Hypothesis of interference to superinfection between bovine spastic paresis and bovine spongiform encephalopathy; suggestions for experimentation, theoretical and practical interest

Sub-acute transmissible spongiform encephalopathies (TSEs) or prion diseases are diseases of little known etiology. The origin of these diseases would appear to be an abnormal protease-resistant prion protein (PrP(res)) which would be infectious by directly inducing its defective conformation to the normal native protein (PrP(C)). This hypothesis does not account for certain aspects of TSEs, such as interference to superinfection: in laboratory animals, inoculation by means of an attenuated strain with a long incubation period protects against later infection by a very virulent strain with a short incubation period. The hypothesis is put forward that there exists a possibility of interference to superinfection between neurodegenerative diseases of unknown origin, thought to be similar to TSEs, and a later infection by a TSE. The study of this interference between bovine spastic paresis (BSP) and bovine spongiform encephalopathy (BSE) could be used as a model for this hypothesis. BSP is a very rare disease among cattle, of unknown etiology; it is curable, in the very early stages, by using tryptophan and especially lithium, potentiated by copper and manganese. An etiology close to that of TSEs has been suggested on several occasions. If interference could be demonstrated between BSP and BSE, interesting data would be provided concerning the etiology, the pathogenesis and possibly the treatment and prevention of these diseases. Notably, such data could lead to the development of a treatment and a prevention with lithium and amino acids precursors of neuromediators (tryptophan, tyrosine, glutamic acid, etc.), as well as the developing of a vaccine to combat TSEs, especially BSE and scrapie.     

Ovine infection with the agents of scrapie (CH1641 isolate) and bovine spongiform encephalopathy: immunochemical similarities can be resolved by immunohistochemistry

Immunochemical ("rapid") tests, which recognize a partly protease-resistant conformer of the prion protein (PrP(res)) are now widely used in Europe for the diagnosis of transmissible spongiform encephalopathies (TSEs). Some of these tests can be used to distinguish natural scrapie from experimental bovine spongiform encephalopathy (BSE) in sheep, on the basis of migration pattern differences of PrP(res) in Western immunoblots. However, PrP(res) from sheep inoculated with CH1641 scrapie gives an immunoblot profile similar to that of sheep inoculated with BSE. Therefore, field scrapie strains similar to CH1641 might be misclassified as ovine BSE in the rapid tests currently employed. This study confirmed that the Western blot similarities (size of the unglycosylated band and distinct reactivity with 6H4 and P4 antibodies) between CH1641 and BSE remained consistent regardless of the PrP genotype of the sheep, but the two infections resulted in accumulation of disease-associated PrP (PrP(d)) that could easily be distinguished by the immunohistochemical "peptide mapping" method. This method, which reveals conformational differences of PrP(d) by the use of a panel of antibodies, indicated that PrP(d) from the CH1641 isolate was truncated further upstream in the N terminus than was PrP(d) from other ovine TSEs, including experimental BSE. In addition, the immunohistochemical "PrP(d) profile method", which defines the phenotype of PrP(d) accumulation in the brain of affected sheep, showed that CH1641 infection leads to much more intra-neuronal and considerably less extracellular PrP(d) than does experimental BSE. The overall results demonstrate that a combined Western blotting and immunohistochemical approach is required to discriminate between different TSE strains in sheep.     

Evaluation of a rapid western immunoblotting procedure for the diagnosis of bovine spongiform encephalopathy (BSE) in the UK.

Bovine brain tissue samples from 625 UK cattle, clinically suspected as bovine spongiform encephalopathy (BSE) cases, were used in a blind analysis to assess a rapid Western immunoblotting technique (Prionics Check; Prionics AG, Zurich), which detects bovine disease-specific protease-resistant prion protein (PrP(Sc)). By means of statutory histopathological examination, 599 of the 625 cattle were confirmed as BSE cases by the demonstration of spongiform encephalopathy, the remaining 26 being classified as negative. Duplicate samples from the same animals were also examined by electron microscopy for the presence of abnormal brain fibrils (scrapie-associated fibrils; SAFs). The Prionics technique showed a high sensitivity, particularly when compared with the fibril detection test; the detection rates were 99.3% and 92.0% respectively, with histopathology being used as the "gold standard". The false negative results by the Prionics test were possibly related to the sampling procedure. Analysis of 50 BSE-positive samples revealed similar glycoprofiles, the majority of PrP(Sc)isoforms being di-glycosylated protein. The Prionics test also detected PrP(Sc)in the four brain samples from the 26 histopathologically negative animals, apparently reducing the specificity of the test to 84.6%; however, confirmatory positive results in these samples were obtained by demonstrating SAF or by immunohistochemical examination, or both. It was concluded that the Prionics test detected PrP(Sc)in a small percentage (0.64%) of clinically suspected BSE cases showing no spongiform change. Since January 2000, the Prionics Western blot test has been introduced as one of the statutory tests for the diagnosis of clinically suspected BSE and scrapie cases in the UK. Copyright Harcourt Publishers Ltd.     

Squirrel monkeys (Saimiri sciureus) infected with the agent of bovine spongiform encephalopathy develop tau pathology

Squirrel monkeys (Saimiri sciureus) were infected experimentally with the agent of classical bovine spongiform encephalopathy (BSE). Two to four years later, six of the monkeys developed alterations in interactive behaviour and cognition and other neurological signs typical of transmissible spongiform encephalopathy (TSE). At necropsy examination, the brains from all of the monkeys showed pathological changes similar to those described in variant Creutzfeldt-Jakob disease (vCJD) of man, except that the squirrel monkey brains contained no PrP-amyloid plaques typical of that disease. Constant neuropathological features included spongiform degeneration, gliosis, deposition of abnormal prion protein (PrP(TSE)) and many deposits of abnormally phosphorylated tau protein (p-Tau) in several areas of the cerebrum and cerebellum. Western blots showed large amounts of proteinase K-resistant prion protein in the central nervous system. The striking absence of PrP plaques (prominent in brains of cynomolgus macaques [Macaca fascicularis] with experimentally-induced BSE and vCJD and in human patients with vCJD) reinforces the conclusion that the host plays a major role in determining the neuropathology of TSEs. Results of this study suggest that p-Tau, found in the brains of all BSE-infected monkeys, might play a role in the pathogenesis of TSEs. Whether p-Tau contributes to development of disease or appears as a secondary change late in the course of illness remains to be determined.     

Animal prion diseases: the risks to human health

Transmissible spongiform encephalopathies (TSEs) or prion diseases of animals notably include scrapie in small ruminants, chronic wasting disease (CWD) in cervids and classical bovine spongiform encephalopathy (C‐BSE). As the transmission barrier phenomenon naturally limits the propagation of prions from one species to another, and the lack of epidemiological evidence for an association with human prion diseases, the zoonotic potential of these diseases was for a long time considered negligible. However, in 1996, C‐BSE was recognized as the cause of a new human prion disease, variant Creutzfeldt‐Jakob disease (vCJD), which triggered an unprecedented public health crisis in Europe. Large‐scale epidemio‐surveillance programs for scrapie and C‐BSE that were implemented in the EU after the BSE crisis revealed that the distribution and prevalence of prion diseases in the ruminant population had previously been underestimated. They also led to the recognition of new forms of TSEs (named atypical) in cattle and small ruminants and to the recent identification of CWD in Europe. At this stage, the characterization of the strain diversity and zoonotic abilities associated with animal prion diseases remains largely incomplete. However, transmission experiments in nonhuman primates and transgenic mice expressing human PrP clearly indicate that classical scrapie, and certain forms of atypical BSE (L‐BSE) or CWD may have the potential to infect humans. The remaining uncertainties about the origins and relationships between animal prion diseases emphasize the importance of the measures implemented to limit human exposure to these potentially zoonotic agents, and of continued surveillance for both animal and human prion diseases.     

The pathogenic mechanisms of prion diseases

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a group of fatal neurodegenerative diseases of humans and animals, including bovine spongiform encephalopathy (BSE) of cattle, scrapie of sheep, and Creutzfeldt-Jakob disease (CJD) of humans. Prion diseases have become an important issue in public health and in the scientific world not only due to the possible relationship between BSE and new variant CJD (nvCJD) but also due to the unique biological features of the infectious agent. Although the nature of the infectious agent and the pathogenic mechanisms of prion diseases are not fully understood, considerable evidence suggests that an abnormal form (PrP(Sc)) of a host prion protein (PrP(C)) may compose substantial parts of the infectious agent and that various factors such as oxidative stress and calcium cytotoxicity are associated with the pathogenesis of prion diseases. Here, we briefly review and discuss the pathogenic mechanisms of prion diseases. These advances in understandings of fundamental biology of prion diseases may open the possibilities for the prevention and treatment of these unusual diseases and also suggest applications in more common neurodegenerative disorders such as Alzheimer's disease (AD) and Parkinson's disease (PD).     

Spread of classic BSE prions from the gut via the peripheral nervous system to the brain

An experimental oral bovine spongiform encephalopathy (BSE) challenge study was performed to elucidate the route of infectious prions from the gut to the central nervous system in preclinical and clinical infected animals. Tissue samples collected from the gut and the central and autonomic nervous system from animals sacrificed between 16 and 44 months post infection (mpi) were examined for the presence of the pathological prion protein (PrP(Sc)) by IHC. Moreover, parts of these samples were also bioassayed using bovine cellular prion protein (PrP(C)) overexpressing transgenic mice (Tgbov XV) that lack the species barrier for bovine prions. A distinct accumulation of PrP(Sc) was observed in the distal ileum, confined to follicles and/or the enteric nervous system, in almost all animals. BSE prions were found in the sympathetic nervous system starting at 16 mpi, and in the parasympathetic nervous system from 20 mpi. A clear dissociation between prion infectivity and detectable PrP(Sc) deposition became obvious. The earliest presence of infectivity in the brain stem was detected at 24 mpi, whereas PrP(Sc) accumulation was first detected after 28 mpi. In summary, our results decipher the centripetal spread of BSE prions along the autonomic nervous system to the central nervous system, starting already halfway in the incubation time.     

All major prion types recognised by a multiplex immunofluorometric assay for disease screening and confirmation in sheep

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.     

Differential diagnosis of infections with the bovine spongiform encephalopathy (BSE) and scrapie agents in sheep.

Scrapie, bovine spongiform encephalopathy (BSE), and variant Creutzfeldt-Jakob disease belong to the group of disorders called transmissible spongiform encephalopathies or prion diseases. The possibility that some sheep may be infected with the BSE agent is of human and animal health concern. Immunohistochemical methods were used to identify specific prion protein (PrP) peptide sequences in specific cell types of the brain and lymphoreticular system (LRS) of sheep with natural scrapie and Suffolk and Romney sheep infected experimentally with the BSE agent. Clinically affected and some pre-clinical cases of BSE infection could be distinguished from scrapie cases by the lesser amount of labelling of PrP containing the 84-102 amino-acid peptide sequences in phagocytic cells of the LRS and brain. Additionally, BSE-infected sheep had higher degrees of intra-neuronal PrP accumulation in the brain, as detected by labelling for a range of PrP peptide sequences. These results suggest that there is strain-dependent processing of PrP in specific cell types within the nervous system and LRS which can be used to distinguish BSE- and scrapie-infected sheep.     

The paraffin-embedded tissue blot detects PrP(Sc) early in the incubation time in prion diseases

With the appearance of bovine spongiform encephalopathy (BSE) and a new variant of Creutzfeldt-Jakob disease (nvCJD) that seems to be caused by BSE, there is an increased need for improvement of diagnostic techniques and recognition of all variants of prion diseases in humans and animals. Publications on the immunohistochemical identification of PrP(Sc) in the tonsils and appendix in the incubation period of nvCJD indicate that new and more sensitive techniques for the detection of PrP(Sc) in various tissues may be a valuable tool for early diagnosis in prion diseases. We developed a new and sensitive technique to detect PrP(Sc) in formalin-fixed and paraffin-embedded tissue, the paraffin-embedded tissue blot (PET blot), and reinvestigated archival brain material from CJD as well as BSE and scrapie. In addition, C57/Bl6 mice experimentally infected with the ME7 strain were investigated sequentially during the incubation time to compare this new technique with conventional methodologies. The PET blot detects PrP(Sc) in idiopathic (sporadic) and acquired prion diseases, even in cases with equivocal or negative immunohistochemistry, and is more sensitive than the conventional Western blot and histoblot techniques. The PET blot makes possible the detection of PrP(Sc) during the incubation period long before the onset of clinical disease and in prion disease variants with very low levels of PrP(Sc). In mice experimentally infected with the ME7 strain, the PET blot detects PrP(Sc) in the brain 30 days after intracerebral inoculation-145 days before the onset of clinical signs. Its anatomical resolution is superior to that of the histoblot technique. It may therefore be of particular interest in biopsy diagnosis. Thus it complements other tissue-based techniques for the diagnosis of prion diseases in humans and animals.     

An autoimmune response causes transmissible spongiform encephalopathies

Misfolded prion protein (PrP) is generally accepted as causing transmissible spongiform encephalopathies (TSEs) by aligning alongside normal host prion protein and inducing it to change to the misfolded configuration. This paper disputes this theory, and proposes that, rather than causing TSEs, misfolded PrP is the result of an autoimmune response to the host PrP, a component both of nerve cells and of lymphocytes. Autoimmunity is initiated by detachment of the phosphotidylinositol glycolipid anchor as a result of exposure to organophosphate pesticides. Once PrP is detached, antibodies are mobilized against it. In some individuals, point mutations, like the codon 129 met-val substitution, have evolved as a self-defence mechanism, causing a change in PrP to the misfolded, protease-resistant form seen in TSEs. Increased PrP production, both in response to nerve damage, and as a component of lymphocytes stimulated to proliferate in response to PrP, produces a positive feedback mechanism, resulting in symptoms of brain destruction.     

Molecular interaction between prion protein and GFAP both in native and recombinant forms in vitro

Gliosis of glial fibrillary acidic protein (GFAP) associated astrocytes is considered to be one of the hallmarks of transmissible spongiform encephalopathies (TSEs). In the present study, remarkable GFAP-PrP(Sc) or GFAP-PrP(C) complexes were separately detected in the brain homogenates of 263 K (Scrapie)-infected or normal hamsters by co-immunoprecipitation assay. To get more exact molecular evidences for interaction between prion protein (PrP) and GFAP, various recombinant PrP or GFAP proteins were expressed using prokaryotic-expressing and in vitro translation system. Using pull down and co-immunoprecipitation assays, reliable molecular interaction between PrP and GFAP was observed, and proteinase K (PK)-digested PrP(Sc) molecules were confirmed to be able to bind the recombinant GFAP specifically as well. The region within PrP that was responsible for interaction with GFAP was narrowed to PK-resistant core of PrP (i.e. aa 91-230). The study of the association of PrP with GFAP supplies the molecular evidence for the observation of co-localization of PrP(Sc) and GFAP in the brains of TSEs and may further provide insight into a potential role of GFAP in the biological function of PrP and the pathogenesis of prion diseases.     

Minimal involvement of the circumventricular organs in the pathogenesis of spontaneously arising and experimentally induced classical bovine spongiform encephalopathy

In sheep infected experimentally with the bovine spongiform encephalopathy (BSE) agent, amplification of infectivity in peripheral organs during early preclinical stages is thought to contribute to high titres of the agent being detected in blood, with subsequent haematogenous neuroinvasion through the circumventricular organs (CVOs). In contrast, little disease-associated prion protein (PrP(d)) or infectivity is detected in the peripheral tissues of cattle during the preclinical and clinical stages of BSE. The aim of this study was to investigate immunohistochemically the role of haematogenous neuroinvasion in cattle with spontaneously arising and experimentally induced BSE. There was almost complete absence of PrP(d) in the peripheral organs of BSE infected cattle. Additionally, there was minimal involvement of the CVOs during preclinical disease and there was progressive caudorostral accumulation of PrP(d) in the brain. These findings do not support haematogenous neuroinvasion in the bovine disease.     

Newly discovered forms of prion diseases in ruminants

Transmissible spongiform encephalopathies (TSEs), are fatal neurodegenerative diseases caused by unconventional agents, the prions. They are characterised by the accumulation in infected tissues of an abnormally folded form of the host-encoded prion protein (PrP). This pathological form is partially resistant to protease digestion, leading to the production of so-called PrP(res) fragments. Different isolates from the same host species may show different eletrophoretic profiles, reflecting the existence of different prion strains. The active surveillance of ruminant TSEs implemented in European countries, based on a large-scale biochemical testing of brain tissue samples from carcasses, has revealed PrP(res) profiles unnoticed so far. Experimental transmission of these atypical cases to various transgenic mouse lines has led to the recognition of a novel scrapie strain in sheep and goats, called Nor98, and of two variant strains of spongiform encephalopathy in cattle. This review is aimed at summarising the current knowledge on these newly recognised forms of ruminants TSEs, and at discussing their possible origin and potential implications in terms of animal and human health.     

Clusterin in bovine spongiform encephalopathy (BSE).

Clusterin mRNA, detected in increased quantities in the cervical spinal cord of cattle with bovine spongiform encephalopathy (BSE), was localized mainly in the neuroglia (including astrocytes) of the lateral and ventral areas of white matter. Axonal degeneration was also observed in these areas. The dorsal horns of the spinal cord in which BSE prion protein (PrP(BSE)) was deposited did not exhibit strong clusterin "up-regulation" but showed increased clusterin immunolabelling with a punctate distribution in the neuropil. Labelling of adjacent sections of the grey matter in BSE-affected spinal cord and thalamus demonstrated that the clusterin was deposited in association with extracellular PrP(BSE).     

Distinct proteinase K-resistant prion protein fragment in goats with no signs of disease in a classical scrapie outbreak

Considerable efforts have been directed toward the identification of small-ruminant prion diseases, i.e., classical and atypical scrapie as well as bovine spongiform encephalopathy (BSE). Here we report the in-depth molecular analysis of the proteinase K-resistant prion protein core fragment (PrP(res)) in a highly scrapie-affected goat flock in Greece. The PrP(res) profile by Western immunoblotting in most animals was that of classical scrapie in sheep. However, in a series of clinically healthy goats we identified a unique C- and N-terminally truncated PrP(res) fragment, which is akin but not identical to that observed for atypical scrapie. These findings reveal novel aspects of the nature and diversity of the molecular PrP(res) phenotypes in goats and suggest that these animals display a previously unrecognized prion protein disorder.     

What can we learn from the oral intake of prions by sheep?

The central nervous system is the ultimate target of prions, the agents responsible for fatal neurodegenerative diseases known as transmissible spongiform encephalopathies (TSEs). The neuro-invasive phase and its related clinical signs take place after a long incubation period. During this asymptomatic phase, however, active transport and replication of the infectious agent take place in peripheral sites. The oral infection route has been extensively studied because of its implication in the recent epidemic of bovine spongiform encephalopathy (BSE) in cattle and of the resulting human cases of variant Creutzfeldt-Jakob disease (vCJD). Rodent models have been useful in studying some aspects of this pathogenesis. Now, new data on the initial steps of oral infection have been obtained in sheep. This species is naturally infected with scrapie by horizontal transmission and there is strong evidence implicating the oral route. Furthermore, the existence of resistant and susceptible genotypes offers the possibility of comparative studies. The data were obtained using surgical and biochemical procedures to modulate the efficiency of oral infection and show that, in sheep, the abnormal prion protein (PrP) associated with the infectious agent crosses the intact intestinal barrier at the level of the enterocytes and then passes rapidly into lymph. These steps are identical in susceptible and resistant sheep. Thereafter, replication takes place in lymphoid structures. Other results in the same study indicate that alimentary fluids almost completely degrade the PrP of the inoculum. Though not directly transposable to human diseases, in which it is not possible to study these early stages, these data allow the elaboration of a simplified concept for the pathogenesis of TSEs. They also suggest that human contamination at the level of the oral cavity might be more important than previously suspected.     

Preclinical diagnosis of scrapie by immunohistochemistry of third eyelid lymphoid tissue

Ovine scrapie is a member of the transmissible spongiform encephalopathies (TSEs), a heterogeneous family of fatal neurologic disorders characterized by deposition of an abnormal isoform (prion protein [PrP] PrP-Sc) of a cellular sialoglycoprotein in neural tissue. PrP-Sc is detectable in some lymphoid tissues of infected sheep months or years before development of clinical disease. Detection of PrP-Sc in these tissues is the basis for live-animal testing. In this study, we characterize the performance of a preclinical diagnostic test for ovine scrapie based on a monoclonal antibody (MAb)-based immunohistochemistry assay of nictitating membrane ("third eyelid")-associated lymphoid tissue. The results of third eyelid immunohistochemistry assay agreed with the scrapie status of the sheep for 41 of 42 clinical suspects with confirmed scrapie and 174 of 175 sheep without scrapie. Third eyelid sampling agreed with the scrapie status for 36 of 41 clinically normal sheep positive for PrP-Sc immunostaining of brain tissue, including 27 sheep with positive biopsy specimens that progressed to clinical disease with confirmed scrapie 3 to 20 months after biopsy. The assay used MAb F89/160.1.5, which binds to residues 142 to 145 of ovine PrP. This antibody can be used in combination with MAb F99/97. 6.1, which binds to residues 220 to 225. One or both MAbs in this cocktail recognize PrP sequences conserved in most mammalian species in which natural TSEs have been reported. Immunohistochemistry assay of routinely formalin-fixed lymphoid tissues with a cocktail of pan-specific MAbs is a practical, readily standardized live-animal and preclinical test for ovine scrapie.     

Experimental bovine spongiform encephalopathy: detection of PrP(Sc) in the small intestine relative to exposure dose and age

European regulations for the control of bovine spongiform encephalopathy (BSE) decree destruction of the intestines from slaughtered cattle, therefore producers have been obliged to import beef casings from countries with a negligible BSE risk. This study applies immunohistochemical and biochemical approaches to investigate the occurrence and distribution of disease-associated prion protein (PrP(Sc)) in the duodenum, jejunum and ileum of cattle orally exposed to a 1 g or 100 g dose of a titrated BSE brainstem homogenate. Samples were derived from animals at various times post exposure. Lymphoid follicles were counted and the frequency of affected follicles recorded. No PrP(Sc) was detected in the duodenum or jejunum of animals exposed to a 1 g dose or in the duodenum of animals receiving a 100 g dose. PrP(Sc) was detected in the lymphoid tissue of the ileum of 1/98 (1.0%) animals receiving the 1 g dose and in the jejunum and ileum of 8/58 (13.8%) and 45/99 (45.5%), respectively, of animals receiving the 100 g dose. The frequency of PrP(Sc)- positive follicles was less than 1.5% per case and biochemical tests appeared less sensitive than immunohistochemistry. The probability of detecting lymphoid follicles in the ileum declined with age and for the 100 g exposure the proportion of positive follicles increased, while the proportion of positive animals decreased with age. Detection of PrP(Sc) in intestinal neural tissue was rare. The results suggest that the jejunum and duodenum of BSE-infected cattle contain considerably less BSE infectivity than the ileum, irrespective of exposure dose. In animals receiving the low exposure dose, as in most natural cases of BSE, the rarity of PrP(Sc) detection compared with high-dose exposure, suggests a very low BSE risk from food products containing the jejunum and duodenum of cattle slaughtered for human consumption.     

Discrimination between scrapie and bovine spongiform encephalopathy in sheep by molecular size, immunoreactivity, and glycoprofile of prion protein

A procedure for discrimination between scrapie and bovine spongiform encephalopathy (BSE) in sheep is of importance for establishing whether BSE has entered the sheep population. Since BSE has not yet been found in sheep at the farm level, such discrimination procedures can be developed only with experimental sheep BSE. Two distinctive molecular features of the prion protein (PrP)-molecular size and glycosylation profile-in proteinase K digests of brain stem tissue from sheep were used here; upon Western blotting, these features led to an unequivocal discrimination among natural scrapie, experimental scrapie, and experimental BSE. The higher electrophoretic mobility of PrP in sheep BSE could be best observed after deglycosylation treatment with N-glycosidase F. A simpler method for confirmation of this size difference involved comparison of the ratios for the binding of two monoclonal antibodies: P4 and 66.94b4. Based on epitope mapping studies with P4 and peptides, it appeared that N-terminal amino acid sequence WGQGGSH was intact only in sheep scrapie digests. Another feature typical for PrP in sheep BSE was the large fraction of diglycosylated PrP (70% or more). These data were obtained for a large group of positive sheep, consisting of 7 sheep with experimental BSE infection (genotypes: six ARQ/ARQ and one AHQ/AHQ), 48 sheep naturally infected with scrapie (six different genotypes), and 3 sheep with primary experimental scrapie infection. Routine tests of slaughter material serve well for the initial detection of both BSE and scrapie. With Western blotting as a rapid follow-up test, a 66.94b4/P4 antibody binding ratio above 1.5 is a practical indicator for serious suspicion of BSE infection in sheep.