NR AREP
AU Brenig,B.; Schütz,E.; Iakoubov,L.; Wemheuer,W.; Schulz-Schaeffer,W.J.; Urnovitz,H.B.
TI Serum nucleic acids directly correlate with BSE risk
QU TSE-Forum, 4. Kongress - Nationale TSE-Forschungsplattform, Düsseldorf 28.10.-29.10.2004, Poster DIA-02
PT Konferenz-Poster
AB
Background: Bovine spongiform encephalopathy (BSE) is a progressive neurodegenerative disease in cattle that presents a public health concern to humans. Current BSE tests are performed only at necropsy (post mortem) and therefore limit efforts to eliminate BSE animals from living herds. The post mortem tests are designed to detect the presence of a mis-folded normal protein, PrPres, in the brain. Because of the lack of natural antibodies to PrPres, immunoassays have limited value for an ante mortem epidemiologic approach. Therefore, a circulating nucleic acids (CNA) test protocol was selected for validation. Serum and plasma nucleic acids were extracted and run in a PCR based differential display assay. This approach of using a PCR based CNA test, specifically, The Göttingen Living Test (GLT), showed a direct correlation of reactivity with BSE-susceptibility.
Test populations: BSE confirmed cows (N=4), BSE cohort cattle (N=176) and PrPres negative normal cattle from local herds or a local slaughterhouse (N=908). The BSE cohorts were associated with nine different BSE confirmed cases.
Sample preparation: Cow serum or plasma was processed within 15 hours of collection and kept frozen until immediately before PCR testing.
CNA PCR: Three µL of the extracted CNA were used in a PCR in a total volume of 20 µL with primers CHX-1F and CHX-1R (1 µM). After the enzymatic amplification, a SybrGreenI derived melting curve was recorded in a MX4000 PCR system. The area under the curve (AUC) of the derived melting function -d(F)/dT between 87 ° and 90 ° was used for analysis. Reactivity of each individual sample was calculated on the basis of an AUC above the detection limit, which is defined as mean + 5 standard deviations above baseline of non-template/normal controls. PCR was repeated in duplicate on retained reactive samples.
Results: All four BSE confirmed samples were repeatedly reactive (RR) in the GLT. The different cohorts had a range of 33% to 91% RR individuals per cohort. The PrPres negative healthy cows had a RR rate of 0.55%. The BSE cohorts had an average RR rate of 61% and were statistically highly different from the normal controls (p<0.001).
Conclusion: The results of this study suggest that using a PCR CNA approach for a BSE ante mortem test is feasible. Furthermore, the high GLT reactivity (61%) of BSE cohorts supports the current practice of culling BSE cohorts. Based on the official German statistics, BSE feeding cohorts are approximately 100 fold more likely to include a PrPres cow than the normal population, which may be attributed to the ingestion of similar contaminated feed or co-exposures to other environmental factors. This study suggests that BSE and BSE co-exposed cohorts share similar host genetic responses to etiopathologic agents associated with BSE, such as PRPres. The isolation or culling of GLT reactive cattle should have a significant impact on the eradication BSE in living herds.
AD Bertram Brenig, Ekkehard Schütz, Wilhelm Wemheuer, Howard B. Urnovitz, Institute of Veterinary Medicine, University of Göttingen, 37073 Göttingen, Germany; Ekkehard Schütz, Howard B. Urnovitz, Chronix Biomedical GmbH, 37073 Göttingen, Germany; Leonid Iakoubov, Chronix Biomedical Inc., San Jose, CA 95112, USA; Walter Schulz-Schaeffer, Dept. of Neuropathology, University of Göttingen, 37075 Göttingen, Germany
SP englisch
PO Deutschland
OR Tagungsband