1 Department of Neurological and Visual Sciences, University of Verona, Piazzale L. A. Scuro, 10, 37134 Verona, Italy
2 CEA Istituto Zooprofilattico del Piemonte, Liguria e Valle dAosta, Via Bologna 148, 10154 Torino, Italy
3 Laboratorio di Biologia delle Malattie Neurodegenerative, Istituto di Ricerche Farmacologiche Mario Negri, Via Eritrea 62, 20157 Milano, Italy
4 Institute of Pathology, Case Western Reserve University School of Medicine, Cleveland, OH 44106-1712, USA
Correspondence
Maria Caramelli
cea{at}to.izs.it
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ABSTRACT |
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INTRODUCTION |
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Over the last 15 years, clinical, genetic and pathological studies of field sheep and goat scrapie, as well as of iatrogenic scrapie, have been reported in Italy (Mechelli & Mantovani, 1988; Capucchio et al., 1998
, 2001
; Vaccari et al., 2001
). However, strain typing and molecular PrPSc characterization studies in Italian scrapie are still lacking.
The aim of this study was to perform a molecular characterization of PrPSc of sheep and goats collected from a single Italian flock with iatrogenic scrapie and to compare physico-chemical properties of PrPSc types with Italian field scrapie.
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METHODS |
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Sample preparation.
Different areas of the brain, including the brainstem, thalamus and cortex, were obtained. Each sample was dissolved in 9 vols of lysis buffer (0·5 % sodium deoxycholate, 0·5 % Nonidet P-40 and 10 mM EDTA in TBS) at pH 4, 7·4 and 8, respectively, and clarified by centrifuging at 1000 g for 10 min. The supernatant was stored at -80 °C until use; the pellet was discarded.
PK and N-glycosidase F treatments.
Protease resistance was assayed by incubating aliquots of brain homogenate with 50 µg PK ml-1 (Boehringer Mannheim) at 37 °C for 1 h. Digestion was blocked by the addition of PMSF to 3 mM. For N-deglycosylation, samples were treated with N-glycosidase F (PNGase F), according to the manufacturer's instruction (Boehringer Mannheim).
Immunoblot analysis.
For SDS-PAGE, sample aliquots were dissolved in sample buffer (3 % SDS, 3 % -mercaptoethanol, 2 mM EDTA, 10 % glycerol and 62·5 mM Tris, pH 6·8) and boiled for 5 min. An equivalent of 0·5 mg of wet tissue was loaded on 12 % SDS-polyacrylamide gels and, after separation, proteins were transferred onto PVDF membranes (Immobilon-P, Millipore) for 2 h at 60 V. Membranes were blocked with 1 % non-fat dry milk in TBST (TBS with 0·1 % Tween 20) and incubated overnight at 4 °C with anti-PrP antibody 8C6 (1 : 1000), which recognizes the amino acid sequence 145167 (Zanusso et al., 1998
). After washing, the membranes were incubated with a peroxidase-conjugated anti-mouse IgG, developed using an enhanced chemiluminescence system (ECL, Amersham) and visualized using Biomax MR films (Eastman Kodak). Films were scanned using a densitometer (GS700, Bio-Rad) and data were analysed using Excel (Microsoft).
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RESULTS |
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Finally, in two sheep from the infected flock (group 3), the isoforms with the 20 and 17 kDa unglycosylated PrPSc fragments were detected in different areas of the brain. As shown in Fig. 1(c), PK digestion of brainstem homogenates generated three bands migrating at 32, 25 and 20 kDa, with glycoform ratios very similar to those detected in the former groups. Conversely, immunoblots of cerebral cortex homogenates showed the presence of PK-resistant PrPSc forms that migrated to about
3 kDa less than the above isoforms, all shifting to the
17 kDa zone following PNGase treatment (Fig. 1c
, lanes 3 and 4). Interestingly, in addition to differences in electrophoretic mobility, PrPSc products obtained from cerebral cortex extracts showed a distinct glycosylation profile, since they were mainly detected as heavily glycosylated forms (Table 1
and Fig. 3
).
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Although a correlation between distinct immunoblot patterns and neuropathological changes was expected in all three groups, the very low spongiform change score detected in the frontal and parietal cortices of all sheep (Caramelli et al., 2001) did not allow such an analysis. Ongoing transmission studies will elucidate the biological relevance of the present biochemical findings.
PrPsc typing in goats with iatrogenic scrapie
Results obtained in goats were comparable to those observed in sheep. In one goat, the PrPSc isoform with the 20 kDa unglycosylated fragment was detected in all areas of the brain (Fig. 2a), the relative percentage of glycoforms overlapping that seen in group 1 sheep (Fig. 3
). In one goat, the 20 and 17 kDa PrPSc co-occurred in all areas of the brain (Fig. 2b
), whereas in four goats, similar to group 3 sheep, the two PrPSc types were detected in the brainstem and in the cerebral cortex, respectively (Fig. 2c
). In summary, both sheep and goats from the vaccinated flock shared two identical PK-resistant PrPSc types, which have only slightly different patterns of glycosylation (Fig. 3
).
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DISCUSSION |
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Current molecular diagnosis of human and animal prion diseases relies strongly on Western blot detection of PrPSc, whereas the characterization of the prion phenotype is based mainly on the size of the core fragment and the ratio of the three PrPSc glycoforms.
Distinct PK cleavage sites of the PrPSc in different prion strains were demonstrated first in two agents responsible for transmissible mink encephalopathy (TME), named hyper (HY), with a 21 kDa PrPSc unglycosylated fragment, and drowsy (DY), with a 19 kDa PrPSc backbone (Bessen & Marsh, 1994). Interestingly, following experimental co-infection with both TME strains, Bartz et al. (2000)
have demonstrated that either the DY or the HY propagates in recipient hamsters, with some animals showing the co-presence of both strains. Similar findings have been reported in human CJD, where two different PrPSc types, with a molecular mass of 19 and 21 kDa, respectively, have been shown to co-occur in some subjects (Parchi et al., 1999
; Puoti et al., 1999
). Taken together, the co-occurrence of two different PrPSc types in iatrogenic scrapie suggests that the pool of mammary gland and brain homogenates used for vaccination might have contained two different prion strains.
It is possible, however, that the 20 kDa isoform represents a brain-derived PrPSc type, whereas the isoform that migrates faster corresponds to a peripherally derived PrPSc type. Consistent with the foregoing hypothesis is the finding that the size of the unglycosylated PrPSc fragment migrates faster in lymphoid organs as opposed to brain tissues from sheep with natural scrapie (Madec et al., 2000).
Recently, PrPSc typing, based primarily on the size of the PK-resistant unglycosylated PrPSc fragment, has been performed in contemporary and archival brain tissues from experimental sheep BSE and sheep scrapie (Hope et al., 1999). According to this study, one isolate of natural scrapie, CH1641, with a fragment size of 22·7 kDa (type C pattern), was found to show a migration pattern similar to that seen with experimental sheep BSE. On the contrary, most of the natural sheep scrapie had a type B pattern (20·2 kDa), with a single sheep showing a type A pattern (24 kDa) shared with the SSBP/1 strain. Our findings in Italian natural sheep scrapie and in animals from the infected flock are consistent with a type B pattern, as to the fragment size and the glycosylation pattern of the 20 kDa isoform. Conversely, the 17 kDa PrPSc species is likely distinct from scrapie-associated PrPSc types described previously, with molecular masses ranging from 19 to 24 kDa (Hope et al., 1999
, 2000).
To date, no consensus exists on the electrophoretic PrPSc pattern of experimental BSE-infected sheep, which has been reported as being both similar (Hope et al., 1999; Baron et al., 2000
) and different (Hill et al., 1998
) from the CH1641 scrapie isolate. To overcome the limitations of conventional molecular PrPSc typing, we propose a combined approach that includes, in addition to analysis of fragment length, the conformational stability of PrPSc at acidic and basic pH.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Baron, T. G. M., Madec, J.-Y., Calavas, D., Richard, Y. & Barillet, F. (2000). Comparison of French natural scrapie isolates with bovine spongiform encephalopathy and experimental scrapie infected sheep. Neurosci Lett 284, 175178.[CrossRef][Medline]
Bartz, J. C., Bessen, R. A., McKenzie, D., Marsh, R. F. & Aiken, J. M. (2000). Adaptation and selection of prion protein strain conformations following interspecies transmission of transmissible mink encephalopathy. J Virol 74, 55425547.
Bessen, R. A. & Marsh, R. F. (1994). Distinct PrP properties suggest the molecular basis of strain variation in transmissible mink encephalopathy. J Virol 68, 78597868.[Abstract]
Bruce, M. E. (1993). Scrapie strain variation and mutation. Br Med Bull 48, 822838.
Bruce, M. E., McConnell, I., Fraser, H. & Dickinson, A. G. (1991). The disease characteristics of different strains of scrapie in Sinc congenic mouse lines: implications for the nature of the agent and host control of pathogenesis. J Gen Virol 72, 595603.[Abstract]
Bruce, M. E., Will, R. G., Ironside, J. W. & 10 other authors (1997). Transmissions to mice indicate that new variant CJD is caused by the BSE agent. Nature 389, 498501.[CrossRef][Medline]
Bruce, M. E., Boyle, A., Cousens, S., McConnell, I., Foster, J., Goldmann, W. & Fraser, H. (2002). Strain characterization of natural sheep scrapie and comparison with BSE. J Gen Virol 83, 695704.
Capucchio, M. T., Guarda, F., Isaia, M. C., Caracappa, S. & Di Marco, V. (1998). Natural occurrence of scrapie in goats in Italy. Vet Rec 143, 452453.[Medline]
Capucchio, M. T., Guarda, F., Pozzato, N., Coppolino, S., Caracappa, S. & Di Marco, V. (2001). Clinical signs and diagnosis of scrapie in Italy: a comparative study in sheep and goats. J Vet Med A Physiol Pathol Clin Med 48, 2331.[Medline]
Caramelli, M., Ru, G., Casalone, C., Bozzetta, E., Acutis, P. L., Calella, A. & Forloni, G. (2001). Evidence for the transmission of scrapie to sheep and goats from a vaccine against Mycoplasma agalactiae. Vet Rec 148, 531536.[Medline]
Foster, J. D., Hope, J. & Fraser, H. (1993). Transmission of bovine spongiform encephalopathy to sheep and goats. Vet Rec 133, 339341.[Medline]
Goldmann, W., Martin, T., Foster, J., Hughes, S., Smith, G., Hughes, K., Dawson, M. & Hunter, N. (1996). Novel polymorphisms in the caprine PrP gene: a codon 142 mutation associated with scrapie incubation period. J Gen Virol 77, 28852891.[Abstract]
Hill, A. F., Desbruslais, M., Joiner, S., Sidle, K. C. L., Gowland, I., Collinge, J., Doey, L. J. & Lantos, P. (1997). The same prion strain causes vCJD and BSE. Nature 389, 448450.[CrossRef][Medline]
Hill, A. F., Sidle, K. C. L., Joiner, S., Keyes, P., Martin, T. C., Dawson, M. & Collinge, J. (1998). Molecular screening of sheep for bovine spongiform encephalopathy. Neurosci Lett 255, 159162.[CrossRef][Medline]
Hope, J., Wood, S. C., Birkett, C. R., Chong, A., Bruce, M. E., Cairns, D., Goldmann, W., Hunter, N. & Bostock, C. J. (1999). Molecular analysis of ovine prion protein identifies similarities between BSE and an experimental isolate of natural scrapie, CH1641. J Gen Virol 80, 14; corrigendum 81, 851.
Madec, J. Y., Groschup, M. H., Calavas, D., Junghans, F. & Baron, T. (2000). Protease-resistant prion protein in brain and lymphoid organs of sheep within a naturally scrapie-infected flock. Microb Pathog 28, 353362.[CrossRef][Medline]
Mechelli, L. & Mantovani, A. (1988). Scrapie in sheep in central and southern Italy. Vet Res Commun 12, 165167.[Medline]
Parchi, P., Giese, A., Capellari, S. & 15 other authors (1999). Classification of sporadic CreutzfeldtJakob disease based on molecular and phenotypic analysis of 300 subjects. Ann Neurol 46, 224233.[CrossRef][Medline]
Prusiner, S. B. (1997). Prion diseases and the BSE crisis. Science 278, 245251.
Puoti, G., Giaccone, G., Rossi, G., Canciani, B., Bugiani, O. & Tagliavini, F. (1999). Sporadic CreutzfeldtJakob disease: co-occurrence of different types of PrPSc in the same brain. Neurology 53, 21732176.
Vaccari, G., Petraroli, R., Agrimi, U. & 8 other authors (2001). PrP genotype in Sarda breed sheep and its relevance to scrapie. Arch Virol 146, 20292037.[CrossRef][Medline]
Zanusso, G., Liu, D., Ferrari, S. & 11 other authors (1998). Prion protein expression in different species: analysis with a panel of new mAbs. Proc Natl Acad Sci U S A 95, 88128816.
Zanusso, G., Farinazzo, A., Fiorini, M., Gelati, M., Castagna, A., Righetti, P. G., Rizzuto, N. & Monaco, S. (2001). pH-dependent prion protein conformation in classical CreutzfeldtJakob disease. J Biol Chem 276, 4037740380.
Received 14 August 2002;
accepted 6 December 2002.
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