Bacteria, Biofilm, and Bio-pesticide BT (Bacillus Thuringiensis)

1
Bacteria, Biofilm, andBio-pesticide BT
(Bacillus Thuringiensis)

• Autism Hypothesis,
• Presented by
• Dr. Anju Usman, MD. and Andrea Lalama,

2
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.

3
Many patients with autistic symptoms have
persistent dysbiosis. Treatment of GI issues
often alleviates symptoms we call
autism.HypothesisPatients 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.

4
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, metal
  toxicity, acid exposure, dehydration salinity,
  phagocytosis, antibiotics, antimicrobial agents
  and the immune system.

Staphylococcus aureus biofilm
5
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.

6
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.

7
Non animated picture of Biofilm/Slime, YUK!
8
Why are they so difficult to treat?

• Remarkably difficult to treat with
  antimicrobials, 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 of quorum sensing molecules.

9
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

10
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.

11
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.

12
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.

13
Biofilm destruction by innate immune system

• Nature. 2002 May 30417(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 from a
  free-living, independent existence to a biofilm
  lifestyle can be devastating, because 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

14
Enzymatic Degradation

• Proc Natl Acad Sci U S A. 2007 Jul
  3104(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.

15
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 bodys antibacterial
action, dissolving biofilm and inhibiting
excessive bacteria maintaining a healthy oral
balance.
16
N-acetyl Glucosamine and Biofilm

• Shanghai Kou Qiang Yi Xue. 2006 Aug15(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

17
Probiotics,IBD, and Biofilms

• J Appl Microbiol. 2007 May102(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 role in inflammatory bowel disease. Because
  bacteria growing in biofilms are more 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.

18
Natural Antimicrobials

Scientists Develop 'Natural' Protection for
Stored Foods Tuesday, August 21, 2007 1200 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.
19
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

20
Our experience

• Positives
• Negatives
• What we have learned.
• ALWAYS work with your doctor especially when
  using chelating agents.

21
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.

22
What NON-GMO 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."

23
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 insectstypically Lepidoptera
(butterflies/moths), Coleoptera (beetles),
or Diptera (flies/mosquitos).
24
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.

25
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.
26
How natural BT works?
BT SPORES
Normal gut bacteria
BT crystalline Toxin 200px.
27
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.

28
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.

29
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.
30
What GMO 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
• Recombinant DNA,
• cell fusion,
• 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."

31
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.
32
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.
33
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 Its NOT organic NOR natural, it
  does NOT act like a natural BT, it is NOT
  selective to just certain insects species.

34
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.

35

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

36
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
37
How Genetically Modified BT works?
ACTIVATION OF Bt ICP IN AN INSECT GUT.
38
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.
39
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."

40
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.

41
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 CCDa 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.

42
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.
43
Compiled evidence on BT
44
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 19851986 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.

45
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.

46
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
AidWASHINGTON, DC, February 16, 2005 (ENS) -
More than 70 environmental,consumer, farmer,
human rights groups and unions from six Central
Americanand Caribbean countries held
simultaneous press conferences today todenounce
the presence of unauthorized genetically modified
organisms (GMOs)in food aid distributed by the
UN World Food Program (WFP), and incommercial
imports of food originating mostly from the
United States.
... StarLink is banned for human consumption due
topossible 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 Ricaand
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.
47
StarLink corn seed was registered and annually
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 foodwith the Cry9C protein
was distributed in their countries. The
organizationsrequested the WFP to immediately
recall all food aid containing GMOs. "It is not
acceptable that a maize which is illegal for
human consumptionworldwide is contained in food
aid distributed in our country. FindingStarLink
four years after it was banned clearly shows that
geneticallymodified foods are not under
control," said Mario Godinez of CEIBA
inGuatemala. "The unwanted presence of unlabeled
GMOs shows that Costa Rica urgentlyneeds a ban
on GMOs," said Fabián Pacheco of the Social
Ecology Associationin Costa Rica. "In order to
protect our population it is of utmostimportance
now more than ever to act with great caution."
48
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.

49
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.

50
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.
51
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
Bacillus thuringiensis Cry9C Protein Corn Used for Feed As Well As Meat, Poultry, Milk, or Eggs Resulting From Animals Fed Such Feed
52
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.
53
Compiled evidence on BT
54
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.
55
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.
56
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.
57
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58
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59
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60
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.
61
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 bacteriums 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 bodys 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.

62

• All TRUTH passes through 3 stages
• 1st - it is ridiculed2nd - it is violently
  opposed3rd - it is accepted as SELF EVIDENT.
  by Arthur
  Schopenhauer

Lets find the truth... and move on
Andrea Lalama
63
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 37152-162 (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 22565-574
  (1999).
• 6. Beegle CC, Yamamoto T. Invitation paper (C.P.
  Alexander Fund) history of Bacillus
  thuringiensis Berliner Research and Development.
  Can Entomol 124587-612 (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 18370-378 (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, NetherlandsKluwer Academic
  Publishers, 2000279-297.
• 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. ltgt Andreas Rang, et al, Detection of RNA
  variants transcribed from the transgene in
  Roundup Ready soybean, Eur Food
• Res Technol 220 (2005) 438443.
• 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., Hernandez M., Moreno-Fierro L and Gustavo A.
  de. La Riva. 2000. Cry1Ac Protoxin from Bacillus
  thuringiensis sp. Kurstaki HD73 Binds to Surface
  Proteins in the Mouse Small Intestines.
  Biochemical and Biophysical Research
  Communications 271, 54-58. Academic Press.
• GEAC. Background Note on Bt Cotton Cultivation in
  India.
• The Safety to Humans of Bacillus thuringiensis
  Insecticidal Sprays A Reassessment. No Spray
  Zone, 2006.

64
References

• URLS USED IN THIS PRESENTATION
• www.soybeans.com/newscfm?docuementid96
• 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/health/w
  mt981109.genfood.html
• http//courses.che.umn.edu/01fscn11021s/general_fo
  od_safety/gmo/gmo.html
• http//www.bt.ucsd.edu/contact.html

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