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| Perspective |
Biotechnology can improve crops by the addition of one or a few genes to make a plant more tolerant to stresses and more resistant to disease and pests. However there is concern that genes introduced into crops may be transferred to nearby weedy relatives or non-target species. Then, there is the risk in depending on one or a few varieties of a transgenic crop plant because this lack of diversity could result in nearly all the plants being susceptible to new stresses.
How It Works
Genetically modified (GM) plants have a piece of inserted genetic material
containing: (1) a gene coding for an insecticidal toxin from Bacillus
thuringiensis (Bt); (2) a gene for antibiotic resistance; and (3) a
gene, usually a viral promoter, which causes the new gene to be expressed
all the time. To date there is no evidence that any of them pose a threat
to human health. Fine tuning of gene expression should diminish the possibility
of detrimental effects and the development of resistance in pest species.
Data on the actual field performance of several genetically engineered crop varieties (canola, corn, cotton, potatoes, soybeans) indicate that, in most cases, the cost of the new resistant seeds is offset by the savings in herbicides and pesticides. Pesticide and herbicide use was cut drastically to 50% or less of that normally used. Possibly offsetting this are natural fluctuations in pest populations leading to lower pesticide cost in some years.
Other Benefits
In addition to preventing insect damage to plants such as corn, the
introduced Bt genes in some corn varieties significantly depress the incidence
of Fusarium ear rot which is spread by corn borers. This, in turn,
decreases levels of Fusarium mycotoxins, in particular fumonisins,
in the harvested corn. Another advantage noted with fields of Newleaf Plus
potatoes (containing genes to combat Colorado potato beetles and potato
leafroll virus) was that reduced pesticide spraying enhanced populations
of spiders and beneficial predatory insects which then kept aphid and mite
populations in check. In this case, some non-target species benefitted
from the planting of transgenic potatoes.
Butterflies
Recently published research on the harmful effects of pollen from Bt
corn on monarch butterfly caterpillars has generated concern about effects
of GM crops on non-target species. Experiments conducted this past summer
demonstrated approximately a fifteen-fold range in toxicity of pollen from
different varieties of Bt corn and this toxicity was gradually lost upon
exposure to sunlight. Pollen grain concentrations on leaves decreased as
distance from the field increased. However, monarch butterflies prefer
to lay their eggs on exposed plants rather than on plants under a canopy
of corn leaves. These preliminary results indicate that pollen from the
most toxic Bt corn variety could be detrimental to monarch populations
living nearby. It appears that there would be minimal impact on populations
living farther away.
Resistance
Pests develop resistance to chemicals used against them and, in time,
they will also become resistant to Bt toxins. Already there are some reports
of tobacco budworm (a cotton pest) being resistant to one or more Bt toxins.
It has been recommended that a plan utilizing refuges (where non-Bt varieties
are planted) be used to delay development of insect resistant populations.
Without refuges, it is estimated that resistant populations would develop
in 12 generations; with 10% of a field planted as refuge, it would take
100 generations to develop a resistant population. Also, refuges planted
around the perimeter of a field would decrease the drift of pollen from
GM plants to weedy relatives, or to plants which are a food source for
nontarget species.
Future Developments
Biotechnology has great promise for crop plants in developing countries.
New transgenic varieties could be resistant to locally important diseases
and pests and have enhanced tolerance for drought or salinity. The nutritional
content of widely used crops, such as rice, could be improved by adding
genes which code for production of vitamin A and some amino acids. These
developments would enhance the productivity of agriculture and improve
the nutritional quality of foods, thereby providing more and better food
for their growing populations.
There are many issues involved in the successful transfer of biotechnology to developing countries. There are many "orphan crops" which are very important in tropical agriculture but are not being studied intensively here because they are not planted over a large area in temperate climates. Agronomists from developing countries are also concerned about the concentration of seed companies in a few hands, and the lack of testing of transgenic crops in tropical environments where insect pests are different, more numerous, and grow faster. Other impediments to use of biotechnology internationally involve legal issues (patents and contracts) and trade secrets, including GURTS (genetic use restriction technologies), such as the terminator gene. Dr. Jefferson of CAMBIA (Center for the Application of Molecular Biology to International Agriculture) states that we need to democratize, decentralize, and diversify research into gene expression of crop plants.
Many consumers are concerned about the introduction of GM foods into the human diet. To some extent, this may reflect a lack of knowledge about the processes and the extensive testing being done to insure the safety of GM crops. But many people who do understand the technology are genuinely concerned about the possibility of introducing allergens into nonallergenic foods and, on a religious level, the manipulation of nature and disruption of the natural order.
Some scientists and industry people have been quick to insist that these fears and concerns are unfounded. But, the BSE crisis in England and recent scares in Europe about dioxin in Belgian foods, contaminants in Coca-Cola, and sewage contamination in France have undermined trust in regulators and "big agriculture." Whether or not people's concerns are scientifically justified, they must be addressed and discussed.
| Research |
| Coming Together: Bacteriology, Immunology, Toxicology |  |
This building will contain 330,000 square feet with 47 laboratories. A preliminary architectural rendering is shown. The estimated cost is $102,000,000. We are trying to raise $50,000,000 from private sources, with matching funds coming from the State of Wisconsin, the Wisconsin Alumni Research Foundation and hopefully federal assistance.
We currently have almost all the intellectual personnel to staff the new laboratories and classrooms, but they are scattered around the campus, some in largely outdated or remodeled-marginal premises. Bringing them together will improve effectiveness by enhancing communication and interaction. Almost 500 scientists will work in the new complex.
More important than size is staff quality and the ability to turn research to practical use. Regarding quality, a recent survey by U.S. News & World Report ranked the UW–Madison microbiology program first in the U.S. among public universities. Finding practical applications for our discoveries is handled by the Wisconsin Alumni Research Foundation (WARF), whose function is patenting and making our research results available to the public. More than one billion dollars worth of products are produced annually under WARF licenses, ranging from anti-clotting drugs to vitamins.
You'll hear more as the project progresses.
[January 2000: UW-Madison Campus Planning Committee approves proposed new building for design stage in fiscal biennium 2001-03.]
| Environmental Toxicology Center | ||
This development presents FRI with a fine opportunity to expand our program by working more closely with ETC in the areas of environmental contaminants and genetically modified foods. In addition, the graduate students within the ETC, who are selected among the best at UW–Madison, will now have expanded opportunities to work at FRI.
The Center was founded in 1971 as an outgrowth of the interest of many UW-Madison faculty members in the problems related to potentially hazardous chemicals in the environment. The Center promotes research on suspected and known environmental toxicants, provides training in toxicology at all university levels and facilitates the exchange of information related to environmental toxicology. The Center is sponsored by the College of Agricultural and Life Sciences (School of Natural Resources) and the Schools of Medicine, Pharmacy, and Veterinary Medicine.
Health-related Toxicology and Toxicants in the Environment
Graduates of this program have a solid foundation in both areas with
a command of skills in one or the other. Graduate students become familiar
with the field through enrollment in a core curriculum and participation
in the Center's seminars and special topics courses. The courses are designed
to provide a framework for experimental methods and information sources.
Through their thesis research projects students learn to study the origin
and fate of chemicals in the environment, and changes induced by toxicants
in the environment.
| Monitoring of Microcystin–Protein Phosphatase Adduct Formation with Immunochemical Methods |
Mechanistically, MCYST-LR, one of the major variants in this group of toxins, is a potent inhibitor of protein phosphatase (PPase) 1 (PP1) and 2A (PP2A). The inhibitor effect is due to its interaction with the enzyme through covalent binding and free form. We have developed a series of specific antibodies against MCYSTs, as well as a sensitive immunoassay for the toxin. In the present study, an immunoblotting method was developed to monitor the adduct formations both in vitro and in vivo. The detection limits for the covalent binding of MCYST-LR with the recombinant protein phosphatase 1 (PP1) and rabbit liver cytosol proteins were found to be 0.1 ng and 0.3 ng per assay, respectively. MCYST-PP1 adducts were detected 30 seconds after the addition of MCYST-LR into the reaction mixture.
Immunoblotting analyses and enzyme-linked immunosorbent assay showed that between 5 min to 16 hr after i.p. injection of a single dose (35 µg/kg) of MCYST-LR into mice, up to 27% of the injected toxin was found covalently bound while 0.2–9.2% existed free form in liver cytosol. Our data suggest that the newly developed method could be used for monitoring MCYST intoxication in humans and animals.
| Purification and Genetic Characterization of a Bacteriocin that Inactivates Clostridium botulinum |
Bacteriocins are bactericidal polypeptides that are lethal against closely related species. Bacteriocins are generally small molecular weight proteins, often heat-resistant, and the structural genes encoding bacteriocins are usually plasmid-linked. Bacteriocins bind specifically to receptors on the surface of target cells and kill the cells by alteration of membrane-bound enzymes, disruption of membrane potential by pore formation, or by enzymatic digestion of RNA and/or DNA. The proteinaceous nature of these antimicrobial molecules as well as their natural occurrence in nature has promoted consideration for their use in foods to prevent microbial foodborne diseases and bacterial food spoilage. Bacteriocins of lactic acid bacteria (LAB) have been intensively investigated due to their potential use in foods as novel biological preservatives and the safe history of LAB in foods.
Boticin B, a bacteriocin produced by C. botulinum 213B, was purified to homogeneity by sequential heat treatment of the culture supernatant at 80ºC, ammonium sulfate precipitation, and C4 reversed-phase chromatography. Boticin B was inhibitory against group I and II C. botulinum and related Clostridium spp., but not against other bacterial species tested. Boticin B is heat stable and retains activity after exposure to 100ºC for 60 min, and is stable over the pH range 4.5–10.0. It is a small acidic peptide that differentiates it from other bacteriocins which are cationic in nature. Boticin B was inactivated by proteinase-K and papain, but was resistant to other proteolytic enzymes, including trypsin, pronase E, endoproteinase Glu-C and Asp-N endoproteinase.
Amino acid analysis indicated a high content of hydrophobic and acidic amino acids, and the absence of basic amino acids. This finding is consistent with the high hydrophobicity of the peptide evident from its elution characteristics during reversed-phase HPLC. Direct amino sequencing of the intact peptide was not successful, except that methionine was identified as the N-terminal amino acid. Chymotryptic fragments were separated by C18 reversed-phase HPLC and three major fragments were sequenced by Edman degradation. The sequences obtained are unique among antimicrobial peptides analyzed in the SWISS-PROT data bank.
The determination of a partial amino acid sequence enabled the design and synthesis of a mixed oligonucleotide (18-mer) that hybridized to a 3.0 kb HindIII fragment of an 18.8 kb plasmid from the C. botulinum strain 213B. The fragment was cloned into pBluescript KS II(+) and subcloned into a clostridial shuttle vector, pJIR1457. DNA sequencing revealed the boticin B structural gene to be an open reading frame encoding 50 amino acids. The plasmid pMVP1113 (pJIR1457 containing the 3.0-kb cloned fragment of the boticin B plasmid) was introduced into C. botulinum strain 62A by conjugative transfer from the E. coli strain S17-1. The C. botulinum strain 62A(pMVP1113) transconjugant expressed boticin B, although at much lower levels than that observed in C. botulinum 213B.
In this study, a bacteriocin having antimicrobial activity against C. botulinum strains was purified and the gene encoding the peptide was cloned and expressed in a clostridial host. Understanding the mechanism of antagonism of C. botulinum 213B against closely related C. botulinum strains may help to understand the intestinal ecology of C. botulinum colonization, to develop a novel strategy to prevent C. botulinum growth in food systems, and to improve challenge studies for assessing the ability of the pathogen to grow in foods.
| Viruses and Protozoa – Detecting the "Other" Foodborne Disease Agents | return to top | |
| Professor Dean O. Cliver left FRI in 1995 to join the University of California–Davis in the School of Veterinary Medicine. He continues as Director of the World Health Organization Collaborating Center for Food Virology, a position he also held here. We asked him to prepare a short article on enteroviruses and protozoa, particularly the present status of the problems. He obliged with the following paper. | ||
The viruses and the protozoa are both strict parasites, so they cannot multiply between hosts, nor can they be enriched in the laboratory before a detection test is applied. Each is transmitted by a fecal-oral cycle; the Norwalk-like viruses are also shed in vomitus. The viruses transmitted via food and water are human-specific, whereas C. parvum and G. lamblia are most often transmitted from other mammals to humans, but also among children in day-care centers. Nonhuman hosts of C. parvum and G. lamblia include wild animals, farm animals, and pets. Some strains of C. parvum appear to be specific for humans.
HAV and the Norwalk-like viruses replicate very slowly or not at all in cell cultures, and thus cannot be detected by that means. C. parvum oocysts and G. lamblia cysts (the transmission forms) are generally detected microscopically, more recently by means of fluorescent antibody. The quest for greater sensitivity, in detecting both groups, has led to the polymerase chain reaction (PCR); because PCR requires DNA, whereas foodborne viruses contain only RNA, PCR detection of these requires reverse transcription (RT). Two drawbacks are inhibition of PCR (and RT, where applicable) by substances in environmental samples and the possibility that agents detected by PCR have already been inactivated (yielding, from a public health standpoint, a false positive result). Further gains in sensitivity require processing larger samples — e.g., the EPA Information Collection Rule specifies samples of source water of 100 to 200 L. Filters that retain the viruses by adsorption, for later elution, and other filters that retain oocysts and cysts mechanically, serve this purpose.
Detection of these agents requires: (1) preparation of a large volume of liquid sample or sample extract, free of solids that interfere with concentration; (2) concentration on a filter and elution in a smaller volume of fluid, perhaps with secondary concentration by precipitation of the agent from this eluate; (3) purification of the agent by collecting it on antibody-coated magnetic beads and washing off extraneous materials; (4) application, where possible, of some treatment that blocks detection of noninfectious units of the agent; and (5) (RT-)PCR testing to detect a portion of the agent's genome. The material amplified by PCR may be used, further, for "genetic fingerprinting."
Clearly, methods for detecting these agents have advanced enormously. Unfortunately, many laboratories are not equipped to perform the new tests.
| Short Subjects |
Two projects have currently been placed:
"Host Defense Mechanisms Against Gastrointestinal Listeriosis," School
of Veterinary Medicine, Professor C. J. Czuprinski. The long-term goal
is to bring about a better understanding of the host defense mechanisms
that protect against gastrointestinal listeriosis. This information could
well apply for other pathogens as well. Since vulnerability to listeria
varies among individuals, knowledge of the mechanism of resistance could
lead to strategies for control of the disease.
"Genetic Engineering of Small Grains for Resistance to Fungal Infestation
and Mycotoxin Production," Department of Agronomy, Heidi Kaeppler, Assistant
Professor. Antifungal genes are currently being delivered into wheat, with
transgenic plants being generated in the next six months. This is the second
year of the project to genetically engineer wheat with the antifungal and
mycotoxin degrading/detoxifying genes and to characterize transgenic plants
and progeny. Fusarium head blight, also known as "scab," has become
a disease for all classes of wheat and barley in the U.S. and worldwide.
This poses a large threat to food and feed safety because scab-infected
grain is usually infected with vomitoxin. A multidisciplinary team has
been formed with FRI, Plant Pathology, Agronomy and the USDA-ARS Cereal
Crops Quality Unit.
| Johnson |
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| Kaspar |
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| Wong |
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| Food Industry |
The egg industry has continually worked to assure the safety of its products. They have pushed for breeder flock testing of SE under the National Poultry Improvement Plan, have supported eggs being listed as a potentially hazardous food (FDA's list), and have worked aggressively with allied industry leaders to develop and implement vaccination programs to minimize human exposure to foodborne pathogens. I have personally sat in rural restaurants with Wisconsin Egg Producers as they hammered out the details for assuring a safe food supply. I have witnessed egg producers working with state regulators in developing advanced refrigeration conditions to assure safe eggs. I know the dedication of our local producers and the pride they have in quality products. The united efforts of the nation's egg producers represent a proactive step in improving the quality and safety of their products.
| World Literature |
Skin prick tests revealed positive reactions to grass pollen, mustard, peanuts, almonds and some fruits in addition to beer, malt, barley, corn, wheat, rye, oats, and rice. Analyses of serum samples from the patient demonstrated the presence of specific IgE antibodies to malt, barley, corn, wheat, rye, oats, and rice. Despite the positive results for wheat, rye and oats, the patient reported no problems associated with eating bread and bakery products. Further immunoblotting tests demonstrated reactions between the patient's serum and proteins from barley, malt and corn extracts. Several allergenic proteins appeared to be more common in the malt than in the barley extracts.
Allergic reactions to beer have rarely been reported in the literature, and most other cases involved simply the development of a rash. This patient has the unfortunate distinction of being severely allergic to both beer and corn products!
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| FRI Newsletter, Winter 1999
index | FRI Communications
| FRI home page | Dept.
Food Microbiology & Toxicology | UW–Madison
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