ISIS Report 28/04/08
Agrobacterium & Morgellons Disease, A GM
Preliminary findings suggest a link between Morgellons Disease and Agrobacterium, a soil bacterium extensively manipulated and used in making GM crops; has genetic engineering created a new epidemic?
Dr. Mae-Wan Ho
and Prof. Joe Cummins
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CDC launch investigation on Morgellons’ Disease
The Centers for Disease Control (CDC) in the United States
announced the launch of an investigation on ‘Morgellons Disease’ in January
2008 , after receiving thousands of complaints from people with this bewildering
condition, which it describes as follows : “Persons who suffer from this
unexplained skin condition report a range of cutaneous (skin) symptoms including
crawling, biting and stinging sensations; granules, threads, fibers, or black
speck-like materials on or beneath the skin, and/or skin lesions (e.g., rashes
or sores). In addition to skin manifestations, some sufferers also report
fatigue, mental confusion, short term memory loss, joint pain, and changes
first became known in 2001, when Mary Leitao created a web site describing
the illness in her young son, which she named after a 17th century medical
study in France describing similar symptoms . Until then, people with Morgellons
Disease have been diagnosed as cases of “delusional parasitosis”, in which
the symptoms are deemed entirely imaginary, and lesions allegedly due to self-inflicted
Indeed, the debate
over Morgellons Disease has continued in the pages of medical and scientific
journals right up to the CDC’s announcement [4-7]
Dr. Michele Pearson,
principal investigator for the CDC said  that the primary goals of the
study are “to learn more about who may be affected with this condition, the
symptoms they experience and to look for clues about factors that might contribute
to the condition,” adding that the condition is “complex”, and “may be due
to multiple factors.”
In response to questions
from journalists at the CDC press conference, Pearson said:
“ We are aware that many
patients have suffered from this condition. And, I can tell you that here
at CDC, we have really been seeing an increasing number of these reports over
the past year or so.”
is to be carried out in conjunction with Kaiser Permanente’s Northern California
Division of Research and the US Armed Forces Institute of Pathology.
Dr. Joe Selby, Director
of the Kaiser Permanente’s Northern California Division of Research, said
the study would proceed in three stages. In the first stage, they will identify
all members who may have seen a Kaiser Permanente physician with symptoms
suggestive of this condition at any point during the 18 months between July
1 2006 and December 31, 2007, and determine whether they meet eligibility
criteria for the study. In stage two, all eligible members will be invited
to complete a comprehensive web based or telephone survey conducted by the
CDC that examines the duration and severity of a variety of symptoms. And
in stage three, those with active symptoms will be invited to the division
of research for an extensive clinical examination including collection of
skin biopsies, blood and urine samples.
In a paper  published
in 2006, researchers from the Morgellons Research Foundation  identified
the states of California, Texas and Florida as having the highest number of
cases of Morgellons disease in the United States. Primary clusters were noted
in Los Angeles and San Francisco (California) and Houston, Dallas
and Austin (Texas). California accounted for 26 percent of cases in the US,
but all 50 US states and 15 other nations, including Canada, the UK, Australia,
and the Netherlands, have reported cases of Morgellons disease. The two main
occupational groups reporting symptoms are nurses and teachers, with nurses
outnumbering teachers three to one. The risk factor common to both groups
is suspected to be the possibility of transmitted infectious agents.
Skin lesions and
fibres may not be readily apparent in all individuals with the disease, as
family members of patients often report similar systemic disease symptoms
without skin symptoms. Families in which all members are affected often have
suspected simultaneous exposure to an inciting agent. Contact with soil or
waste products appears to be associated with the disease. Cases have been
reported in cats and dogs, as well as horses.
What finally prompted CDC to investigate the disease? The Morgellons Research
Foundation  was set up in 2002 in honour of Mary Leitao, the Foundation’s
executive director. It publicises the plight of patients with similar conditions
and operates a registry of afflicted families. The Foundation also funds scientific
research. It has a Medical Advisory Board of seven with M.D. degree and two
with nursing degrees. In addition, it has a Board of Nursing with five other
nurses, and a Scientific Advisory Board of six scientists, all with Ph.D. degree;
one of which is Vitaly Citovsky. It may have been Citovsky’s discovery last
year that finally persuaded the CDC to announce an investigation.
The Agrobacterium connection
Vitaly Citovsky is a professor
of molecular and cell biology at Stony Brook University in New
York (SUNY). He is a world authority on the genetic modification of cells
by Agrobacterium, a soil bacterium
causing crown gall disease in plants, that has been widely used in creating
genetically modified (GM) plants since the 1980s because of its ability to
transfer a piece of its genetic material, the T-DNA on its tumour-inducing
(Ti) plasmid to the plant genome (see later for details).
Citovsky’s team took
scanning electron microscope pictures of the fibres in or extruding from the
skin of patients suffering from Morgellons disease, confirming that they are
unlike any ordinary natural or synthetic fibres (see Fig. 1, assembled from
Citovsky’s website ).
Figure 1. Scanning electron microscope
images of fibres from skin biopsies of patients with Morgellons Disease -
a, white fibre with calcite,
scale bar 10 mm; b, green fibre with alumina ‘rock’ protruding,
scale bar 20 mm; c,
various ribbon-like, cylindrical and faceted fibres all coated with minerals,
scale bar 10 mm; d,
skin lesion with fibres stabbing through the epidermis, scale bar 300 mm
They also analysed
patients for Agrobacterium DNA.
Skin biopsy samples from Morgellons patients were subjected to high-stringency
polymerase chain reaction (PCR) tests for genes encoded by the Agrobacterium chromosome and also for Agrobacterium virulence (vir) genes and T-DNA on its Ti plasmid. They
found that “all Morgellons patients screened to date have tested positive
for the presence of Agrobacterium,
whereas this microorganism has not been detected in any of the samples derived
from the control, healthy individuals.” Their preliminary conclusion is that
“Agrobacterium may be involved in the etiology
and/or progression” of Morgellons Disease.
The unpublished findings
have been posted on a website  since January 2007. They were further publicized
in the “first ever” Morgellons conference in Austin Texas, attended by 100
in March 2008 . A growing list of people are registered with Morgellons
Disease, totalling 12 106 worldwide recorded by Morgellons Research Foundation
, as of 12 April 2008.
San Francisco physician, Raphael Stricker, one of only a few doctors who believe
the disease is real, said . “There’s almost always some history of exposure
to dirt basically either from gardening or camping or something.” He is one
of the co-authors on the Agrobacterium research done in SUNY, which reported
finding Agrobacterium DNA in all 5 Morgellons patients studied. Stricker
suggests it is transmitted by ticks, like Lyme disease, and in a recent survey
of 44 Morgellons patients in San Francisco, 43 of them also tested positive
for the bacterium causing Lyme disease. Another factor consistent with Agrobacterium
being a causative agent, if not the causative agent, is that when patients
are treated with antibacterials for their Lyme disease, remission of Morgellons
symptoms is seen in most of them .
Stricker also told
his audience that Agrobacterium
lives in the soil, and is known to cause infections in animals and human beings
with compromised immune systems. It can cause skin lesions when injected into
Swiss mice, a strain that is immune deficient, he said.
At this point, the
findings on the Agrobacterium
connection are still preliminary, as only seven patients have been studied.
Nevertheless, the implications are far-reaching if this connection is confirmed,
as existing evidence (reviewed below) suggests a link between Agrobacterium and genetic engineering in
the creation of new disease agents, and it is paramount for the CDC investigation
to include this aspect, if only to rule it out.
Agrobacterium and the genetic engineering connection
not only infects human and other animal cells, it also transfers genes into
them. It was SUNY professor Citovsky and his team that made the discovery
some years ago . Until then, the genetic engineering community had assumed
that Agrobacterium did not infect animal cells,
and certainly would not transfer genes into them.
was found to transfer T-DNA into the chromosomes of human cells.
In stably transformed HeLa
cells, the integration occurred at the right border of the T-DNA, exactly
as would happen when it is being transferred into a plant cell genome, suggesting
that Agrobacterium transforms
human cells by a mechanism similar to that involved in transforming plants
cells (see Box 1). Human cancer cells, neurons and kidney cells were all transformed
with the Agrobacterium T-DNA.
Commenting on this research in 2001, Joe Cummins had warned of hazards to
laboratory and farm workers  (i-sis news11/12)
The Agrobacterium vector system for gene transfer
Since the discovery in the
1970s that Agrobacterium can
transfer genes into plants causing crown gall disease, the soil bacterium
has been developed into a vector for inserting desirable genes into the
plant genome to create transgenic (GM) plants .
Agrobacterium transfers T-DNA – a small region
of approximately 5 to 10 percent of a resident tumour-inducing (Ti) or root-inducing
(Ri) plasmid – into numerous species of plants; and as later turns out,
also to fungi, algae, and even animal and human cells [13, 14] (see main
Transfer requires three major elements : T-DNA border direct repeat sequences
of 25 base pairs that flank the T-DNA and delineate the region transferred into
the host, the virulence (vir) genes located on the Ti/Ri plasmid, and
various genes on the bacterial chromosome. Plant genes are also involved in
the successful integration of T-DNA . The T-DNA contains oncogenes (cancer
genes or gene for forming tumours) and genes for synthesizing opines; none of
which is essential for T-DNA transfer, so they can be deleted and replaced with
genes of interest and selectable markers.
vir genes and T-DNA region need not be on
the same replicating plasmid. This gave rise to the binary vector systems
in which T-DNA and the vir
genes are located on separate replicating units. The T-DNA containing unit
is the binary vector and contains also the origin(s) of replication that
could function both in E. coli and
Agrobacterium tumefaciens, and antibiotic
resistance marker genes used to select for the presence of the binary vector
in bacteria. The replicating unit containing the vir
genes is the ‘helper’ plasmid. Strains of Agrobacterium
harbouring the two separate units are considered ‘disarmed’ if they do not
contain oncogenes that could be transferred to a plant.
The association of
Morgellons Disease with dirt and soil where Agrobacterium
lives, the widespread use of Agrobacterium
in genetic engineering of plants, and the ability of Agrobacterium to infect human cells, all point towards a possible
role of genetic engineering in the aetiology of Morgellans disease via Agrobacterium.
manipulation of Agrobacterium
does have the potential to transform it into an aggressive human pathogen.
Genetic engineering is nothing if not enhanced and facilitated horizontal
gene transfer and recombination, which is widely acknowledged to be the main
route for creating new pathogens. Mae-Wan Ho was among an international panel
of scientists have raised this very issue in 1998, calling for a public enquiry
into the possible contributions of genetic engineering biotechnology to the
aetiology of infectious diseases which has greatly increased since genetic
engineering began in the 1970s .
data of Morgellons Disease are very incomplete, and the Morgellons Research
Foundation’s registry of more than 12 000 families afflicted worldwide is
almost certainly only a fraction of the emerging epidemic. Still, it is significant
that the majority of the cases are in the United States, the
first country to release GM crops and remaining the top producer ever since.
There are other findings
implicating Agrobacterium in
transgenic plants released into the environment, particularly during the early
years of field trials, when knowledge was poor and safety measures not as
stringent as they may be today.
Agrobacterium persists in transgenic plants and is a vehicle for gene
By the late 1990s, the Agrobacterium vector system became very widely
used, and many GM crops created were commercially released.
Scientists at the
Kinsealy Research and Development Centre in Dublin, Ireland, and the Scottish
Crop Research Institute in Dundee, Scotland, were concerned that the inserted
genes in plants would spread to wild populations by cross-pollination or by
horizontal gene transfer to unrelated species, which was by then well-documented
in the scientific literature.
They considered it “imperative” to address the risk posed in using Agrobacterium
as a tool in genetic engineering , given its ability to transfer genes to
plants. The transformation procedure involves inoculating the cells or tissue
explants with Agrobacterium and co-cultivation the plant cells and bacterium
for a short period, followed by the elimination of the bacterium with antibiotics.
However, if all the
bacteria were not eliminated, then “release of these plants may also result
in release of the Agrobacterium
[with the foreign genes]”, which will serve as a vehicle for further gene
escape, at least to other Agrobacterium
strains naturally present in the soil.
antibiotics have been used to eliminate Agrobacterium
following transformation, the researchers stated that “very few authors actually
test to ensure that the antibiotics succeed.”
The difficulty is compounded because the bacterium can remain latent within
the plant tissue. So putting transgenic plant material into culture medium without
antibiotics and finding no Agrobacterium is no guarantee that the transgenic
plant is free of the bacterium, as was often assumed.
In their study, they investigated the ability of antibiotics to eliminate Agrobacterium
tumefaciens after transformation in three model systems: Brassica (mustard),
Solanum (potato), and Rubus (raspberry). The antibiotics carbenicillin,
cefataxime and ticaracillin were used respectively to eliminate the bacterium
at four times the minimum bactericidal concentration, as recommended. They found
that none of the antibiotic succeeded in eliminating Agrobacterium.
levels increased from 12 to 16 weeks to such an extent that
transgenic Solanum cultures
senesced and died. Contamination in shoot material decreased over 16 to 24
weeks possibly because only the apical node was used in further culture, but
even that did not eliminate Agrobacterium
from all the samples; 24 percent remained contaminated at 24 weeks.
The binary vector
was also present under non-selective conditions up to 6 months after transformation,
where approximately 50 percent of contaminated material still harboured bacterial
cells with the binary vector at high levels of about 107 colony
forming units per gram. The researchers pointed out: “Here is where the possibility
of gene escape arises. The presence of the disarmed Agrobacterium in the tissue would not be
a problem if the binary vector had been lost, but now its survival and spread
are real possibilities.” The binary vector contains the foreign genes as well
as antibiotic resistance marker gene(s).
There is no limit
to the foreign genes that can be inserted into the binary vector. A few years
earlier, a research group in Israel had inserted a viroid that causes disease
in citrus fruits into the disarmed Ti plasmid of Agrobacterium and used that to infect and transform several
plant species including tomato (Lycopersicon
esculentum) Gynura aurantiaca,
avocado (Persea americana),
and grapefruit (Citrus paradisi)
grafted on Troyer citrange (Pancirus trifoliate
x C. sinensis) . Extracts
prepared from tissues of the infected plants 38-90 days after inoculation
were plated on selective media and found to contain large amounts of the engineered
The researchers warned
of “newly formed combinations of persistently transmitted viruses” coupled
with “the opportunistic and systemically moving Agrobacterium vector infectious to a wide
host range might eventually cause infection and damage to crop plants or natural
vegetation” that are “not presently visited by the traditional vectors of
the virus disease.”
In other words, Agrobacterium persisting in transgenic plants
released into the environment has the potential to spread new diseases, and
to plants that normally would not be infected by the disease agents. At the
time, the researchers did not know that Agrobacterium
would also infect animals and humans, and could spread new diseases to them
Have these warnings been heeded by other researchers?
There is no evidence they have been taken on board. Agrobacterium has since been shown to transform at least 80
different non-plant species including yeasts and other fungi, algae, mammalian
and human cells, also the gram positive bacterium Streptomyces lividans. In a recent review, the researchers
stated : “Future research has to show whether Agrobacterium-mediated transformation contributed to horizontal
gene transfer between microorganisms in the rhizosphere.”
But there is already evidence suggesting that Agrobacterium can indeed
engage in horizontal gene transfer with a wide range of bacteria in the soil.
(For more on horizontal gene transfer see  Horizontal Gene Transfer
from GMOs Does Happen, SiS 38)
Agrobacterium gene transfer mechanisms similar to conjugation in bacteria
Ho first alerted regulators to the potential of Agrobacterium contaminating
GM plants to facilitate the escape of transgenes in 2003 (see Living with the Fluid Genome
 and The Case for A GM-Free
Sustainable World  ISIS publications). By then, Gayle Ferguson
and Jack Heinemann at the University of Canterbury, Christchurh, New Zealand,
had already pointed out in a review that the process whereby Agrobacterium
injects T-DNA into plant cells strongly resembles conjugation, the normal mating
process between bacteria .
Conjugation, mediated by certain bacterial plasmids, depends on a sequence
called the origin of transfer (oriT) on the DNA transferred. All other
functions - called tra for trans-acting functions - can be supplied from
unlinked sources. Thus, ‘disabled’ plasmids with no trans-acting functions,
can nevertheless be transferred by helper plasmids, the same as the binary vector
system of Agrobacterium (Box 1). The resemblance does not stop there.
The left and right
borders of T-DNA are similar to oriT
and can be replaced by it. Furthermore, the disarmed T-DNA binay vector, lacking
oncogenes as well as virulence genes, can
be helped by similar genes belonging to many other pathogenic bacteria.
The trans-kingdom gene transfer apparatus of Agrobacterium
and the conjugative systems of bacteria are both involved in transporting
macromolecules, not just DNA but also protein.
plants with contaminating Agrobacterium
 “have a ready route for horizontal gene escape, via Agrobacterium, helped by the ordinary conjugative
mechanisms of many other bacteria that cause diseases, which are present in
the environment.” In the process, new and exotic disease agents could be created.
Investigations on the role of Agrobacterium in Morgellons Disease urgently
The investigation launched
by the CDC needs to clarify the role of Agrobacterium in the aetiology of Morgellons Disease as a matter
of urgency. This should include:
- Molecular characterization of Agrobacterium DNA sequences in Morgellans
- Design of suitable probes for diagnostic purposes and for monitoring soil
samples and other suspected sources of infection
- Introduction of stringent tests for Agrobacterium contamination for
all transgenic plants already released or about to be released into the environment.