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.

Postmortem diagnosis of preclinical and clinical scrapie in sheep by the detection of disease-associated PrP in their rectal mucosa.

Samples of tissue from the central nervous system (cns), the lymphoreticular system (lrs) and the rectal mucosa of a large number of scrapie-exposed sheep, with and without signs of clinical disease, were examined immunohistochemically for evidence of disease-associated prion protein (PrP(d)). The rectal mucosa has received almost no attention so far in scrapie diagnosis, despite its abundant rectoanal mucosa-associated lymphoid tissue, and its accessibility. The scrapie-confirmed cases included 244 with clinical disease, of which 237 (97.1 per cent) were positive in the rectal mucosa, and 121 apparently healthy sheep, of which 104 (86 per cent) were positive in the rectal mucosa. PrP(d) was detected in 86.4 to 91.5 per cent of the other lrs tissues of the healthy sheep examined and in 77.7 per cent of their cns tissues. The stage of infection, therefore, affected the probability of a positive result in the rectal mucosa, whereas the breed, PrP genotype, age and sex had little or no independent effect. Accumulations of PrP(d) were observed in the rectal mucosa and other lrs tissues of vrq/arr sheep with preclinical and clinical scrapie, albeit with a lower frequency and magnitude than in sheep of other PrP genotypes. Western immunoblotting analyses of samples of rectal mucosa gave the characteristic PrP glycoprofile, with a sensitivity similar to that of immunohistochemistry.

Lymphocyte markers in the bovine foetus.

The ontogeny of lymphocyte surface markers was studied in thymus, retropharyngeal lymph nodes, spleen, bone marrow, and liver of 48 bovine foetuses, 80-280 days of gestation. The markers examined were the spontaneous (E) rosette against sheep erythrocytes (SRBCs) in foetal calf serum (E/FCS), in medium supplemented with gelatin (E/CFG), and in medium supplemented with dextran (E/dextran), the erythrocyte-antibody (EA) rosette for immunoglobulin (Fc) receptors, the erythrocyte-antibody-complement (EAC) rosette for complement receptors, and the immunofluorescence test (IFT) for surface-membrane immunoglobulins (SmIg). E-rosette-forming cells (RFCs) occurred in the thymus in substantial numbers throughout the period studied, in spleen and lymph nodes only after 160 days, and in bone marrow and liver rarely. The dynamics of E rosettes formed in the three supplements suggested that each supplement might be supporting rosette formation by different populations of cells, albeit considerable overlap might be expected. Organ distribution and development of EA and EAC RFCs did not parallel the expected ontogeny of any class of lymphocytes. Cells carrying SmIg developed mainly in spleen and lymph nodes. Towards the end of gestation some cells in liver also had SmIg.

Bovine spongiform encephalopathy: detection and quantitation of fibrils, fibril protein (PrP) and vacuolation in brain.

Bovine spongiform encephalopathy (BSE) is a new disease of cattle which has considerable homology with scrapie, the archetype of the transmissible spongiform encephalopathies. Abnormal brain fibrils, called scrapie associated fibrils (SAF), are specific ultrastructural markers for these diseases. Fibril detection was compared with histopathological diagnosis in the brains of 167 cattle; 157 clinically suspect BSE and 10 clinically normal. Fibrils were detected in samples of pooled brain regions of 67/144 in which vacuolar changes of BSE were confirmed, but absent in the remaining 23 brains, in which no vacuolation was found, including those from the clinically normal cattle and 13 with alternative neuropathological diagnoses. When eight defined anatomic regions from the brains of another 22 affected cows were examined, the sensitivity of fibril detection was greater than 90% for the brain stem areas. Fibril prevalence in these areas approximated to severity of vacuolar changes. When the same defined regions from four of the affected cows were assayed for fibril protein (PrP) by western blotting, the density of immuno-labelling generally correlated with the fibril prevalence. This study confirms the specificity of fibril detection for BSE, shows that the ease of fibril detection depends on anatomic region sampled and suggests an association between PrP accumulation and vacuolar changes in certain neuroanatomic areas.

Correlation between enhanced E-rosette formation and spontaneous incorporation of thymidine by bovine thymus lymphocytes.

Bovine thymus lymphocytes were centrifuged at low speed through 27% BSA and separated into five equal fractions. Enhanced formation of E rosettes and 10-20 fold increases in spontaneous uptake of tritiated (3H) thymidine occurred among the buoyant cell populations with less or no increases among cells from lower fractions. It is suggested that BSA stimulates blastogenesis and that the increases capacity for E-rosette formation is due to cell surface alterations among the activated cells or cells affected by their products.

Diagnosis of preclinical and subclinical scrapie in a naturally infected sheep flock utilizing currently available postmortem diagnostic techniques.

Scrapie is a naturally occurring transmissible encephalopathy of sheep and goats. Currently available methods for diagnosis are the presence of characteristic histopathologic changes and detection of an abnormal form of prion protein (PrPres) in the brains of affected animals. This study documents preclinical and subclinical scrapie in a flock of 16 sheep utilizing histopathology, immunohistochemistry (IHC), western blot, and electron microscopy (for scrapie-associated fibrils) for confirmation of the disease. Prior to necropsy, none of the sheep showed signs of clinical scrapie. Based on the results of histopathology and positive PrPres tests, 3 ewes were found to have subclinical scrapie. An additional ewe, which did not have histopathologic changes in the brain but was positive by IHC and western blot,was considered a preclinical case of scrapie. None of the sheep had amyloid in the brain stem.

Idiopathic disseminated intracytoplasmic neuronal vacuolation in a neonatal Holstein calf born in the USA.

Histopathologic, immunohistochemical, and ultrastructural evaluations were made of a 6-day-old Holstein calf with severe vacuolation of the neuronal perikarya that was widely distributed throughout the central nervous system. No evidence of storage material within the vacuoles was revealed by histopathologic and ultrastructural examinations. Immunohistochemical and electron microscopic examinations were negative for protease-resistant prion protein and scrapie-associated fibrils, respectively. These results indicate that the clinical signs in this calf were not associated with transmissible spongiform encephalopathy. Neuronal vacuolation has not previously been documented in calves.

Preliminary findings on the experimental transmission of chronic wasting disease agent of mule deer to cattle.

To determine the transmissibility of chronic wasting disease (CWD) to cattle and to provide information about clinical course, lesions, and suitability of currently used diagnostic procedures for detection of CWD in cattle, 13 calves were inoculated intracerebrally with brain suspension from mule deer naturally affected with CWD. Between 24 and 27 months postinoculation, 3 animals became recumbent and were euthanized. Gross necropsies revealed emaciation in 2 animals and a large pulmonary abscess in the third. Brains were examined for protease-resistant prion protein (PrP(res)) by immunohistochemistry and Western blotting and for scrapie-associated fibrils (SAFs) by negative-stain electron microscopy. Microscopic lesions in the brain were subtle in 2 animals and absent in the third case. However, all 3 animals were positive for PrP(res) by immunohistochemistry and Western blot, and SAFs were detected in 2 of the animals. An uninoculated control animal euthanized during the same period did not have PrP(res) in its brain. These are preliminary observations from a currently in-progress experiment. Three years after the CWD challenge, the 10 remaining inoculated cattle are alive and apparently healthy. These preliminary findings demonstrate that diagnostic techniques currently used for bovine spongiform encephalopathy (BSE) surveillance would also detect CWD in cattle should it occur naturally.

Comparison of scrapie-associated fibril detection and Western immunoblotting for the diagnosis of natural ovine scrapie.

Detergent- and proteinase K-treated extracts of grey matter were prepared from four regions of the brains of 106 sheep with scrapie, diagnosed clinically and by the demonstration of spongiform encephalopathy. The extracts were examined by electron microscopy for the presence of scrapie-associated fibrils and by Western immunoblotting for the disease-specific abnormal prion protein (PrPSc). As a diagnostic method, Western immunoblotting proved to be more sensitive than electron microscopy, the detection rates in the 106 sheep being 97 and 91% respectively (medulla), 99 and 76% (cerebellum), 95 and 88% (frontal cerebral cortex) and 93 and 61% (occipital cerebral cortex). Neither fibrils nor PrPSc could be detected in comparable brain extracts from 25 control sheep which had shown no clinical or histopathological evidence of scrapie.

The reproducibility of scrapie-associated fibril and PrPSc detection methods after long-term cold storage of natural ovine scrapie-affected brain tissue.

A pool of grey matter (medulla/brain stem, cerebellum and frontal cerebral cortex) was prepared from the brains of 16 sheep with scrapie, diagnosed clinically and by the demonstration of spongiform encephalopathy. Aliquots from the pool of tissue were finely chopped or homogenized and stored at +4 degrees C or -70 degrees C, after undergoing one of several specific pre-treatments (storage with or without protease inhibitors or, alternatively, with or without the cryoprotectant, dimethyl sulphoxide). At intervals over a period of 2 years, the stored extracts were examined by electron microscopy for the presence of scrapie-associated fibrils (SAFs) and by Western immunoblotting for the disease-specific abnormal protein PrPSc. Throughout the 2-year period, SAFs and PrPSc were detected in the majority of all stored tissue extracts under all combinations of tissue preparation and pre-treatment. The combined detection rates for SAFs and PrPSc were 91% at +4 degrees C and 94% at -70 degrees C. There was no significant difference between the results obtained by the two detection methods and no specific combination of preparation method and pre-treatment was superior to any other. Storage of the samples at -70 degrees C appeared to give better results than storage at +4 degrees C, particularly with regard to fibril detection. For logistical reasons and ease of processing, and to avoid the effects of autolysis on recognizable brain regions, long-term storage at -70 degrees C, without any pre-treatment, would appear to be the method of choice.

Comparison of detergent and protease enzyme combinations for the detection of scrapie-associated fibrils from the central nervous system of sheep naturally affected with scrapie.

Standardized samples of tissue from the central nervous system of four sheep naturally affected with scrapie and from four healthy control sheep were subjected to a centrifugal extraction technique used to obtain scrapie-associated fibrils; the latter were then demonstrated by negative-contrast transmission electron microscopy. This regime was used to evaluate the fibril yield obtained from the 25 possible combinations of five different detergents and five different proteolytic enzymes. N-lauroylsarcosine detergent was found to be the most efficient detergent for all five enzymes, followed by sulphabetaine 3-14. Sodium dodecyl sulphate detergent was successful only in combination with a subtilisin Carlsberg enzyme. Octylglucoside and nonidet P40 detergents did not produce fibrils with any of the enzymes. Proteinase K was the least efficient of the five enzymes when used in combination with N-lauroylsarcosine; subtilisin Carlsberg, clostripain, pronase and trypsin enzymes all gave higher fibril yields. A combination of N-lauroylsarcosine detergent and subtilisin Carlsberg proteolytic enzyme gave the highest fibril yield.

Comparison of biochemical extraction techniques for the detection of scrapie-associated fibrils in the central nervous system of sheep naturally affected with scrapie.

Standardized samples of brain material from four sheep naturally affected with scrapie and from four healthy control sheep were subjected to six different extraction techniques used for the detection of scrapie-associated fibrils by negative-contrast transmission electron microscopy. The six methods were compared in respect of fibril yield and clarity of ultrastructure. The simplest method consisting of a single N-lauroylsarcosine detergent extraction and differential centrifugation, followed by proteinase K enzyme digestion, gave the best overall results. The use of proteinase and nuclease inhibitors made no apparent difference to the yield or ultrastructural clarity of fibrils. Density gradient centrifugation appeared to reduce tungstate stain penetration and often obscured the ultrastructural clarity. The results suggested that the preferred technique could be improved by the use of a double homogenization stage at the beginning of the procedure and by adding an ultrasonic disintegration step to resuspend the final pellet prior to tungstate staining.

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.

Scrapie associated fibril detection on decomposed and fixed ovine brain material.

Samples of cerebral cortex from eight scrapie affected sheep and two unaffected control sheep were stored for up to nine days at temperatures ranging from 18 degrees C to 29 degrees C. Scrapie associated fibrils (SAF) could be detected in proteinase K treated brain extracts from all the eight scrapie affected animals after five days storage and in six out of the eight after nine days storage. SAF could not be detected in any brain extracts from the two control animals. Formol saline fixed brain material from a further six scrapie affected and two clinically normal sheep, were also subjected to an extraction technique used to detect fibrils. No characteristic SAF were observed in any of these fixed samples. Long filamentous structures were observed in four of the fixed scrapie affected brain extracts and in one of the fixed unaffected control brain extracts.

Scrapie associated fibril detection from formaldehyde fixed brain tissue in natural cases of ovine scrapie.

The medulla oblongata of the brains of 71 scrapie-suspect cases were routinely fixed in 10 per cent formal saline and assessed for vacuolation on HE-stained sections. A pool of fresh brain material was also dissected from each animal and extracts prepared for the routine detection of scrapie-associated fibrils by negative stain transmission electron microscopy. The remaining formaldehyde fixed medulla samples, which were not used for the histological examination, were coded and subjected to a pretreatment with sodium borohydride and then processed using the routine fibril detection procedure. Of the 71 samples tested 46 were considered positive by all three test procedures. Sixteen samples were negative for all three tests. Four samples were positive by histopathological examination and positive for fibrils using fresh tissue, but fibrils could not be detected in the fixed tissue preparations. Conversely, there were five fixed samples in which fibrils could be detected which were negative for the other two tests. The fibrils observed in fixed preparations were indistinguishable from those observed in fresh tissue extracts. The sensitivity of the test for fibril detection using fixed tissue was 92 per cent and the specificity 76 per cent. It is concluded that scrapie-associated fibrils can be recovered from formaldehyde fixed tissue, as presented for routine histopathological examination, and therefore the method has potential in the retrospective analysis of archived brain tissue where only fixed material was stored.

Comparative study of electron microscopical techniques for the detection of scrapie-associated fibrils.

Samples of cervical spinal cord and four anatomical regions of the brains of 12 sheep with natural scrapie and six control sheep were examined by electron microscopy, after the tissues had been stored at 4 degrees C and -20 degrees C. The tissues were tested for the presence of scrapie-associated fibrils by a centrifugal extraction technique and by a touch-grid technique. The touch-grid technique was no better than the centrifugal extraction technique for the detection of fibrils. Structures which could have been classified as tubulofilaments were detected in touch-grid preparations without detergent treatment. With the centrifugal extraction technique there was a significant reduction of the fibril scores in some of the tissue extracts stored at -20 degrees C, but not in any of the extracts stored at 4 degrees C. There was, however, a reduction in the fibril scores when the final extracted pellets were stored at 4 degrees C. The stability of the fibrils on the test grids was unaffected by six months storage at room temperature but the clarity of their ultrastructure did deteriorate. Poor hydrophilic spread of the sample on the test grids did not have a significant effect on the fibril scores.

The distribution of scrapie-associated fibrils in neural and non-neural tissues of advanced clinical cases of natural scrapie in sheep.

The distribution of scrapie-associated fibrils (SAFs) throughout four brain regions, the pituitary gland, along the whole length of the spinal cord and in the sciatic nerve was assessed in 10 sheep terminally affected by scrapie and in four control sheep. Tonsils, retropharyngeal, broncho-mediastinal and mesenteric lymph nodes, the distal ileum, proximal colon and spleen were also examined for fibrils in all 14 sheep. Fibrils were detected in all four brain regions and throughout the length of the spinal cord in nine of the scrapie affected sheep. SAFs were not detectable in any of the sciatic nerve samples tested. In one of the 10 clinically affected sheep only minimal lesions were found by histopathology and fibrils were detected only from the cerebrum and one spinal cord region (taken at the C1 C2 vertebrae). Fibrils were not detected in the tonsils or retropharyngeal lymph nodes but were detected in other non-neural tissues of some of the scrapie-affected sheep. These tissues included pituitary gland, broncho-mediastinal and mesenteric portal lymph nodes, distal ileum, proximal colon and spleen. Fibrils could not be detected in any of the tissues taken from the four control sheep.

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.

Natural scrapie: detection of fibrils in extracts from the central nervous system of sheep.

Extracts from the cervical spinal cord and from the medulla, thalamus, cerebellum and cerebral cortex of the brains of 10 sheep, histopathologically confirmed as cases of scrapie, were examined by electron microscopy for the presence of scrapie-associated fibrils. Characteristic fibrils were observed in all the extracts except for that from the thalamus of one sheep. No fibrils were found in any extracts from three control sheep. A comparison of these results with a similar study of 22 cases of bovine spongiform encephalopathy (BSE) suggests that in cases of scrapie the area of the brain chosen for the detection of fibrils is less critical than in cases of BSE, in which fibrils are more readily extracted from areas of the brain stem.

Parapox infection in grey seals (Halichoerus grypus) in Cornwall.

In the winter of 1991/92 there was an outbreak of parapox infection in grey seals (Halichoerus grypus) around the coast of Cornwall. Pups were cared for at a seal rehabilitation centre and the infection occurred in most of them. The presence of parapox virus was confirmed by electron microscopy. The clinical and pathological findings, together with details of the morphology of the virus, are compared with those in previous outbreaks in North America.