Duration: 10/01/2000 to 09/30/2005

Administrative Advisor(s):

NIFA Reps:

Non-Technical Summary

Statement of Issues and Justification

Mycotoxin contamination of food and feed grains is a serious economic problem for grain producers and processors in the United States because grain contamination impacts markets. The reason there is a market effect is that some of the mycotoxins adversely affect animal and human health. The fungi that produce mycotoxins in grains commonly include Fusarium Aspergillus and Penicillium species. The primary focus of this project is on the fusaria that infect grains However, other mycotoxins also occur in grains and associated studies on other toxigenic fungi and their metabolites are needed to understand the overall problem of mycotoxins in cereal grains. This regional project is very interdisciplinary in nature and includes members from Plant Pathology, Veterinary Pathobiology, Veterinary Clinical Medicine, Veterinary Biosciences Food Science and Human Nutrition, Animal Science and Industry, Veterinary Medical Diagnostic Labs, Food Microbiology and Toxicology Departments as well as representatives from Ontario's Ministry of Agricultural, Food and Rural Affairs and the mycotoxin unit at NCAUR/USDA/ARS.

JUSTIFICATION

Mycotoxins are secondary metabolites of fungi that can produce harmful effects when ingested by animals or humans. Some of the toxic effects are easy to recognize such as acute aflatoxicosis (aflatoxm), equine leukoencephalomalacia and porcine pulmonary edema (fumonisin), estrogenism (zearalenone), and feed refusal (vomitoxin), whereas other effects are insidious and difficult to diagnose. The trichothecenes, exceeding 100 in number, are examples of toxins with health effects that are not easily recognized except at extremely high concentrations. Mycotoxins also may cause suppression of the immune system (e.g. aflatoxins and T-2 toxin) and thus redispose animals to infectious disease. This regional research project deals primarily, but not exclusively, with the effects of Fusarium mycotoxins on animal health, but with the underlying assumption that these toxins are consumed by, and therefore hazardous to humans. Therefore, issues of food safety are of primary importance to this project.

Mycotoxins play a global role in human and animal health; some of their effects are well documented while others are not. Examples of major Fusarium mycotoxins with known biological activity include the fumonisins (neurotoxicity, hepatotoxicity, cardiotoxicity, carcinogenesis), T-2 toxin (hemorrhage, enteritis, immune suppression), deoxynivalenol (decreased weight gain), zearalenone (estrogenism) and fusarochromanone (bone deformation). The National Toxicology Program (National Institutes of Health) and WHO (World Health Organization) lists zearalenone as a carcinogen in mice but not in rats. The economically important mycotoxins that are possible human carcinogens include the aflatoxins, fumonisins, and ochratoxins. Many of the other known mycotoxins are acutely toxic or have subacute effects on animal production. The economic impact of mycotoxins is difficult to determine, primarily because of the subclinical effects on animals. Many of the diseases encountered in animals are idiopathic and some may be due to mycotoxins, or to their interaction with other disease causing agents.

In the Midwest region of the United States, losses due to Fusarium Head Blight (SCAB, FHB), in wheat and barley, with the accompanying production of vomitoxin can be devastating, as evidenced by the severe outbreaks in North Dakota, South Dakota, Minnesota, Manitoba, Indiana, Ohio, Illinois and Michigan. The effects include crop production losses, and the inability to use the crop for human (milling and baking) and animal food. The economic and health risks associated with mycotoxins are presently presently poorly defined because mycotoxin contamination affects all segments of the production, marketing and utilization of grains. Part of the reason for this uncertainty is due to yearly climatic changes (the insidious nature of toxic effects, lack of diagnostic procedures for some mycotoxicoses and the multiplicity of crops affected. However, different routes and levels of mycotoxin exposure may affect the outcome of the toxicity and clinical diagnosis. These variables must be defined so that reliable toxicity information can be documented. Toxicological, metabolic and residue determinations are part of this cooperative effort.

Cooperation among NC-129 scientists extends into the Southeast (Georgia) where Fusarium fungi exist in crops in association with Aspergillus flavus and the aflatoxins. The Fusarium Research Center at Pennsylvania State University provides an invaluable service in maintaining a collection of Fusarium isolates, which are made available to researchers worldwide. The states and the USDA/ARS are cooperating to provide mass spectral as well as chemical and immunochemical analyses of various mycotoxins on research and diagnostic specimens in grains and animal tissues.

No single Experiment Station in the North Central Region has the resources in personnel, expertise, money or time to investigate more than a few mycotoxin problems at one time. Therefore a regional project is needed for this research. Because of the predominance of cereal crops grown in this region, the Fusarium mycotoxins in cereal grains are the major thrust of this project. However, there is a lack of toxicological information regarding mycotoxins in general. Cooperation among the members of NC-129 includes sharing of toxins, technical expertise, information on current problems, and priorities and collaborative studies. Large animal studies are expensive and generally not affordable except through collaborative projects within the region. Furthermore, to obtain maximum utilization of such animals, a multidisciplinary team is needed. This condition is met through cooperative efforts among members of the NC-129 Committee. The application of technology to isolate and identify fungi and their toxins has not advanced as rapidly as the need for this information. Another important function of this project is the validation of new analytical technology. Although these technologies may not be developed collaboratively, their evaluation through cooperative research and testing results in useful techniques that can be quickly adopted for widespread use. The NC-129 project is a logical approach for such cooperative studies.

Current investigations by members of this committee on the biosynthesis of Fusarium mycotoxins using genetic and molecular technologies, may lead to understanding and control of the associated plant diseases and formation of mycotoxins in these commodities. The goal of the collaborative research is to understand the health effects of mycotoxins and ultimately to control and hopefully eliminate mycotoxins from occurring in our foods.

Related, Current and Previous Work

There are four active Regional Research or Information Exchange Projects on mycotoxins in addition to NC-129. IEG 51 in the Southern Region covers Mycotoxins in Food and Feed Grains. NC-129 and IEG 51 cooperate closely and meet together about every three years. The research efforts in these two groups complement each other effectively because both groups address different areas of mycotoxicology. The Southern group primarily stresses aflatoxins while the NC group stresses the Fusarium mycotoxins. NC 213 also addresses grain quality and covers Marketing and Delivery of Quality Cereals and Oilseeds. NCR 184.Management of Head Scab of Small Grains, is primarily concerned with vomitoxin contamination and pre-harvest disease control issues. These groups are all diverse and communication between groups is common and effective. These research groups are the primary regional and interregional research efforts on mycotoxins in the Experiment Station system. The various USDA/ARS research efforts are included in all of the regional efforts because the USDA/ARS has ongoing research projects on Fusarium and Aspergillus mycotoxins. Many times the projects are cooperative efforts between the Experiment Stations and the USDA/ARS. The CRIS database lists a total of 59 projects when searched with the keyword mycotoxin. There are very few projects that are not already in one of the mycotoxin-related Regional Projects or Information Exchange Groups. The projects that are not covered in any of these groups are plant breeding programs for control of diseases caused by fusaria or aspergilli that can produce mycotoxin contamination of the harvested product. The plant breeders cooperate with regional project members in almost all cases so there are effective communication and information mechanisms. There is little duplication of research in the projects in the CRIS database and the researchers from the various states have developed productive multistate research projects in many areas of mycotoxin research. The strengths of mycotoxin regional research efforts are easy to document. Since the mycotoxin problems impacting food safety are often related to weather patterns it is critical that multistate studies and surveys be current and complementary

Objectives

1. Determine the effects of mycotoxins produced in cereal grains on animal and ultimately human health
   
2. Develop new techniques and improve current assays for identification and quantification f mycotoxins in cereal grains
   
3. Establish integrated strategies to manage and to prevent mycotoxin contamination in cereal grains
   
4. Define the metabolic pathways and regulatory pathways of mycotoxin biosynthesis.
   
5. 
   

Methods

Objective 1. Determine the effects of mycotoxins produced in cereal grains on animal and ultimately human health.

Contributing stations will cooperate to determine the effects of the fumonisins the tnchothecenes, the fusarins, beauvericin, moniliformin, and fusaric acid on animal health and to determine the interactions of these mycotoxins in cereal derived foods. This goal will be achieved by focusing on: a) preparing adequate mycotoxins for toxicological studies-b) determining the health effects of mycotoxins by toxicological studies; c) determining the mechanisms of toxicity of selected mycotoxins; d) evaluating diagnostic criteria for mycotoxicoses, through recognition of health and production. These studies will provide abasistor developing guidelines to insure that food intended for human and animal consumption is safe.

1.1 Toxicology Studies

Toxicology of mycotoxins will be evaluated by Missouri, NCAUR, Iowa, Illinois and Nebraska. Missouri will supply corn culture material for preparation of the fumonisins, aflatoxins, ochratoxin A, T-2 toxin, DAS, moniliformin and beauvericin. NCAUR will purify fumonisins, beauvericin and moniliformin from the culture materials. Missouri will focus on toxicology studies using swine, chickens and turkeys. Iowa will focus on studies using swine, horses and rats. In collaboration, Missouri, USDA/NCAVR, Michigan, and Illinois will determine in swine the mechanism of cardiotoxicity and the effect of long-term low dose effects of fumonisins on the cardiovascular system including cholesterol and homocysteine levels. The dose-response/no effect level for fumonisin B, induced cardiovascular changes in swine needs to be determined. The long-term cardiovascular studies will provide information regarding the potential human health effect. Illinois will also complete their investigations of fumonisin treated horses. A dose response relationship will be determined and correlation between target tissues and alterations in sphingoid bases will be examined. The pathogenesis of the neurologic disease in horses will be explored using MRI and more traditional methods. NCAUR and Missouri will collaborate on trichothecene toxicology studies. Nebraska will focus on the molecular mechanisms of disease development by fumonisin. Iowa will provide cooperating stations with information on initiating events in carcinogenesis, through placental glutathione tranferase foci and other serum indicators. Kansas will focus on the toxicological effects of beauvericin and fusaproliferin (or combination) in cattle. Michigan will work on a toxicity model for deoxynivalenoi using human tissue culture cells. This approach was validated by comparing the effects of DON in a mouse tissue culture system with live animals.

1.2Diagnostics

Iowa, NCAUR, Missouri, Michigan and Wisconsin will work on diagnostic criteria. NCAUR will provide analysis of mycotoxins in tissues and fluids. North Dakota will provide analysis and/or confirmation of mycotoxins in foods. Wisconsin, Michigan, Illinois, and NCAUR are developing monoclonal antibodies and recombinant antibodies to evaluate tissues and fluids for mycotoxins with ELISA and immunohistochemical techniques. The effects of stress, heat, disease, and nutritional status on diagnostic criteria for Fusarium mycotoxins will be evaluated at Missouri and Wisconsin.

Objective 2. Develop new techniques and improve current assays for identification and quantification of mycotoxins in cereal grains.

The general public is concerned about food safety and the prevention of mycotoxin contamination in cereal grains that enter the food chain. Therefore, rapid and accurate assays for mycotoxins are important to the grain industry, animal producers, overseas buyers and inspection agencies. Methodology currently centers on fumonisins, however, of equal importance area flatoxins, zearalenones, deoxynivalenoi, nivalenol, moniliformin, beauvericin and other mycotoxins, many of which are not routinely assayed. Early detection, of these toxins in cereal grains, at time of harvest or prior to entering commercial channels, requires a rapid screening test for common toxins (e.g. DON) that can be used in elevators and field stations.

2.1 Immunological Techniques

Previously, antibody-based assays were developed using conventional antibody techniques, including polyclonal and monoclonal antibodies with specific binding affinities for an array of mycotoxins. In collaboration, Michigan, North Dakota and Virginia will investigate the use of recombinant antibody technology to develop a new generation of antibody assays. Using a technique called phage display we can screen recombinant antibody libraries for binding specificity to targeted mycotoxins. In addition, phase display libraries of short peptide sequences may provide a totally new way to identify specific sequences that bind to mycotoxins. Recombinant antibodies that are bispecific and/or bifunctional may be adapted to multioxin assays. NCAUR will continue to work with Illinois in the development of novel mycotoxin antibodies, will be developing biosensors based upon new antibodies, and will be cooperating with Georgia in the evaluation of these biosensors.

2.2 Spectral Techniques

Analytical methods for the detection of multiple mycotoxins (fumonisins, moniliformin, beauvericin and other mycotoxins) present in the same commodity are also needed. To develop these assays, new technologies will be studied, including capillary zone electrophoresis, mass spectrometry, micro-capillary continuous flow fast atom bombardment/MS microcapillary electrospray and ionspray/MS. North Dakota will develop Negative Chemical Ionization techniques by GC/MS for detection of ppb/ppt levels of trichothecenes in body tissues and fluids. This service may play a critical role in identifying animal exposure to mycotoxins and will be available to all NC129 collaborators.

Methods for the detection of fumonisins, as well as sphingosine and sphinganine in urine, or tissues of animals, are needed to ascertain exposure and metabolism in animals. Greater emphasis is being placed on the water soluble (e.g. fumonisin) and polar adducts of the mycotoxins and hence techniques using microcapillary HPLC interfaced to a mass spectrometer are needed. The latter methods do not use derivatives such as the trimethylsilyl ethers but need to be interfaced to a mass spectrometer. Derivatization techniques for GC/MS or GC/ECD need to be studied extensively. The analysis of deoxynivalenol (DON), 3-acetyl-DON, 15-acetyl-DON and nivalenol in cereal grain is important. Routine and reliable analyses are presently limited to DON.

Cooperative efforts between Minnesota, Missouri, North Dakota, NCAUR, and Georgia will be directed towards preparing reference samples of cereal grains with known mycotoxin levels for interlaboratory validation of methods. Kansas, Georgia, Michigan, Wisconsin, Minnesota, NCAUR, North Dakota, and Iowa will collaborate to integrate chromatographic and immunological techniques for single and multimycotoxin analysis. NCAUR and Minnesota will use capillary electrophoresis for detection of minute quantities of mycotoxins in animal tissues and fluids.

2.3 Sampling Techniques

With collaboration from North Dakota, Michigan will continue research on estimating heterogeneity of the distribution of deoxynivalenol in harvested wheat and barley. Confidence interval estimates vary from year to year, and a method to rapidly determine the confidence intervals at the beginning of a harvest season is needed. This project involves identifying trucks of wheat/barley potentially contaminated with deoxynivalenol, and performing intensive probe sampling of the truck. The goal is to determine how movement of the grain between storage points affects the heterogeneity of toxin distribution, and to develop sampling protocols that take into account the heterogeneity.

Objective 3. Establish integrated strategies to manage and to prevent mycotoxin contamination in cereal grains.

3.1 Surveys

Surveys are necessary to determine the incidence, frequency and scope of toxigenic fungi and their toxins in cereal grains. This is a unique opportunity for individual states to assist in surveys each year, so that reliable mycotoxin data can be acquired quickly for use by scientists, economists and others who utilize cereal grains. Technical Committee members reside and conduct research and surveys in various areas of the Region encompassed in the NC-129 and their crop interests vary. We need to develop effective multistate survey methods to identify the mycotoxigenic fungi infecting cereal grains and the environmental factors involved in mycotoxin outbreaks. Guidelines will be established for sampling each type of crop (preharvest and postharvest). Kansas, Indiana, Michigan, and Georgia have initiated annual surveys for toxigenic fungi and mycotoxins in cereal grains. Within the U. S. there are a variety of cereal crops (corn, wheat, oats, barley, rye, etc.) that are vulnerable to invasion by Fusarium and Aspergillus species with potential toxin production. Certain crops tend to predominate in various parts of the North Central Region, i.e. hard wheat in Kansas, Nebraska, North Dakota, South Dakota, and Minnesota; soft wheat in Indiana, Illinois, and Missouri, and corn predominantly in Nebraska, Iowa, Minnesota, Illinois, and Indiana. Substantial quantities of corn are also found in Wisconsin, Michigan, and Ohio with significant increases in Kansas, Missouri, South Dakota, and Ohio. NC129 members from Minnesota and Michigan, North Dakota, and Wisconsin also have collaborations with members of NCR 184 to facilitate surveys for DON associated with Fusarium Head Blight of wheat and barley. The goals of this collaboration will be 1) to evaluate fungicide chemistry, application timing, and application techniques on reducing Fusarium head blight severity and levels of deoxynivalenol in infected wheat; 2) to collaborate with a wheat breeders and pathologists in the region to evaluate wheat germplasm under different environments to identify sources of resistance to Fusarium graminearium; 3) to study the potential of anti-deoxynivalenol specific recombinant antibody, and a peptide mimic of deoxynivalenol, as resistance factors in transgenic wheat.

Fusarium disease epidemiology will be studied, with the cooperation of plant breeders to compare varieties for susceptibility at different locations in the U. S. and to develop models for predicting outbreaks of infection and toxicity. For this proposal, NCAUR will coordinate the effort to determine the role of fumonisins in pathogenicity, and to determine if strains disrupted in fumonisin biosynthesis are good candidates for biological control agents. Indiana will collaborate with the NCAUR group by providing additional mutant strains of F. moniliforme. NCAUR will also determine if the mating locus of G. zeae, which allows the fungus to produce sexual spores, plays a critical role in head scab epidemics. Elucidating the mechanisms that regulate sensitivity to fumonisin could be useful information for use in molecular plant breeding.

3.2 Fusarium species

Pennsylvania State University has traditionally provided identification service for Fusarium species to members of the NC-129 Committee. They also maintain cultures of Fusarium moniliforme and F. proliferatum that are known to be high producers of fumonisins. These have been made available to all members of the NC-129 Committee and this collaboration needs to continue. Wisconsin and Minnesota also maintain reference cultures of mycotoxin producing Fusarium species.

3.3 Detoxification

Michigan, Nebraska, Kansas, Illinois, and North Dakota will collaborate on grain processing as a means of detoxification. Missouri will evaluate the efficiency of adsorbent clays and crude enzyme preparations to ameliorate the toxic effects of Fusarium mycotoxins in poultry. Kansas will test the effect of extrusion processing on fumonisins in corn and hydrolyzed fumonisins in alkali-cooked corn, as well as their toxic effects when fed to rats. Chemical reactions including enzymatic and binding of mycotoxins, with special emphasis on processes for human foods, will be investigated by Iowa. The effects of nutrients on mycotoxin toxicity will be studied by Iowa, Missouri and Wisconsin.

3.4 Toxin reduction through disease management

Michigan, North Dakota, Minnesota, Canada and Ohio will collaborate on uniform fungicide trial nurseries for FHB on wheat and barley and on the development of regional variety nursery trials to identify and validate sources of resistance. New approaches to resistance will involve the development of transgenic plants using a variety of gene sources, including recombinant antibody, and peptide mimics of deoxynivalenol. Iowa will continue work with Bt transgenic corn that has consistently shown lower fungal infection and toxin production. Georgia will cooperate with the University of Florida on planting date and cultivar studies with Bt corn by measuring the aflatoxin and fumonisin content of the harvested product as well as determining the mycoflora. Illinois will continue field testing to determine if transgenic plants the express higher chitinase and B-1. 3-glucanase activity have lower aflaloxin levels when inoculated with A. flavus and Fusarium.

Objective 4. Define the metabolic pathways and regulatory pathways of mycotoxin biosynthesis

NC-129 scientists are working to better understand the biosynthesis and regulation of aflatoxins, fumonisins and trichothecenes through collaborations between NCAUR, Michigan, Indiana. Nebraska, and Wisconsin. The NCAUR group has made significant progress in elucidating the biosynthesis of fumonisins and trichoihecenes. Although the present knowledge about the biosynthesis and regulation of fumonisin is incomplete, NCAUR group has isolated the gene encoding the polyketide synthase (fum5) and determined that fum5 is located in a cluster of genes responsible for fumonisin biosynthesis. The NCAUR will continue to elucidate the genes in the cluster and determine their functions. Indiana has isolated a gene (fic1) involved in the regulation of fumonisin production when F moniliforme grows on corn. Indiana will determine the upstream and down stream components of this regulation pathway. The Nebraska group, which has expertise in the area of molecular signal transduction, will cooperate in this effort. Indiana has inserted into F moniliforme a gene-reporter construct consisting of the fum5 promoter fused to the B-glucuronidase (GUS) gene from E. coli. Mutants affected in the regulation of fumonisin biosynthesis will be generated in this strain by restriction enzyme-mediated integration (REMI). Genomic approaches are also being used to identify genes involved in the regulation of fumonisin biosynthesis. Indiana will construct a subtractive library with eDNAs for fumonisin producing and non-producing cultures of F moniliforme. Initially 500 clones from the library will be sequenced. cDNA sequences with similarity to known regulatory genes will be candidates for gene disruption to determine any involvement in fumonisin biosynthesis. Indiana, Nebraska and NCAUR will collaborate in these studies.

Measurement of Progress and Results

Outputs

Outcomes or Projected Impacts

Milestones

(0):0

Projected Participation

View Appendix E: Participation

Outreach Plan

Organization/Governance

Chair - Establish location of meeting and coordinate the date with the Administrative Adviser's approval. Notify technical committee members of dates, times and location of meeting and assist members in making accommodations. Call the meetings to order and preside during the meeting. Will become the Retiring Chair following Annual Meeting adjournment.

Vice-Chair - Will function as the Chair in his/her absence. Becomes the Chair immediately following the Annual Meeting. Is responsible for writing, getting approval and disseminating the Annual Report.

Secretary - Will take minutes for all meetings of the Executive Meeting and the Annual Meeting at which he/she is elected. Is responsible for disseminating copies of the minutes to all Technical Committee members following approval by the Administrative Adviser. Becomes Vice chair for the next Annual Meeting.

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