Bacteria, Biofilm,

and
Bio-pesticide BT
(Bacillus Thuringiensis)
Autism Hypothesis,
Presented by
Dr. Anju Usman, MD. and Andrea
Lalama,
First part of the Presentation

• What is Biofilm?
• What is the
implication of biofilm
production in ASD?
• How are they
formed?
• Where do they grow?
• Possible Treatments.
 Many patients with autistic symptoms have
persistent dysbiosis. Treatment of GI issues often
alleviates symptoms we call autism.

Hypothesis:
Patients with autism, who have toxic metal burdens and toxic
chemical burdens (Bt toxin), are likely to grow resistant
organisms in their GI tract. This resistance to treatment is
perpetuated by the production of biofilms. Treatment of
biofilm will help to eradicate dysbiotic flora and improve
symptoms we call autism.
 What is Biofilm?
• A biofilm is a collection of microbial
communities enclosed by a matrix of
extracellular polymeric substance (EPS) and
separated by a network of open water
channels.

• These communities adhere to manmade and

natural surfaces, such as metals and teeth,
typically at a liquid-solid interface . Their
architecture is an optimal environment for
cell-cell interactions, including the
intercellular exchange of genetic material,
communication signals, and metabolites,
which enables diffusion of necessary
nutrients to the biofilm community.

• The matrix is composed of a negatively

charged polysaccharide substance, held
together with positively charged metal ions
(calcium, magnesium, and iron).

• The matrix in which microbes in a biofilm are

embedded protects them from UV exposure,
Staphylococcus aureus biofilm metal toxicity, acid exposure, dehydration
salinity, phagocytosis, antibiotics,
antimicrobial agents and the immune system.
 How is Biofilm formed?
5 stages of biofilm development.

• Stage 1, initial attachment; stage 2, irreversible attachment; stage 3,

maturation I; stage 4, maturation II; stage 5, dispersion.
• Each stage of development in the diagram is paired with a
photomicrograph of a developing P. aeruginosa biofilm.
 Where do they grow?
• Biofilm formation appears common near polluted and
toxic areas and environments.

• Account for more than 80% of all microbial infections of

the human body.

• Device-related infections, intravenous catheters,

joint prostheses

•Human body –
pancreatic/biliary tracts, lungs,
sinuses, adenoids, tonsils and
the intestinal tract….
Non animated picture of
Biofilm/Slime, YUK!
Why are they so difficult to
treat? difficult to treat with antimicrobials,
Remarkably
resistant to doses of antimicrobials 100- to 1000-fold
over the minimum lethal dose for microbes outside of
biofilms.

•Antibiotics do not penetrate polysaccharide matrix.

•Highly resistant to both immunological and non-

specific defense mechanisms of the body.

•Difficult to diagnose, difficult to culture.

•Microbes impart genetic material to one another to

maintain resistance.

•Colonies communicate with one another thru the use

What type of biofilm control
strategies have been studied?
• What are potential treatment options?
• EDTA
• Fe chelating compounds
• Enzymes - mucous degrading
• Probiotics
• Fermented Foods
• High dose Antibiotics
 The Efficacy of EDTA Against Biofilm
Bacteria (Kim, 2005)
• Biofilms = complex communities of micro-organisms
attached to surfaces held together by EPS (extracellular
polysaccharides, that are negatively charged and held
together by positively charged cations, specifically Fe2+,
Ca2+, and Mg2+.
• EDTA complexes with cations in the extracellular matrix.
• Neither Vancomycin or EDTA alone detached Staph
biofilm.
• EDTA plus Vancomycin together caused biomass removal.
 Chelator-Induced Dispersal and Killing
of Pseudomonas aeruginosa Cells in a
Biofilm (Banin, 2005)
• EDTA is a potent Pseudomonas biofilm disrupter.
• 1000x killing when EDTA combined with Gentamicin.
• EDTA causes dispersal and killing of biofilm cells.
• Ca, Fe, and Mg protect biofilm.
• When Ca or Fe are added, killing and detachment are
completely blocked.
 Iron Chelating Compounds
• Outer membrane proteins(OMP) are expressed
when iron is restricted.
– If OMP are not expressed, the immune system is not
alerted appropriately, and can not illicit a normal
immune response.

• Transferrin and Lactoferrin

– Synthesized by host to inhibit bacterial growth by
sequestering free Iron.
– Pathogenic bacteria secrete iron chelators
(siderophores) to compete with transferrin and
lactoferrin for Iron.
 Biofilm destruction by innate immune
system
Nature. 2002 May 30;417(6888):552-5.

A component of innate immunity prevents bacterial biofilm development.

Singh PK, Parsek MR, Greenberg EP, Welsh MJ.

Antimicrobial factors form one arm of the innate immune system, which protects mucosal surfaces from
bacterial infection. These factors can rapidly kill bacteria deposited on mucosal surfaces and
prevent acute invasive infections. In many chronic infections, however, bacteria live in biofilms,
which are distinct, matrix-encased communities specialized for surface persistence. The transition
because
from a free-living, independent existence to a biofilm lifestyle can be devastating,
biofilms notoriously resist killing by host defence
mechanisms and antibiotics. We hypothesized that the
innate immune system possesses specific activity to
protect against biofilm infections. Here we show that
lactoferrin, a ubiquitous and abundant constituent of human
external secretions, blocks biofilm development by the
opportunistic pathogen Pseudomonas aeruginosa. This occurs at lactoferrin concentrations below
those that kill or prevent growth. By chelating iron, lactoferrin stimulates twitching, a specialized
form of surface motility, causing the bacteria to wander across the surface instead of forming cell
clusters and biofilms. These findings reveal a specific anti-biofilm defence mechanism acting at a
critical juncture in biofilm development, the time bacteria stop roaming as individuals and aggregate
into durable communities.
PMID: 12037568 [PubMed - indexed
 Enzymatic Degradation
Proc Natl Acad Sci U S A. 2007 Jul 3;104(27):11197-202. Epub 2007 Jun
25.

Dispersing biofilms with engineered enzymatic bacteriophage.

Lu TK, Collins JJ.

Synthetic biology involves the engineering of biological organisms by using modular and generalizable
designs with the ultimate goal of developing useful solutions to real-world problems. One such
problem involves bacterial biofilms, which are crucial in the pathogenesis of many clinically important
infections and are difficult to eradicate because they exhibit resistance to antimicrobial treatments and
removal by host immune systems. To address this issue, we engineered bacteriophage
to express a biofilm-degrading enzyme during infection to
simultaneously attack the bacterial cells in the biofilm and the
biofilm matrix, which is composed of extracellular polymeric
substances. We show that the efficacy of biofilm removal by this
two-pronged enzymatic bacteriophage strategy is significantly
greater than that of nonenzymatic bacteriophage treatment. Our
engineered enzymatic phage substantially reduced bacterial biofilm cell counts by approximately 4.5
orders of magnitude ( approximately 99.997% removal), which was about two orders of magnitude
better than that of nonenzymatic phage. This work demonstrates the feasibility and benefits of using
engineered enzymatic bacteriophage to reduce bacterial biofilms and the applicability of synthetic
biology to an important medical and industrial problem.
“Normal mouthwashes can only
clean the surface, which is why
bad breath returns quickly and
gum disease is a constant
problem. With the new patented
technology in Biotene PBF
mouthwash, you can dissolve the
biofilm, expose hidden bacteria
colonies and kill germs.

In addition, Biotene PBF contains

the proven LP3 salivary enzyme
system to strengthen the body’s
antibacterial action, dissolving
biofilm and inhibiting excessive
bacteria – maintaining a healthy
oral balance.”
N-acetyl Glucosamine and Biofilm
Shanghai Kou Qiang Yi Xue. 2006 Aug;15(4):407-10. Links

Effects of chitosans with different molecular weights on

Streptococcus sanguis biofilm
Ma R, Zhu M, Liu Z.

PURPOSE: To investigate the effects of chitosan on Streptococcus sanguis

biofilm. METHODS: Streptococcus sanguis biofilm was formed on saliva-
coated glass (SCG) in a flow culture system, then exposed to 2% chitosans
with different molecular weights (5 cps, 80 cps, 600 cps) for 3, 10, 30
minutes. Confocal laser scanning microscope and Vital/Dead fluorescent
staining technique (vital stained green, dead stained red) were combined to
observe the biofilm thickness, bacterial density. Analysis of variance was
used for PMID:
RESULTS the biofilm thickness and bacterial density reduced significantly
after treatment with 2% chitosan. Low molecular weight chitosan
seems most effective at detaching biofilms.
16955169 [PubMed - in process]
 Probiotics,IBD, and Biofilms
J Appl Microbiol. 2007 May;102(5):1187-96.
Microbial biofilms in the human gastrointestinal tract.
Macfarlane S, Dillon JF.

The human gastrointestinal tract contains rich and diverse microbiotas along its length. However, while extensive
studies have been made on lumenal bacterial communities in the gut, less work has been carried out on
organisms growing in biofilms, where individual groups of bacteria exist in a multiplicity of different microhabitats
and metabolic niches associated with the mucosa, the mucus layer and particulate surfaces in the gut lumen.
Bacteria and yeasts also occur in biofilms attached to artificial surfaces and devices implanted in the host, such
as in patients being fed via enteral tubes. Although we are just beginning to investigate the composition and
metabolic activities of these structures, increasing evidence suggests that they are important to the host in both
health and disease. There is mounting interest in mucosal biofilms in the colon, especially with respect to their
bacteria growing in biofilms are more
role in inflammatory bowel disease. Because
resistant to antibiotics than unattached organisms, it is often
difficult to modify the structure and composition of these
communities, or to eradicate them from the body. However,
recent work has shown that there is considerable potential to
alter the species composition of mucosal biofilms in a
beneficial way using synbiotics.
 Natural Antimicrobials
Scientists Develop 'Natural' Protection for Stored Foods

Tuesday, August 21, 2007; 12:00 AM

TUESDAY, Aug. 21 (HealthDay News) -- Natural methods of preventing food

contamination and spoilage could greatly expand the shelf life of products,
food scientists at Rutgers University in New Jersey say.

The researchers used natural antimicrobial agents

developed from sources such as cloves, oregano, thyme
and paprika to create biodegradable polymers or plastics
designed to prevent the formation of bacterial biofilms on
food surfaces and packaging.
 Biofilm Protocol
• Step 1 Lysis/Detachment (empty stomach)
– Enzyme (polysaccharidase, disaccharidase)
– Disodium EDTA (oral) or Apple Cider Vinegar
– Lactoferrin
– NAG (chitosan)
• Step 2 Killing
– Anti-bacterial, Anti-fungal and/or Anti-viral agents
• Step 3 Clean up
– Fiber, insoluble/soluble
– Activated Charcoal
– Modified Citrus Pectin
• Step 4 Rebuilding/Nourishing the Gut Lining
– Fermented Foods
– Probiotics
– Pre-biotics
– Healing, nutritional foods
 Our experience
• Positives

• Negatives

• What we have learned.

• ALWAYS work with your doctor especially when
using chelating agents.
Second part of the Presentation
• Natural Bacillus
Thuringiensis (NON-GMO
BT)
• Bio-pesticide BT (GMO)
• My Hypothesis
– Compiled evidence on BT
– My Hypothesis and opinion
References.
 What NON-GM O stands
for?
• NON Genetically Modified Organism

In other words NON-GMO is any natural

organism that has been left intact as God
originally created and has not been changed
or altered in any way by human intervention.
Sometimes the organism are also called
organic.
• Organic Definition is : "The use of
genetically engineered organisms
or their products are prohibited in
any form or at any stage in organic
production, processing, or handling."
What is Natural Bacillus Thuringiensis (BT)?
A Short History of Bacillus
Thuringiensis (BT)
Bt is a naturally occurring soil bacterium.
It was first detected in 1902 in the
Dying larvae of Bombyx mori by Ishiwata,
who reported his finding in the book:
"Pathology of the Silkworm".
It was first isolated from the larvae of
Ephestia kuehniella by Berliner in 1911
after he noted that it had the capacity to kill
certain insects in their larva stage.1
Natural Bt is highly specific, with toxicity limited to only some
species of one of the major groups of insects—typically
Lepidoptera (butterflies/moths), Coleoptera (beetles), or
Diptera (flies/mosquitos).
What is Natural Bacillus Thuringiensis (BT)?
• Bacillus thuringiensis is a gram-positive, spore-
forming bacterium which, during sporulation,
produces protein crystals (CRY). It is
characterized as a widespread insect
pathogen, and its insecticidal activity is
attributed to the parasporal crystals.
• A variety of strains have been isolated from
different habitats and, to date, more than
100 crystal protein genes have been
sequenced.
What is Natural Bacillus Thuringiensis (BT)?

• The toxicity of these crystal

proteins against certain
insects and their
high specificity
led to the
development of
bio-insecticides
for the control of pest insect species among
the orders Lepidoptera, Diptera, and
Coleoptera.
 How natural BT works?

BT SPORES

Normal gut
bacteria

BT crystalline Toxin 200px.

 How natural BT works?
Mode of Action

• The naturally Bacillus Thuringiensis is only effective when eaten by

specific family of insects with a specific (usually alkaline) gut pH
and the specific gut membrane structures required to bind the toxin.
(typically butterflies, moths, beetles, flies and mosquitoes).
• Not only must the insect have the correct and be at a susceptible
stage of development, but the bacterium must be eaten in sufficient
quantity.
• When ingested by a susceptible insect, the spores feed on natural
intestinal flora then it burst releasing the protein toxin (Crystalline
protein) damaging the gut lining (the intestinal walls), leading to a
kind of leaky gut condition.
• Affected insects stop feeding and die from the combined effects of
starvation, tissue damage and gastrointestinal infections by other
pathogens like bacteria and funguses.
• The natural Bt spores do not usually spread to other insects or
cause disease outbreaks on their own as occurs with many pathogens.
 What means GMO?
Genetically Modified Organisms.
• Traditional methods of genetic modification include selective crossbreeding
and hybridization. Other methods include interspecies and intergeneric
protoplast fusion, in vitro gene transfer techniques, somaclonal selection,
haploid doubling, and mutagenesis (McHughen, 2000). Rather than using
the term “GMO,” then, the scientific community prefers “genetically
engineered,” “genetically transformed,” “rDNA technology,” “gene splicing,”
or simply “transgenic.”
• Recombinant DNA technology goes beyond traditional cross
breeding techniques, making possible an exchange of
traits from different species, even among plants,
animals and bacteria.
 What means GMO?
Genetically Modified Organisms.

Bt corn, for example, was produced by incorporating genetic

material from a bacterium (Bacillus Thuringiensis) into the genetic
material of corn.
 What GM O stands for?
Currently U.S. National Organic Standards Board definition of
genetic engineering:
"Made with techniques that alter the molecular or cell biology
of an organism by means that are not possible under natural
conditions or processes. Genetic engineering includes:
3. Recombinant DNA,
4. cell fusion,
5. micro- and macro-encapsulation, and the following results
when achieved by recombinant techniques:
A. Gene deletion and doubling,
B. Introducing a foreign gene, and
C. Changing the positions of genes.

It shall not include traditional breeding, conjugation,

fermentation, hybridization, in-vitro fertilization, or tissue
culture."
 What is Bio-Pesticide BT ?
According to an article by
Jacobs in the “Proceedings of
the Society of Applied
Bacteriology (1950,13 p83)”,
Bt seems to have been used
for the first time as a
microbial bio-pesticide
against
Lepidopterous larvae in 1938, thereby giving Bt a
role in food production and that it has had ever
since.
How Genetically Modified BT works?

• Once consumed, Bt
products are
activated in the
alkaline gut of
insects, thus
making them very
safe to mammals.
Food Drug and Cosmetic Act
FDA 402(a)(1) - a food is adulterated
if it contains any poisonous or
deleterious substance which may
render the food injurious to health.
 What is Bio-Pesticide BT ?

• One way to avoid spraying pesticides on the corn has

been found and is currently being used in 30 percent of
the corn crops in the United States this year. It is a
genetically altered corn plant that produces an insect
toxin called Bt. Bt is a toxin produced by a bacteria called
Bacillus thuringiensis. The Bt toxin gene was taken
from the bacteria and then placed in the corn plant.
• The microbial biopesticide are genetically engineering which means BT
biopesticide is a GMO.
• BT bio-pesticide It’s NOT organic NOR natural, it does NOT act like a
natural BT, it is NOT selective to just certain insect’s species.
 What is Bio-Pesticide BT ?

Laboratory Tests of Acute Toxicity

Each of the more than 800 strains of Bacillus
thuringiensis may exhibit different toxicity to
insects, rodents and humans... The earliest
tests done regarding Bt's toxicity were
conducted using Bt var. thuringiensis, a Bt strain
known to contain a second toxin called beta-
exotoxin... Beta-exotoxin also causes genetic
damage to human blood cells...currently
being made to register beta-exotoxin as an
insecticide in the United States.
 How Genetically Modified BT works?

Bt insecticides, whether in the form of a spray or a Bt crop, do not function on

contact as most chemical insecticides do, but rather, as midgut toxins.

•In the case of Bt sprays:

Parasporal crystals ingested by insect larvae feeding on plant surfaces dissolve and the
insecticidal proteins are activated by proteases in the juices of the midgut, which
typically are alkaline (pH 8-10.5).
•In Bt crops (genetically modified crops-GMO):
The plant tissues produce specific ICPs in a soluble form. In either case, the active
ICP then traverses the peritrophic membrane and binds to specific receptors on the
midgut epithelium, forming pores and leading to loss of the transmembrane potential, cell
lysis, leakage of the midgut contents, paralysis, and death of the insect.3
 How Genetically Modified BT works?
Most widely used organic pesticide requires help to kill
The world's most widely used organic insecticide, a plucky bacterium known
as Bacillus thuringiensis or Bt for short, requires the assistance of other
microbes to perform its insect-slaying work, a new study has found. Writing
in the Sept. 26 issue of the Proceedings of the National Academy of
Sciences (PNAS), a team of researchers from the University of Wisconsin-
Madison reports that without the help of the native bacteria that colonize the
insect gut, Bt is unable to perform its lethal work.
The startling new insight into the workings of one of the most important and
environmentally friendly weapons in the human arsenal against insect pests
has significant implications not only for the control of insects in agriculture,
forestry and human health, but for understanding microbial disease in
humans and other animals.
"The take-home message is that we've shown that the mechanism of
killing for Bacillus thuringiensis is facilitated by the normal gut
community," says Nichole Broderick, a UW-Madison graduate student
and the lead author of the PNAS study. "This is a mechanism that was
not previously known."

Sept. 25, 2006

by Terry Devitt
 How Genetically Modified BT works?
ACTIVATION OF Bt ICP IN AN INSECT GUT.
 Compiled evidence on BT
“Because the living bees that the scientists were
able to study carried almost every virus and
parasite known to infect honeybees, researchers
are working on the idea that the insects' immune
systems have failed. Reducing the body's ability
to fight disease allows infection by a host of
pathogens...It could be that one disease, perhaps
a new type of lurgy, invites the others to infect
the bee, or that a pesticide performs this
role.”

The economist magazine, science and technology April 2007.

 Compiled evidence on BT
• "The German Television ZDF reported on
Sunday May 21 that a German researcher
found a gene transfer from genetically
engineered rapeseed to bacteria and fungi in
the gut of honey bees. Prof. Hans-Hinrich
Kaatz from the Institute for Bienenkunde
(Institute for bee research) at the University of
Jena experimented during the last three years
with honey bees on an experimental field with
transgenic rapeseed in Saxony, Germany."
 Compiled evidence on BT
• "The DNA of bacteria and yeast taken from
bees' guts contained the same modified
genes as those added to the plants whose
pollen the bees had fed on....
...At any rate we still maintain that the bees
intestinal flora and bacteria has been altered
by ingestion of the genetically modified
pollens and toxins causing digestive
problems, immune supression and ultimately
leading to a higher incidence of infection by
parasite or virus.
 Compiled evidence on BT
The bee epidemic,
Agriculture department of USA:..
CCD epidemic which threatens 33% of world food production at length in To Bee Or Not To
Bee. “we have indentified a bee epidemic called CCD or Colony Collapse Disorder”

An update on the situation and some validation for our position that GENETICALLY
MODIFIED crops are at the root of the cause.

GMOs, chemicals and pesticides are also cited as possible causes in the CCD…a
University of Florida study.

One of the researchers, Jamie Ellis, points out that chemical use in bee hives, chemical
toxins present in the environment and GMOs, that can actually pass in their pollen and
nectar the chemicals from the insecticide bath given to seeds prior to planting, could
produce a combined effect that stresses the immune systems of the bees making them
more susceptible to parasitic infections.

And according to an insider, the PSU report states...that they found pesticides, herbicides and
fungicides in the pollen in high enough amounts to cause alarm. This may indicate that the
food crop itself may be toxic.
 Compiled evidence on BT
“To our knowledge, this is the first report of immune responses occurring in farm
workers exposed to Bt-containing pesticides. Molecular genetic probes to identify
Bt organisms isolated from these workers confirmed that both skin and antibody
reactions were directed against the same Btk strain that was present in the
commercial product used during current spray operations.
Exposure to Bt sprays may lead to allergic skin sensitization and induction of IgE
and IgG antibodies, or both”.10

“Our concern over the virulence potential of these organisms focuses on evidence that
demonstrates the close genetic similarities between B. thuringiensis organisms and
B.cereus and B.anthracis pathogens reports on putative infections arising from
various B. thuringiensis subspecies and recent epidemiologic evidence of Bernstein et
al.”
Compiled evidence on BT
 Compiled evidence on BT
Cases of occupational allergies to Bt products have been reported and
confirmed in a recent study of farm workers, but only a small fraction
might be attributable to Cry proteins.
Also, bacterial enzymes used in detergents reportedly caused adverse
reactions in occupational settings and among consumers before
preventive measures were introduced.
Soy proteins released to the air at grain-loading docks caused
community outbreaks of asthma in Spain in 1985–1986 and in New
Orleans.
Stability with processing and digestion would not be relevant to
assessment of the potential for inhalation allergenicity.
Proteins may also have antinutritional properties; they may
decrease absorption of nutrients. An example of this is lectins
that are present in many plants and are harmful unless cooked.
The U.S. EPA and the FDA are aware of the existence of such
proteins in GMOs.
 Compiled evidence on BT
• The StarLink corn controversy
StarLink was a variety of Bt corn patented
by (a subdivision of Aventis, acquired by
Bayer AG in 2002), intended for use in
animal feed.
U.S. regulatory authorities permitted the commercial sale of
StarLink seed, with the stipulation that crops produced must not
be used for human consumption. This restriction was based on the
possibility that a small number of people might develop an allergic
reaction because the version of the Bt protein used in StarLink is
less rapidly digested than other Bt varieties.
StarLink corn was subsequently found
in food destined for consumption by
humans, with an episode involving
Taco Bell taco shells being
particularly well publicized.
 Compiled evidence on BT
Prohibited Gene-Altered Corn Found in Latin American & Caribbean Food
Aid Shipments
From: Environmental News, Service 2/16/05
Banned as Human Food, StarLink Corn Found in Food Aid
WASHINGTON, DC, February 16, 2005 (ENS) - More than 70 environmental,
consumer, farmer, human rights groups and unions from six Central American
and Caribbean countries held simultaneous press conferences today to
denounce the presence of unauthorized genetically modified organisms (GMOs)
in food aid distributed by the UN World Food Program (WFP), and in
commercial imports of food originating mostly from the United States.
... StarLink is banned for human consumption due to
possible allergic reactions to the genetically altered protein it contains...
In total over 50 samples of maize and soy from food aid in Nicaragua,
Honduras, El Salvador, Guatemala, and from commercial imports
in Costa Rica
and Dominican Republic were sent to Genetic ID, an
independent U.S.
laboratory, to verify whether GMOs were present.
GMOs were found in more than 80 percent of all
samples sent to the Laboratory.
renewed for domestic animal feed and non-food,
industrial use in the USA in 1998, 1999 and 2000.
But the groups in Central America and
Caribbean are concerned that food
with the Cry9C protein was distributed in
their countries. The organizations
requested the WFP to immediately recall all food aid containing
GMOs.
"It is not acceptable that a maize which is illegal for
human consumption
worldwide is contained in food aid distributed in our
country. Finding
StarLink four years after it was banned clearly shows
that genetically
modified foods are not under control," said Mario Godinez
of CEIBA in
Guatemala.
"The unwanted presence of unlabeled GMOs shows that Costa Rica
urgently
needs a ban on GMOs," said Fabián Pacheco of the Social Ecology
Association
in Costa Rica. "In order to protect our population it is of utmost
 Compiled evidence on BT
Corn sent by the UN and the US as help to
Central African nations was also found to
contain some StarLink corn. The nations
involved refused to accept the aid.
The southern portion of the U.S. corn belt
planted the greatest amount of StarLink
corn. It is this portion of the U.S. where
corn borer damage creates the greatest
economic loss to farmers.
Greenpeace, which opposes genetic
engineering in general, responded with a
movement to ban the production and
distribution of StarLink corn.
 Compiled evidence on BT
Mortality in Sheep Flocks after grazing on Bt Cotton fields
Warangal District, Andhra Pradesh
Report of the Preliminary Assessment, April, 2006
The preliminary information gathered from meeting shepherds
across 3 mandals, strongly suggests that the sheep mortality was
due to a toxin, and most likely Bt toxin from the foliage...The post-
mortem symptoms as observed by the shepherds, suggest severe
irritation of the intestines and associated organs (bile duct,
liver) connected to the absorption and assimilation of food and
processing of toxins.... The symptoms appear to be a
generalized immune response to toxins or organisms
producing toxins in the gut of the animal and thus suggest
death due to a phyto-toxin, most probably Bt toxin.... Since the
toxin may bind to intestinal proteins, there is a chance that if
the sheep were exclusively eating the Bt crop matter, they
would have in effect concentrated the toxin in their intestines
due to the binding properties.
 Compiled evidence on BT

On 17 July 2002, it reported that “British researchers have

demonstrated for the first time that genetically modified DNA
material from crops is finding its way into human gut
bacteria, raising potentially serious health questions”.

In an article in Nature Biotechnology in February 2004 (Vol 22, no. 2, pp 170-172), John Heritage of
the University of Leeds and one of the researchers in the Duggan et al study said “on
balance, the data presented in the paper support the conclusion that gene flow
from transgenic plants to the gut microflora does occur. Furthermore, because transfer
events seem to have occurred in three of the seven subjects examined, it may be that transgenic
gene transfers are not as rare as suggested by the UK GM Science Review Panel”. He said that the
risks of horizontal gene transfer should be assessed in the approval process for GMOs.
 Compiled evidence on BT
History of Plant-Pesticides Evaluated for Use in Human Food and/or Animal Feed
All plant-pesticides that have been approved for use in food and feed to date have
originated from sources not known to be food allergens and thus were not expected to
be food allergens. The following chart presents a list of the proteins in plant-pesticides
that have been approved for direct human consumption in food as of
September 1999.
Plant-Pesticide Protein Approved Dietary Use

Watermelon Mosaic Virus-2 Coat Protein All Food Commodities

Zucchini Yellow Mosaic Virus Coat Protein All Food Commodities
Potato Virus Y Coat Protein All Food Commodities
Papaya Ringspot Virus Coat Protein All Food Commodities

Cucumber Mosaic Virus Coat Protein All Food Commodities

Potato Leaf Roll Virus Replicase Gene All Food Commodities
Bacillus thuringiensis Cry3A Protein Potatoes
Bacillus thuringiensis Cry1Ac Protein All Plant Raw Agricultural Commodities
Bacillus thuringiensis Cry1Ab Protein All Plant Raw Agricultural Commodities

Corn Used for Feed; As Well As Meat,

Bacillus thuringiensis Cry9C Protein
Poultry, Milk, or Eggs Resulting From
Animals Fed Such Feed
 Compiled evidence on BT
Corn and cotton have been genetically
engineered to express the bacterial toxin
Bacillus thuringiensis, or Bt. This transgenic
trait allows plants to manufacture within their
cells a crystalline protein that is toxic to most
Lepidopteran insects (moths and butterflies).
Some 183 million acres of Bt transgenic corn
and cotton have been planted since 1996,
representing 27 percent of total GE crop
acreage. (Benbrook, 2004)
In 1999, 29 million acres of Bt corn, potato
and cotton were grown globally.
Compiled evidence on BT
 Compiled evidence on BT

THERE WERE MORE CASES OF LEVEL ONE AUTISM ADDED IN 2001 THEN IN ALL OF
1994, 1995, AND 1996 COMBINED

IT TOOK 25 YEARS (1970-1995) TO ADD 6,527 CASES...

IT HAS TAKEN ONLY 3 YEARS (1999-2001) TO ADD AN
ADDITIONAL 6,596 NEW CASES.
 Compiled evidence on BT
• Potato
A very important crop to Peru and third world countries
throughout the world is the potato. The potato (Solanum
tuberosum) originated in South America and was brought to
Europe by the Spaniards in the 16th century.
By the 21st century the potato has become the second most wide cultivated crop
and the fourth most important food crop in the world.
One single medium sized potato contains half the daily adult requirement of vitamin
C, more protein than maize, and nearly twice the calcium of maize.
Until recently, the first line of defense against the potato tuber moth was heavy
doses of chemical pesticides.
-But leading corporations in the genetically engineering bio-pesticides
visited Peru and south America offering their “NEW improved BT
biopesticide”
The potato tuber moth is now controlled with Bt pesticides.
Then they offer genetically modified organisms, such as the Bt potato.
 Compiled evidence on BT
Can Bt live in Humans?
This is an interesting question. There are no absolutely definitive studies addressing this
issue, but there is a good deal of circumstantial evidence that Bt can and does
survive and grow in humans:
• The culture media used to grow Bt in the lab is the same media used to grow other
human pathogenic bacteria.
• The conditions for growth of Bt (pH 7.4, temperature 37oC, moist environment)
are found in humans.
• Humans develop antibodies to the Bt organism.
• Even four months after a single exposure, Bt organisms of the same strain as the
pesticide used in the exposure can be cultured from nasal swabs. It is unlikely that
the original spores would still be present after this period of time.
• There are a few studies that show Bt can and does cause gastroenteritis in
humans and that you can recover culturable Bt from nursery workers feces,
indicating that Bt can live and grow in the intestinal tract.
• When humans are infected in this way, the immune system of healthy
individuals probably fights off and eventually destroys the invading cells.
However, there are some indications that Bt is able to survive for quite some
time at a level that does not cause any overt signs of disease.

Adopted from www.nosprayzone.org.

 Compiled evidence on BT

This paper states that the difference between

Bt and B. cereus is virtually nil. B. cereus
causes acute gastroenteritis and meningitis.
A complete physical map of a Bacillus thuringiensis chromosome
Carlson-CR; Kolsto-AB J-Bacteriol. 1993 Feb; 175(4): 1053-60
Bacillus thuringiensis is the source of the most widely used biological pesticide, through its production of
insecticidal toxins. The toxin genes are often localized on plasmids. We have constructed a physical map
of a Bacillus thuringiensis chromosome by aligning 16 fragments obtained by digestion with the restriction
enzyme NotI. The fragments ranged from 15 to 1,350 kb. The size of the chromosome was 5.4 Mb. The
NotI DNA fingerprint patterns of 12 different B. thuringiensis strains showed marked variation. The cryIA-
type toxin gene was present on the chromosome in four strains, was extrachromosomal in four strains,
and was both chromosomal and extrachromosomal in two strains. A Tn4430 transposon probe hybridized
to 5 of the 10 cryIA-positive chromosomal fragments, while cryIA and the transposon often hybridized to
different extrachromosomal bands. Ten of the strains were hemolytic when grown on agar plates
containing human erythrocytes. Nine of the strains were positive when assayed for the presence of
Bacillus cereus enterotoxin.
We conclude that B. thuringiensis is very closely related to B. cereus and that the
distinction between B. cereus and B. thuringiensis should be reconsidered.
 My hypothesis
Based on the evidence I have found it is my opinion that autism can only
develop in children that has a “predisposition”.
The so call “predisposition” based on my observation and evidence is
living in the gastrointestinal track. The trigger o the
It is my believe that the “predisposition” is caused by a bacterium’s toxins
known as Bacillus thuringiensis CRY proteins, giving other pathogens
like the ones found in vaccine or heavy metals and pollutants the
opportunity to overload their body’s immune system until our children
develop all the characteristics that we know as “autism”.
The exposure to BT toxins can occur during pregnancy or the children
acquires early in their lives from exposure to bio-pesticide BT in food or
transgenic crops BT.
My believe is that if we shut down the “MAIN” problem, the “predisposition”
our children can recover from the overload of combined problems they
have.
More research needs to be done to out rule this very strong possibility.
Andrea Lalama.
All TRUTH passes through 3 stages:

1st - it is ridiculed
2nd - it is violently opposed
3rd - it is accepted as SELF EVIDENT.
by Arthur Schopenhauer

“Let’s find the truth... and move on”

Andrea Lalama
•
References
1.(Z.Angew Entomologie 1915,2, p29)
• 2.The Canadian Biotechnology Advisory Committee Project Steering Committee on the
Regulation of Genetically Modified Foods
• 3.American Academy of Microbiology
• 4.Tayabali AF, Seligy VL. Cell integrity markers for in vitro evaluation of cytotoxic responses to
bacteria-containing commercial insecticides. Ecotoxicol Environ Saf 37:152-162 (1997).
• 5. Seligy VL, Rancourt JM. Antibiotic MIC/MBC analysis of Bacillus-based commercial insecticides:
use of bioreduction and DNA-based assays. J Ind Microbiol Biotechnol 22:565-574 (1999).
• 6. Beegle CC, Yamamoto T. Invitation paper (C.P. Alexander Fund): history of Bacillus
thuringiensis Berliner Research and Development. Can Entomol 124:587-612 (1992).
• 7. Seligy VL, Beggs RW, Rancourt JM, Tayabali AF. Quantitative bioreduction assays for
calibrating spore content and viability of commercial Bacillus thuringiensis insecticides. J Ind
Microbiol Biotechnol 18:370-378 (1997).
• 8. Helgason E, Okstad OA, Caugant DA, Johansen HA, Fouet A, Mock M, Hegna I, Kolsto AB.
Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis--one species on the basis of genetic
evidence. Appl Environ Microbiol 66(6):2627-2630 (2000).
• 9. Seligy VL, Douglas GR, Rancourt JM, Tayabali AF, Otvos I, van Frankenhuyzen K, Dugal J,
Rousseau G, Szabo AG. Comparative performance of conventional and molecular dosimetry
methods in environmental biomonitoring: assessment using Bacillus-based commercial
biopesticides as models. In. Rapid Methods for the Analysis of Biological Materials in the
Environment (Stopa PJ, Bartoszcze MA, eds). NATO ASI Series. Dordrecht, Netherlands:Kluwer
Academic Publishers, 2000;279-297.
• 10.Immune Responses in Farm Workers after Exposure to Bacillus thuringiensis Pesticides1.
Leonard Bernstein,1 Jonathan A. Bernstein,1 Maureen Miller,1 Sylva Tierzieva,1 David L
Bernstein,1 Zana Lummus,1 MaryJane K. Selgrade,2 Donald L. Doerfler,3 and Verner L. Seligy4
'Division of Immunology, Department of Internal Medicine, University of Cincinnati College of
Medicine, Cincinnati, Ohio, USA; 2Experimental Toxicology Division; 3Biostatistics and Research
Support Staff, National Health and Environmental Effects Research Laboratory, U.S.
Environmental Protection Agency, Research Triangle Park, North Carolina, USA; 4Environmental
and Occupational Toxicology Division, Environmental Health Centre, Health Protection Branch,
Department of Health Canada, Ottawa, Ontario, Canada.
• 11. <> Andreas Rang, et al, “Detection of RNA variants transcribed from the transgene in
Roundup Ready soybean,” Eur Food
• Res Technol 220 (2005): 438–443.
• Genetic Roulette, by Jeffrey Smith.
• Australian Government, Department of Environment and Heritage. 2005. Summary of the
Ecological Impacts of GM Cotton on soil biodiversity report. www.deh.gov.aus
• Vazquez-Padron R.I.,Gonzales-Cabrera J, Garcia-Tovar, C., Neri bazan L., Lopez-Revilla R.,
 References
URL’S USED IN THIS PRESENTATION:
www.soybeans.com/newscfm?docuementid=96
www.fmi.org/industry/biotech/
www.aphis.usda.gov/biotechnology/faqs.html
www.ifst.org/hottop10.htm
www.fmi.org/media/bg/biotech.html
www.fda.gov/bbs/topics/consumer/geneg.html
www.extension.iastate.edu/feci/argmo/
www.204202.137.114/onair/worldnewstonight/he
alth/wmt981109.genfood.html
http://courses.che.umn.edu/01fscn11021s/general_food_safety/gmo/gmo.html
•http://www.bt.ucsd.edu/contact.html

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