, while PrPBSE depositions were analyzed by immunohistochemistry (IHC) and by protein misfolding cyclic amplification (PMCA). We were able to show that as early as 8 mpi the thoracic spinal cord as well as the parasympathetic nodal ganglion of these animals contained PrPBSE and BSE infectivity. This shows that the centripetal prion spread starts early after challenge at least in this age group, which represents an essential piece of information for the risk assessments for food, feed, and pharmaceutical products produced from young calves.

Epidemiological verification of the mechanism of occurrence of atypical L-type bovine spongiform encephalopathy.

Since 2004, a novel bovine spongiform encephalopathy (BSE), distinct from the conventional 'classical BSE' (C-BSE), has been reported as an atypical BSE. Atypical BSE is detected mostly in aged cattle, and it is suggested that atypical BSE may occur spontaneously. Relaxation of the relevant countermeasures such as feed ban, which prevents the use of bovine meat-and-bone meal as feed, has been discussed in recent years owing to the decrease in C-BSE cases. If atypical BSE occurs spontaneously without exposure to an agent called abnormal prion protein (PrPSc ), complete removal of these measures will be difficult. In this study, we verified the possibility that L-BSE, which is a subtype of atypical BSE, occurs spontaneously. We first hypothesized that L-BSE occurs only through the process of infection via oral exposure. If the hypothesis was true, the infection of L-BSE would be mostly limited to calves under 1 year of age due to their high susceptibility, and the feed ban would effectively reduce the number of infected calves by birth cohort. Thus, we created a mathematical model to estimate the number of infected calves by birth cohort and compared the effectiveness of the feed ban on C-BSE and L-BSE. The number of tested animals and detected cases in nine European countries were used for this analysis. Our results showed that the estimated number of infected calves in the birth cohort indicated that feed ban was less effective on L-BSE. This result supports the alternative hypothesis that at least a part of the L-BSE can occur without infection via oral exposure. Our results suggest that the complete abolition of countermeasures, such as feed ban, should be discussed carefully. As for the occurrence mechanism, although there remains uncertainty to reach conclusions, it is reasonable to assume that L-BSE can occur spontaneously at present.

Classical BSE prions emerge from asymptomatic pigs challenged with atypical/Nor98 scrapie

Pigs are susceptible to infection with the classical bovine spongiform encephalopathy (C-BSE) agent following experimental inoculation, and PrPSc accumulation was detected in porcine tissues after the inoculation of certain scrapie and chronic wasting disease isolates. However, a robust transmission barrier has been described in this species and, although they were exposed to C-BSE agent in many European countries, no cases of natural transmissible spongiform encephalopathies (TSE) infections have been reported in pigs. Transmission of atypical scrapie to bovinized mice resulted in the emergence of C-BSE prions. Here, we conducted a study to determine if pigs are susceptible to atypical scrapie. To this end, 12, 8–9-month-old minipigs were intracerebrally inoculated with two atypical scrapie sources. Animals were euthanized between 22- and 72-months post inoculation without clinical signs of TSE. All pigs tested negative for PrPSc accumulation by enzyme immunoassay, immunohistochemistry, western blotting and bioassay in porcine PrP mice. Surprisingly, in vitro protein misfolding cyclic amplification demonstrated the presence of C-BSE prions in different brain areas from seven pigs inoculated with both atypical scrapie isolates. Our results suggest that pigs exposed to atypical scrapie prions could become a reservoir for C-BSE and corroborate that C-BSE prions emerge during interspecies passage of atypical scrapie.

Understanding Intra-Species and Inter-Species Prion Conversion and Zoonotic Potential Using Protein Misfolding Cyclic Amplification.

Prion diseases are fatal neurodegenerative disorders that affect humans and animals, and can also be transmitted from animals to humans. A fundamental event in prion disease pathogenesis is the conversion of normal host prion protein (PrPC) to a disease-associated misfolded form (PrPSc). Whether or not an animal prion disease can infect humans cannot be determined a priori. There is a consensus that classical bovine spongiform encephalopathy (C-type BSE) in cattle transmits to humans, and that classical sheep scrapie is of little or no risk to human health. However, the zoonotic potential of more recently identified animal prion diseases, such as atypical scrapie, H-type and L-type BSE and chronic wasting disease (CWD) in cervids, remains an open question. Important components of the zoonotic barrier are (i) physiological differences between humans and the animal in question, (ii) amino acid sequence differences of the animal and human PrPC, and (iii) the animal prion strain, enciphered in the conformation of PrPSc. Historically, the direct inoculation of experimental animals has provided essential information on the transmissibility and compatibility of prion strains. More recently, cell-free molecular conversion assays have been used to examine the molecular compatibility on prion replication and zoonotic potential. One such assay is Protein Misfolding Cyclic Amplification (PMCA), in which a small amount of infected tissue homogenate, containing PrPSc, is added as a seed to an excess of normal tissue homogenate containing PrPC, and prion conversion is accelerated by cycles of incubation and ultrasonication. PMCA has been used to measure the molecular feasibility of prion transmission in a range of scenarios using genotypically homologous and heterologous combinations of PrPSc seed and PrPC substrate. Furthermore, this method can be used to speculate on the molecular profile of PrPSc that might arise from a zoonotic transmission. We discuss the experimental approaches that have been used to model both the intra- and inter-species molecular compatibility of prions, and the factors affecting PrPc to PrPSc conversion and zoonotic potential. We conclude that cell-free prion protein conversion assays, especially PMCA, are useful, rapid and low-cost approaches for elucidating the mechanisms of prion propagation and assessing the risk of animal prions to humans.

A new model for sensitive detection of zoonotic prions by PrP transgenic Drosophila

Prions are transmissible protein pathogens most reliably detected by a bioassay in a suitable host, typically mice. However, the mouse bioassay is slow and cumbersome, and relatively insensitive to low titers of prion infectivity. Prions can be detected biochemically in vitro by the protein misfolding cyclic amplification (PMCA) technique, which amplifies disease-associated prion protein but does not detect bona fide prion infectivity. Here, we demonstrate that Drosophila transgenic for bovine prion protein (PrP) expression can serve as a model system for the detection of bovine prions significantly more efficiently than either the mouse prion bioassay or PMCA. Strikingly, bovine PrP transgenic Drosophila could detect bovine prion infectivity in the region of a 10−12 dilution of classical bovine spongiform encephalopathy (BSE) inoculum, which is 106-fold more sensitive than that achieved by the bovine PrP mouse bioassay. A similar level of sensitivity was observed in the detection of H-type and L-type atypical BSE and sheep-passaged BSE by bovine PrP transgenic Drosophila. Bioassays of bovine prions in Drosophila were performed within 7 weeks, whereas the mouse prion bioassay required at least a year to assess the same inoculum. In addition, bovine PrP transgenic Drosophila could detect classical BSE at a level 105-fold lower than that achieved by PMCA. These data show that PrP transgenic Drosophila represent a new tractable prion bioassay for the efficient and sensitive detection of mammalian prions, including those of known zoonotic potential.

Canine D163-PrP polymorphic variant does not provide complete protection against prion infection in small ruminant PrP context

E/D163 polymorphism of dog prion protein (PrP) has been recently proposed as the variant responsible for canid prion resistance. To further investigate the protective role of this variant against prion replication, the transgenic mouse model OvPrP-Tg532 expressing sheep/goat PrP carrying the substitution D162 (equivalent to D163 position of dog PrP) was generated and intracranially inoculated with a broad collection of small ruminant prion strains. OvPrP-Tg532 mice showed resistance to classical bovine spongiform encephalopathy (BSE) from sheep and some classical scrapie isolates from sheep and goat but were susceptible to ovine atypical L-BSE and numerous classical scrapie isolates. Strikingly, some of these classical scrapie isolates showed a shift in their prion strain properties. These results suggest that other PrP residues apart from E/D163 variant of dog PrP or factors distinct than PrP may participate in prion resistance of canids and that different factors may be required for D162 sheep PrP to provide effective protection to sheep against ruminant prions.

The ultrastructure of infectious L-type bovine spongiform encephalopathy prions constrains molecular models.

Bovine spongiform encephalopathy (BSE) is a prion disease of cattle that is caused by the misfolding of the cellular prion protein (PrPC) into an infectious conformation (PrPSc). PrPC is a predominantly α-helical membrane protein that misfolds into a β-sheet rich, infectious state, which has a high propensity to self-assemble into amyloid fibrils. Three strains of BSE prions can cause prion disease in cattle, including classical BSE (C-type) and two atypical strains, named L-type and H-type BSE. To date, there is no detailed information available about the structure of any of the infectious BSE prion strains. In this study, we purified L-type BSE prions from transgenic mouse brains and investigated their biochemical and ultrastructural characteristics using electron microscopy, image processing, and immunogold labeling techniques. By using phosphotungstate anions (PTA) to precipitate PrPSc combined with sucrose gradient centrifugation, a high yield of proteinase K-resistant BSE amyloid fibrils was obtained. A morphological examination using electron microscopy, two-dimensional class averages, and three-dimensional reconstructions revealed two structural classes of L-type BSE amyloid fibrils; fibrils that consisted of two protofilaments with a central gap and an average width of 22.5 nm and one-protofilament fibrils that were 10.6 nm wide. The one-protofilament fibrils were found to be more abundant compared to the thicker two-protofilament fibrils. Both fibrillar assemblies were successfully decorated with monoclonal antibodies against N- and C-terminal epitopes of PrP using immunogold-labeling techniques, confirming the presence of polypeptides that span residues 100-110 to 227-237. The fact that the one-protofilament fibrils contain both N- and C-terminal PrP epitopes constrains molecular models for the structure of the infectious conformer in favour of a compact four-rung β-solenoid fold.

Classical scrapie in small ruminants is caused by at least four different prion strains

The diversity of goat scrapie strains in Europe has recently been studied using bioassays in a wide collection of rodent models, resulting in the classification of classical scrapie into four different categories. However, the sole use of the first passage does not lead to isolate adaptation and identification of the strains involved and might therefore lead to misclassification of some scrapie isolates. Therefore, this work reports the complete transmission study of a wide collection of goat transmissible spongiform encephalopathy (TSE) isolates by intracranial inoculation in two transgenic mouse lines overexpressing either small ruminant (TgGoat-ARQ) or bovine (TgBov) PrPC. To compare scrapie strains in sheep and goats, sheep scrapie isolates from different European countries were also included in the study. Once the species barrier phenomenon was overcome, an accurate classification of the isolates was attained. Thus, the use of just two rodent models allowed us to fully differentiate at least four different classical scrapie strains in small ruminants and to identify isolates containing mixtures of strains. This work reinforces the idea that classical scrapie in small ruminants is a prion disease caused by multiple different prion strains and not by a single strain, as is the case for epidemic classical bovine spongiform encephalopathy (BSE-C). In addition, the clear dissimilarity between the different scrapie strains and BSE-C does not support the idea that classical scrapie is the origin of epidemic BSE-C.

Met166 -Glu168 residues in human PrP β2-α2 loop account for evolutionary resistance to prion infection.

AimsThe amino acid sequence of prion protein (PrP) is a key determinant in the transmissibility of prion diseases. While PrP sequence is highly conserved among mammalian species, minor changes in the PrP amino acid sequence may confer alterations in the transmissibility of prion diseases. Classical bovine spongiform encephalopathy (C‐BSE) is the only zoonotic prion strain reported to date causing variant Creutzfeldt‐Jacob disease (vCJD) in humans, although experimental transmission points to atypical L‐BSE and some classical scrapie isolates as also zoonotic. The precise molecular elements in the human PrP sequence that limit the transmissibility of prion strains such as sheep/goat scrapie or cervid chronic wasting disease (CWD) are not well known.MethodsThe transmissibility of a panel of diverse prions from different species was compared in transgenic mice expressing either wild‐type human PrPC (MDE‐HuTg340) or a mutated human PrPC harbouring Val166‐Gln168 amino acid changes (VDQ‐HuTg372) in the β2‐α2 loop instead of Met166‐Glu168 wild‐type variants.ResultsVDQ‐HuTg372 mice were more susceptible to prions than MDE‐HuTg340 mice in a strain‐dependent manner.ConclusionsMet166‐Glu168 amino acid residues present in wild‐type human PrPC are molecular determinants that limit the propagation of most prion strains assayed in the human PrP context.

Absence of classical and atypical (H- and L-) BSE infectivity in the blood of bovines in the clinical end stage of disease as confirmed by intraspecies blood transfusion.

While the presence of bovine spongiform encephalopathy (BSE) infectivity in the blood of clinically affected sheep has been proven by intraspecies blood-transfusion experiments, this question has remained open in the case of BSE-affected cattle. Although the absence of infectivity can be anticipated from the restriction of the agent to neuronal tissues in this species, evidence for this was still lacking. This particularly concerns the production and use of medicinal products and other applications containing bovine blood or preparations thereof. We therefore performed a blood-transfusion experiment from cattle in the clinical end stage of disease after experimental challenge with either classical (C-BSE) or atypical (H- and l-) BSE into calves at 4–6 months of age. The animals were kept in a free-ranging group for 10 years. Starting from 24 months post-transfusion, a thorough clinical examination was performed every 6 weeks in order to detect early symptoms of a BSE infection. Throughout the experiment, the clinical picture of all animals gave no indication of a BSE infection. Upon necropsy, the brainstem samples were analysed by BSE rapid test as well as by the highly sensitive Protein Misfolding Cyclic Amplification (PMCA), all with negative results. These results add resilient data to confirm the absence of BSE infectivity in the donor blood collected from C-, H- and l-BSE-affected cattle even in the final clinical phase of the disease. This finding has important implications for the risk assessment of bovine blood and blood products in the production of medicinal products and other preparations.