ARTICLE |
Correspondence to: Sabine O.S. Debeer, AFSSA, Laboratoire d'Etudes et de Recherches en Pathologie Bovine et Hygiène des Viandes, Unité Virologie-ATNC, 31 Avenue Tony Garnier, 69364 Lyon Cedex 07, France. E-mail: s.debeer@lyon.afssa.fr
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Summary |
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Bovine spongiform encephalopathy (BSE) is a transmissible neurodegenerative disease of cattle. Clinical diagnosis can be confirmed by investigation of both spongiform changes and abnormal prion protein (PrPsc), a marker considered specific for the disease. Tissue autolysis, often unavoidable in routine field cases, is not compatible with histological examination of the brain even though PrPsc is still detectable by immunoblotting. To determine how autolysis might affect accurate diagnosis using PrPsc immunohistochemistry, we studied 50 field samples of BSE brainstem (obex) with various degrees of autolysis. We demonstrated that the antigen-unmasking pretreatments necessary for PrPsc immunohistochemistry were compatible with the preservation of autolyzed brain sections and that PrPsc detection was unaffected by autolysis, even though anatomic markers were sometimes lost. In tissue samples in which anatomic sites were still recognizable, PrPsc accumulation was detected in specific gray matter nuclei. In samples with advanced autolysis, PrPsc deposits were still observed, at least at the cellular level, as an intraneuronal pattern. We found that the sensitivity of PrPsc immunohistochemistry as a diagnostic method for BSE was undiminished even by severe tissue autolysis. (J Histochem Cytochem 49:15191524, 2001)
Key Words: autolysis, BSE, diagnosis, immunohistochemistry, prion, PrP
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Introduction |
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TRANSMISSIBLE SPONGIFORM ENCEPHALOPATHIES (TSEs) belong to a group of neurodegenerative diseases that includes CreutzfeldJakob disease (CJD) in humans, scrapie in sheep, and bovine spongiform encephalopathy (BSE) in cattle. BSE was first reported in England in 1986 (
For all these reasons, in this study we undertook to test these limits on 50 brains (of which 46 were BSE-positive cases) with varied autolysis status. Using monoclonal antibody (MAb) SAF84 (
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Materials and Methods |
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Brain Samples
Brains were collected from cattle aged 24 months and over, acquired through the French BSE surveillance program on cattle at risk, i.e., from cattle dead on farm or subjected to emergency slaughter because of sickness or accident. Sub-samples of each of these brains were frozen for biochemical analysis (brainstem) and were also fixed for immunohistochemical analysis (medulla oblongata at the level of the obex).
Forty-four brains that were positive using the Prionics test (
For every brain sample, the degree of autolysis was noted and graded as shown in Table 1. Grading was based on the macroscopic appearance of the sample (clean sections, amorphous, pieces, liquid state) as well as the ability to identify the area of the obex macroscopically when the samples were cut coronally before embedding. In addition, autolysis degree was also assessed on subjective histological criteria such as survival of tissue architecture, vascular retraction, and undefined cell borders, as used elsewhere (
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Tissue Preparation
All brain samples were maintained in fixative (buffered 10% formalin) for a minimum of 1 week. Then they were placed in tissue-embedding cassettes (samples showing an advanced state of autolysis were filtered using a nylon bag and the filtrates placed in embedding cassettes) and, after incubation in a bath of 98100% formic acid (Merk Eurolab; Darmstadt, Germany) for 1 hr, they were processed through graded ethanol to chloroform and embedded in paraffin. Sections from blocks were cut to a thickness of 5 µm, collected on treated glass slides (Starfrost; Medite Histotechnic, Burgdorf, Germany), and kept in an oven overnight at 55C.
Immunohistochemistry
The sections were subjected to pretreatments to enhance antigen retrieval and allow distinction of the pathological form of PrP from its normal cellular isoform (PrPc) (
After a wash, nonspecific antigenic sites were blocked using 1% blocking reagent (Roche Boehringer Mannheim) for 30 min at 37C. Then blocking agent was replaced by MAb SAF84 (courtesy of J. Grassi; CEA-Saclay, France) at a concentration of 0.5 µg/ml, and the sections were incubated in this for 1 hr at 37C. After washing, the secondary antibody (biotinylated goat anti-mouse; Southern Biotechnologies, Birmingham, AL) was applied at a dilution of 1:200 for 1 hr at 37C. The diluent buffer for both primary and secondary antibodies was 0.1 M PBS, pH 7.4, containing 0.001% Triton X-100. The slices were then incubated with peroxidase-labeled avidinbiotin complex (Vectastain Elite ABC; Vector Laboratories, Burlingame, CA) for 30 min at RT. Finally, peroxidase activity was revealed using a solution of diaminobenzidine intensified with nickel chloride (DAB; Zymed, San Francisco, CA). The slides were counterstained using aqueous hematoxylin solution, mounted using Eukitt, and observed under a microscope (Zeiss; Oberkochen, Germany) coupled to an image analysis workstation (Biocom; Les Ulis, France).
Omission of primary antibodies was used to check for nonspecific background staining in BSE tissues with and without autolysis and known to produce PrPsc immunolabeling.
The specificity of positive PrPsc immunolabeling was also assessed using BSE-negative bovine brain tissue, with and without autolysis.
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Results |
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In the sections from BSE-positive tissues that were adequately preserved, pathological PrPsc was detected in gray matter only, white matter being devoid of any immunostaining. Staining was particularly marked in the nucleus of the solitary tract (NST), the dorsal nucleus of the vagus nerve (DNV), the olivary nucleus (ON), the spinal tract of the trigeminal nerve (STN), the cuneate nucleus (CN), and the reticular formation (RF) (Fig 1A and Fig 1B). In particular, no background staining was observed. In BSE-negative control sections with adequate preservation, neither PrPsc nor background immunostaining was detected (Fig 1D).
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In BSE-positive tissues with obvious autolysis, PrPsc deposits were distinguished within the obex in specific sites at a higher magnification (NST, DNV, ON, STN, CN, RF; Fig 1C), whereas in the autolyzed BSE-negative cases neither PrPsc nor background immunostaining was seen (Fig 1E).
The 46 brain samples from the French BSE surveillance programs were classified into categories A to E according to the degree of autolysis and also the extent to which it was possible to identify the anatomic site. The categories were defined as follows (see Table 1): Category A represents 13 samples which were identifiable as obex and two as spinal cord, which showed no autolysis. Obex samples (n=10) with slight autolysis were grouped into Category B. Category C contained one spinal cord and nine obex samples with distinct autolysis. In all these categories, PrPsc deposits were seen in the obex in the anatomic sites described above, with no labeling elsewhere (Fig 2A12C1).
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In the three samples of spinal cord, PrPsc deposits were confined to the gray matter and no immunolabeling was seen in the white matter (Fig 2, Asp1-Asp2). Intact samples (n=8) with advanced autolysis, which were not identifiable anatomically, were grouped into Category D. Category E (n=3) represents the samples received in a liquid state and impossible to identify anatomically. In these two last categories, PrPsc deposits were discernible macroscopically on the slide (Fig 2D1 and 2D2), even from samples originally received in a liquid state (Fig 2E1 and 2E2).
At the cellular level in Categories AC, deposition of PrPsc was scattered within the neuropil in the NST, ON, or DNV, appearing as small granules (Fig 2A2 and 2B2) or as a granular delineation of neuronal processes in the RF (Fig 2C2). In addition, in this area PrPsc deposits were seen either within the cytoplasm of neurons (Fig 2, Asp3-E3) or in a perineuronal manner (Fig 2B3 and 2C3).
In Categories D and E, cells were most often very poorly preserved and PrPsc deposits were seen, either within these cells (Fig 2D3 and 2E3) or widespread within the remaining histological and cellular structures (Fig 2D2 and 2E2).
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Discussion |
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Histopathological diagnosis of BSE, based mainly on the recognition of spongiform vacuolation in the gray matter neuropil, may not be possible if the tissue sample is poorly preserved or autolyzed. It has been shown that, because of its great resistance to degradation (
In well-preserved BSE-positive brain samples at the level of the obex, PrPsc deposits were found in this study to be restricted to the gray matter, notably NST, DNV, ON, STN, CN, and RF, and this was in accordance with previous studies (
It was possible to evaluate the "mechanical" resistance of the autolyzed brain sections to pretreatments that are necessary in the PrPsc IHC protocol, because they enhance PrPsc detection and at the same time reduce or abolish the detectability of normal PrPc (
Among the gray matter nuclei accumulating PrPsc deposits, all were not systematically found to be positively labeled. This may reflect different stages in the progression of the disease at which the different nuclei are not all affected in the same time. However, the tissues analyzed here were chosen according to the degree of autolysis only and not according to the intensity of PrPsc accumulation. Therefore, we did not attempt to investigate the effects of autolysis on the intensity of PrPsc immunolabeling, which would have required tissues in which the autolytic process was controlled.
Nevertheless, in each of those BSE cases that gave weak Prionics test results (two from Categories A and B, two others from Category D), a few cells (three to ten) still undoubtedly contained PrPsc deposits, Category A and B tissues being weaker than Category E tissues. This indicated that IHC was sensitive even in weak cases. Alternatively, it has applications as a complementary method, especially to clarify cases weakly positive by a biochemical method. In a context in which methods are needed to evaluate the true prevalence of BSE in cattle, the present study demonstrates that PrPsc immunohistochemistry is a reliable diagnostic tool regardless of tissue quality and even in cases with small amounts of PrPsc.
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Acknowledgments |
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Supported by grants from the European Commission (FAIR 98-7021) and the French Ministry of Agriculture and Fisheries. Sabine Debeer was financially supported by the EU (FAIR 98-7021).
We gratefully thank Dr E. Monks (Veterinary Research Laboratories, Dublin, Ireland) for critical reading of the manuscript and valuable suggestions. We extend our grateful thanks to S. Philippe (Statistician, AFSSA-Lyon, France) for valuable comments and suggestions.
Received for publication April 27, 2001; accepted July 11, 2001.
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Literature Cited |
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---|
Bell JE (1996) Neuropathological diagnosis of human prion disease. PrP immunocytochemical techniques. In Bakker HRRM, ed. Prion Disease. Totowa, NJ, Humana Press, 59-83
Bell JE, Gentleman SM, Ironside JW, McCardle L, Lantos PL, Doey L, Lowe J, Fergusson J, Luthert P, McQuaid S, Allen , IV (1997) Prion protein immunocytochemistryUK five centre consensus report. Neuropathol Appl Neurobiol 23:26-35[Medline]
Bolton DC, McKinley MP, Prusiner SB (1982) Identification of a protein that purifies with the scrapie prion. Science 218:1309-1311[Medline]
Borras D, Pumarola M, Ferrer I (2000) Neuronal nuclear DNA fragmentation in the aged canine brain: apoptosis or nuclear DNA fragility? Acta Neuropathol (Berl) 99:402-408[Medline]
Bruce M, Chree A, McConnell I, Foster J, Pearson G, Fraser H (1994) Transmission of bovine spongiform encephalopathy and scrapie to mice: strain variation and the species barrier. Phil Trans R Soc London 343:405-411. [B][Medline]
Demart S, Fournier JG, Creminon C, Frobert Y, Lamoury F, Marce D, Lasmezas C, Dormont D, Grassi J, Deslys JP (1999) New insight into abnormal prion protein using monoclonal antibodies. Biochem Biophys Res Commun 265:652-657[Medline]
Farquhar CF, Somerville RA, Ritchie LA (1989) Post-mortem immunodiagnosis of scrapie and bovine spongiform encephalopathy. J Virol Methods 24:215-222[Medline]
Fix AS, Garman RH (2000) Practical aspects of neuropathology: a technical guide for working with the nervous system. Toxicol Pathol 28:122-131[Medline]
Lignereux Y (1986) Atlas Stéréotaxique de l'Encéphale de la Vache Frisonne (Bos taurus L.). Anatomie. Toulouse, Université Paul Sabatier
Merz P, Somerville R, Wisniewski H, Iqbal K (1981) Abnormal fibrils from scrapie-infected brain. Acta Neuropathol (Berlin) 51:63-74
Orge L, Simas J, Fernandes A, Ramos M, Galo A (2000) Similarity of the lesion profile of BSE in Portuguese cattle to that described in British cattle. Vet Rec 147:486-488[Medline]
Prusiner SB, Füzi M, Scott MR, Serban D, Serban H, Taraboulos A, Gabriel J-M, Wells GAH, Wilesmith JW, Bradley R, DeArmond SJ, Kristensson K (1993) Immunologic and molecular biologic studies of prion proteins in bovine spongiform encephalopathy. J Infect Dis 167:602-613[Medline]
Schaller O, Fatzer R, Stack M, Clark J, Cooley W, Biffiger K, Egli S, Doherr M, Vandevelde M, Heim D, Oesch B, Moser M (1999) Validation of a Western immunoblotting procedure for bovine PrPSc detection and its use as a rapid surveillance method for the diagnosis of bovine spongiform encephalopathy (BSE). Acta Neuropathol (Berl) 98:437-443[Medline]
Scott AC, Wells GAH, Stack MJ, White H, Dawson M (1990) Bovine spongiform encephalopathy: detection and quantitation of fibrils, fibril protein (PrP) and vacuolation in brain. Vet Microbiol 23:295-304[Medline]
Simmons MM, Harris P, Jeffrey M, Meek SC, Blamire IWH, Wells GAH (1996) BSE in Great Britain: consistency of the neurohistopathological findings in two random annual samples of clinically suspect cases. Vet Rec 138:175-177[Medline]
Taylor DM (2000) Inactivation of transmissible degenerative encephalopathy agents: a review. Vet J 159:10-17[Medline]
Van Everbroeck B, Pals P, Martin J-J, Cras P (1999) Antigen retrieval in prion protein immunohistochemistry. J Histochem Cytochem 47:1465-1470
Wells GAH, Hawkins SA, Green RB, Austin AR, Dexter I, Spencer YI, Chaplin MJ, Stack MJ, Dawson M (1998) Preliminary observations on the pathogenesis of experimental bovine spongiform encephalopathy (BSE): an update. Vet Rec 142:103-106[Medline]
Wells GAH, Scott AC, Johnson CT, Gunning RF, Hancock RD, Jeffrey M, Dawson M, Bradley R (1987) A novel progressive spongiform encephalopathy in cattle. Vet Rec 121:419-420[Medline]
Wells GAH, Spencer YI, Haritani M (1994) Configurations and topographic distribution of PrP in the central nervous system in bovine spongiform encephalopathy: an immunohistochemical study. Ann NY Acad Sci 724:350-352[Medline]
Wells GAH, Wilesmith JW, McGill IS (1991) Bovine spongiform encephalopathy: a neuropathological perspective. Brain Pathol 1:69-78[Medline]