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Ausgehend von der retrospektiven Betrachtung des Vorkommens von klassischen und atypischen BSE-Fallen wird die Fallzahlentwicklung zusammen mit den im Rahmen des gesundheitlichen Verbraucherschutzes durchgefuhrten Masahmen beleuchtet. Dieser Ansatz ermoglicht die Identifizierung der Masnahmen, die wesentlich zur Verringerung der BSE-Falle beigetragen haben konnten. Durch diese Identifizierung wird es moglich, die zurzeit diskutierten Vorschlage zur Anderung der Masnahmen zu qualifizieren.

To obtain a more detailed understanding of the prevalence of classical scrapie infections in a heavily affected German sheep flock (composed of 603 sheep and 6 goats), we analysed 169 sheep and 6 goats that carried the genotypes susceptible to the disease and that were therefore culled following discovery of the index case. The initial tests were performed using the Biorad TeSeE ELISA and reactive results were verified by official confirmatory methods (OIE-immunoblot and/or immunohistochemistry (IHC)) to demonstrate the deposition of scrapie-associated PrPSc in the brain stem (obex). This approach led to the discovery of 40 additional subclinically scrapie-infected sheep. Furthermore, peripheral lymphatic and nervous tissue samples of the 129 sheep and 6 goats with a negative CNS result were examined by IHC in order to identify any preclinical infections which had not already spread to the central nervous system (CNS). Using this approach we found 13 additional sheep with PrPSc depositions in the gut-associated lymph nodes (GALT) as well as in the enteric nervous system. Moreover, in most of these cases PrPSc was also deposited in the spleen and in the retropharyngeal and superficial cervical lymph nodes. Taken together, these results show a 30.3% infection prevalence in this scrapie-affected flock. Almost 7.4% of the infected animals harboured PrPSc exclusively in the peripheral lymphatic and nervous tissue and were therefore missed by the currently used testing strategy.

Transmissible spongiform encephalopathy strains can be differentiated by their behavior in bioassays and by molecular analyses of the disease-associated prion protein (PrP) in a posttranslationally transformed conformation (PrP Sc ). Until recently, isolates from cases of bovine spongiform encephalopathy (BSE) appeared to be very homogeneous. However, a limited number of atypical BSE isolates have recently been identified upon analyses of the disease-associated proteinase K (PK) resistance-associated moiety of PrP Sc (PrP res ), suggesting the existence of at least two additional BSE PrP res variants. These are defined here as the H type and the L type, according to the higher and lower positions of the nonglycosylated PrP res band in Western blots, respectively, compared to the position of the band in classical BSE (C-type) isolates. These molecular PrP res variants, which originated from six different European countries, were investigated together. In addition to the migration properties and glycosylation profiles (glycoprofiles), the H- and L-type isolates exhibited enhanced PK sensitivities at pH 8 compared to those of the C-type isolates. Moreover, H-type BSE isolates exhibited differences in the binding of antibodies specific for N- and more C-terminal PrP regions and principally contained two aglycosylated PrP res moieties which can both be glycosylated and which is thus indicative of the existence of two PrP res populations or intermediate cleavage sites. These properties appear to be consistent within each BSE type and independent of the geographical origin, suggesting the existence of different BSE strains in cattle. The choice of three antibodies and the application of two pHs during the digestion of brain homogenates provide practical and diverse tools for the discriminative detection of these three molecular BSE types and might assist with the recognition of other variants.

Prion diseases are characterised by the conversion of a cellular prion protein (PrP C ) by its misfolded, hence pathogenic, isoform (PrP Sc ). The efficiency of this transition depends on the molecular similarities between both interaction partners and on the intrinsic convertibility of PrP C . Transgenic mice expressing chimeric murine/ovine PrP C (Tgmushp mice) are susceptible to BSE and/or scrapie prions of bovine or ovine origin while transgenic mice expressing similar murine/bovine PrP C chimera (Tgmubo mice) are essentially resistant. We have studied this phenomenon by cell-free conversion on procaryotically expressed chimeric PrP C . Mouse passaged scrapie or BSE PrP Sc was used as a seed and the conversion reaction was carried out under semi-native conditions. The results obtained in this assay were similar to those of our in vivo experiments. Since mubo- and mushp-PrP C differ only at four amino acid positions (S96G, N142S, Y154H and Q185E), single or double point mutations of mushp-PrP C were examined in the cell-free conversion assay. While the scrapie Me7 prion induced conversion was largely reduced by the N142S and Q185E but not by the S96G and Y154H mutation, the BSE induced conversion was retained in all mutants. Newly formed PrP res exhibited strain specific characteristics, such as the localisation of the proteinase K cleavage site, even in the chimeric PrP C mutants. We therefore postulate that the efficiency of the conversion of chimeric PrP C depends on the amino acid sequence as well as on prion strain specific effects.

To elucidate the still-unknown pathogenesis of bovine spongiform encephalopathy (BSE), an oral BSE challenge and sequential kill study was carried out on 56 calves. Relevant tissues belonging to the peripheral and central nervous system, as well as to the lymphoreticular tract, from necropsied animals were analysed by highly sensitive immunohistochemistry and immunoblotting techniques to reveal the presence of BSE-associated pathological prion protein (PrPSc) depositions. Our results demonstrate two routes involving the autonomic nervous system through which BSE prions spread by anterograde pathways from the gastrointestinal tract (GIT) to the central nervous system (CNS): (i) via the coeliac and mesenteric ganglion complex, splanchnic nerves and the lumbal/caudal thoracic spinal cord (representing the sympathetic GIT innervation); and (ii) via the Nervus vagus (parasympathetic GIT innervation). The dorsal root ganglia seem to be subsequently affected, so it is likely that BSE prion invasion of the non-autonomic peripheral nervous system (e.g. sciatic nerve) is a secondary retrograde event following prion replication in the CNS. Moreover, BSE-associated PrPSc was already detected in the brainstem of an animal 24 months post-infection, which is 8 months earlier than reported previously. These findings are important for the understanding of BSE pathogenesis and for the development of new diagnostic strategies for this infectious disease.

Intensive active surveillance has uncovered two atypical German BSE cases in older cattle which resemble the two different atypical BSE phenotypes that have recently been described in France (designated H-type) and Italy (designated L-type or BASE). The H-type is characterized by a significantly higher molecular size, but a conventional glycopattern of the proteinase K treated abnormal prion protein (PrP(Sc)), while the L-type PrP(Sc) has only a slightly lower molecular size and a distinctly different glycopattern. In this paper we describe the successful transmission of both German atypical BSE cases to transgenic mice overexpressing bovine PrP(C). Upon challenge with the L-type, these mice developed BSE after a substantially shorter incubation period than any classical BSE transmission using these mice to date. In contrast, the incubation period was distinctly prolonged when these mice were challenged with the H-type. PrP(Sc) accumulated in the brains of these mice were of the same atypical BSE type that had been used for the transmission. These atypical cases suggest the possible existence of sporadic BSE cases in bovines. It is thus feasible that the BSE epidemic in the UK could have also been initiated by an intraspecies transmission from a sporadic BSE case.

Following the implementation of a large scale transmissible spongiform encephalopathies (TSE) surveillance programme of small ruminants, evidence for a natural transmission of bovine spongiform encephalopathy (BSE) to a French goat has been found. During the years 2002-2004, a massive TSE rapid testing programme on >250,000 small ruminants was carried out in Germany. In this national survey, 186 scrapie-affected sheep were found which originated from 78 flocks. The majority of these cases were of the classical TSE type (115 sheep belonging to 14 outbreaks). However, 71 cases coming from 64 flocks were of the novel atypical scrapie type. According to the regulation EU 999/2001, all TSE cases in small ruminants have to be examined by strain typing methods to explore any possibility of the existence of BSE cases in the field sheep population. Here we report on a biochemical typing strategy (termed FLI-test), which includes the determination of molecular masses, antibody binding affinities and glycosylation pattern of the TSE induced abnormal prion protein. Based on this typing approach none of the analysed German classical TSE outbreaks (total number of analysed sheep: 36) displayed biochemical features indicative for a BSE infection. However, in two cases distinct but BSE-unrelated PrP(Sc) types were found, which alludes to the existence of different scrapie strains in the German sheep population.

The intensified surveillance of scrapie in small ruminants in the European Union (EU) has resulted in a substantial increase of the number of diagnosed cases. Four rapid tests which have passed the EU evaluation for BSE testing of cattle are also recommended currently and used for the testing of small ruminants by the EU authorities. These tests include an indirect ELISA (cELISA), a colorimetric sandwich ELISA (sELISA I), a chemiluminescent sandwich ELISA (sELISA II), and a Western blot (WB). To this point, the majority of samples have been screened by using either sELISA I (predominantly in Germany) or WB (predominantly in France). In this study, it is shown that a number of the German and French scrapie cases show inconsistent results using rapid and confirmatory test methods. Forty-eight German sheep, 209 French sheep and 19 French goat transmissible spongiform encephalopathy (TSE) cases were tested. All cases were recognised by the sELISA I and either one of the confirmatory methods (scrapie-associated fibrils (SAF)-immunoblot or immunohistochemistry). Surprisingly, three rapid tests failed to detect a significant number of scrapie cases (29 in France and 24 in Germany). The possible reasons for these inconsistent reaction patterns of scrapie cases are discussed. Similar discrepancies have not been observed during rapid testing of cattle for BSE, the disease for which all diagnostic methods applied have been evaluated.

The accumulation of pathological prion protein is used as a diagnostic marker for transmissible spongiform encephalopathies. According to European Union (EU) regulations cattle older than 30 months of age (Germany, France, Italy, and Spain by national law >24 months) and slaughtered for human consumption must be tested by using rapid tests for bovine spongiform encephalopathy (BSE). Likewise fallen stock and clinically affected animals must be tested. This article gives a short overview of the incidence of BSE in Europe. The diagnostic hierarchy, i.e., the officially approved methodology for the confirmation of suspect rapid test cases, and the organization of the numerous laboratories involved in this large-scale testing for BSE are described. Special emphasis is given to necessary quality control measures currently in place for BSE rapid testing laboratories and to measures intended to assure a consistent performance of the commercially available rapid test kits.

This is the first report of cases of scrapie in Poland. The disease was an atypical phenotype, diagnosed in two aged sheep which were found dead. Brainstem samples from both animals were positive on the applied ELISA rapid test, while the confirmatory immunoblot indicated abnormal banding patterns of protease resistant prion protein (PrP(res)). The genotypes of these sheep were ALRQ/ALHQ and ALRQ/ALRR. The absence of premonitory clinical signs, the advanced age of the affected sheep, the higher concentration of PrP(res) in the cerebellum relative to the obex, the unusual banding profile of the prion protein and its relatively low resistance to proteolytic degradation confirmed the diagnosis of atypical scrapie (Nor98-like) in both cases.

The physiological role of the cellular prion protein (PrP (c)) is still not fully understood. Current evidence strongly suggests that PrP (c) overexpression in different cell lines sensitizes cells to apoptotic stimuli through a p53 dependent pathway. On the other hand, an expression of PrP (c) in PrP (c)-deficient cells undergoing apoptosis exhibited repeatedly antiapoptotic effects. Therefore, the presence/absence and/or the level of PrP (c) expression seem to be critical for the fluctuation between PrP (c)'s pro- and antiapoptotic properties. The present study examined whether an overexpression of PrP (c) itself, without addition of any apoptotic agent, can lead to proteome changes that might account for the higher responsiveness to apoptotic stimuli. Beyond this, we examined whether the sole introduction of PrP (c) into PrP (c)-deficient cells could be sufficient to up-regulate antiapoptotic proteins capable of mitigating apoptosis. For this purpose, we used two cell lines, one expressing [human embryonic kidney (HEK) 293 cells] and the other lacking (mouse neuronal PrP (c)-deficient cells) endogenous PrP (c). Protein profiling following transient PrP (c) overexpression in HEK 293 cells revealed a major PrP (c) involvement in regulation of proteins participating in energy metabolism and cellular homeostasis, whereas transient introduction of PrP (c) into mouse neuronal PrP (c)-deficient cells resulted mainly in the regulation of proteins involved in protection against oxidative stress and apoptosis. In addition, we report for the first time that PrP (c) overexpression influenced the regulation of several proteins known to have contributory roles in the pathogenesis of Alzheimer disease (AD). Revealing the correlation between presence/absence and/or different levels of PrP (c) expression with the regulation of certain cellular proteins might further contribute to our understanding of the complex role of PrP (c) in cell physiology.

Bovine spongiform encephalopathy (BSE) is a transmissible fatal neurodegenerative disease in cattle with an average incubation time of five years. The first BSE case in an indigenous cow was detected in Germany in November 2000. This was almost eight years after the huge BSE epidemic in the United Kingdom had peaked, and several years after many EU member states had seen their first BSE case. In the 1990s, BSE had been diagnosed in six imported animals in Germany. However, after the implementation of an active surveillance programme using BSE rapid testing systems, 399 indigenous German BSE cases have been found up to the end of July 2006. The birth cohorts of 1995-1997 contribute to the vast majority of the first 250 German cases that were diagnosed between 2000 and 2003. However, the most recent German BSE cases belong primarily to the birth cohorts 1998-2000 which is indicative of a recycling of BSE infectivity at that time. Moreover, there were two BSE cases in cattle born in spring 2001, i.e. after the meat and bone meal feed ban had come into effect on 2nd December 2000. In this article, we describe the dynamics of the German BSE epidemic and compare these data with those of other countries that observed larger numbers of cases.

The persistence of infectious biomolecules in soil constitutes a substantial challenge. This holds particularly true with respect to prions, the causative agents of transmissible spongiform encephalopathies (TSEs) such as scrapie, bovine spongiform encephalopathy (BSE), or chronic wasting disease (CWD). Various studies have indicated that prions are able to persist in soil for years without losing their pathogenic activity. Dissemination of prions into the environment can occur from several sources, e.g., infectious placenta or amniotic fluid of sheep. Furthermore, environmental contamination by saliva, excrements or non-sterilized agricultural organic fertilizer is conceivable. Natural transmission of scrapie in the field seems to occur via the alimentary tract in the majority of cases, and scrapie-free sheep flocks can become infected on pastures where outbreaks of scrapie had been observed before. These findings point to a sustained contagion in the environment, and notably the soil. By using outdoor lysimeters, we simulated a contamination of standard soil with hamster-adapted 263K scrapie prions, and analyzed the presence and biological activity of the soil-associated PrP(Sc) and infectivity by Western blotting and hamster bioassay, respectively. Our results showed that 263K scrapie agent can persist in soil at least over 29 months. Strikingly, not only the contaminated soil itself retained high levels of infectivity, as evidenced by oral administration to Syrian hamsters, but also feeding of aqueous soil extracts was able to induce disease in the reporter animals. We could also demonstrate that PrP(Sc) in soil, extracted after 21 months, provides a catalytically active seed in the protein misfolding cyclic amplification (PMCA) reaction. PMCA opens therefore a perspective for considerably improving the detectability of prions in soil samples from the field.

International audience; The aim of this study was to analyze the epidemiology and prion protein (PrP) genetics in scrapie-affected sheep flocks in Germany. For this purpose, 224 German scrapie cases in sheep diagnosed between January 2002 and February 2006 were classified as classical or atypical scrapie and the amino acids at codons 136, 141, 154 and 171 were determined. Likewise, representative numbers of flock mates were genotyped. Significant epidemiological differences were observed between classical and atypical scrapie cases in regard to the numbers of scrapie-affected sheep within a flock, the sizes of flocks with only a single scrapie-positive sheep or more than one scrapie-positive sheep and the age distribution of the scrapie-positive sheep. Sheep with the ARQ/ARQ genotype had by far the highest risk for acquiring classical scrapie, but the risk for atypical scrapie was the highest for sheep carrying phenylalanine (F) at position 141 (AF$_{141}$RQ) and/or the AHQ haplotype. However, atypical scrapie also occurred with a notable frequency in sheep with the PrP haplotypes ARR and/or ARQ in combination with Leucine at position 141 (AL$_{141}$RQ). Furthermore, six atypical scrapie-positive sheep carried the PrP genotype ARR/ARR. The high proportion of sheep flocks affected by atypical scrapie underscores the importance of this scrapie type.

Atypical neuropathological and molecular phenotypes of bovine spongiform encephalopathy (BSE) have recently been identified in different countries. One of these phenotypes, named bovine “amyloidotic” spongiform encephalopathy (BASE), differs from classical BSE for the occurrence of a distinct type of the disease-associated prion protein (PrP), termed PrPSc, and the presence of PrP amyloid plaques. Here, we show that the agents responsible for BSE and BASE possess different biological properties upon transmission to transgenic mice expressing bovine PrP and inbred lines of nontransgenic mice. Strikingly, serial passages of the BASE strain to nontransgenic mice induced a neuropathological and molecular disease phenotype indistinguishable from that of BSE-infected mice. The existence of more than one agent associated with prion disease in cattle and the ability of the BASE strain to convert into the BSE strain may have important implications with respect to the origin of BSE and spongiform encephalopathies in other species, including humans., Author Summary Twenty years after the identification of bovine spongiform encephalopathy (BSE), the origin of the causal agent is still unknown. This issue is of fundamental importance, since knowledge of the origin of the BSE agent is essential for prevention of future outbreak of the disease, or variants thereof, in cattle and other mammals. In this study, we show that an atypical form of spongiform encephalopathy of cattle, termed BASE, is caused by a prion strain distinct from that of classical BSE. Noteworthy, this newly characterized prion strain has the ability to convert into the classical BSE strain upon serial transmission to inbred mouse lines. According to these results, BASE, which is regarded as a sporadic form of prion disease in cattle, may be the origin of BSE, following conversion of the causal agent in an intermediate host. These findings may have major implications with respect to the origin of BSE epidemic and spongiform encephalopathies in other species, including humans.

Variant Creutzfeldt-Jakob disease (vCJD) is an at present inevitably lethal neurodegenerative disease which can only be diagnosed definitely post mortem. The majority of the approximately 200 victims to date have resided in the UK where most contaminated beef materials entered the food chain. Three cases in the UK demonstrated that vCJD can be transmitted by blood transfusion. Since BSE and vCJD have spread to several countries outside the UK, it appears advisable that specific risk assessments be carried out in different countries and geographic areas. This review explains the approach adopted by Germany in assessing the risk and considering precautionary measures. A fundamental premise is that the feeding chain of cattle and the food chain have been successfully and permanently cleared from contaminated material. This raises the question of whether transmissions via blood transfusions could have the potential to perpetuate vCJD in mankind. A model calculation based on actual population data showed, however, that this would not be the case. Moreover, an exclusion of transfusion recipients from blood donation would add very little to the safety of blood transfusions, but would have a considerable impact on blood supply. Therefore, an exclusion of transfusion recipients was not recommended in Germany.

After the implementation of an active surveillance programme for scrapie in sheep in the EU, the number of diagnosed classical scrapie cases rose sharply and a novel kind of so-called atypical scrapie case was discovered. These atypical scrapie cases display unusual features concerning the distribution of the abnormal prion protein (PrPSc) in the brain, a distinct electrophoretic profile of PrPSc and an inconsistent reaction pattern in the currently used rapid tests. In this report, PrPSc of two German atypical sheep scrapie cases was characterized by epitope mapping using a panel of 18 monoclonal antibodies that were directed against epitopes located throughout the prion protein. This analysis suggests that PrPSc derived from atypical scrapie cases and treated with proteinase K is largely composed of an 11 kDa fragment (previously referred to as the 12 kDa band) and of polymeric fragments thereof. The 11 kDa band corresponds to a prion protein fragment spanning approximately aa 90–153 and may therefore represent a novel PrPSc type.

The term of 'TSE infections in small ruminants' summarises BSE as well as classical and the recently discovered atypical scrapie infections in sheep and goats. There are fundamental differences between the TSE infections in small and large ruminants. Other than in bovines the TSE pathogenesis in small ruminants implies that various peripheral tissues become infectious long before the onset of clinical symptoms. At least in sheep, classical scrapie is efficiently transmitted horizontally within affected flocks. On the other hand, BSE poses a distinctly higher zoonotic risk than scrapie. Therefore, regulatory measures for the protection of animals and humans from a BSE infection must be substantially different for large and small ruminants. While culling of the birth and feeding cohort of a BSE affected cattle is considered to be effective to prevent any further BSE cases in the affected herd, an effective BSE and classical scrapie eradication programme in small ruminants requires a much more stringent eradication strategy and the rendering of all susceptible animals. The situation became even more complicated when atypical scrapie cases with divergent transmission and pathogenesis characteristics and with a novel biochemical phenotype of the infectious agent came into play. The discovery of these atypical scrapie cases has initiated a discussion about the suitability of the current TSE eradication measures in sheep (which are selective breeding and genotype based culling), in particular when such cases were also found in sheep carrying the believed scrapie resistant genotypes.

Transgenic mice expressing bovine prion protein (PrP)(C) (Tgbov XV mice) display remarkably shorter incubation times for cattle-derived bovine spongiform encephalopathy (BSE) infectivity than do nontransgenic mice. To verify that this phenomenon reflects increased sensitivity, we challenged Tgbov XV mice and conventional RIII mice with a BSE brain-stem homogenate of known infectivity titer in cattle. An end-point titration experiment in Tgbov XV mice revealed their superior sensitivity, which exceeded that of RIII mice by at least 10,000-fold and even that of cattle by approximately10-fold. Moreover, Tgbov XV mice were challenged with various tissues from cattle with end-stage clinical BSE, and infectivity was found only in the central and peripheral nervous system and not in lymphatic tissues; the only exception was the Peyer's patches of the distal ileum, which most likely are the site of entry for BSE infectivity. These results provide further indication that the pathogenesis of BSE in cattle is fundamentally different from that in sheep and mice, due to an exclusive intraneuronal spread of infectivity from the gut to the central nervous system.

The susceptibility of sheep to scrapie infection is influenced by prion gene alleles, which are modulated by polymorphic variations corresponding to amino acid positions 136, 154 and 173 of the prion protein (PrP). As no unquestioned report of a diseased sheep carrying homozygous alleles encoding alanine, arginine and arginine (PrPARR) at these sites has been published to date, sheep of this genotype are believed to be scrapie resistant. After the introduction of large-scale rapid testing for scrapie, a number of so-called ‘atypical’ scrapie cases have been found in Germany and elsewhere. Among those cases were two supposedly scrapie-resistant sheep. Brain samples from these animals tested positive for abnormal PrP (PrPSc) in one of four rapid tests available. Moreover, scrapie-associated fibril (SAF)-immunoblotting and immunohistochemistry, which are the generally accepted diagnostic techniques for scrapie, revealed prominent PrPSc deposition in the cerebellum. SAF immunoblotting also revealed PrPSc deposition in the obex, frontal cortex and brainstem. Transmission experiments to investigate the infectivity of scrapie propagated from these sheep have been initiated.

The conversion of cellular prion protein (PrP C ) into its pathological isoform (PrP S c ) conveys an increase in hydrophobicity and induces a partial resistance to proteinase K (PK). Interestingly, co-incubation with high copper ion concentrations also modifies the solubility of PrP C and induces a partial PK resistance which was reminiscent of PrP S c . However, concerns were raised whether this effect was not due to a copper-induced inhibition of the PK itself. We have therefore analyzed the kinetics of the formation of PK-resistant PrP C and excluded possible interference effects by removing unbound copper ions prior to the addition of PK by methanol precipitation or immobilization of PrP C followed by washing steps. We found that preincubation of PrP C with copper ions at concentrations as low as 50 μM indeed rendered these proteins completely PK resistant, while control substrates were proteolyzed. No other divalent cations induced a similar effect. However, in addition to this specific stabilizing effect on PrP C , higher copper ion concentrations in solution (>200 μM) directly blocked the enzymatic activity of PK, possibly by replacing the Ca 2 + ions in the active center of the enzyme. Therefore, as a result of this inhibition the proteolytic degradation of PrP C as well as PrP S c molecules was suppressed.

Prion protein (PrP) genotypes were determined in eight sheep that have been tested positive for atypical scrapie from purebred or crossbred Merinoland sheep flocks in Germany and compared with the PrP genotypes of their flock mates. Two restriction fragment length polymorphism (RFLP) analyses were developed to determine all PRNP haplotypes occurring by variations at codons 136, 154 and 171. At least one copy of the A(136) H(154) Q(171) (AHQ) allele was found in all scrapie-positive sheep while the frequency of AHQ varied from over 23% to less than 3% in the whole flocks. There was a significant association between PrP genotype and a positive scrapie diagnosis over all flocks, suggesting a high scrapie susceptibility of PrP genotypes including the AHQ allele, at least in sheep of Merinoland type. These results argue that sheep with the AHQ allele are not generally less susceptible to scrapie and support the hypothesis that the influence of this allele on scrapie susceptibility may vary from flock to flock depending on genetic and/or epidemiological factors. This has to be considered when strategies for the eradication of scrapie in sheep are based on PrP genotypes.

We here report on the first case of scrapie in Bavaria. The post mortem of a six year old ewe of the breed German grey Heath with a suspected subclinical calcinosis revealed typical histopathological signs of a spongiform encephalopathy and immunohistochemistry was positive for PrPSc in the cervical part of the spinal cord. Eight out of 18 sheep of the herd showed clinical symptoms.

The susceptibility of sheep to scrapie is known to involve, as a major determinant, the nature of the prion protein (PrP) allele, with the VRQ allele conferring the highest susceptibility to the disease. Transgenic mice expressing in their brains three different ovine PrP VRQ -encoding transgenes under an endogenous PrP-deficient genetic background were established. Nine transgenic (tgOv) lines were selected and challenged with two scrapie field isolates derived from VRQ-homozygous affected sheep. All inoculated mice developed neurological signs associated with a transmissible spongiform encephalopathy (TSE) disease and accumulated a protease-resistant form of PrP (PrPres) in their brains. The incubation duration appeared to be inversely related to the PrP steady-state level in the brain, irrespective of the transgene construct. The survival time for animals from the line expressing the highest level of PrP was reduced by at least 1 year compared to those of two groups of conventional mice. With one isolate, the duration of incubation was as short as 2 months, which is comparable to that observed for the rodent TSE models with the briefest survival times. No survival time reduction was observed upon subpassaging of either isolate, suggesting no need for adaptation of the agent to its new host. Overexpression of the transgene was found not to be required for transmission to be accelerated compared to that observed with wild-type mice. Conversely, transgenic mice overexpressing murine PrP were found to be less susceptible than tgOv lines expressing ovine PrP at physiological levels. These data argue that ovine PrP VRQ provided a better substrate for sheep prion replication than did mouse PrP. Altogether, these tgOv mice could be an improved model for experimental studies on natural sheep scrapie.

Prion diseases are closely linked to the conversion of host-encoded cellular prion protein (PrPC) into its pathological isoform (PrPSc). PrP conversion experiments in scrapie infected tissue culture cells, transgenic mice, and cell-free systems usually require unique epitopes and corresponding monoclonal antibodies (MAbs) for the immunological discrimination of exogenously introduced and endogenous PrP compounds (e.g., MAb 3F4, which is directed to an epitope on hamster and human but not on murine PrP). In the current work, we characterize a novel MAb designated L42 that reacts to PrP of a variety of species, including cattle, sheep, goat, dog, human, cat, mink, rabbit, and guinea pig, but does not bind to mouse, hamster, and rat PrP. Therefore, MAb L42 may allow futurein vitroconversion and transgenic studies on PrPs of the former species. The MAb L42 epitope on PrPCincludes a tyrosine residue at position 144, whereas mouse, rat, and hamster PrPs incorporate tryptophane at this site. To verify this observation, we generated PrP expression vectors coding for authentic or mutated murine PrPCs (i.e., codon 144 encoding tyrosine instead of tryptophan). After transfection into neuroblastoma cells, MAb L42 did not react with immunoblotted wild-type murine PrPC, whereas L42 epitope-tagged murine PrPCwas strongly recognized. Immunoblot and fluorescence-activated cell sorting data revealed that tagged PrPCwas correctly posttranslationally processed and translocated to the cell surface.

The sensitivity of Western blot detection of PrPres using two different extraction procedures on brain material of 30 scrapie-affected sheep was compared. Whereas PrPres could be detected in all sheep after extraction with the first method, 30% did not give any signal after extraction with the second method. However, the second method, when positive, permitted the detection of PrPres from smaller amounts of infected brain tissue. When used with the ruminant specific monoclonal antibody p4, the second method gave positive signals corresponding to less than 12.5 microg of scrapie-infected brain, that, up to now, is the highest sensitivity described for PrPres detection from naturally infected ruminant brains. The overall results showed highly variable levels of PrPres between sheep and are presented in relation to breed, survival time and animal genotype data. Further progress can thus be expected for PrPres detection in prion diseases, if more efficient extraction procedures and more sensitive immunological reagents are used. Such technical improvements could contribute to more accurate diagnosis in animals affected naturally.

International audience; Until today most prion strains can only be propagated and the infectivity content assayed by experimentally challenging conventional or transgenic animals. Robust cell culture systems are not available for any of the natural and only for a few of the experimental prion strains. Moreover, the pathogenesis of different transmissible spongiform encephalopathies (TSE) can be analysed systematically by using experimentally infected animals. While, in the beginning, animals belonging to the natural host species were used, more and more rodent model species have been established, mostly due to practical reasons. Nowadays, most of these experiments are performed using highly susceptible transgenic mouse lines expressing cellular prion proteins, PrP, from a variety of species like cattle, sheep, goat, cervidae, elk, hamster, mouse, mink, pig, and man. In addition, transgenic mice carrying specific mutations or polymorphisms have helped to understand the molecular pathomechanisms of prion diseases. Transgenic mouse models have been utilised to investigate the physiological role of PrP$^{\rm C}$, molecular aspects of species barrier effects, the cell specificity of the prion propagation, the role of the PrP glycosylation, the mechanisms of the prion spread, the neuropathological roles of PrP$^{\rm C}$ and of its abnormal isoform PrP$^{\rm D}$ (D for disease) as well as the function of PrP Doppel. Transgenic mouse models have also been used for mapping of PrP regions involved in or required for the PrP conversion and prion replication as well as for modelling of familial forms of human prion diseases.