The Reproducibility of Scrapie-associated Fibriland PrPScDetection Methods after Long-termCold Storage of Natural Ovine Scrapie-affectedBrain Tissue

J. Comp. Path. 1999 Vol. 120, 357–368

The Reproducibility of Scrapie-associated Fibril and PrPSc Detection Methods after Long-term Cold Storage of Natural Ovine Scrapie-affected Brain Tissue W. A. Cooley, L. A. Davis, P. Keyes and M. J. Stack Veterinary Laboratories Agency, Central Veterinary Laboratory, Woodham Lane, New Haw, Weybridge, Surrey KT15 3NB, UK Summary 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°C or −70°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°C and 94% at −70°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°C appeared to give better results than storage at +4°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°C, without any pre-treatment, would appear to be the method of choice.

Introduction Scrapie in sheep and goats is a naturally occurring infectious neurodegenerative disease which is invariably fatal. It is the archetypal member of a group of similar diseases, termed transmissible spongiform encephalopathies (TSEs), for which no causative agent has yet been identified. The group includes bovine spongiform encephalopathy (BSE) and human Creutzfeldt-Jakob disease (CJD) (for reviews see: DeArmond, 1993; Prusiner, 1993; Diringer et al., 1994). The traditional method of diagnosis of TSEs is based on the evaluation of clinical signs and the detection of characteristic vacuolation of neurons and neuropil by histopathological examination of formaldehyde-fixed tissue from the central nervous system (CNS) (Wells et al., 1987, 1991; Bell and Ironside, 1993; Fraser, 1993). New techniques, based on the use of fresh, unfixed tissue, 0021–9975/99/040357+12 $12.00/0

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include the detection of scrapie-associated fibrils (SAFs) (Merz et al., 1981, 1983, 1984) and prion rods (Prusiner et al., 1982, 1983). Both are distinct morphological markers found in TSE-affected CNS tissues under different preparation conditions, and are identified by negative contrast transmission electron microscopy. They are regarded as different forms of the same structure and do not differ in their protein composition or antigenicity (Merz et al., 1987). It has been suggested that the morphological differences between SAFs and prion rods are dependent on the purification method employed (Liberski et al., 1991; Liberski and Brown, 1993). The method of biochemical purification used in our laboratory for the demonstration of SAFs from fresh CNS tissue is N-lauroylsarcosine detergent extraction followed by differential centrifugation and proteinase K enzyme digestion (Stack et al., 1996). The main constituent of SAFs and prion rods is a disease-specific, protease resistant, neuronal membrane glycoprotein (molecular mass 27–30 kDa), termed the prion protein (Bolton et al., 1982) or PrPSc (Chesebro et al., 1985; Oesch et al., 1985). The protein is considered to be an abnormal isoform of a host cellular glycoprotein (PrPC, 33–35 kDa) present in many normal tissues (Oesch et al., 1985). PrPC, unlike PrPSc, is completely digested by proteinase K. Both PrPC and PrPSc can be extracted from fresh tissue and detected by immunoblotting with sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) (Laemmli, 1970), followed by Western immunoblotting with an appropriate antiserum (Towbin et al., 1979). In this technique, a method of purification similar to that employed for SAFs is used. Since 1986, SAF detection has been used in the UK for scrapie diagnosis, and as an adjunct to histopathological testing for the diagnosis of BSE and other animal spongiform encephalopathies (European Commission for Agriculture, 1994; Stack et al., 1996). A modified Western immunoblotting technique (Beekes et al., 1995), based on a rapid extraction procedure, has also been used in our laboratory for the diagnosis of natural ovine scrapie (Cooley et al., 1998). Storage of CNS tissue before testing by electron microscopy or immunoblotting is a logistic essential, and cold storage at −70°C is the recognized method of choice for biological materials, particularly those containing viruses. However, one of the main problems in detecting SAF and PrPSc after storage is the reproducibility of a small proportion of positive results, with the related consequences of false negative diagnoses (Stack et al., 1991; Wells et al., 1994). It has been suggested that, although SAFs and PrPSc are relatively protease-resistant, naturally occurring protease enzymes released in CNS tissue through mechanical manipulation (dissection or homogenization) may adversely affect SAF or PrPSc detection after prolonged storage in the frozen state. Furthermore, CNS tissue is normally stored at −70°C without any cryoprotection; this may also contribute to false negative results. This report describes the use of two pre-treatments for chopped or homogenized grey matter from scrapie-positive brain tissue, the preparations being subsequently stored at either +4°C or −70°C. The two pre-treatments were (1) a “cocktail” of protease enzyme inhibitors (Kascsak et al., 1987), and (2) a commonly used cryoprotectant, dimethyl sulphoxide (DMSO). Electron

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microscopical examination for SAFs and Western immunoblotting for PrPSc were then carried out at intervals over a 2-year period. Materials and Methods Animals, Tissues and Procedures Sheep (n=16), of the Finn Dorset breed, showing clinical signs of scrapie, were obtained as two groups of eight from a single flock. Logistically, 16 animals could not be processed on a single day, and the animals were therefore treated as two separate batches. The following procedure was carried out on the first eight animals (batch 1) to supply tissue for storage at +4°C, and repeated with the second group of eight animals (batch 2) to supply tissue for storage at −70°C. The whole brain was removed at necropsy from each animal in batch 1, and a portion of the medulla taken at the obex was fixed in 10% formol saline for histopathological examination. The remainder of each brain was then used to provide a single pool of grey matter (taken from the medulla/brain stem, cerebellum and frontal cerebral cortex region). This pool of tissue was finely chopped, thoroughly mixed, and divided into eight aliquots (nos 1–8), each weighing 14 g. Aliquots 1–4 were left in the chopped state and suspended in 28-ml volumes of various pre-treatment fluids (see below); aliquots 5–8 were homogenized (for 15 min in a Silverson blender) in 28-ml volumes of similar fluids. Aliquots 1 and 5 were suspended in a solution of 1 mM NaHCO2, 1 mM MgCl2 and 0·5 mM KCl containing 5 lg/ml of each of the five protease inhibitors phenylmethylsulphonyl fluoride, pepstatin A, antipain, leupeptin and benzimedine hydrochloride (Rubenstein et al., 1986; Kascsak et al., 1987). The same solution but without inhibitors was used for aliquots 2 and 6 (controls). Aliquots 3 and 7 were suspended in the cryoprotectant DMSO (final v/v concentration of 7·5%) and aliquots 4 and 8 (controls) in distilled water. After pre-treatment, each of the eight aliquots were dispensed into seven Universal containers (6 ml/container). These were then stored at +4°C. The eight aliquots derived from batch 2, having been prepared in exactly the same way on another day, were stored at −70°C (see above). At intervals over a period of 2 years, one sample of each aliquot was removed and divided into two 3-ml volumes for testing (after appropriate extraction; see below) for (1) SAFs, by electron microscopy, and (2) PrPSc, by Western immunoblotting. Demonstration of SAFs The samples (3 ml) for SAF-detection from each pre-treatment were homogenized with 0·5 ml of 50% N-lauroylsarcosine detergent and after differential centrifugation were subjected to enzymatic digestion with proteinase K (Stack et al., 1993, 1996). The final centrifuged pellets obtained from the SAF extraction procedure were each dispersed in 50 ll of sterile deionized water, and dried on to formvar/carbon-coated support grids which had been subjected to plasma glow discharge. The grids were then negatively stained with 2% phosphotungstic acid (pH 6·6) and coded, so that the particular pre-treatment was unknown to the observer. Examination of the grids for SAFs was carried out in a Philips CM10 electron microscope with an accelerating voltage of 80 kV at magnifications of >×25 000. The support grids used throughout the study were 3 mm in diameter, with 332 grid squares (Gilder Grids, Grantham, Lincolnshire, England). A sample was scored as a positive if characteristic fibrils were identified during a 20-min search of the prepared grid. The scoring system used to indicate the number of SAFs present was as follows: −, none; +, <5; ++, 5–100; +++, >100. Demonstration of PrPSc The remaining 3-ml volume (equivalent to 1 g of brain) of each sample was extracted and examined for PrPSc as described by Stack et al. (1996). The samples had been

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coded and the pre-treatments were revealed only after the results had been recorded. Immunolabelling was carried out with polyclonal antiserum 971 G1 (raised in rabbits against a synthetic peptide of bovine PrP residues 230–244). Statistical Methods The percentages of positive results were compared with StatXact software (Mehta and Patel, 1991), which computes exact probabilities for non-parametric tests. McNemar’s Test for paired samples was used to compare the two detection methods at each storage temperature. The effects of the pre-treatments and of homogenization as opposed to chopping were tested for each detection method by pooling the results for each temperature and using Fisher’s Exact Test. For each temperature and method a linear by linear association test was used to test for trends with storage time.

Results Histopathological Examination Vacuolar changes diagnostic of scrapie were detected in the medulla of 16 clinically suspect sheep. Histopathological confirmation was based on presence of multiple or single vacuolation of the neuronal cytoplasm and presence of sufficient spongiform changes to indicate spongiform cephalopathy (Wells and McGill, 1992).

the the the en-

Electron Microscopy (EM) for SAFs, and Western Immunoblotting Analysis (WBA) for PrPSc Characteristic SAFs (Fig. 1) were identified and a fibril detection score was obtained for the samples declared positive by EM. Samples were declared positive by WBA if bands corresponding to the characteristic labelling pattern for abnormal PrPSc protein (molecular mass 27–30 kDa) were present in proteinase K-treated samples, and negative if no labelling was present. A representative immunoblot is shown in Fig. 2. The SAF and WBA results for the samples tested over the 2-year period are shown in Table 1 (+4°C) and Table 2 (−70°C). At both storage temperatures, throughout the 2-year period, SAF- and WBA-positive results were recorded in most, but not all, of the samples, regardless of the preparation method and pre-treatment used. Comparison of the efficacy of the two detection methods showed no significant difference (P>0·45) at either storage temperature. At +4°C, 90% and 92% of samples gave positive results by EM and WBA, respectively; the corresponding figures at −70°C were 96% and 91%, respectively. Comparison of the results obtained for the two pre-treatments (protease inhibitors or DMSO), and for the homogenized versus chopped samples, also showed no significant differences (P>0·19). However, there was a marked variability in the recorded SAF scores, and at −70°C the trend towards progressively stronger positives was significant (P=0·002). There were also some differences in the clarity and morphology of the fibrils observed by EM during the 2-year period (Fig. 1). The intensity of staining of the WBA bands (and therefore the quantity of PrPSc protein) varied, but only a positive or negative result was recorded. A greater percentage

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Fig. 1.

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Negatively stained SAFs recovered from brain extracts. (a) Day 0, chopped with no pre-treatment, stored at −70°C. (b) Six months, chopped with DMSO, stored at +4°C. (c) One year, homogenized with no pre-treatment, stored at −70°C. (d) Two years, homogenized with proteinase inhibitors, stored at +4°C. EM. ×100 000.

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Fig. 2.

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Representative immunoblot of brain aliquots extracted with (+) or without (−) proteinase K treatment after storage for one year at −70°C. Lane 1: homogenized without proteinase inhibitors (PI). Lane 2: chopped with DMSO. Lane 3: homogenized without DMSO. Lane 4: chopped without PI. Lane 5: chopped with PI. Lane 6: homogenized with PI. Lane 7: homogenized with DMSO. Lane 8: chopped without DMSO. Immunolabelling was carried out with polyclonal antiserum 971 G1.

of SAF-positive results was recorded in the samples stored at −70°C (96%) than in those stored at +4°C (91%), but for WBA there was little difference (92% and 91%, respectively). Because the material came from different groups of animals, however, differences associated with storage temperature could not be assessed statistically. Discussion Reproducibility is an important factor in any diagnostic test. For the detection of SAFs and PrPSc a reliable method of storing tissue is a necessity. This study was designed to evaluate the optimum storage conditions for CNS grey matter to be used in the diagnosis of natural sheep scrapie. The results showed that, over a 2-year period, and in all the combinations of pre-treatment and storage conditions tested, SAFs and PrPSc could be detected from standardized samples of CNS tissue from the brains of naturally affected sheep. Comparison of the two detection methods employed showed no difference in sensitivity. The addition of the cryoprotectant DMSO, as used for biological material containing viruses, appeared to be unnecessary, as also was the presence of protease inhibitors. The results showed that if endogenous protease enzymes were released when the tissue was mechanically damaged, or during longterm storage, they had little or no effect on the subsequent detection of SAF and PrPSc. No discernible difference in positive results was detected between homogenized and chopped tissue; this aspect was investigated because cold storage of tissue extracts in a semi-prepared state is sometimes a useful prelude to the examination of purified SAF/PrPSc preparations. Autolysis and decomposition were encountered with all the pre-treatment samples stored at +4°C, and there was also bacterial contamination of some samples, particularly towards the end of the 2-year storage period. The results suggested that for fibril detection, storage at −70°C was preferable. There

EM WBA EM WBA EM WBA EM WBA EM WBA EM WBA EM WBA EM WBA

Test method 2 weeks +++ + ++ + +++ + ++ + +++ + + + ++ + + +

0 weeks +++ NT + NT +++ NT + NT ++ NT ++ NT + NT + NT

++ − + + ++ + − − +++ + +++ + ++ + + +

1 month ++ + − + − + − + +++ + + + + + + +

3 months + + + + + + + + +++ + +++ + + + ++ +

6 months

Results† after storage for

∗ Confirmed histopathologically. NT=Not tested. † See Materials and Methods for SAF scoring system (− to +++); WBA results are either positive (+) or negative (−).

Chopped without proteinase inhibitors

Chopped with proteinase inhibitors

Chopped without DMSO

Chopped with DMSO

Homogenized without proteinase inhibitors

Homogenized with proteinase inhibitors

Homogenized without DMSO

Homogenized with DMSO

Treatment

++ + + + + − + + ++ − + + + + − +

1 year

+++ + ++ + + + +++ + ++ + +++ + ++ + + +

2 years

Table 1 Detection of SAFs by EM and PrPSc by WBA in pre-treated grey matter brain extracts from scrapie-affected∗ sheep after storage at +4°C

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EM WBA EM WBA EM WBA EM WBA EM WBA EM WBA EM WBA EM WBA

Test method 2 weeks + + + + + + + + − + + + + + + +

0 weeks + + + + + + + + + + + + + + + +

+ + + + +++ + ++ + + + ++ + + + + +

1 month − + + + ++ + +++ + + + ++ + + + + +

3 months + + +++ + +++ + +++ + ++ − +++ − ++ + ++ −

6 months

Results† after storage for

∗ Confirmed histopathologically. † See Materials and Methods for SAF scoring system (− to +++); WBA results are either positive (+) or negative (−).

Chopped without proteinase inhibitors

Chopped with proteinase inhibitors

Chopped without DMSO

Chopped with DMSO

Homogenized without proteinase inhibitors

Homogenized with proteinase inhibitors

Homogenized without DMSO

Homogenized with DMSO

Treatment

+++ − +++ + +++ + +++ + +++ + +++ + +++ + +++ +

1 year

++ + ++ + +++ + +++ + ++ − + + + + + +

2 years

Table 2 Detection of SAFs by EM and PrPSc by WBA in pre-treated grey matter brain extracts from scrapie-affected∗ sheep after storage at −70°C

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was little difference between the two temperatures for samples tested by WBA. The fact that SAFs and PrPSc were still detectable after 2 years’ storage at +4°C corroborates previous studies (Scott et al., 1992; Race et al., 1994; Stack et al., 1995). This study again demonstrated that PrPSc is a durable marker for the TSEs, and that brain tissue can be stored in various conditions and temperatures without serious detriment to diagnostic procedures. However, although this study substantiated previous findings on the value of SAF-detection (Dawson et al., 1987; Gibson et al., 1987; Rubenstein et al., 1987; Scott et al., 1987) and WBA (Farquhar et al., 1989; Mohri et al., 1992) for the diagnosis of TSEs, the problem of false negative results (Stack et al., 1991; Wells et al., 1994) was not completely eliminated. Negative results for both the SAF and WBA tests occurred in an apparently unconnected and random pattern. In the case of SAF-detection, the scoring system showed considerable variation in the numbers of SAFs observed in the positive samples, with a trend in the samples maintained at −70°C towards stronger positives after prolonged storage (Table 2). The morphology and clarity of the fibrils, and the intensity of the WBA staining, showed some variation throughout the study, but this did not affect the results. Previous studies have demonstrated that the choice of specific brain regions and the pooling procedure is important in the diagnosis of natural sheep scrapie by SAF detection (Stack et al., 1991, 1996) and WBA (Cooley et al., 1998). In the present study, the PrPSc in some brain areas may have become diluted to an undetectable level by the inclusion of brain areas lacking the abnormal protein. Natural ovine scrapie cases may exhibit different lesion profiles (Fraser, 1993), and lower levels of PrPSc and SAFs are detected in sheep than in experimentally infected mice (Gibson et al., 1987; Rubenstein et al., 1987). This, coupled with a variation in the relative amounts of grey and white matter in certain brain areas (Hope et al., 1988), and an uneven distribution of PrPSc, may have created difficulties in obtaining a uniform concentration of the abnormal protein in all samples. The study showed that storage conditions did not appear to affect the reproducibility of results and that the most likely explanation for false positives lay in differences in PrPSc concentration and SAFs between individual tissue samples (Stack et al., 1995). It is possible, although unlikely, that operator error accounted for all the false negative results. Shortage of material precluded repetition of tests. Repeated freezing and thawing of stored samples may have an effect on SAF and PrPSc protein detection (Stack et al., 1995), but the frozen samples in the present study were thawed only once, just before testing. In conclusion, storage at +4°C and homogenization of samples are best avoided if the subsequent identification of specific tissues or brain areas is required for further studies. Nevertheless, this study showed that the detection of SAFs by EM and of PrPSc by WBA was not affected by long-term storage. The pre-treatments offered no advantage. Short-term temporary storage at temperatures below freezing, such as −20°C, is unlikely to have any adverse effect on subsequent SAF and PrPSc detection methods; in general, however, storage at −70°C would be the method of choice.

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Acknowledgements This work was funded by the Ministry of Agriculture, Fisheries and Food, UK. The authors thank the following staff at the Central Veterinary Laboratory: Mr M. Dawson for the supply of animals; members of the workgroups headed by Mrs Y. Spencer and Mr G. Wells for the histopathological assistance; Mr R. Sayers for the statistical analysis; and the staff of the Electron Microscopy Unit for excellent technical support.  1999 Crown

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Received, August 14th, 1998 Accepted, November 26th, 1998