Altered trafficking of abnormal prion protein in atypical scrapie: prion protein accumulation in oligodendroglial inner mesaxons.

AIMS: Prion diseases exist in classical and atypical disease forms. Both forms are characterized by disease-associated accumulation of a host membrane sialoglycoprotein known as prion protein (PrPd ). In classical forms of prion diseases, PrPd can accumulate in the extracellular space as fibrillar amyloid, intracellularly within lysosomes, but mainly on membranes in association with unique and characteristic membrane pathology. These membrane changes are found in all species and strains of classical prion diseases and consist of spiral, branched and clathrin-coated membrane invaginations on dendrites. Atypical prion diseases have been described in ruminants and man and have distinct biological, biochemical and pathological properties when compared to classical disease. The purpose of this study was to determine whether the subcellular pattern of PrPd accumulation and membrane changes in atypical scrapie were the same as those found in classical prion diseases. METHODS: Immunogold electron microscopy was used to examine brains of atypical scrapie-affected sheep and Tg338 mice. RESULTS: Classical prion disease-associated membrane lesions were not found in atypical scrapie-affected sheep, however, white matter PrPd accumulation was localized mainly to the inner mesaxon and paranodal cytoplasm of oligodendroglia. Similar lesions were found in myelinated axons of atypical scrapie Tg338-infected mice. However, Tg338 mice also showed the unique grey matter membrane changes seen in classical forms of disease. CONCLUSIONS: These data show that atypical scrapie infection directs a change in trafficking of abnormal PrP to axons and oligodendroglia and that the resulting pathology is an interaction between the agent strain and host genotype.

Goats with aspartic acid or serine at codon 146 of the PRNP gene remain scrapie-negative after lifetime exposure in affected herds in Cyprus.

The results of the study reported here are part of an ongoing integrated research programme aimed at producing additional, robust, evidence on the genetic resistance to classical scrapie in goats, with particular reference to codon 146. The study targeted animals aged ⩾6 years, which were born and raised in infected herds and were being culled for management reasons. A total of 556 animals were tested, and all positive animals (n = 117) were of the susceptible NN genotype. A total of 246 goats heterozygous or homozygous for putatively resistant alleles (S146 and D146) were screened with no positive results. The outcome of this study supports the hypothesis that the D146 and S146 alleles could be used as the basis for a nationwide strategy for breeding for resistance in the Cypriot goat population.

Does the Presence of Scrapie Affect the Ability of Current Statutory Discriminatory Tests To Detect the Presence of Bovine Spongiform Encephalopathy?

Current European Commission (EC) surveillance regulations require discriminatory testing of all transmissible spongiform encephalopathy (TSE)-positive small ruminant (SR) samples in order to classify them as bovine spongiform encephalopathy (BSE) or non-BSE. This requires a range of tests, including characterization by bioassay in mouse models. Since 2005, naturally occurring BSE has been identified in two goats. It has also been demonstrated that more than one distinct TSE strain can coinfect a single animal in natural field situations. This study assesses the ability of the statutory methods as listed in the regulation to identify BSE in a blinded series of brain samples, in which ovine BSE and distinct isolates of scrapie are mixed at various ratios ranging from 99% to 1%. Additionally, these current statutory tests were compared with a new in vitro discriminatory method, which uses serial protein misfolding cyclic amplification (sPMCA). Western blotting consistently detected 50% BSE within a mixture, but at higher dilutions it had variable success. The enzyme-linked immunosorbent assay (ELISA) method consistently detected BSE only when it was present as 99% of the mixture, with variable success at higher dilutions. Bioassay and sPMCA reported BSE in all samples where it was present, down to 1%. sPMCA also consistently detected the presence of BSE in mixtures at 0.1%. While bioassay is the only validated method that allows comprehensive phenotypic characterization of an unknown TSE isolate, the sPMCA assay appears to offer a fast and cost-effective alternative for the screening of unknown isolates when the purpose of the investigation was solely to determine the presence or absence of BSE.

Allelic variants at codon 146 in the PRNP gene show significant differences in the risk for natural scrapie in Cypriot goats.

Previous studies have shown the association between the polymorphisms serine (S) or aspartic acid (D) at codon 146 of the PRNP gene and resistance to scrapie. All goats aged >12 months (a total of 1075 animals) from four herds with the highest prevalence of scrapie in the country were culled and tested, of which 234 (21·7%) were positive by either the rapid test or immunohistochemistry (IHC) for any of the tissues tested. The odds of scrapie infection occurring in NN146 goats was 101 [95% credible interval (CrI) 19-2938] times higher than for non-NN146 or unknown genotypes. IHC applied to lymphoreticular tissue produced the highest sensitivity (94%, 95% CrI 90-97). The presence of putatively resistant non-NN146 alleles in the Cypriot goat population, severely affected by scrapie, provides a potential tool to reduce/eradicate scrapie provided that coordinated nationwide breeding programmes are implemented and maintained over time.

Experimental classical bovine spongiform encephalopathy: definition and progression of neural PrP immunolabeling in relation to diagnosis and disease controls.

Tissues from sequential-kill time course studies of bovine spongiform encephalopathy (BSE) were examined to define PrP immunohistochemical labeling forms and map disease-specific labeling over the disease course after oral exposure to the BSE agent at two dose levels. Study was confined to brainstem, spinal cord, and certain peripheral nervous system ganglia-tissues implicated in pathogenesis and diagnosis or disease control strategies. Disease-specific labeling in the brainstem in 39 of 220 test animals showed the forms and patterns observed in natural disease and invariably preceded spongiform changes. A precise temporal pattern of increase in labeling was not apparent, but labeling was generally most widespread in clinical cases, and it always involved neuroanatomic locations in the medulla oblongata. In two cases, sparse labeling was confined to one or more neuroanatomic nuclei of the medulla oblongata. When involved, the spinal cord was affected at all levels, providing no indication of temporal spread within the cord axis or relative to the brainstem. Where minimal PrP labeling occurred in the thoracic spinal cord, it was consistent with initial involvement of general visceral efferent neurons. Labeling of ganglia involved only sensory ganglia and only when PrP was present in the brainstem and spinal cord. These experimental transmissions mimicked the neuropathologic findings in BSE-C field cases, independent of dose of agent or stage of disease. The model supports current diagnostic sampling approaches and control measures for the removal and destruction of nervous system tissues in slaughtered cattle.

Protective effect of the T112 PrP variant in sheep challenged with bovine spongiform encephalopathy.

Sheep with an ARQ/ARQ PRNP genotype at codon positions 136/154/171 are highly susceptible to experimental infection with bovine spongiform encephalopathy (BSE). However, a number of sheep challenged orally or intracerebrally with BSE were clinically asymptomatic and found to survive or were diagnosed as BSE-negative when culled. Sequencing of the full PRNP gene open reading frame of BSE-susceptible and -resistant sheep indicated that, in the majority of Suffolk sheep, resistance was associated with an M112T PRNP variant (TARQ allele). A high proportion (47 of 49; 96%) of BSE-challenged wild-type (MARQ/MARQ) Suffolk sheep were BSE-infected, whereas none of the 20 sheep with at least one TARQ allele succumbed to BSE. Thirteen TARQ-carrying sheep challenged with BSE are still alive and some have survival periods equivalent to, or greater than, reported incubation periods of BSE in ARR/ARR and VRQ/VRQ sheep.

Abnormal prion protein is associated with changes of plasma membranes and endocytosis in bovine spongiform encephalopathy (BSE)-affected cattle brains.

AIMS: Transmissible spongiform encephalopathies (TSEs) or prion diseases are fatal neurodegenerative diseases of man and animals characterized by vacuolation and gliosis of neuropil and the accumulation of abnormal isoforms of a host protein known as prion protein (PrP). It is widely assumed that the abnormal isoforms of PrP (PrP(d), disease-specific form of PrP) are the proximate cause of neurodegeneration. METHODS: To determine the nature of subcellular changes and their association with PrP(d) we perfusion-fixed brains of eight bovine spongiform encephalopathy (BSE)-affected cows and three control cattle for immunogold electron microscopy at two different neuroanatomical sites. RESULTS: All affected cattle presented plasma membrane alterations of dendrites and astrocytes that were labelled for PrP(d). PrP(d) on membranes of dendrites and occasionally of neuronal perikarya was associated with abnormal endocytotic events, including bizarre coated pits and invagination of the plasma membrane. BSE-affected cattle also presented excess and abnormal multivesicular bodies, sometimes associated to the plasma membrane perturbations. In contrast, two TSE-specific lesions, vacuolation and rare tubulovesicular bodies, were not labelled for PrP(d) as were a number of other nonspecific lesions, such as autophagy and dystrophic neurites. At least two different morphological pathways to vacuoles were recognized. CONCLUSIONS: When compared with other TSEs, these changes are common to those of sheep and rodent scrapie and shows that there are consistent membrane toxicity properties of PrP(d). This toxicity involves an aberration of endocytosis. However, it is by no means clear that the lesions are of sufficient severity to result in clinical deficits.

Tissue distribution of bovine spongiform encephalopathy infectivity in Romney sheep up to the onset of clinical disease after oral challenge.

Sixty Romney sheep of three prion protein genotypes were dosed orally at six months of age with an inoculum prepared from the brains of cattle clinically affected with BSE, and 15 sheep were left undosed as controls. They were randomly assigned within genotype to groups and were sequentially euthanased and examined postmortem at intervals of six or 12 months, depending on their predicted susceptibility. Tissue pools prepared from the three, four or five dosed animals in each group were inoculated into groups of 20 RIII mice as a bioassay for infectivity. Separate inocula were prepared from the matched control sheep killed at each time. In the ARQ/ARQ sheep killed four months after inoculation, infectivity was detected in the Peyer's patch tissue pool, and at 10 months it was detected in the spleen pool; from 16 months, infectivity was detected in a range of nervous and lymphoreticular tissues, including the spinal cord pool, distal ileum excluding Peyer's patches, liver, Peyer's patches, mesenteric and prescapular lymph nodes, spleen, tonsil and cervical thymus. No infectivity was detected in the tissue pools from the ARQ/ARR and ARR/ARR sheep killed 10 months or 22 months after infection.

A stochastic model to estimate the prevalence of scrapie in Great Britain using the results of an abattoir-based survey.

In 1997/1998, an abattoir survey was conducted to determine the likely exposure of the human population to transmissible spongiform encephalopathy (TSE) infection in sheep submitted for slaughter in Great Britain. The survey examined brain material from 2809 sheep processed through British abattoirs. Sampling was targeted by age: 45% of animals tested were > or =15 months old. All samples of adequate quality (98%) were tested for signs of scrapie infection using histopathology and scrapie-associated fibril (SAF) detection and 500 were tested using immunohistochemistry (IHC). No conclusive positive animals were found using either histology or IHC. Ten animals were positive by SAF. Standard statistical analyses suggest (with 95% confidence) that the prevalence of detectable (by histopathology) infection in the slaughter population was < or =0.11%. However, the incubation period of scrapie is long (usually around 2-3 years) and none of the tests used in the survey is capable of detecting scrapie infection in the early stages of infection. We present an age-structured stochastic model incorporating parameters for the incubation period of scrapie, prevalence of infection by age and test sensitivity. Using the model, we demonstrate that the negative results obtained for all samples using IHC and histopathology are consistent with a true prevalence of infection in the slaughter population of up to 11%. This suggests that up to 300 of the animals tested might have been infected but the infection was not sufficiently advanced in these animals to be detectable by IHC or histopathology. The survey was designed to detect a prevalence of 1% with a precision of +/-0.5% and a confidence level of 95% in each age group assuming that diagnostic tests were 100% specific and sensitive from a known stage in the incubation period. The results of the model demonstrate that to estimate a true prevalence of scrapie infection of 1% with an accuracy of +/-0.5% would have required a far larger sample size. An accurate estimate of the required sample size is complicated by uncertainty about test sensitivity and the underlying infection dynamics of scrapie. A pre-requisite for any future abattoir survey is validation of the diagnostic tests used in relation to both stage of incubation and genotype. Sampling in the <15-month age group was of no value in this survey because the diagnostic tests used were thought to be ineffective in most of the animals in this age group.

Lafora disease in the cow?

Lafora disease in man is an autosomal recessive defect which affects carbohydrate metabolism and results in a progressive, ultimately fatal neurological condition. It is characterized histologically by intraneuronal cytoplasmic polyglucosan inclusions (Lafora bodies). Similar inclusions have been seen in association with neurological signs in other species, including the dog, the cockatiel and the cow. Polyglucosan bodies, however, are not always considered to be disease specific, and have also been reported as an age-related change in cats, dogs and man. The only recorded case in cattle to date has been a single animal in the USA. The present report records a case study of two animals with Lafora inclusion bodies, together with a survey of the occurrence of non-specific polyglucosan bodies in aged cattle. It is concluded that the inclusions in the two putative cases of Lafora disease were not non-specific age-related changes, and that these cases represent the first report of the disease in cattle in the UK.

Distribution of vascular amyloid in scrapie-affected sheep with different genotypes.

Vascular amyloidosis in the brain is a pathological feature of ovine scrapie. Its occurrence varies between sheep, but whether this variation reflects differences in the host or the infecting scrapie strain (or both) is not clear. To investigate whether amyloidosis, like vacuolation and PrPsc distribution, is associated with genotype, the brains from 131 sheep representing a range of genotypes commonly associated with scrapie were examined histologically and immunohistochemically. Vascular amyloidosis was absent in 66 sheep, 59 of which were of the ARQ/ARQ genotype and seven the ARQ/AHQ genotype. In contrast, it was found in four of 39 ARQ/VRQ sheep (10.2%) and in 10 of 26 VRQ/VRQ sheep (38.4%). The distribution of amyloid was highly variable, but the most severely affected areas were the lateral geniculate nuclei (five cases) and the ventral thalamic nuclei (four cases). No amyloidosis was found in the medulla or in the basal nuclei. From this preliminary study it was concluded that amyloidosis is relatively rare in sheep with scrapie. Moreover, its occurrence appeared to depend on the presence of at least one valine at codon 136.

Histological observations on the brains of symptomless 7-year-old cattle.

The histological changes in the brains of 506 clinically normal 7-year-old cattle, which were part of a cohort study on maternal transmission of bovine spongiform encephalopathy, are described. Vacuolation of the white matter, of unknown aetiology, located particularly in the substantia nigra, was a frequent finding. Vacuolated neurons were commonly observed in the red nucleus (64.3% of the animals) and in the habenular nucleus (50.1%). Spheroids were found in 10.8% of the brains, most frequently in the vestibular nuclei. Cellular inflammatory infiltrates in association with blood vessels occurred in 30% of the animals at various locations in the brain; their aetiology remains uncertain, but they may have reflected subclinical or latent infections. Mineralization of the wall of blood vessels, with proliferation of the intima, was observed frequently in vessels of the internal capsule and was probably associated with ageing. The description of histological findings in the brain of symptomless adult cattle in the present study provides a useful background for diagnostic bovine neuropathology.

Distinction of scrapie phenotypes in sheep by lesion profiling.

Major determinants of the pathological phenotype of natural scrapie are considered to be the agent strain and host prion protein (PrP) genotype, but the relationship between these is far from clear. Little is known about the strains that produce natural scrapie. A method of brain vacuolation profiling was developed which enables this aspect of disease phenotype to be characterized in detail. This method distinguished at least two distinct pathological phenotypes in sheep of a single genotype (ARQ/ARQ) from different flocks in the UK. Great similarity was also demonstrated between one of these phenotypes and the phenotype of sheep from a flock in Sardinia. The profile of four sheep of the same ARQ/ARQ genotype experimentally infected with bovine spongiform encephalopathy (BSE) was determined for comparison. It would appear from these preliminary observations that the application of lesion profiling techniques to ovine transmissible spongiform encephalopathy (TSE) may contribute to the definition of a particular scrapie phenotype within a flock. It may, therefore, have potential for improving our understanding of current TSE phenotypes in sheep, with regard to the possibility of identifying those of bovine origin.

Simple method for the perfusion-fixation of adult bovine brain.

It is widely accepted that the vascular perfusion of a tissue leads to its optimal fixation for ultrastructural examination, but the technique has been applied with little success in large animals such as cattle. This paper describes a simple and highly reproducible method for the perfusion-fixation of adult bovine brain. The carotid arteries are cannulated and the perfusate is introduced by means of a peristaltic pump. The inclusion of toluidine blue in the fixative makes it possible to assess the success of the technique, and prevents the costly and time-consuming processing of material which is suboptimally fixed. The method is potentially valuable for the examination of a wide range of neurological conditions in large domestic animals at an ultrastructural level in optimally fixed material.

Correlation between the results of a histopathological examination and the detection of abnormal brain fibrils in the diagnosis of bovine spongiform encephalopathy.

A statistical comparison was made between the results of the statutory neurohistopathological method for the post mortem diagnosis of bovine spongiform encephalopathy (BSE) and the detection of abnormal brain fibrils (SAF). A total of 773 suspect cases was examined by both methods; it comprised 531 animals born before the feeding of ruminant-derived protein to ruminant species was prohibited and 242 born after the ban. The relative sensitivities and specificities of the methods were calculated for the diagnosis of clinically suspected BSE. The agreement between the results of the methods was excellent for 331 of the cases born before the ban and for all the cases born after it. In these cases the samples were not autolysed. For the 200 cases in which autolysis was recorded there was poor agreement between the diagnostic methods and this was attributed to an apparently reduced specificity of the histopathological diagnosis in the autolysed material. Despite the potentially greater specificity of fibril detection in the diagnosis of scrapie-like disease, this study indicates that a reliance on fibril detection alone may result in some false negative diagnoses, probably owing to the inadequate sampling of the tissues.

Evaluation of the effects of controlled autolysis on the immunodetection of PrP(Sc) by immunoblotting and immunohistochemistry from natural cases of scrapie and BSE.

Seventeen clinically suspect scrapie sheep, and twelve suspected BSE-affected cattle were confirmed using routine histopathological examination by the detection of characteristic spongiform change in the medulla brain region taken at the level of the obex. Three sheep and four cows acquired as controls showed no spongiform change. Five aliquots of brain tissue from each of four brain regions were taken (cerebellum, medulla, frontal cerebral cortex and occipital cerebral cortex) from each of the 36 animals. One aliquot was frozen at -70 degrees C, the others were subjected to one of four autolysis regimes at 3 or 7 days at 25 degrees C or 37 degrees C. All samples were tested by Western immunoblotting for detection of PrP(Sc) using the Prionics - Check test (Prionics AG, Zurich, Switzerland). Further samples of medulla from 15 suspect scrapie cases, 10 healthy sheep, 13 suspect BSE cows and 5 healthy cows, were taken adjacent to the obex, and subjected to autolysis at 37 degrees C for 6, 12, 24 and 48 hours before being fixed in 10 per cent formal saline and subsequently examined by a routine immunohistochemical technique for detection of PrP(Sc) protein. The abnormal protein could not be detected in any of the control animals by either technique. PrP(Sc) could be detected by Western immunoblotting in at least one brain area from all the positive animals after autolysis for 7 days at 37 degrees C. The protein could be detected by immunohistochemistry in all cases which were positive by histopathological examination using all autolysis conditions. From the results of this study it is concluded that autolysis does not significantly compromise the diagnosis of scrapie or BSE by either of these diagnostic methods.

BSE in Great Britain: consistency of the neurohistopathological findings in two random annual samples of clinically suspect cases.

Two annual, random samples of clinically suspect cases of bovine spongiform encephalopathy (BSE) were taken in 1992-93 (year 1, 1500 cases) and 1993-94 (year 2, 1000 cases). From each sample, 100 positive cases were examined in detail to establish the severity of the vacuolation in 17 specific neuroanatomical locations. The resultant 'lesion profiles' were compared with the profile obtained from a similar sample of BSE-affected cattle from early in the epidemic (1987-89); the comparison showed that the distribution and severity of vacuolation in BSE has remained unchanged. The cases not confirmed as BSE on histological examination (172 in year 1 and 162 in year 2) were examined for evidence of any alternative neurohistological diagnosis. As in previous studies, the majority of these cases showed no significant lesions (61.6 and 61.7 per cent). The remainder consisted of bilateral focal spongiosis of unknown significance (26.7 and 21.0 per cent), inflammatory conditions (8.1 and 11.1 per cent) and a small number of cases with tumours, cerebrocortical necrosis or idiopathic brainstem neuronal chromatolysis. No evidence was found of any cases of BSE with an atypical distribution of lesions. These findings support the theory that the BSE epidemic is sustained by a single, stable strain of the BSE agent, and confirm that the existing statutory diagnostic criteria continue to be appropriate.

Clinicopathological findings in sheep from Sardinia showing neurological signs of disease.

Histopathological and bacteriological examinations were performed on 178 brains from Sardinian sheep which were showing neurological signs. The sheep represented the total number of sheep with neurological syndromes submitted for diagnostic investigations over a three-year period in Sardinia. Scrapie was detected in 57 cases, cerebrocortical necrosis in 25, intoxication by a typical Mediterranean plant (Cistus species) was suspected in 25, coenurosis was detected in 11 cases, Listeria monocytogenes in eight cases and focal symmetrical encephalomalacia in six cases. Non-suppurative inflammatory changes were observed in three of the brains and suppurative changes were noted in two. Lesions restricted to the spinal cord were found in three cases. In the remaining 38 cases there were no significant neuropathological changes.

A cohort study to examine maternally-associated risk factors for bovine spongiform encephalopathy.

This long-term cohort study, initiated in July 1989, was designed to examine maternally-associated risk factors for bovine spongiform encephalopathy (BSE), forming part of the epidemiological research programme to assess the risks of non-feedborne transmission of BSE. In this study, the incidence of BSE in offspring of cows which developed clinical signs of BSE is compared with that in offspring, born in the same calving season and herd, of cows which had reached at least six years of age and had not developed BSE. All offspring were allowed to live to seven years of age. The results indicate a statistically significant risk difference between the two cohorts of 9.7 per cent and a relative risk of 3.2 for offspring of cows which developed clinical BSE. However, there is some evidence that this enhanced risk for offspring of BSE cases declined the later the offspring was born, but was increased the later the offspring was born in relation to the stage of the incubation period of the dam. The results presented cannot distinguish between a genetic component and true maternal transmission or a combination of both risks, but they do not indicate either that the BSE epidemic will be unduly prolonged or that the future incidence of BSE in Great Britain will increase significantly.

Scrapie surveillance in Great Britain: results of an abattoir survey, 1997/98.

A randomised sample of 2,809 apparently healthy sheep, 55 per cent of them less than 15 months of age, which were slaughtered for human consumption at abattoirs in Great Britain in 1997/98, was taken to establish the prevalence of scrapie infection. The medulla oblongata of each sheep was examined histopathologically at the level of the obex, and fresh brain tissue was examined for scrapie-associated fibrils (SAF) to establish whether there was evidence of scrapie. In addition, histological sections of the medulla from 500 of the sheep were immunostained with an antiserum to PrP, and the same technique was also applied to any animal found positive or inconclusive by the histological or SAF examinations. Any sheep which was positive by any of these diagnostic methods was also examined by Western immunoblotting, for the detection of the disease-specific protein PrP(Sc). A total of 2,798 sheep (99.6 per cent) were negative by all the methods applied. Ten animals were SAF-positive but negative by all the other methods, and in one animal there was immunohistochemical staining which could not be interpreted unequivocally as disease-specific. A mathematical model was used to estimate the prevalence of scrapie infection in the national slaughtered sheep population which would be consistent with these results. By this model, the absence of unequivocally substantiated cases of scrapie in the sample was consistent with a prevalence of infection in the slaughter population of up to 11 per cent.