Untitled Document

Before BSE was identified, a simple grid-touch negative-staining method by electron microscope (EM) had been developed to demonstrate both Nemavirrus particels (NVP) and scrapie-associated fibrils (SAF). Full details of this work were published in 1987 (Narang, Asher and Gajdusek, Tubulofilaments in Negatively Stained Scrapie Infected Brains: Relationship to Scrapie-associated Fibrils. Proc Natl. Acad. Sci. USA-8 4:7730-7734, 1987). Subsequently many more papers were published describing in detail the simple technique using CJD human brains. I was invited to AFRC and MRC Neuropathogenesis Unit, Edinburgh in September 1987 to demonstrate the test. Preparation and examination was carried out on six randomised scrapie and normal brains and correct results were recorded on all six brains within two and a half hours from start to finish. MAFF were aware of this test because I presented the test at the 74th Meeting of the British Neuropathological Society on the 14th and 15th January 1988.

A definitive diagnosis of SE's can be made by showing the presence of NVP and SAF by a simple grid-touch negative-staining method by electron microscopy (EM). Full details of the work were published in 1987 (Proc Natl. Acad. Sci. USA-84:7730-7734, 1987). Subsequently many more papers were published describing in detail the simple technique using CJD human brains.

In a blind study, 18 hamsters were divided into 2 groups of 9 each. Group 1 hamsters were infected with the scrapie agent and group 2 were not infected and acted as controls. One hamster from each group was killed on a rota as follows: 3, 5, 7, 10, 14, 18, 21, 24, and 28 days. Examination of grids prepared from both left and right side of the brain revealed NVP and SAF in the right side of scrapie-infected hamster brains from 10 days post inoculation, and both the NVP and SAF were observed from 18 days from post inoculation from both sides of the brain. No NVP/SAF were seen in normal control hamsters.

In about 1990 when I suggested a random test and that I could collect cattle brain materials from local abattoirs, I was told by Ray Bradley that it would be illegal for me to do this and the only way of obtaining such material was through MAFF. I was therefore not able even to carry on work on this test using private funding which had been offered to me.

On many occasions in 1988 and subsequently I proposed at least random postmortem testing on cattle but random BSE testing was then judged to be of low priority because MAFF considered that a visual examination of animals, noting the overt symptoms of clinical cases before slaughter, was adequate.

Had random testing been started when I suggested the same it we would have enabled to discover the true extent of the disease and it could have been eradicated. In a study undertaken for "World in Action" in 1995 obtained 30 cattle heads from abattoirs in the Midlands. I tested 28 brains and established that 8 of the cattle tested which must have appeared healthy at slaughter, actually had BSE. The BSE would be detected by my test even though they were sub-clinical, symptom free cases. To date, MAFF has no such test of its own.

Mice inoculation studies have revealed that CJD blood has 1,000 units of infectivity per ml. I have developed a simple method to concentrate the agent from CJD patients' urine and I have by EM demonstrated the presence of both NVF and SAF similar to those seen in brain samples of CJD victims. I have used this CJD urine test on a number of live subjects who have subsequently died and were confirmed to have had CJD by the Surveillance Unit in Edinburgh.

I have developed a simple method to concentrate the agent from CJD patients' urine and I have by EM demonstrated the presence of both NVF and SAF similar to those seen in brain samples of CJD victims. I have used this CJD urine test on a number of live subjects who have subsequently died and were confirmed to have had CJD by the Surveillance Unit in Edinburgh.

Shortly after I was asked by the parents of a victim to carry out a urine test on the victim Dr Duke of MRC in October 1995 contacted the victim's relatives. Dr Duke told the relatives that the test had not been approved and suggested to the relatives that they could take a legal action against me. They did not agree with this suggestion and I duly carried out the test. Another victim's parents approached me after doctors had told them repeatedly that he was too young to be suffering from CJD and that his symptoms were not those of CJD. In January 1996, the urine test confirmed that he had CJD. Following this, his mother insisted upon the post mortem, which her doctors were advising was not necessary because they were unlikely to learn anything. Five weeks after the victim's death the post mortem confirmed CJD. In the light of this experience, it must be expected that other young individuals who have died of CJD have not been subject to a post mortem and are therefore not included in the definite statistics.

After I carried out the first urine test in 1995 on Victim 12, Dr Will of the Surveillance Unit told me that more urine specimens from CJD victims would be made available. Eventually in 1997 MRC funded a project in Leeds University to verify the urine test. However, MRC failed to supply urine specimens.

An ion-capture technique was used to demonstrate amyloid precursor protein or its segments in clinically diagnosed cases of Alzheimer's Disease. This involves concentration of APP from urine samples. The concentrate was then subjected to Western blotting. Using APP antibody it was possible to demonstrate one, two or all three APP segments from urine of Alzheimer's Disease cases but not from healthy individuals (Fig 1), this test may provide a significant contribution for diagnostic work. Similarly this technique could be applied using PrP antibody to demonstrate protease-resistant protein/SAF in BSE and CJD by Western blotting, or concentrated samples can be used to prepare grids for electron microscopic examination (Fig 2).

The urine test on Peter Hall. His parents approached me after doctors had told them repeatedly that he was too young to be suffering from CJD and that his symptoms were not those of CJD. In January 1996 the urine test confirmed that he had CJD. Following this his mother insisted upon the post mortem. which her doctors were advising was not necessary because they were unlikely to learn anything. Five weeks after Peter's death the post mortem confirmed CJD. In the light of this experience it must be expected that other young individuals who have died of CJD have not been subject to a post mortem and are therefore not included in the definite statistics. After I carried out the first urine test in 1995 on Victim 12, Dr Will of the Surveillance Unit told me that more urine specimens from CJD victims would be made available to me. Eventually in 1997 MRC funded a project in Leeds University to verify the urine test.

I have developed an alternative method to electron microscopy, western blotting to detect CJD in humans. The science involved in this technique is well understood. This method is more user friendly and is also cheaper. Hundreds of specimens can be tested within one working day and this process would be very useful for the purpose of testing blood donors. Since March 1997 I have been funded by MRC to carry out work using EM techniques. Initially they told me at a meeting with MAFF that they would also purchase western blotting equipment to develop the urine test. However, they are now very reluctant to allow me to develop western blotting at the same time and I never got the go ahead with the test.

  Figure 1          Figure 2         Figure 3

        Click on pictures to enlarge

Had any of these various tests been funded or supported in any way by Government bodies, it would have been possible to set up a slaughter house test for diagnosing sub-clinical BSE in cattle that had not yet shown the symptoms of the disease, but which were entering the national diet. In this way the disease could have been eradicated as I had explained on many occasions at the time.