Agrobacterium & Morgellons Disease, A GM Connection?

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Agrobacterium & Morgellons Disease, A GM Connection?

 

 

by Dr. Mae-Wan Ho and Prof. Joe Cummins

 

_http://www.globalresearch.ca/index.php?context=va & aid=9891_

(http://www.globalresearch.ca/index.php?context=va & aid=9891)

 

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?

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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 [1],

after

receiving thousands of complaints from people with this bewildering condition,

which it describes as follows [2]: “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 in

visions.â€

Morgellons Disease 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 [3]. Until then,

people with Morgellons Disease have been diagnosed as cases of “delusional

parasitosisâ€, in which the symptoms are deemed entirely imaginary, and

lesions

allegedly due to self-inflicted wounds.

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 [1] 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

“

complexâ€, and “may be due to multiple factors.â€

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.â€

CDC’s investigation 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 [6] published in 2006, researchers from the Morgellons Research

Foundation [3] 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 [3] 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 [8]).

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 [8] since January

2007. They were further publicized in the “first ever†Morgellons

conference in

Austin Texas, attended by 100 in March 2008 [9]. A growing list of people

are registered with Morgellons Disease, totalling 12 106 worldwide recorded by

Morgellons Research Foundation [3], as of 12 April 2008.

San Francisco physician, Raphael Stricker, one of only a few doctors who

believe the disease is real, said [9]. “There’s almost always some history

of

exposure to dirt basically either from gardening or camping or something.â€

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 [6].

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

 

Agrobacterium 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 [10]. Until then, the genetic engineering community

had assumed that Agrobacterium did not infect animal cells, and certainly

would not transfer genes into them.

Agrobacterium 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 [11] (_i-sis news11/12_

(http://www.i-sis.org.uk/isisnews/i-sisnews11.php) )

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 [12].

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 text).

Transfer requires three major elements [13]: 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 [15]. 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.

Furthermore, the 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.

Extensive genetic 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

[16].

The epidemiological 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 escape

 

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 [17], 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]â€

, which will serve as a vehicle for further gene escape, at least to other

Agrobacterium strains naturally present in the soil.

Although various antibiotics have been used to eliminate Agrobacterium

following transformation, the researchers stated that “very few authors

actually

test to ensure that the antibiotics succeed.â€

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 ba

cterium at four times the minimum bactericidal concentration, as recommended.

They found that none of the antibiotic succeeded in eliminating

Agrobacterium.

The contamination 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) [18]. 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 bacteria.

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.â€

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 as well.

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 [14]: “Future research

has to show whether Agrobacterium-mediated transformation contributed to

horizontal gene transfer between microorganisms in the rhizosphere.â€

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 [19] _Horizontal Gene Transfer from

GMOs Does Happen_ (http://www.i-sis.org.uk/horizontalGeneTransfer.php) , 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_ (http://www.i-sis.org.uk/fluidGenome.php) [20] and _The Case

for A GM-Free Sustainable World_ (http://www.indsp.org/ISPreportSummary.php)

[21] 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 [22].

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.

Thus, transgenic plants with contaminating Agrobacterium [20] “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

needed

 

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 Disease patients

* 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.