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ISIS Press Release 22/08/05
ISIS Exclusive
Living Test for Mad Cow Disease
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
The fully
referenced article is posted on ISIS members’ website. Details here
Mad Cow Disease and variant CJD
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.
Box
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 [1]. 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 [2]. Nevertheless, infected cattle have appeared in Canada,
Japan, Israel, Oman and the Falkland Islands; and in the United States
at the end of 2003 [3].
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 [1].
(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
[4].) 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 [5]. 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).
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Living test depends on specific genetic markers
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 [8]. 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.
Circulating nucleic acids and chronic diseases
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.
BSE diagnosis in both sick cows and healthy BSE-exposed cows
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.
What is the basis of the diagnosis?
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.
What really caused BSE?
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 [10]; 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 [11]. 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.
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