Abstract
BackgroundIn the framework of the Bovine Spongiform Encephalopathy (BSE) surveillance programme, samples with non-conclusive results using the OIE confirmatory techniques have been repeatedly found. It is therefore necessary to question the adequacy of the previously established consequences of this non-conclusive result: the danger of failing to detect potentially infected cattle or erroneous information that may affect the decision of culling or not of an entire bovine cohort. Moreover, there is a very real risk that the underreporting of cases may possibly lead to distortion of the BSE epidemiological information for a given country.In this study, samples from bovine nervous tissue presenting non-conclusive results by conventional OIE techniques (Western blot and immunohistochemistry) were analyzed. Their common characteristic was a very advanced degree of autolysis. All techniques recommended by the OIE for BSE diagnosis were applied on all these samples in order to provide a comparative study. Specifically, immunohistochemistry, Western blotting, SAF detection by electron microscopy and mouse bioassay were compared. Besides, other non confirmatory techniques, confocal scanning microscopy and colloidal gold labelling of fibrils, were applied on these samples for confirming and improving the results.ResultsImmunocytochemistry showed immunostaining in agreement with the positive results finally provided by the other confirmatory techniques. These results corroborated the suitability of this technique which was previously developed to examine autolysed (liquified) brain samples. Transmission after inoculation of a transgenic murine model TgbovXV was successful in all inocula but not in all mice, perhaps due to the very scarce PrPsc concentration present in samples.Electron microscopy, currently fallen into disuse, was demonstrated to be, not only capable to provide a final diagnosis despite the autolytic state of samples, but also to be a sensitive diagnostic alternative for resolving cases with low concentrations of PrPsc.ConclusionsDemonstration of transmission of the disease even with low concentrations of PrPsc should reinforce that vigilance is required in interpreting results so that subtle changes do not go unnoticed. To maintain a continued supervision of the techniques which are applied in the routine diagnosis would prove essential for the ultimate eradication of the disease.
Highlights
In the framework of the Bovine Spongiform Encephalopathy (BSE) surveillance programme, samples with non-conclusive results using the OIE confirmatory techniques have been repeatedly found
The samples included in this study were 5 bovine nervous tissues corresponding to the Spanish active surveillance programme which had presented a non-conclusive result by immunohistochemistry and Western blotting routinely applied for confirming BSE cases
All the confirmatory techniques established by the OIE were applied on problem non-conclusive samples and were modified in order to increase the sensitivity to detect foreseeable low concentrations of PrPsc
Summary
In the framework of the Bovine Spongiform Encephalopathy (BSE) surveillance programme, samples with non-conclusive results using the OIE confirmatory techniques have been repeatedly found. Samples from bovine nervous tissue presenting non-conclusive results by conventional OIE techniques (Western blot and immunohistochemistry) were analyzed. Their common characteristic was a very advanced degree of autolysis. Bovine Spongiform Encephalopathy (BSE) belongs to a group of neurodegenerative diseases termed Transmissible Spongiform Encephalopathies (TSEs). It is characterized by a chronic course (4–5 years of incubation period) and lethal end [1]. PrPsc detection is possible due to its partial resistance to proteinase K digestion and shows a characteristic glycoform pattern by applying Western blotting techniques. PrPsc deposits appear along with neuronal loss and gliosis, even prior to the clinical symptoms [7] which mainly consist of apprehension or aggressive behavior, ataxia, trouble standing up, decreased milk production and weight loss [8]
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