Early spread of scrapie from the gastrointestinal tract to the central nervous system involves autonomic fibers of the splanchnic and vagus nerves.

Although the ultimate target of infection is the central nervous system (CNS), there is evidence that the enteric nervous system (ENS) and the peripheral nervous system (PNS) are involved in the pathogenesis of orally communicated transmissible spongiform encephalopathies. In several peripherally challenged rodent models of scrapie, spread of infectious agent to the brain and spinal cord shows a pattern consistent with propagation along nerves supplying the viscera. We used immunocytochemistry (ICC) and paraffin-embedded tissue (PET) blotting to identify the location and temporal sequence of pathological accumulation of a host protein, PrP, in the CNS, PNS, and ENS of hamsters orally infected with the 263K scrapie strain. Enteric ganglia and components of splanchnic and vagus nerve circuitry were examined along with the brain and spinal cord. Bioassays were carried out with selected PNS constituents. Deposition of pathological PrP detected by ICC was consistent with immunostaining of a partially protease-resistant form of PrP (PrP(Sc)) in PET blots. PrP(Sc) could be observed from approximately one-third of the way through the incubation period in enteric ganglia and autonomic ganglia of splanchnic or vagus circuitry prior to sensory ganglia. PrP(Sc) accumulated, in a defined temporal sequence, in sites that accurately reflected known autonomic and sensory relays. Scrapie agent infectivity was present in the PNS at low or moderate levels. The data suggest that, in this scrapie model, the infectious agent primarily uses synaptically linked autonomic ganglia and efferent fibers of the vagus and splanchnic nerves to invade initial target sites in the brain and spinal cord.

Effect of repeated oral infection of hamsters with scrapie.

The development of a transmissible spongiform encephalopathy upon uptake of the infectious agent in feed was studied in the model system scrapie in hamsters. Compared to single dosing, repeated dosing caused disease at a considerable higher incidence. The risk of infection was higher when the time interval between repetitive dosing was short. There was a statistically significant trend of clearance of infectivity with time.

Evidence for an alternative direct route of access for the scrapie agent to the brain bypassing the spinal cord.

Scrapie is a disease which occurs naturally in sheep and goats and belongs to a group of neurodegenerative disorders known as transmissible spongiform encephalopathies, or TSEs. There is currently no cure for TSEs, and the causative agent has not yet been identified. Numerous experiments, however, have addressed the pathogenetic process following a TSE infection. In this paper we present a study of the spread of the scrapie agent after intraperitoneal infection of hamsters. The accumulation of TSE-specific amyloid protein, TSE-AP (also known as PrP), was used as a marker for infectivity. The data suggested three points of agent entry into the spinal cord: the most important one between thoracic vertebrae T7-9, and two minor ones in the lower cervical spinal cord and between vertebrae T13-L2. Further, strong evidence was found for the existence of a direct route of access to the brain which bypasses the spinal cord and most likely terminates in the medulla oblongata. The indication of an alternative pathway to the brain was confirmed by the data from orally infected hamsters. The spleen appeared to play a potential, but non-essential role in pathogenesis after intraperitoneal infection in our animal model.

Highly infectious purified preparations of disease-specific amyloid of transmissible spongiform encephalopathies are not devoid of nucleic acids of viral size.

An efficient purification protocol for infectivity causing a transmissible spongiform encephalopathy (TSE) is described. From fractions purified by this protocol about 3 x 10(8) LD50 but only 3 ng of nucleic acids per gram of brain material can be isolated from all TSE-affected brains (hamster, human, sheep, cattle). By PAGE such fractions from brains of infected and control hamsters contained only one distinct nucleic acid band of 1.5 kg together with some broader smear of nucleic acid material. Although distilled water was used for such purifications, quite often a similar nucleic acid band was isolated from blanks containing no brain material. In all instances this material proved to be DNA. The result challenges the potentially important claim that purified infectious preparations of TSE-specific amyloid are free of nucleic acids of viral size. Nucleic acids isolated by other groups from diseased brain were not detected in preparations isolated by the new protocol. The application of this purification protocol in future studies will be helpful to decide whether TSEs are caused by agents containing nucleic acid or by protein only.

[Creutzfeldt-Jakob disease in Austria]. / Die Creutzfeldt-Jakob-Krankheit in Osterreich.

Between 1969 and 30th June 1996, Creutzfeldt-Jakob disease (CJD) was definitively diagnosed in 88 Austrian patients by autopsy and/or biopsy. The number of diagnosed cases has steadily increased in recent years (average incidence in 1969-1985: 0.18 per million; 1986-1994: 0.67 per million; 1995: 1.25 per million; estimate for 1996: 1.7 per million). The percentage of patients older than 70 years increased until 1989 and has decreased slightly since then. One patient received a lyophilised dura transplant 11 years before death. Another patient gave a history of intramuscular injections of bovine RNA (Regeneresen) extracted from various organs including brain administered over a ten-year period. One female patient had familial CJD with a glutamate-->lysine mutation at codon 200 of the prion protein (PrP) gene PRNP. The ages at death are distributed symmetrically around the median of 64 years. Two female patients died at the unusually young ages of 27 and 30 years. Median duration of disease was 4.5 months; 77% of the patients died within 6 months after onset of the disease. Retrospectively, 83% of the patients fulfilled the clinical criteria of probable or possible CJD. Neuropathologically, each patient showed accumulation of immunocytochemically detectable pathological PrP in the central nervous system. No patient had the neuropathological profile of the new CJD variant recently described in the U.K. The recent rise in diagnosed CJD in Austria probably reflects increased awareness and recognition rather than a real increase. Since bovine spongiform encephalopathy (BSE) has not been observed in Austria, our data argue against an association between the rise of incidence of CJD and BSE.

Sequential appearance and accumulation of pathognomonic markers in the central nervous system of hamsters orally infected with scrapie.

Both infectivity and TSE-specific amyloid protein (also referred to as protease resistant- or prion protein, PrP) are pathognomonic markers for transmissible spongiform encephalopathies (TSE). This paper presents a new densitometric method for the quantification of TSE-specific amyloid protein and its application to studying the pathogenesis of scrapie in Syrian hamsters after infection with scrapie strain 263K. A first study established a close correlation between infectivity and TSE-specific amyloid protein with a doubling time of 2-2.6 days in the brain and cervical spinal cord for both markers. The ratio of infectivity and TSE-specific amyloid protein was relatively constant at a mean value of about 10(6) protein molecules per infectious unit during the course of infection. A subsequent study addressed the temporal-spatial spread of infection in the central nervous system by tracing the accumulation of the pathological protein. The pathogenetic process was first detected in the spinal cord between vertebrae T4 and T9, and then showed an anterograde and retrograde spread with a rate of 0.8-1.0 mm/day. There were also some indications for a possible alternative route of spread of infection from the periphery to the brain, other than via the spinal cord. Involvement of the spleen did not appear essential for the early pathogenesis in hamsters orally infected with the 263K strain of scrapie.

Creutzfeldt-Jakob disease in Austria.

Between 1969 and 30 September 1995, 79 Austrian patients had Creutzfeldt-Jakob disease (CJD) diagnosed neuropathologically by necropsy or biopsy. The annual incidence has significantly increased in recent years (average 0.18 per million in 1969-85, and 0.67 per million in 1986-94; estimate for 1995: 1.5 per million). Also, the percentage of patients with CJD over 70 years at death increased significantly until 1989 but is since in decline. There is no regional clustering, familial occurrence, or recognised iatrogenic risk. One patient had a 10 year history of intramuscular injection of purified bovine RNA preparation (Regeneresen) from various organs including the brian. The ages at death are symmetrically distributed around the median of 64 years. The median duration of disease is four months. Most patients (76%) died within six months of onset. Retrospectively, 86% of patients fulfilled clinical criteria of probable or possible CJD. Neuropathology showed the classic triad of spongiform change, astrogliosis, and neuronal loss in most cases. Two cases did not show unequivocal tissue alterations, but anti-PrP immunocytochemistry detected PrP deposits also in these cases. It is concluded that the recent rise in incidence of CJD in Austria most likely reflects increased awareness and diagnosis of CJD rather than a real increase. As bovine spongiform encephalopathy (BSE) has not been reported in Austria, the data do not support a link between a rise in incidence of sporadic CJD and BSE.

[Consensus report: tissue handling in suspected Creutzfeldt-Jakob disease and other spongiform encephalopathies (prion diseases) in the human. European Union Biomed-1 Concerted Action]. / Konsensusbericht: Gewebsbehandlung bei Verdacht auf Creutzfeldt-Jakob- Krankheit und andere spongiforme Enzephalopathien (Prionen-Krankheiten) des Menschen. European Union Biomed-1 Concerted Action.

Despite many sensational and intimidating reports in the mass media, transmissible spongiform encephalopathies (prion diseases) are not contagious in the usual sense. Successful transmission requires both specific material (an affected individual's tissue, from or adjacent to CNS) and specific modes (mainly penetrating contact with the recipient). Nevertheless, specific safety precautions are mandatory to avoid accidental transmission and to decontaminate any infectivity. The autopsy is essential for definite diagnosis of these disorders. Recommendations are given here for safe performance of the autopsy, for neuropathology service and appropriate decontamination; they are based on the current literature and on precautions taken in most laboratories experienced in handling tissue from transmissible spongiform encephalopathies. In essence, special care must be taken to avoid penetrating wounds, possible contamination should be kept to a minimum, and potentially infectious material must be adequately decontaminated by specific means. The full English text of this Consensus Report was published in Brain Pathology 5: 319-322 (1995).

Proposed link between transmissible spongiform encephalopathies of man and animals.

A link between scrapie and Creutzfeldt-Jakob disease (CJD) is likely to exist. Based on old observations on scrapie, new experiments on bovine spongiform encephalopathy, and modern reviews on CJD, my proposal fits general rules of virus transmission.

Western blot mapping of disease-specific amyloid in various animal species and humans with transmissible spongiform encephalopathies using a high-yield purification method.

SAF-protein, an amyloid, is the main constituent of scrapie-associated fibrils (SAF) and a specific marker for transmissible spongiform encephalopathies (TSE). Using an improved extraction method and Western blot detection, the disease-specific amyloid was found in various parts of the central nervous system of hamsters orally infected with scrapie, of squirrel monkeys orally infected with kuru, sporadic Creutzfeldt-Jakob disease (CJD) and scrapie, of human patients with sporadic CJD, of a sheep with natural scrapie and of a cow with bovine spongiform encephalopathy (BSE). In human CJD samples, the concentration of TSE-specific amyloid was estimated to be 1000- to 10 000-fold lower than in the central nervous system of hamsters with scrapie. The extraction method has a yield of 70% and allows Western blot detection of the TSE-specific amyloid in samples representing 1-10 micrograms of brain tissue from intracerebrally infected hamsters, as well as in individual spleens from hamsters with terminal scrapie infected by the intracerebral, oral or intraperitoneal route. A 20-100 mg sample of material is sufficient for the extraction of the pathological protein from different rodent, monkey, ovine, bovine and human tissues. The results reported here demonstrate the potential suitability of the method for the routine diagnosis of TSE as well as for the detailed analysis of distribution patterns of the TSE-specific amyloid in experimental approaches to the investigation of these diseases.

The original Gerstmann-Sträussler-Scheinker family of Austria: divergent clinicopathological phenotypes but constant PrP genotype.

We present new data on the original Austrian kindred with Gerstmann-Sträussler-Scheinker disease (GSS) which encompasses currently 221 members in 9 generations. The mode of inheritance is autosomal dominant. Predominant clinical features are slowly progressive ataxia and late impairment of higher cerebral functions. In contrast, a recent case with proven P102L mutation of the PRNP gene had rapidly developing dementia and severe cortical damage indistinguishable from the clinicopathological phenotype of Creutzfeldt-Jakob disease (CJD). PRNP codon 129 was homozygous for methionine in both the historic and recent cases. Neuropathology confirms spongiosis of variable degree and numerous protease resistant/prion protein (PrP) amyloid plaques scattered throughout most of the brain as constant features in this family. Some amyloid deposits are surrounded by dystrophic neurites with accumulation of phosphorylated neurofilaments and abnormal organelles, reminiscent of Alzheimer-type plaques. Severe telencephalic damage and a synaptic-type fine granular immunoreactivity in laminar distribution in the cortex with anti-PrP after hydrated autoclaving of sections were seen only in the recent patient. In conclusion, factors in addition to the PRNP genotype at codons 102 and 129 must play a role in determining clinicopathological characteristics of this inherited brain amyloidosis.

Tissue handling in suspected Creutzfeldt-Jakob disease (CJD) and other human spongiform encephalopathies (prion diseases)

Despite many sensational and intimidating reports in the mass media, transmissible spongiform encephalopathies (prion disease) are not contagious in the usual sense. Successful transmission requires both specific material (an affected individual's tissue, from or adjacent to CNS) and specific modes (mainly penetrating contact with the recipient). Nevertheless, specific safety precautions are mandatory to avoid accidental transmission and to decontaminate any infectivity. Autopsy is essential for definite diagnosis of these disorders. Recommendations are given here for performance of the autopsy, for neuropathology service and appropriate decontamination; they are based on the current literature and on precautions taken in most laboratories with experience in handling tissue from transmissible spongiform encephalopathies. In particular, special care must be taken to avoid penetrating wounds, possible contamination should be kept to a minimum, and potential infectious material must be adequately decontaminated by specific means.

Small virus-like structure in brains from cases of sporadic and familial Creutzfeldt-Jakob disease.

We have previously observed small virus-like particles in the brain of hamsters with experimental scrapie. Here we report that small virus-like structures can be isolated from brains of patients with Creutzfeldt-Jakob disease and identified by electronmicroscopy.

Bovine spongiform encephalopathy in Germany.

Bovine spongiform encephalopathy (BSE) has been described as an epidemic central nervous disorder in cattle from the United Kingdom. The disease is thought to have emerged by an interspecies transmission of the scrapie agent of sheep to cattle, after feeding scrapie-contaminated meat and bone meal (MBM). The disease has caused substantial economic losses for the British cattle industry. Because of strict veterinary regulations for the import of adult British cattle by the European Union and for MBM by most of the member states the spread of BSE to continental Europe could be efficiently controlled, and only few cases have been described outside the UK. Here we report the first German case of BSE diagnosed in a Scottish Highland cow. The affected cow was imported into Germany before the import ban for cattle from the UK was implemented. BSE was confirmed by histopathology, immunohistochemistry, animal experiments, immunoblotting and by electron microscopic detection of scrapie-associated fibrils (SAFs).