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Bacteria, Biofilm, and Bio-pesticide BT ( B acillus T huringiensis ) Autism Hypothesis, Presented by Dr. Anju Usman, MD. and Andrea Lalama,

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Presentation on theme: "Bacteria, Biofilm, and Bio-pesticide BT ( B acillus T huringiensis ) Autism Hypothesis, Presented by Dr. Anju Usman, MD. and Andrea Lalama,"— Presentation transcript:

1 Bacteria, Biofilm, and Bio-pesticide BT ( B acillus T huringiensis ) 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. 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.

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? 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. 5 stages of biofilm development.

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 are potential treatment options? EDTA Fe chelating compounds Enzymes - mucous degrading Probiotics Fermented Foods High dose Antibiotics What type of biofilm control strategies have been studied?

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 May 30;417(6888): A component of innate immunity prevents bacterial biofilm development. Singh PKSingh PK, Parsek MR, Greenberg EP, Welsh MJ.Parsek MRGreenberg EPWelsh 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: [PubMed - indexed

14 Enzymatic Degradation Proc Natl Acad Sci U S A Jul 3;104(27): Epub 2007 Jun 25. Dispersing biofilms with engineered enzymatic bacteriophage. Lu TKLu TK, Collins JJ.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 % 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 body’s antibacterial action, dissolving biofilm and inhibiting excessive bacteria – maintaining a healthy oral balance.”

16 N-acetyl Glucosamine and Biofilm Shanghai Kou Qiang Yi Xue Aug;15(4): Links Effects of chitosans with different molecular weights on Streptococcus sanguis biofilm Ma RMa R, Zhu M, Liu Z.Zhu MLiu 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 [PubMed - in process]

17 Probiotics,IBD, and Biofilms J Appl Microbiol May;102(5): Microbial biofilms in the human gastrointestinal tract. Macfarlane SMacfarlane S, Dillon JF.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; 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.

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 BTCompiled evidence on BT –My Hypothesis and opinionMy Hypothesis References.

22 What NON-GMO stands for? NON G enetically M odified O rganism 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 insects—typically 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 crystalline Toxin 200px. Normal gut bacteria BT SPORES

27 How natural BT works? Mode of Action 1.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). 2.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. 3.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. 4.Affected insects stop feeding and die from the combined effects of starvation, tissue damage and gastrointestinal infections by other pathogens like bacteria and funguses. 5.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: 1.Recombinant DNA, 2.cell fusion, 3.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.Bacillus thuringiensis 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.

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 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 ). 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?

36 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 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.

42 Compiled evidence on BT “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.” Exposure to Bt sprays may lead to allergic skin sensitization and induction of IgE and IgG antibodies, or both”. 10 “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.

43 Compiled evidence on BT

44 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.

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 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.

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 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 importance 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 In an article in Nature Biotechnology in February 2004 (Vol 22, no. 2, pp ), 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. 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”.

51 Compiled evidence on BT Plant-Pesticide ProteinApproved Dietary Use Watermelon Mosaic Virus-2 Coat ProteinAll Food Commodities Zucchini Yellow Mosaic Virus Coat ProteinAll Food Commodities Potato Virus Y Coat ProteinAll Food Commodities Papaya Ringspot Virus Coat ProteinAll Food Commodities Cucumber Mosaic Virus Coat ProteinAll Food Commodities Potato Leaf Roll Virus Replicase GeneAll Food Commodities Bacillus thuringiensis Cry3A ProteinPotatoes Bacillus thuringiensis Cry1Ac ProteinAll Plant Raw Agricultural Commodities Bacillus thuringiensis Cry1Ab ProteinAll 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 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.

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)Benbrook, 2004 In 1999, 29 million acres of Bt corn, potato and cotton were grown globally.

53 Compiled evidence on BT

54 IT TOOK 25 YEARS ( ) TO ADD 6,527 CASES... IT HAS TAKEN ONLY 3 YEARS ( ) TO ADD AN ADDITIONAL 6,596 NEW CASES. THERE WERE MORE CASES OF LEVEL ONE AUTISM ADDED IN 2001 THEN IN ALL OF 1994, 1995, AND 1996 COMBINED

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 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: 1.The culture media used to grow Bt in the lab is the same media used to grow other human pathogenic bacteria. 2.The conditions for growth of Bt (pH 7.4, temperature 37oC, moist environment) are found in humans. 3.Humans develop antibodies to the Bt organism. 4.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. 5.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. 6.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. Compiled evidence on BT Adopted from

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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 Feb; 175(4): 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 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.

62 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

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 37: (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: (1999). 6. Beegle CC, Yamamoto T. Invitation paper (C.P. Alexander Fund): history of Bacillus thuringiensis Berliner Research and Development. Can Entomol 124: (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: (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): (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; 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 Summary of the Ecological Impacts of GM Cotton on soil biodiversity report. 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 Cry1Ac Protoxin from Bacillus thuringiensis sp. Kurstaki HD73 Binds to Surface Proteins in the Mouse Small Intestines. Biochemical and Biophysical Research Communications 271, 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 URL’S USED IN THIS PRESENTATION: alth/wmt genfood.html al_food_safety/gmo/gmo.html


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