Exploring the cause of initially reactive bovine brains on rapid tests for BSE

Abstract

Bovine spongiform encephalopathy (BSE) is an invariably fatal prion disease of cattle. The identification of the zoonotic potential of BSE prompted safety officials to initiate surveillance testing for this disease. In Canada, BSE surveillance is primarily focused on high risk cattle including animals which are dead, down and unable to rise, diseased or distressed. This targeted surveillance results in the submission of brain samples with a wide range of tissue autolysis and associated contaminants. These contaminants have the potential to interfere with important steps of surveillance tests resulting in initially positive test results requiring additional testing to confirm the disease status of the animal.The current tests used for BSE screening in Canada utilize the relative protease resistance of the prion protein gained when it misfolds from PrPC to PrPSc as part of the disease process. Proteinase K completely digests PrPC in normal brains, but leaves most of the PrPSc in BSE positive brains intact which is detected using anti-prion antibodies. These tests are highly reliable but occasionally give rise to initially reactive/false positive results. Test results for these reactive samples were close to the positive/negative cut-off on a sub set of test platforms. This is in contrast to all of the previous Canadian positive samples whose numeric values on these same test platforms were 10 to 100 fold greater than the test positive/negative cut-off. Here we explore the potential reason why a sample is repeatedly positive on a sub-set of rapid surveillance tests, but negative on other test platforms.In order to better understand and identify what might cause these initial reactions, we have conducted a variety of rapid and confirmatory assays as well as bacterial isolation and identification on BSE positive, negative and initially reactive samples. We observed high levels of viable bacterial contamination in initially reactive samples suggesting that the reactivity may be related to bacterial factors. Several bacteria isolated from the initially reactive samples have characteristics of biofilm forming bacteria and this extracellular matrix might play a role in preventing complete digestion of PrPC in these samples.

Presumptive BSE cases with an aberrant prion protein phenotype in Switzerland, 2011: Lack of prion disease in experimentally inoculated cattle and bovine prion protein transgenic mice

“…In the latter, skulls of cattle were defrosted with hot steam in winter to facilitate sampling of the medulla oblongata. In Canada, individual decomposed brainstem samples of fallen stock revealed a similar PrP res profile in Western immunoblot as the Swiss cases (Dudas, James, Anderson, & Czub, 2015). In that study, the authors further characterized the status of the samples using a variety of test platforms, including ones that were not relying on PK digestion, such as an affinity ligand-based immunoassay (IDEXX HerdChek BSE Antigen Test) and an ELISA test that used mild PK digestion in combination with PrP res precipitation (Bio-Rad TeSeE ELISA).…”

Conventional and State-of-the-Art Detection Methods of Bovine Spongiform Encephalopathy (BSE)

“…A weak signal may show a single diglycosylated band or a weak signal for diglycosylated and monoglycosylated bands with the absence of an unglycosylated band [ 21 , 76 ]. In very weak positive samples, the ultracentrifugation step prior to loading the samples onto an SDS-PAGE gel considerably enhances the concentration of PrP Sc , leading to a much more intense Western blot signal [ 88 ]. The use of the correct brain sample is important for correct diagnosis.…”

“…In field conditions, brainstem samples are often autolyzed and contaminated with bacteria and other debris (feces, soil, hair). Obviously, these factors have an impact on the quality of the sample and affect the performance of the diagnostic test and the results generated [ 88 ]. Meloni et al revealed that four rapid tests (Enfer TSE Kit, Bio-Rad TeSeE test, Prionics ® -Check-LIA test, and IDEXX HerdCheck BSE Antigen Test Kit EIA) can be considered as well-running diagnostics tools regardless of tissue quality [ 100 ].…”

Atypical BSE: Current Knowledge and Knowledge Gaps

“…As in atypical BSE, new types could have different characteristics and pose different risks to the health of animals and humans. Several countries have reported low level reactivity in samples submitted for BSE surveillance [32][33][34] . There is suggestion that these cases represent a yet to be identified prion disease of cattle 32) , but also proposed alternative explanations 33) .…”

“…Several countries have reported low level reactivity in samples submitted for BSE surveillance [32][33][34] . There is suggestion that these cases represent a yet to be identified prion disease of cattle 32) , but also proposed alternative explanations 33) . To definitively determine if these animals are infected with a prion disease that is transmissible, brain homogenates were intra-cranially inoculated into transgenic mice and cattle.…”