Canada Close to Live Testing Methods

Canadian and German scientists have discovered that a single protein can accurately distinguish between healthy cows and those with mad cow disease, or bovine spongiform encephalopathy (BSE). This finding should hopefully lead to the development of a test for diagnosing BSE in live cattle, says lead researcher David Knox from the University of Manitoba, Winnipeg.

First detected in the UK in 1985, BSE is a degenerative neurological disease that affects cows. Like scrapie in sheep and Creutzfeld-Jakob Disease (CJD) in humans, BSE is a type of a transmissible spongiform encephalopathy (TSE) disease, caused by the build-up in the brain of an abnormal protein known as a prion.

Following the BSE epidemic among UK cows in the 1980s and 1990s and the discovery that humans could contract the so-called new variant CJD by eating BSE-infected meat, many countries implemented monitoring programmes to detect cows with BSE. This usually involves testing for the presence of prions in the brains of all cows older than a certain age or showing possible signs of BSE.

Understandably, this kind of test can only be conducted after the cows have been killed, which is sort of shutting the stable door after the cow has bolted. In contrast, a test that could detect BSE in live cows would permit the routine testing of all cattle. This would allow any BSE outbreak to be detected at an earlier stage and give much greater confidence that a country's cow population was entirely free of BSE.

Unfortunately, although prions are found at high concentrations in brains and spinal cords, they're present at much lower concentrations in easily testable bodily fluids such as cerebrospinal fluid, blood and urine. This has helped to stymie attempts to develop just such a live test.

But detecting prions is not the only way to diagnose BSE. Like any other disease, BSE induces a whole host of biochemical changes in infected cows, which should be reflected in the presence of characteristic protein biomakers in their bodily fluids. So Knox and his colleagues set out to find these biomarkers.

Using two-dimensional gel electrophoresis, they searched for proteins present at different concentrations in urine from four healthy and four BSE-infected cows. Finding that 56 of the over 1,300 protein spots on each of the subsequent gels were present at different concentrations in the two groups, Knox and his colleagues then used a range of multivariate statistical techniques to determine which spots were best at distinguishing between healthy and infected cows.

This uncovered a single protein spot that was able to distinguish between the two groups with perfect accuracy. Analysing this protein using mass spectrometry revealed it to be the glycoprotein clusterin, which is commonly found in a wide range of different biological tissues. Unfortunately, high concentrations of clusterin have already been linked to neurodegenerative diseases such as Alzheimer's disease, suggesting that it might not make a particularly specific BSE biomarker.

However, Knox and his team actually detected two versions of clusterin in cow urine, perhaps reflecting two different post-translational modifications of the protein, and only one of these versions was able to distinguish between the healthy and infected groups. This raises the possibility that this particular version of clusterin could make a more accurate biomarker.

Furthermore, by analysing proteins in the cow urine at six different times during the course of the BSE infection, Knox and his team discovered that changes in the concentrations of 16 of the protein spots accurately matched the progression of the disease.

'Our work shows that it is possible to identify biomarkers in urine that could be useful in the diagnosis and monitoring of disease progression in BSE,' says Knox. In addition to developing a live BSE test, Knox hopes that this work will lead to live tests for other TSE diseases, including CJD


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