A new diagnostic test that claims to detect Mad Cow Disease in living animals before symptoms appear also raises questions on the cause of the disease Dr. Mae-Wan Ho
It has been 20 years since Mad Cow Disease (bovine spongiform encephalopathy, BSE) appeared in Britain, killing more than180 000 cattle, and causing the mass slaughter of a further 5 million. The disease has jumped species to human beings, resulting in some 160 known cases worldwide of the fatal variant Creutzfeldt-Jakob Disease (vCJD); although the precise extent of the CJD epidemic is suspected to be 20 times worse than appears (see Box).
The disease agent, according to the current establishment view, is a highly unusual misfolded protein, prion, which both causes and transmits the disease. Prion proteins are present in the brain tissues of all healthy animals in the correctly folded form. However, on being exposed to the misfolded form, the correctly folded normal protein becomes misfolded, causing it to aggregate into dense fibres, clogging up the cells and triggering a degenerative disease that turns the brain into a sponge.
There has been little progress in diagnosis or treatment for either BSE or vCJD. The only available tests are those done post mortem on brain tissue from slaughtered animals, based on detecting the misfolded prion protein that’s found after the disease has progressed to a late stage. This not only underestimates the cases of BSE, but can also allow infected cattle to pass into the human food chain. A tiny amount of misfolded prion protein may be sufficient to make a healthy animal’s own correctly folded prion protein to misfold.
A number of laboratories have been trying to develop a test that can detect BSE in live animals before the disease symptoms appear; nearly all based on improving the sensitivity of detecting prion proteins.
A brief history of Mad Cow Disease
Mad Cow Disease first appeared in Britain in the mid1980s, where it was officially diagnosed in 1986 as bovine spongiform encephalopathy (BSE), as it turns the brain into a sponge-like mass . It killed over 180 000 cattle and devastated the British beef industry and farmers. Humans have contracted variant Creutzfeldt-Jakob Disease (vCJD), a disease most closely similar to BSE, by eating meat from infected animals.
From Britain the epidemic spread to the rest of Europe infecting over 4 200 cattle in 19 countries by mid-2003. As the disease has jumped species barriers, infecting and killing humans, the European authorities have destroyed more than 5 million potentially infected cattle as a precautionary measure. Since 1996, cattle over 30 months old have been banned from entering the food chain, a measure that is thought to remove over 99 percent of infected cattle . Nevertheless, infected cattle have appeared in Canada, Japan, Israel, Oman and the Falkland Islands; and in the United States at the end of 2003 .
By 2003, more than 150 people have contracted vCJD: 143 in the UK, 6 in France, 2 in Canada and one each in Ireland, Italy and the US . (The figure for UK has increased to 157 at the end of July 2005, and cases of human contracting vCJD from blood transfusions have been discovered .) Variant CJD tends to strike young people, is invariably fatal and takes about 14 months to kill its victim. Classic CJD strikes mainly the elderly. Recent evidence suggests that BSE can cause both classic as well as variant CJD, which may explain the rising numbers of CJD cases in Europe, and the disturbing trend to younger CJD cases in the US. Several autopsy studies in the US suggest that 3 to 13% of patients diagnosed with Alzheimer’s or dementia are actually CJD cases; thus, at least 120 000 CJD cases may go undetected and excluded from official statistics . Similarly, a team of UK scientists found that 3 out of 12 674 stored appendix and tonsil samples showed evidence of infection, which gives an estimate of about 3 800 individuals in the UK who would test positive [6, 7].
Mad Cow Disease, CJD and related diseases – including chronic wasting disease spreading among the US deer and elk population – are associated with misfolded proteins called prions that aggregate to form dense tangled fibres in the brain cells, thereby killing them. Prions are highly resistant to heat, chemicals and radiation treatments, and cannot be inactivated with disinfection measures used to kill ordinary disease agents such as viruses and bacteria. The misfolded prion proteins are believed to be both the cause of BSE and the infectious agents transmitting the disease, and that feeding cattle with rendered remains of sheep affected with a related disease, scrapie, led to the outbreak of the BSE epidemic (but see main text).
In July 2005, a company in Gottingen, Germany, published a peer-review paper in the journal, Clinical and Diagnostic Laboratory Immunology, reporting a diagnostic test for BSE in live animals, which does not depend on detecting the prion protein. Instead, the “Gottingen Living Test (GLT)”, as it is called, depends on detecting “unique, specific gene markers” that are in 100 percent of cows with confirmed BSE, and in 100 percent of groups of associated high-risk animals, i.e., cows in the same feeding cohorts as those with BSE . In contrast, only 0.6 percent of the control group of healthy animals tested positive on the GLT. This suggests that the test could be used to identify animals that are at risk of developing BSE in BSE eradication and surveillance programmes. The GLT will enable animals at-risk to be removed from the food chain while still alive, thereby reducing both the threat to human health and the economic impact on the cattle industry.
The development of GLT involved the collaboration between the Institute of Veterinary Medicine in Georg-August University, Gottingen, a leading research institute in BSE, and Chronix Biomedical, a genomics company whose core technology – protected by patent - is based on developing tests for detecting and monitoring a new class of markers for chronic illnesses: circulating nucleic acids (CNAs).
CNAs are RNA and DNA detected in biological fluids free of cells or cellular material and found to be useful for the staging of some chronic illnesses. Most CNA lab diagnostics are based on amplifying either RNA or DNA with primers (probes) for single-copy functional coding regions of genes associated with infectious agents such as West Nile virus, HIV, hepatitis B virus.
In contrast, some CNA studies have focussed on endogenous repeat sequences found in the genome. Dr. Howard Urnovitz, the CEO of Chronix Biomedical, found three out of three sick veterans of the 1991 Gulf War had the same repetitive sequences, including short Alu repetitive sequences in their CNAs (“Dynamic genomics & environmental health”, SiS 19). Similarly, repetitive sequences in CNAs were associated with the clinical status of individual multiple myeloma patients.
For BSE diagnosis, a specific polymerase chain reaction (PCR) probe was used that amplified the tail-end of a bovine genome short interspersed nuclear element (SINE), Bov-tA, about 285 000 copies of which are present in the genome, often at the 3’(tail-end) unstranslated region of genes. The probe detected multiple CNAs, ranging from less than 150 to 350bp, found only in the sera of BSE-confirmed cows and among high-risk cows exposed to BSE in the same feeding cohorts. None of the bands was amplified from the sera of healthy control animals.
The PCR products from two BSE cases and six BSE cohort animals were cloned and sequenced. The range of fragment sizes was from 105 to 304bp, with an average size of 210bp. A stretch of about 80bp - found in nearly all the clones (150 out of 163) – was part of Bov-tA, as expected. However, this 80bp piece has other bits of sequences attached downstream, which, though they also appear to belong to the bovine genome, are not found in the bovine genome as contiguous sequences.
The researcher team analysed a further four confirmed BSE cases, eight unrelated cohorts consisting of a total of 135 animals that were diagnosed BSE-negative by the standard prion tests, and 176 healthy control cows, which included 148 samples from a slaughterhouse processing cattle from the same area where the BSE cases developed (to avoid a regional bias), and 28 samples from a BSE non-exposed healthy control herd. The BSE cases tested 100 percent positive by the same PCR diagnosis, i.e., 4 out of 4, while only 1 out of 176 healthy controls tested positive, or 0.6 percent. The 8 BSE-cohorts tested 33 percent to 91 percent positive, with an average of 63 percent positive. This was a very significant result, as these BSE-cohort cows were diagnosed BSE-negative by the standard tests for prion proteins in brain tissue after they were slaughtered.
According to data provided by the German Ministry of Consumer Safety, Nutrition and Agriculture, the likelihood of detecting a prion positive animal among cohorts of BSE cattle is more than 100-fold greater than in healthy, non-cohort cattle. This figure matches the finding in the present report: 63 percent of cohorts reacting positive compared to 0.6 percent positive in noncohort healthy cattle.
In a further field study, an additional 669 samples from a slaughterhouse were tested. These samples originated from 257 different farms. Only four samples were found to be repeatedly positive (0.59%), which confirms the results with the previous 176 normal control cattle.
The diagnosis depends on amplifying circulating nucleic acids all of which contain a fragment of a particular repetitive element (Bov-tA, a member of SINE) present in the bovine genome. The expression of SINE elements is associated with cell stress, as previous work by other researchers has indicated. For example, cells stressed by exposure to the toxic drugs cycloheximide or puromycin rapidly and for a short while increased the abundance of Alu-containing RNA (Alu is a SINE specific to primates including humans). Thus, finding SINE-containing CNAs in both BSE and BSE-exposed cohorts suggest that cell stress may be involved in BSE; and further, that detection of specific cell-stress CNAs could offer an early diagnosis of impending disease.
The sequences attached to the SINE sequence in the BSE-associated CNAs appear to be rearranged or scrambled bovine genome sequences. This is consistent with the strong involvement of SINE sequences in recombination events.
The results suggest, therefore, that exposure to toxic agents causing cell-stress has led to activation of repetitive elements in the genome involved in recombination, and extensive scrambling of genome sequences in animals that had developed BSE and others “exposed to BSE”.
This research does appear to be the first living test for BSE or very nearly so. To really clinch the test, it would be necessary to see if the healthy BSE-exposed animals which tested positive will actually go on to develop BSE. But in the absence of any other contender test for the disease, most farmers and regulators might be willing to accept the present test at least as an indicator for BSE-exposure, so that animals at-risk can be removed from the food chain, thereby reducing both the threat to human health and the economic impact on the cattle industry. Another argument in its favour is that in the absence of such a test, the whole herd would have had to be slaughtered as a precautionary measure in any case.
This research also raises important questions over the cause of BSE. There are many scientists who remain doubtful of the official account that prion proteins are the infectious agent ; as this runs counter to the conventional wisdom that all known infectious agents such bacteria and viruses contain genetic material - RNA or DNA – which is crucial for infectivity.
There is also doubt as to whether the BSE epidemic was caused by feeding cattle with improperly treated meat and bone meal feed containing the related scrapie agent from sheep remains. Organic farmer Mark Purdey in Britain reviewed extensive epidemiological and biochemical evidence contradicting the official view on the origin and cause of BSE . This evidence suggests instead that BSE was triggered by the widespread use of the organophosphate insecticide Phosmet following the Warble Fly Order issued by UK’s then Ministry of Agriculture, Fisheries and Food in 1984, coupled with the industrial pollution of agricultural land by manganese, which appears to be involved in the misfolding of prion proteins.
Purdey’s hypothesis is consistent with the cell-stress circulating nucleic acids found in BSE-diagnosed and BSE-exposed animals reported by Chronix Biomedical. Further research should be done to see if BSE-specific cell-stress CNAs correlates with exposure of cattle herds to toxic agents such as organophosphate insecticides and manganese.
Article first published 22/08/05
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