Experimental Bovine Spongiform Encephalopathy Research Articles

Interspecies transmission to bovinized transgenic mice uncovers new features of a CH1641-like scrapie isolate

In animal prion diseases, including bovine spongiform encephalopathy (BSE) in cattle, chronic wasting disease in cervids, and scrapie in sheep and goats, a disease-associated isoform of prion protein (PrPd) accumulates in the brains of affected animals. Although the CH1641 scrapie isolate was experimentally established in the UK, a few natural CH1641-like scrapie cases have been reported in France and the UK. The molecular mass of the unglycosylated protease-resistant core of PrPd (PrPres) is known to be similar between CH1641-like scrapie and experimental BSE in sheep. We previously established an experimental CH1641-like scrapie isolate (Sh294) from a natural classical scrapie case. Here, we demonstrated that the Sh294 isolate was independent of both classical and atypical BSEs by cross-species transmission to bovine PrP overexpressing (TgBoPrP) mice and wild-type mice. Interestingly, we found that the Sh294 isolate altered its host range by the transmission to TgBoPrP mice, and we succeeded in the first stable reproduction of CH1641-like scrapie specific PrPres banding patterns with the ~12-kDa small C-terminal fragment in wild-type mice. This study provides new insight into the relationship between CH1641-like scrapie isolates and BSEs. In addition, interspecies transmission models such as we have demonstrated here could be a great help to investigate the origin and host range of animal prions.

Experimental sheep BSE prions generate the vCJD phenotype when serially passaged in transgenic mice expressing human prion protein

The epizootic prion disease of cattle, bovine spongiform encephalopathy (BSE), causes variant Creutzfeldt-Jakob disease (vCJD) in humans following dietary exposure. While it is assumed that all cases of vCJD attributed to a dietary aetiology are related to cattle BSE, sheep and goats are susceptible to experimental oral challenge with cattle BSE prions and farmed animals in the UK were undoubtedly exposed to BSE-contaminated meat and bone meal during the late 1980s and early 1990s. Although no natural field cases of sheep BSE have been identified, it cannot be excluded that some BSE-infected sheep might have entered the European human food chain. Evaluation of the zoonotic potential of sheep BSE prions has been addressed by examining the transmission properties of experimental brain isolates in transgenic mice that express human prion protein, however to-date there have been relatively few studies. Here we report that serial passage of experimental sheep BSE prions in transgenic mice expressing human prion protein with methionine at residue 129 produces the vCJD phenotype that mirrors that seen when the same mice are challenged with vCJD prions from patient brain. These findings are congruent with those reported previously by another laboratory, and thereby strongly reinforce the view that sheep BSE prions could have acted as a causal agent of vCJD within Europe.

Increased susceptibility of transgenic mice expressing human PrP to experimental sheep bovine spongiform encephalopathy is not due to increased agent titre in sheep brain tissue

Bovine spongiform encephalopathy (BSE) in cattle and variant Creutzfeldt–Jakob disease in humans have previously been shown to be caused by the same strain of transmissible spongiform encephalopathy agent. It is hypothesized that the agent spread to humans following consumption of food products prepared from infected cattle. Despite evidence supporting zoonotic transmission, mouse models expressing human prion protein (HuTg) have consistently shown poor transmission rates when inoculated with cattle BSE. Higher rates of transmission have however been observed when these mice are exposed to BSE that has been experimentally transmitted through sheep or goats, indicating that humans may potentially be more susceptible to BSE from small ruminants. Here we demonstrate that increased transmissibility of small ruminant BSE to HuTg mice was not due to replication of higher levels of infectivity in sheep brain tissue, and is instead due to other specific changes in the infectious agent.

Complex proteinopathy with accumulations of prion protein, hyperphosphorylated tau, α-synuclein and ubiquitin in experimental bovine spongiform encephalopathy of monkeys.

Proteins aggregate in several slowly progressive neurodegenerative diseases called 'proteinopathies'. Studies with cell cultures and transgenic mice overexpressing mutated proteins suggested that aggregates of one protein induced misfolding and aggregation of other proteins as well - a possible common mechanism for some neurodegenerative diseases. However, most proteinopathies are 'sporadic', without gene mutation or overexpression. Thus, proteinopathies in WT animals genetically close to humans might be informative. Squirrel monkeys infected with the classical bovine spongiform encephalopathy agent developed an encephalopathy resembling variant Creutzfeldt-Jakob disease with accumulations not only of abnormal prion protein (PrP(TSE)), but also three other proteins: hyperphosphorylated tau (p-tau), α-synuclein and ubiquitin; β-amyloid protein (Aβ) did not accumulate. Severity of brain lesions correlated with spongiform degeneration. No amyloid was detected. These results suggested that PrP(TSE) enhanced formation of p-tau and aggregation of α-synuclein and ubiquitin, but not Aβ, providing a new experimental model for neurodegenerative diseases associated with complex proteinopathies.

Atypical Scrapie Prions from Sheep and Lack of Disease in Transgenic Mice Overexpressing Human Prion Protein

Public and animal health controls to limit human exposure to animal prions are focused on bovine spongiform encephalopathy (BSE), but other prion strains in ruminants may also have zoonotic potential. One example is atypical/Nor98 scrapie, which evaded statutory diagnostic methods worldwide until the early 2000s. To investigate whether sheep infected with scrapie prions could be another source of infection, we inoculated transgenic mice that overexpressed human prion protein with brain tissue from sheep with natural field cases of classical and atypical scrapie, sheep with experimental BSE, and cattle with BSE. We found that these mice were susceptible to BSE prions, but disease did not develop after prolonged postinoculation periods when mice were inoculated with classical or atypical scrapie prions. These data are consistent with the conclusion that prion disease is less likely to develop in humans after exposure to naturally occurring prions of sheep than after exposure to epizootic BSE prions of ruminants.

Infectious titres of sheep scrapie and bovine spongiform encephalopathy agents cannot be accurately predicted from quantitative laboratory test results

It is widely accepted that abnormal forms of the prion protein (PrP) are the best surrogate marker for the infectious agent of prion diseases and, in practice, the detection of such disease-associated (PrP(d)) and/or protease-resistant (PrP(res)) forms of PrP is the cornerstone of diagnosis and surveillance of the transmissible spongiform encephalopathies (TSEs). Nevertheless, some studies question the consistent association between infectivity and abnormal PrP detection. To address this discrepancy, 11 brain samples of sheep affected with natural scrapie or experimental bovine spongiform encephalopathy were selected on the basis of the magnitude and predominant types of PrP(d) accumulation, as shown by immunohistochemical (IHC) examination; contra-lateral hemi-brain samples were inoculated at three different dilutions into transgenic mice overexpressing ovine PrP and were also subjected to quantitative analysis by three biochemical tests (BCTs). Six samples gave 'low' infectious titres (10⁶·⁵ to 10⁶·⁷ LD₅₀ g⁻¹) and five gave 'high titres' (10⁸·¹ to ≥ 10⁸·⁷ LD₅₀ g⁻¹) and, with the exception of the Western blot analysis, those two groups tended to correspond with samples with lower PrP(d)/PrP(res) results by IHC/BCTs. However, no statistical association could be confirmed due to high individual sample variability. It is concluded that although detection of abnormal forms of PrP by laboratory methods remains useful to confirm TSE infection, infectivity titres cannot be predicted from quantitative test results, at least for the TSE sources and host PRNP genotypes used in this study. Furthermore, the near inverse correlation between infectious titres and Western blot results (high protease pre-treatment) argues for a dissociation between infectivity and PrP(res).

Selection of Distinct Strain Phenotypes in Mice Infected by Ovine Natural Scrapie Isolates Similar to CH1641 Experimental Scrapie

A few cases of transmissible spongiform encephalopathies in sheep have been described in France in which the protease-resistant prion protein (PrP(res)) exhibited some features in Western blot of experimental bovine spongiform encephalopathy in sheep. Their molecular characteristics were indistinguishable from those produced in the CH1641 experimental scrapie isolate. Four of these CH1641-like isolates were inoculated intracerebrally into wild-type C57Bl/6 mice. In striking contrast to previous results in ovine transgenic mice, CH1641 transmission in wild-type mice was efficient. Several components of the strain signature, that is, PrP(res) profile, brain distribution, and morphology of the deposits of the disease-associated prion protein, had some similarities with "classical" scrapie and clearly differed from both bovine spongiform encephalopathy in sheep and CH1641 transmission in ovine transgenic mice. These results on CH1641-like isolates in wild-type mice may be consistent with the presence in these isolates of mixed conformers with different abilities to propagate and mediate specific disease phenotypes in different species.

Neuroanatomical Distribution of Disease-Associated Prion Protein in Experimental Bovine Spongiform Encephalopathy in Cattle after Intracerebral Inoculation

The pathologic disease-associated prion protein (PrP(Sc)) has been shown to be expressed in the central nervous system of Holstein cattle inoculated intracerebrally with 3 sources of classical bovine spongiform encephalopathy (BSE) isolates. Several regions of the brain and spinal cord were analyzed for PrP(Sc) expression by immunohistochemical and Western blotting analyses. Animals euthanized at 10 months post-inoculation (mpi) showed PrP(Sc) deposits in the brainstem and thalamus, but no vacuolation; this suggested that the BSE agent might exhibit area-dependent tropism in the brain. At 16 and 18 mpi, a small amount of vacuolation was detected in the brainstem and thalamus, but not in the cerebral cortices. At 20 to 24 mpi, when clinical symptoms were apparent, heavy PrP(Sc) deposits were evident throughout the brain and spinal cord. The mean time to the appearance of clinical symptoms was 19.7 mpi, and the mean survival time was 22.7 mpi. These findings show that PrP(Sc) accumulation was detected approximately 10 months before the clinical symptoms of BSE became apparent. In addition, the 3 sources of BSE prion induced no detectable differences in the clinical signs, incubation periods, neuroanatomical location of vacuoles, or distribution and pattern of PrP(Sc) depositions in the brain.

Neuropathological changes correlate temporally but not spatially with selected neuromodulatory responses in natural scrapie

Neuropathological changes classically associated with sheep scrapie do not always correlate with clinical disease. We aimed to determine if selected neuromodulatory responses were altered during the course of the infection as it has been described in Creutzfeldt-Jakob disease and experimental bovine spongiform encephalopathy. Hemi-brains from healthy sheep and natural scrapie cases at two stages of infection were examined for biochemical alterations related to the expression of type I metabotropic glutamatergic receptors (mGluR(1) ) and type I adenosine receptors I (A(1) R), and of selected downstream intermediate signalling targets. Immunohistochemistry for different scrapie-related neuropathological changes was performed in the contralateral hemi-brains. PrP(d) deposition, spongiform change, astrocytosis and parvalbumin expression were significantly altered in brains from clinically affected sheep compared with preclinical cases and negative controls; the latter also showed significantly higher immunoreactivity for synaptophysin than clinical cases. Between clinically affected and healthy sheep, no differences were found in the protein levels of mGluR(1) , while phospholipase Cβ1 expression in terminally ill sheep was increased in some brain areas but decreased in others. Adenyl cyclase 1 and A(1) R levels were significantly lower in various brain areas of affected sheep. No abnormal biochemical expression levels of these markers were found in preclinically infected sheep. These findings point towards an involvement of mGluR(1) and A(1) R downstream pathways in natural scrapie. While classical prion disease lesions and neuromodulatory responses converge in some affected regions, they do not do so in others suggesting that there are independent regulatory factors for distinct degenerative and neuroprotective responses.

Paraffin-embedded tissue blot as a sensitive method for discrimination between classical scrapie and experimental bovine spongiform encephalopathy in sheep

The paraffin-embedded tissue (PET) blot was modified for use as a tool to differentiate between classical scrapie and experimental bovine spongiform encephalopathy (BSE) in sheep. Medulla (obex) from 21 cases of classical scrapie and 6 cases of experimental ovine BSE were used to develop the method such that it can be used as a tool to differentiate between BSE and scrapie in the same way that differential immunohistochemistry (IHC) has been used previously. The differential PET blot successfully differentiated between all of the scrapie and ovine BSE cases. Differentiation was permitted more easily with PET blot than by differential IHC, with accurate observations possible at the macroscopic level. At the microscopic level, sensitivity was such that discrimination by the differential PET blot could be made with more confidence than with differential IHC in cases where the immunohistochemical differences were subtle. The differential PET blot makes use of harsh epitope demasking conditions, and, because of the differences in the way prion protein is processed in different prion diseases, it can serve as a new, highly sensitive method to discriminate between classical scrapie and experimental BSE in sheep.

Molecular Discrimination of Sheep Bovine Spongiform Encephalopathy from Scrapie

Sheep CH1641-like transmissible spongiform encephalopathy isolates have shown molecular similarities to bovine spongiform encephalopathy (BSE) isolates. We report that the prion protein PrPSc from sheep BSE is extremely resistant to denaturation. This feature, combined with the N-terminal PrPSc cleavage, allowed differentiation of classical scrapie, including CH1641-like, from natural goat BSE and experimental sheep BSE.

Universal White Blood Cell Reduction in Europe: Has Transmission of Variant Creutzfeldt-Jakob Disease Been Prevented?

Universal white blood cell (WBC) reduction was introduced in Europe to prevent transmission of variant Creutzfeldt-Jakob disease (vCJD) by transfusion. Findings from rodent models indicate that WBC reduction should not prevent vCJD transmission because the residual plasma infectivity suffices to infect transfusion recipients even under optimistic infectivity assumptions. Although infectivity in human blood may not partition in the manner in which it is distributed in rodents, prion-reduction filters remove the residual plasma infectivity in rodent models. Precautionary introduction of prion filtration in the UK--for patients without dietary exposure to bovine spongiform encephalopathy and in the absence of a reported case of vCJD transmission attributable to infectivity residing in plasma--is consistent with the (already in place for such subjects) precautionary importation to the UK of fresh frozen plasma from low-risk countries. Thus, implementation of prion filtration in the UK does not imply that universal WBC reduction--as currently practiced in Europe--does not abrogate transmission of vCJD. Because neither a human case of vCJD transmission through transfusion of WBC-reduced red blood cells nor a case of experimental bovine spongiform encephalopathy transmission by WBC-reduced transfusion to sheep has been reported, it cannot be concluded that ordinary WBC reduction is ineffective in preventing transfusion transmission in humans. Accordingly, universal WBC reduction for the prevention of vCJD in Europe should continue.

Characterization of Strains of Ovine Transmissible Spongiform Encephalopathy with a Short PrP d Profiling Method

Scrapie is the transmissible spongiform encephalopathy (TSE) that naturally affects sheep and goats; these species are also susceptible to experimental infection with the bovine spongiform encephalopathy (BSE) agent. Discrimination between different strains of sheep scrapie and ovine BSE has been achieved by descriptive and quantitative profiling of deposits of the disease-associated prion protein (PrPd) in different areas of the brain, but this process is time-consuming and difficult to standardize between laboratories. The present paper describes an alternative PrPd profiling method that is less demanding and addresses these difficulties. It is based on the scoring of similar 14 PrPd types in 11 precisely defined areas of the telencephalon. When applied to 48 archived cases of experimental sheep BSE, SSBP/1, CH1641 and natural scrapie, it gave comparable results to the original profiling method, previously conducted on the same brains, and allowed differentiation between the different infectious sources. This new 'short PrPd profiling' method has the advantages of being less time-consuming and easier to standardize, so that it can be readily adopted by different laboratories to provide comparable results.

Prion Protein Expression and Processing in Human Mononuclear Cells: The Impact of the Codon 129 Prion Gene Polymorphism

BackgroundSo far, all clinical cases of new variant Creutzfeldt-Jakob disease (vCJD), thought to result from the Bovine Spongiform Encephalopathy (BSE) prion agent, have shown Methionine–Methionine (M/M) homozygosity at the M129V polymorphism of the PRNP gene. Although established, this relationship is still not understood. In both vCJD and experimental BSE models prion agents do reach the bloodstream, raising concerns regarding disease transmission through blood transfusion.Methodology/Principal FindingsWe investigated the impact of the M129V polymorphism on the expression and processing of the prion protein in human peripheral blood mononuclear cells (PBMCs) from three blood donor populations with Methionine-Methionine (M/M), Valine-Valine (V/V) and M/V genotypes. Using real-time PCR, ELISA and immunoblot assays we were unable to find differences in prion protein expression and processing relating to the M129V polymorphism.Conclusions/SignificanceThese results suggest that in PBMCs, the M129V PrP polymorphism has no significant impact on PrP expression, processing and the apparent glycoform distribution. Prion propagation should be investigated further in other cell types or tissues.

Three serial passages of bovine spongiform encephalopathy in sheep do not significantly affect discriminatory test results

During the 1980s, bovine spongiform encephalopathy (BSE)-contaminated meat and bonemeal were probably fed to sheep, raising concerns that BSE may have been transmitted to sheep in the UK. The human disease, variant Creutzfeldt-Jakob disease, arose during the BSE epidemic, and oral exposure of humans to BSE-infected tissues has been implicated in its aetiology. The concern is that sheep BSE could provide another source of BSE exposure to humans via sheep products. Two immunological techniques, Western immunoblotting (WB) and immunohistochemistry (IHC), have been developed to distinguish scrapie from cases of experimental sheep BSE by the characteristics of their respective abnormal, disease-associated prion proteins (PrP(d)). This study compares the WB and IHC characteristics of PrP(d) from brains of primary, secondary and tertiary experimental ovine BSE cases with those of cattle BSE and natural sheep scrapie. Discrimination between experimental sheep BSE and scrapie remained possible by both methods, regardless of the route of challenge.

Immunohistochemical and biochemical characteristics of BSE and CWD in experimentally infected European red deer (Cervus elaphus elaphus)

BackgroundThe cause of the bovine spongiform encephalopathy (BSE) epidemic in the United Kingdom (UK) was the inclusion of contaminated meat and bone meal in the protein rations fed to cattle. Those rations were not restricted to cattle but were also fed to other livestock including farmed and free living deer. Although there are no reported cases to date of natural BSE in European deer, BSE has been shown to be naturally or experimentally transmissible to a wide range of different ungulate species. Moreover, several species of North America's cervids are highly susceptible to chronic wasting disease (CWD), a transmissible spongiform encephalopathy (TSE) that has become endemic. Should BSE infection have been introduced into the UK deer population, the CWD precedent could suggest that there is a danger for spread and maintenance of the disease in both free living and captive UK deer populations. This study compares the immunohistochemical and biochemical characteristics of BSE and CWD in experimentally-infected European red deer (Cervus elpahus elaphus).ResultsAfter intracerebral or alimentary challenge, BSE in red deer more closely resembled natural infection in cattle rather than experimental BSE in small ruminants, due to the lack of accumulation of abnormal PrP in lymphoid tissues. In this respect it was different from CWD, and although the neuropathological features of both diseases were similar, BSE could be clearly differentiated from CWD by immunohistochemical and Western blotting methods currently in routine use.ConclusionRed deer are susceptible to both BSE and CWD infection, but the resulting disease phenotypes are distinct and clearly distinguishable.

Detection of Pathologic Prion Protein in the Olfactory Bulb of Natural and Experimental Bovine Spongiform Encephalopathy Affected Cattle in Great Britain

To investigate the relative involvement of the olfactory region in classical bovine spongiform encephalopathy (BSE), immunohistochemical labeling of prion protein scrapie (PrP(Sc)) was scored in the brainstem, frontal cerebral cortex, and olfactory bulb of cattle with natural and experimental clinical cases of BSE in Great Britain. The intensity of immunolabeling was greatest in the brainstem, but PrP(Sc) was also detected in the olfactory bulb and the cerebral cortex. A diffuse, nonparticulate labeling, possibly due to abundance of cellular PrP, was consistently observed in the olfactory glomeruli of the cases and negative controls. Involvement of the olfactory bulb in BSE and other naturally occurring TSEs of animals raises speculation as to an olfactory portal of infection or a route of excretion of the prion agent.

Pathogenesis of experimental bovine spongiform encephalopathy (BSE): estimation of tissue infectivity according to incubation period.

This paper reports the results of tissue infectivity assays of bovine spongiform encephalopathy (BSE) agent in orally exposed cattle at stages during the incubation period. Estimations of the titre of infectivity in central nervous system (CNS), certain peripheral nerve ganglia and distal ileum tissue were made according to time post exposure from the relationship between incubation period and dose for RIII mice and C57bl mice using data from titrations of brain material from cases of BSE. The rate of increase of infectivity in the bovine CNS was then estimated, taking into account these tissue infectivity titres, the variability of the brain titre of clinical field cases of BSE, and the probability density of the expected number of months before clinical onset of each infected bovine. The doubling time for CNS was shown to equal 1.2 months. The titre in the thoracic dorsal root ganglia (DRG) was, on average, approximately 1 log units less than CNS, and cervical DRG approximately 0.5 log less than thoracic DRG. The pattern of increase of infectivity in the distal ileum is that of an initial increase up to 14–18 months post exposure, followed by a decrease, which is likely to be highly variable between animals. These results will be informative for future risk assessments of BSE, especially in relation to reviewing current control measures.

Neuroanatomical distribution of abnormal prion protein in naturally occurring atypical scrapie cases in Great Britain

Scrapie belongs to a group of diseases known as the transmissible spongiform encephalopathies or prion diseases. Two different categories of naturally occurring scrapie have been identified: classical scrapie, which was first recorded around 1750, and atypical scrapie or 'Nor-98', which was first identified in Norway in 1998. The molecular characteristics of atypical scrapie have been well defined, but detailed descriptions of the neuropathological phenotype are rare since the majority of cases have been detected through active surveillance programmes where only brainstem and cerebellum are collected for statutory diagnosis. In order to characterise the neuropathology of naturally occurring atypical scrapie in sheep, we examined multiple brain levels from 15 whole brains from field cases of atypical scrapie, both clinical suspects and fallen stock, collected in Great Britain between 2004 and 2006. We found that the distribution of disease-associated prion protein (PrP(Sc)) and vacuolation in atypical scrapie cases are very different to both classical scrapie and experimental bovine spongiform encephalopathy in sheep. Immunolabelling for PrP(Sc) is mild and restricted at the obex and more intense and widespread rostrally, particularly in the cerebellum, substantia nigra, thalamus and basal nuclei. Intracellular immunolabelling types are not seen, but distinctive white matter immunolabelling is widespread. Vacuolation associated with PrP(Sc) deposits was not observed in the brainstem neuroanatomical areas commonly affected in classical scrapie and bovine spongiform encephalopathy, but was instead most prominent in the cerebellar cortex and neocortex. This is the largest comprehensive descriptive study of atypical scrapie pathology to date, and provides baseline data against which other natural or experimental cases can be compared. It also reinforces the current recommendation to collect cerebellum in addition to brainstem to enable confident confirmation of this distinct disease phenotype within surveillance programmes.

A Histopathologic and Immunohistochemical Review of Archived UK Caprine Scrapie Cases

In 2005, a prion disease identified in a goat from France was reported to be consistent with disease from the bovine spongiform encephalopathy (BSE) agent. Subsequent retrospective examination of UK goat scrapie cases led to the identification of one potentially similar, but as yet unconfirmed, case from Scotland. These findings strengthened concerns that small ruminant populations exposed to the BSE agent have become infected. The lack of data relating specifically to scrapie in goats has been contributory to past assumptions that, in general, sheep and goats respond similarly to prion infections. In this study, brain material from 22 archived caprine scrapie cases from the UK was reviewed by histopathology and by immunohistochemical examination for accumulations of disease-specific prion protein (PrP(Sc)) to provide additional data on the lesions of caprine scrapie and to identify any BSE-like features. The vacuolar change observed in the goats was characteristic of transmissible spongiform encephalopathies in general. PrP(Sc) immunohistochemical morphologic forms described in scrapie and experimental BSE infections of sheep were demonstrable in the goats, but these were generally more extensive and variable in PrP(Sc) accumulation. None of the cases examined showed a PrP(Sc) immunohistochemical pattern indicative of BSE.