RAPID COMMUNICATION |
Correspondence to: Juan J. Badiola, National Reference Centre for Transmissible Spongiform Encephalopathies, Dept. of Pathological Anatomy of the School of Veterinary Medicine, Miguel Servet 177, 50013 Zaragoza, Spain. E-mail: badiola@posta.unizar.es
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Summary |
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Although detection of the abnormal isoform of prion protein (PrPsc), the specific feature of transmissable spongiform encephalopathies (TSEs), has been previously demonstrated on formalin-fixed autolytic tissue, no samples with autolysis as severe as tested here (i.e., liquid state) have previously been tested. It is inevitable that a small but significant proportion of brains, especially in summer due to delays in postmortem examination, undergo an extremely severe autolysis that makes samples unsuitable for diagnosis by conventional techniques. In this study, 25 bovine samples were diagnosed by applying immunocytochemistry on the corresponding liquid fraction. Four additional portions of brainstem (positive and negative sheep and cattle) were subjected to one of the autolysis regimens at 56C or environmental conditions for up to 80 days and were analyzed with the same methodology. No abnormal protein could be detected in any of the control animals. PrPsc accumulation was observed by immunocytochemistry in all cases that were positive by either immunohistochemistry on the corresponding filtrates or by Prionics Western blotting, showing an excellent agreement between the methodology assessed and these routine techniques. The results of this study demonstrate immunocytochemistry as a useful tool for diagnosis in liquid-state samples, solving a most relevant problem in BSE and scrapie epidemiology. (J Histochem Cytochem 51:1518, 2003)
Key Words: autolysis, liquid state, BSE, scrapie, immunocytochemistry, PrPsc
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Introduction |
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THE DIAGNOSIS of sheep scrapie and bovine spongiform encephalopathy (BSE), as well as other transmissible spongiform encephalopathies (TSEs), is based on the histopathological finding of classical vacuolation located in specific areas of the brainstem (
The detection of the abnormal isoform of prion protein (PrPsc), TSE's specific feature (
However, the question of whether the disease specific protein is also detectable in fully autolyzed tissues (i.e., liquid state) has not been yet studied. To our knowledge, only a few experiments have been performed on such samples but have always been applied on formalin-fixed filtrates obtained from the autolyzed tissue (
In this study, 25 positive and negative samples were used to assess whether immunocytochemistry using monoclonal antibodies (L42 and 6H4) is able to detect PrPsc in liquid-state neural tissue. Furthermore, the effects of controlled autolysis on the results obtained by this technique were assessed in this study. In this last experiment, scrapie samples were also included because of the relevance of analysis of these animals in the new ovine surveillance program.
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Materials and Methods |
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Samples
Five brainstem samples from suspect cattle aged over 24 months submitted by several regional diagnostic laboratories for BSE confirmation were used. Twenty control negative samples of the same age were provided by the Regional Laboratory for TSEs in Aragón.
All samples presented a very advanced degree of autolysis (mostly liquid state) and were obtained through the Spanish BSE Surveillance Programme, i.e., dead cattle on farm, during the summer of 2001. One subsample of each of them was analyzed using Prionics Western immunoblotting (
Controlled Autolysis
A portion of undamaged brainstem comprising 1 g of tissue was taken caudally to the obex belonging to one positive and one negative BSE cases and one positive and one negative scrapie cases (all of them previously confirmed by immunohistochemistry) and placed in containers. The samples were then subjected to controlled treatments as follows. One sample from each animal was incubated at 56C and another one subjected to the environment (in a screened outdoor enclosure in full sunlight; the ambient temperature exceeded 37C on several days). After incubation or exposure, respectively, an aliquot or swab (in cases when the liquid state was not yet present) was recovered twice a week for 80 days, for PrPsc detection by immunocytochemistry according to the methodology described below.
Immunocytochemistry
A swab from each sample was diluted and vortexed in 1 ml of TBS (0.5 M Tris, 8% NaCl; pH 7.6) and a 40-µl subaliquot from this dilution, together with two subsequent serial decimal dilutions, was dispensed onto Vectabond-pretreated glass slides to optimize the dilution for microscopic assessment. After 24 hr of drying at 56C, samples were immersed in formalin 10% for 1 hr and subjected to the same protocol applied for immunohistochemistry, as described below.
PrPsc detection using a monoclonal antibody was performed following pretreatment as previously described (
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Results |
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PrPsc deposits, presenting a granular type in all cases, were detected in five samples with both monoclonal antibodies by immunocytochemistry (Fig 1A). Because of the advanced degree of autolysis in all samples analyzed, neither protein accumulation was identifiable anatomically, nor were tissue components (neurons or neuropil) distinguished. Consequently, it was not possible to assess whether immunolabeling was confined to gray matter or whether it extended to white matter, and most affected nuclei were impossible to distinguish. No positive staining was observed in 20 samples (Fig 1B), even though background immunostaining was observed in some of them (nonspecific staining was always distinguished from the characteristic granule-positive deposits). There was excellent agreement between these results and those obtained by immunohistochemistry on fixed tissue filtrates and by Prionics WB on fresh subsamples. A single disagreement arose when the Prionics immunoblotting technique was used. One truly positive sample gave a positive result by immunocytochemistry but a negative result by Prionics WB. Nevertheless, PrPsc was also detected when concentration and no proteinase K digestion was followed.
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Concerning the controlled autolysis experiment, PrPsc was still detectable on the positive samples by immunocytochemistry even after 80 days of environmental exposure, although decreasing with time. No PrPsc was detected in the sample from the control animal at day 0 or at any other interval during this treatment. Moreover, there was no difference in the detection of PrPsc in the samples exposed compared with the others heated at 56C.
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Discussion |
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On the basis of the results obtained in this study, immunocytochemistry is a useful tool for diagnosis in very severely autolyzed samples, particularly those in a liquid state, which present such difficulty for analysis. Several experiments have confirmed that neither immunohistochemistry (
The specificity of immunolabeling is validated for the absence of any PrPsc deposits in BSE-negative samples. The greater difficulty of interpretation posed by the degree of background staining in the autolyzed material, corresponding to some samples examined, did not compromise the accurate final diagnosis because nonspecific staining could never be confused as a positive deposit because of the characteristic granular type of the latter. In spite of experiments previously performed that had shown no background labeling (
Furthermore, the resistance of the autolyzed material to all pretreatments for antigen unmasking has been confirmed in this experiment. Formic acid incubation, autoclaving, and proteinase K digestion were compatible, as previously described (
On the other hand, the strong agreement between the routine methods (Prionics WB and IHC) and the technique assessed in this study concerning all the samples analyzed confirms the accuracy of the latter on liquid samples, the main objective of the experiment. The one disagreement found between them (although disappearing as indicated above) might suggest a failure of the Prionics immunoblotting technique in such severely autolyzed material. Nevertheless, further studies will be necessary to overcome this potential problem.
This work should encourage laboratories to address the problem of those cattle and sheep brains that present extreme difficulties for TSE analysis because of a severe degree of autolysis. It is important to note that such difficulty involves a consequent loss of data for epidemiological studies about such a relevant disease. Furthermore, the results recorded here should also provide confirmation of immunocytochemistry as an appropriate diagnostic tool for such samples.
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Acknowledgments |
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Supported by a grant from the European Commission (FAIR 98-7021).
We gratefully thank Dr Groschup for kindly supplying antibody, regional laboratories for TSEs for providing the samples, and the technical staff (National Reference Centre and Regional Laboratory in Aragón) for assistance in processing the samples.
Received for publication August 9, 2002; accepted September 11, 2002.
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Literature Cited |
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