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Brief Communications |
Correspondence: 1Corresponding Author: Stefanie Czub, Canadian Food Inspection Agency, BSE Reference Laboratory, Box 640, Lethbridge, Alberta T1J 3Z4, Canada. czubs{at}inspection.gc.ca
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Abstract |
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Key Words: Bovine spongiform encephalopathy • diagnosis • immunohistochemistry • prion
Bovine spongiform encephalopathy (BSE) is a fatal neurological disease of adult cattle that was first recognized in Great Britain in 1986,16 and is one of the subacute spongiform encephalopathies associated with unconventional transmissible agents or prions.9 The agent of BSE is considered the cause of variant Creutzfeldt–Jakob disease (vCJD) in humans.2,4,12 The rapid and accurate diagnosis of BSE is therefore of critical importance to control measures for human and animal health protection. When BSE surveillance was implemented in 1992 in Canada, it was based on testing mature cattle with clinical signs compatible with BSE collected at provincial and federally inspected abattoirs, from rabies-negative submissions, and from private practitioners' submissions. At the initiation of the current study (2001), the U.K. BSE surveillance program comprised passive surveillance of all cattle presenting with clinical signs compatible with BSE, irrespective of age, together with active surveillance that targeted all cattle aged 30 months or over at death or slaughter (fallen animals, on-farm casualties, and healthy slaughtered). The surveillance program later included cattle aged 24–30 months slaughtered for human consumption but presenting as casualty animals or as animals segregated at antemortem inspection as unfit. The principal diagnostic approach for confirmation of BSE was originally neuropathological examination for morphological changes of spongiform encephalopathy.18 After recognition of the diagnostic importance of the disease-associated form of the prion protein (PrPSc), immunochemical methods using polyclonal or monoclonal antibodies became standard approaches. For screening purposes these have evolved principally as high throughput ELISA or Western blot formats, but immunohistochemistry (IHC) provides a confirmatory and complementary approach that characterizes PrPSc distribution in brain to inform on disease phenotype. Because the antibodies used for IHC cannot distinguish between the cellular form of prion protein (PrPC) and PrPSc, demonstration of disease-specific immunolabeling is critically dependent on epitope demasking pretreatments and on the pattern of antibody binding. For this reason, it is important that the application and interpretation of PrP IHC for BSE diagnosis includes an understanding of the disease-specific topography and cellular patterns of the immunolabelling under defined assay conditions. In this study, the performance of a commercially available IHC assaya for the disease-associated prion protein in formalin-fixed tissue was assessed in an international collaboration between the National Centre for Foreign Animal Diseases (NCFAD; CFIA, Winnipeg, Manitoba, Canada) and the Veterinary Laboratories Agency (VLA; Weybridge, U.K.).
Samples of brain were collected over a 3-year period (1998–2001) from cattle that exhibited neurological signs at the time of slaughter or death. The diagnosis of all cases included in this evaluation was based initially on histological examination in the laboratory of origin. The study included 100 cases considered BSE positive and 500 cases considered BSE negative. Positive cases demonstrated histological evidence of spongiform changes suggestive of BSE.18 The negative cases showed no histologic evidence of spongiform encephalopathy and no evidence of PrP-Sc when screened by immunohistochemistry using MAb R145 (U.K. samples), a C-terminal specific anti-PrP rat monoclonal antibody produced at the VLA and raised against a bovine PrP peptide sequence, residues 221–23315 or 6H4 (Canadian samples), a murine monoclonal antibody raised against recombinant bovine PrP and recognizing an epitope corresponding to residues 156–164.6 Four hundred of the 500 negative samples were provided by NCFAD and 100 by the VLA. All VLA samples represented the medulla oblongata at the level of the obex. Of the 400 Canadian samples, 35% were from the medulla at obex, 46% from the medulla oblongata (nonobex), and the remaining 19% were from other areas of the brain, including the cerebellum. Samples were fixed and processed with minor protocol differences between the NCFAD and VLA laboratories, including fixation (10% neutral buffered formalin overnight vs. 10% formol saline for 48 hr), formic acid decontamination (2 hr vs. 1 hr), and embedding medium (paraffinb or paraffin/plasticc). Four consecutive 5-µm sections from each block were mounted on positively chargedd or treatede slides. Sections were coded and exchanged between laboratories. Each laboratory immunolabeled 1 set of 600 slides. All test kits were from the same lot number and were supplied to each laboratory by the manufacturer. The kit includes: a citrate buffer for antigen retrieval by hydrated autoclaving,3 a proprietary proteinase K (PK) reagent of undisclosed concentration, primary monoclonal antibody F99/97.6.18 (a murine IgG1 binding the epitope QYQRES, residues 220–225 of the bovine PrP 5-octapeptide repeat molecule), antibody diluent, antimouse–IgG–biotinylated secondary antibody, peroxidase-labeled streptavidin, and diaminobenzidine (DAB) as the substrate-chromogen. Each laboratory independently selected a single BSE case as a positive control and a single non-BSE case as a negative control; both controls were included in each run with 38 test samples. Both laboratories immunolabeled the sections using the same generic protocol, including pretreatment with 3% (v/v) hydrogen peroxide in methanol to quench endogenous peroxidase, antigen retrieval with acid (undiluted 98% formic acid) for 5 min followed by 3 rinses with Tris buffered saline (TBS, pH 7.6) and hydrated autoclaving (121°C for 20 min with the kit-supplied citrate buffer). Slides were cooled, placed onto facing plastic coverplates,f which provide a constant 100-µl volume incubation chamber at the slide surface and a 2-ml reservoir above the slide for wash solution, and transferred to a slide rackg designed to hold the assembly vertically. Care was taken to remove air bubbles from the assembly before application of reagents. Wash bufferh was added to the reservoir and the sections were washed by gravity flow of the solution. The kit-supplied PK solution was added to the sections and incubated for 60 (VLA) or 90 (NCFAD) sec. Preliminary optimization assays demonstrated that this step was time and temperature dependent and performed most reliably if the solution was removed from the refrigerator within 5–10 min of use. The PK pretreatment caused partial digestion of the tissue sections in some cases, although this did not affect the final observations. Sections were immunolabeled with sequential 10-min incubations at room temperature with primary antibody at 1 or 10 µg/ml, antimouse–IgG–biotin, and streptavidin–horseradish peroxidase complex with intervening wash buffer rinses. Five hundred microliters of DAB was added as the enzyme substrate for 3–5 min (endpoint determined macroscopically by NCFAD) or 10 min (standardized by VLA). Deionized water was added and the coverplates were removed. The slides were washed for 2 min in running tap water, counterstained with Mayer's hematoxylin, and blued in Scott's Tap Wateri (NCFAD) or tap water (VLA). After thoroughly washing in running tap water for 4 min, the slides were dehydrated and placed into xylene, then permanently mounted with mounting media.
Two pathologists and 2 technicians evaluated each coded slide using brightfield light microscopy. Samples were considered positive if there was evidence of chromogen deposits conforming to disease-associated patterns of labeling17,18 in the dorsal nucleus of the vagus nerve and the solitary tract nucleus, the regions previously described as target areas for PrPSc accumulation.18 Disease-specific labeling in samples immunolabeled with this protocol was as described previously for BSE using other antibodies3,17,18 and corresponds to some, but not all, forms of immunolabeling reported in sheep with scrapie5,7,11 using polyclonal antisera and other antibodies and in deer and elk with chronic wasting disease,13,14 in a variety of manual and automated protocols. Multifocal particulate labeling (also termed "punctate"5 or "synapse-like"18) predominated, with variably sized, irregular particles distributed in a random fashion through the neuropil without obvious association with morphologically defined cells or structures. This labeling was most commonly evident as separate fine particles but was sometimes also aggregated (Fig. 1A). Perineuronal labeling appeared similar to the particulate labeling, but was clearly distributed in close association with the neuronal membrane around the perikaryon (Fig. 1B) and bordering neurites. Throughout the sections there was glial-associated labeling (Fig. 1C). The most obvious cell-associated disease-specific labeling was a granular labeling of the perikaryonal cytoplasm of neurons. This labeling was either diffusely distributed throughout the cytoplasm or localized, sometimes with a perinuclear location. Labeled granules were usually round and variable in size (Fig. 1D). The specific labeling patterns observed in the hypoglossal nucleus were predominantly neuronal with some perineuronal particulate labeling. The dorsal nucleus of the vagus nerve demonstrated mainly coarse granular neuropil labeling and occasional intraneuronal labeling. In the reticular formation, linear particulate and coalescing labeling patterns were evident. Samples were considered negative if no chromogen deposits were observed or if the disease-associated patterns of labeling were absent.
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Acknowledgments |
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a. Bovine Spongiform Encephalopathy Antigen Test Kit, Immunohistochemistry, VMRD Inc., Pullman, WA.
b. ParaplastTM Media, McCormick Scientific, St. Louis, MO.
c. FibroWax, Van Loenen Instruments, Zaandam, The Netherlands.
d. ProbeOn Plus Slides, Fisher Scientific Company, Ottawa, Ontario, Canada.
e. VECTABONDTM Reagent, Vector Laboratories, Burlingame, CA.
f. Shandon CoverplateTM, Thermo Fisher Scientific Inc., Waltham, MA.
g. Shandon Sequenza® Slide Rack, Thermo Fisher Scientific Inc., Waltham, MA.
h. Tris-Buffered NaCl Solution with Tween 20, pH 7.6, Dako Canada Inc., Mississauga, Ontario, Canada.
i. Surgipath Medical Industries, Richmond, IL.
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