ARS Food Safety, (animal and plant products) National Program (108)

The Agricultural Research Service (ARS) food safety research program significantly expanded to $91.07 million in 2001, with a budget increase of $2.87 million for preharvest research and $3.94 million for postharvest research. The new funds were directed for research in the areas of: antibiotic resistance; pathogen control during preslaughter, transportation, slaughter and processing of meat and poultry; pathogen and residue detection during processing and storage; control of Salmonella enteritidis infection in laying hens, and pathogen control in shell eggs; pathogen control in fruits and vegetables; and control of plant toxins and heavy metals.

During fiscal year (FY) 2001, research programs were continued with the National Alliance for Food Safety to specifically address issues on Listeria monocytogenes and E. coli O157:H7. This included the initiation of a program to develop a microarray for Listeria. Research programs were initiated with The Institute for Genomic Research (TIGR) to provide annotated genomic sequences of the bacterial pathogens Listeria monocytogenes and Campylobacter jejuni.

In FY 2001, ARS held several workshops to help relate the research progress in the program to customer and stakeholder needs. ARS, in conjunction with Cooperative State Research, Education, and Extension Service (CSREES), held the Fair 2002 Conference in Washington, D.C. The purpose of the meeting was to assess the research needs of a variety of audiences, from Federal regulatory agencies, producers, processing and handling companies, retailers, consumers, and consumer agencies. The Annual ARS Food Safety and Inspection Service (FSIS) Research Program Planning Meeting held at the National Conservation and Training Center, Sheperdstown, West Virginia, brought together ARS scientists and FSIS staff for a research progress update, and discussion of FSIS needs for meeting their regulatory responsibilities. Representatives from several other Federal agencies with food safety programs also particpated; for example, the USDACSREES, Food and Drug Administration Center for Food Safety and Applied Nutrition (FDACFSAN), Canadian Food Inspection Agency, Health Canada, and the European Commission headquartered in Brussels.

The Annual Aflatoxin Elimination Workshop held in Yosemite Park, California, updated the peanut, corn, cottonseed, and tree nut industries on ARS supported research progress made to control aflatoxin and fumonisins in these commodities. The Annual Fresh Fruit and Vegetable Research Planning Workshop held at the National Conservation and Training Center, Shepherdstown, West Virginia, brought together ARS scientists, FDA CFSAN scientists and staff, and university collaborator researchers for a research progress update, and discussion of FDA needs for meeting their regulatory responsibilities. The Food Safety and Animal Health National Programs conducted a USDA ARS Bovine Spongiform Encephalopathy (BSE) workshop at Beltsville, Maryland, to identify targeted approaches to reduce the introduction of BSE into U.S. cattle populations. Participants in the meeting included representatives from other United States and United Kingdom agencies with animal health and food safety programs, producers, and national commodity organizations.

The National Food Safety Team conducted a joint ARS Institute of Food Research (IFR)Biotechnology and Biological Sciences Research Council, Norwich, United Kingdom Food Safety Pathogen Workshop in July at Norwich, to identify specific program areas for collaboration between ARS and scientists within the European Union (EU). Major actions resulting from the meeting were the establishment of an Memorandum of Understanding between ARS and the IFR, and continued agreement to expand the cooperative research programs between ARS and IFR. Members of the National Food Safety Team also participated in a joint ARSEuropean Commission (EC) Food Safety Workshop in Brussels, Belgium, to identify specific program areas for collaboration between the United States and scientists within the EC. The major action resulting from the meeting was a report outlining expansion of cooperative research programs, specifically in areas of emerging pathogen research, probiotics, risk analysis, and predictive modeling, and Transmissible Spongiform Encephalopathy (TSE’s).

Program Components

In FY 2001 a majority of the remaining research (Current Research Information Systems CRIS) projects within the National Food Safety Program underwent external review through a new Congressionally mandated review process referred to as OSQR (Office Scientific Quality Review). A panel provided an indepth critique of the 5year research project plans. The peer review panels were comprised of scientific peers with expert knowledge of the fields of science pertinent to the research being reviewed. The review criteria and project plan design policies assure that ARS research scientists develop carefully conceived project plans that focus on three key elements of research planning: merit and significance as it aligns with the National Program Action Plan; approach and procedures; and probability of success.

Microbial Pathogens

Determining how to reduce microbial pathogens in food products, throughout food operations from farm to fork, is the most urgent food safety problem today. The major pathogens addressed were the bacterial pathogens Salmonella, Campylobacter, E. coli 0157:H7 and related EHEC, Shigella, Listeria, Yersinia, Aeromonas, Arcobacter, Vibrio, and Clostridium; the parasites Cryptosporidium and Toxoplasma; and various viruses, hepatitis A Norwalk and Norwalklike. New research programs were initiated in antibiotic resistance; pathogen control during preslaughter, transportation, slaughter and processing of meat and poultry; pathogen and residue detection during processing and storage; control of Salmonella enteritidis infection in laying hens, and pathogen control in shell eggs; pathogen control in fruits and vegetables.

Small group of multiresistant Salmonella also can secrete a cytotoxin. Antibiotic resistance of human pathogens sometimes present in food may create serious human health problems. Therefore, it is important to determine the relationship of resistance factors with toxicity of the bacteria. ARS scientists found that a small group of multiresistant Salmonella are capable of secreting a cytotoxin which is a natural peptide antibiotic (bacteriosin), thus the hypervirulent abilities of this group of multiresistant Salmonella could be due to their ability to damage cells within their host. This finding will help scientists understand the various types of antibiotic resistance, and helps provide the basis for strategies to prevent the expansion and reduce the incidence of antibiotic resistance in bacteria.

Holding pens a major risk factor for Salmonella contamination of pork. Sources of Salmonella infection in market pigs during transport and holding prior to slaughter must be identified in order to develop effective strategies to prevent Salmonella contamination of pork and pork products. Since swine are routinely placed in holding pens at the slaughterhouse for at least 2 hours prior to slaughter to improve the quality of their meat, ARS scientists tested market swine, culled sows, and the holding pen environment for Salmonella and other pathogens. They found that the number and variety of Salmonella strains carried by the swine increased significantly while in the holding pens, thus demonstrating conclusively that holding pens pose a significant risk for Salmonella contamination of pork.

Reproducible experimental model of E coli O157:H7. A consistent and reproducible experimental model of E coli O157:H7 infection in older calves is an important requirement for experiments to test specific vaccines and other intervention strategies to reduce E coli O157 colonization and shedding. ARS scientists inoculated weaned calves with E coli 0157:H7 followed by an injection of dexamethazone, a compound that is used to prevent inflammation. They found that the dexamethazonetreated calves were colonized more often and had greater intestinal damage than untreated calves. This experimental infection model will be useful for determining the effectiveness of specific vaccines for reducing E coli O157:H7 shedding in cattle, thus helping to assure the safety of beef.

Multi antibiotic resistance from naturally occurring bacteriocin. Resistance of some foodborne pathogens to five or more antibiotics can be a serious problem in treating both animals and humans, and the medical community believes that antibiotic use in animals is a serious contributing factor. An ARS scientist found an E. coli bacteria expressing a natural peptide antibiotic (bacteriocin) which can induce a multiresistant phenotype Salmonella. These results demonstrate that bacteria in the same ecosystem, that is in the gastrointestinal tract, can induce antibiotic resistance in Salmonella. This finding demonstrates that, since the feeding of antibiotics is not the only factor which can induce antimicrobial resistance, control of AR development will be more complicated than originally envisioned.

Sodium chlorate selectively kills foodborne pathogens. The swine and cattle industries need intervention strategies that protect animals from infection with Salmonella and E. coli O157:H7. ARS research with cattle and swine demonstrated that sodium chlorate administered orally selectively kills pathogenic E coli and Salmonella without harming beneficial bacteria or normal gut function. The selective killing is based on the fact that (1) E. coli and Salmonella possess the ability to respire anaerobically on nitrate, an activity lacking among most beneficial anaerobes, and, (2) respiratory nitrate reductases coincidentally reduce chlorate to the cytotoxic chlorite. This discovery can speed the development of a practical, efficacious, costeffective, and commercially viable supplementation strategy that can significantly reduce concentrations of food borne pathogens in cattle and swine, thus enhancing the microbiological safety of beef and pork for consumers.

Faster detection of food borne pathogens. Detection and quantification of pathogenic microorganisms in foods is vital for ensuring a safe food supply. Effective testing methods must meet a number of criteria including speed, high sensitivity and selectivity, ruggedness, and cost. During the past year, ARS scientists have developed a number of new methods that will have significant impact on regulatory agencies and the food industry:

Pathogen modeling and its use in risk assessment. Predictive microbiology is increasingly being used to estimate the effect of multiple variables on the growth and survival of bacterial pathogens in food. This information is also an integral component of microbial risk assessments, which in part, identify control points in the food production system that can be used to reduce the risk of food borne diseases. The USDA FSIS risk assessment of E. coli O157:H7 in ground beef and ground beef products identified a lack of knowledge about the effect of competitive microflora on the behavior of E. coli O157:H7. Studies by ARS scientists showed that without competitive microflora the O157:H7 growth rate and maximum population density was similar to that described in the USDA ARS Pathogen Modeling Program prediction. However, no lag phase was observed indicating a food matrix effect on pathogen behavior. As a result, further studies will have to be performed, and O157:H7 risk assessment document will have to be revisited.

Removal of pathogens from food surfaces. Pathogenic bacteria may contaminate the surfaces of foods, compromising the food supply. ARS scientists developed an innocuous steamvacuum process which kills bacteria on the surface of solid foods without significant loss of quality. While the process can be applied to many foods including red and white meats and fish, it has found the greatest utility to reduce contamination in readytoeat foods, and fresh fruits and vegetables. Bacterial kills of > 99.999 percent are possible, which fulfills the FDA and FSIS pathogen reduction requirements for these commodities.

New technology for killing pathogens and spoilage organisms on minimally processed foods. The reduction of microbial populations on foods is vital to the safety, quality, and shelf life of minimally processed foods. ARS scientists have evaluated the utility and effectiveness of using gaseous chlorine dioxide to disinfect fresh fruits, vegetables, and seeds. Using a variety of products including lettuce, cantaloupe, strawberries, and alfalfa seeds, gas phase application of chlorine dioxide showed significant promise for general industry use.

HACCP in redmeat processing plants. Hazard Analyses and Critical Point (HACCP) Systems are being introduced into redmeat processing plants to assist in eliminating pathogens from the raw meat supply. To be effective these preventive control systems require sciencebased identification of critical control points and novel intervention strategies. ARS scientists tracked inplant contamination from animal to finished carcass by E. coli O157:H7/NM strains using genetic fingerprints. The majority of the contamination was traceable to cattle within the same lot, and contamination found later in the processing line was attributable to contamination that occurred earlier in processing. These results provide the industry with the evidence and knowledge to direct new intervention strategies to the very early steps of individual animal processing.

Monitoring decontamination of broiler carcasses. Identification and separation of broilers contaminated by feces and ingesta is critical to protect the consumer from a potential source of bacterial pathogens. To protect the consumer, the FSIS has a zero tolerance regulation for feces and ingesa prior to the carcass entering the chiller. Currently, inspections are conducted by trained personnel, however, there is always the potential for human error. ARS scientists have now invented and developed an online visioning system which can detect contamination in real time. The invention, currently under patent, has the potential to provide science based inspection of carcasses for online zero tolerance compliance.

The objectives of the chemical residue program are to reduce the risks of chemical residues from animal drugs, food additives, herbicides and pesticides, and environmental contaminants that are potentially present in foods. A major goal is to develop reliable, effective, accurate, userfriendly, costeffective residue detection methodology which requires minimal amounts of organic solvents to detect these residues.

Endogenous hormones sorbed to silt loam in columns. Endogenous hormones excreted by food producing animals may be harmful in the environment if present in sufficient concentrations. ARS scientists applied testosterone and estradiol to silt loam soil or sand columns. Most of the testosterone and estradiol (80 percent and 96 percent respectively) was sorbed to the top five centimeters of the loam soil column. At least 2 metabolites were found in the sorbed material, but most of the testosterone and estradiol (87 percent and 85 percent) was not sorbed on a similar sand column. These results will help scientists devise methods to facilitate faster degradation to decrease the presence of these hormones in the environment.

Improved chemical analysis. Pesticides, nutrients, and veterinary drugs in foods are routinely monitored by regulatory agencies such as the FDACenter Veterinary Medicine and FSIS. Many of the current laboratorybased chemical assays use substantial quantities of organic solvents for extraction and cleanup of extracts prior to analysis. This has a significant impact on personnel safety and waste disposal in the environment. ARS scientists using pressurized water have developed an extraction and integrated cleanup method for the selective removal of pesticides from animal tissue. The coextraction of interfering matric components is minimal using this method, allowing chromatographic and/or spectrophotometric methods to be applied to quantify the amount of pesticide in food matrices. This technology is being implemented by regulatory laboratories both for research, and routine high throughput sample analysis.

The presence and potential for the presence of mycotoxins in crops is not only a direct food safety problem, but it threatens the competitiveness of U.S. agriculture in the world market. Major goals were to control aflatoxin in peanuts, corn, cottonseed, tree nuts, and figs; fumonisins in corn; and deoxynivalenol in wheat and barley through an understanding of the biology of plantfungus interactions and toxin production in the field.

Proteins identified in corn which inhibit infection by Aspergillus flavus. Methods are needed to prevent aflatoxin contamination of crops before harvest, since the presence of this toxin, in even very low amounts, makes foods and feeds unacceptable for animal and human consumption. ARS scientists have discovered proteins in corn kernels that probably act together to inhibit infection by A. flavus, the fungus that produces aflatoxin. Levels of these proteins were elevated in maize varieties, demonstrating resistance to invasion by A. flavus in the laboratory or in the field relative to those protein levels found in susceptible varieties. The identification of these resistancerelated kernel proteins has led to identification of the genes encoding these proteins. With resistance related genes identified, both genetic engineering and marker assisted selection can more quickly increase resistance of maize to aflatoxin contamination, and thus help to ensure the safety of human food.

Aflatoxin protection factor in walnuts. The occurrence of aflatoxin in tree nuts creates a potential human health hazard, and severely decreases the value of the nuts to the producers. ARS scientists identified a mycotoxin protective factor in the Tulare variety of walnut which completely suppressed aflatoxin production. These scientists also demonstrated that the resistance factor is confined almost entirely to the thin, paperlike skin (pellicle) surrounding the kernel of the nut. This is the first example of a commercial variety of any crop plant known to be contaminated by aflatoxins that shows complete resistance. Since the pellicle is inherited from the maternal parent, it should be possible to develop new walnut crosses with predictable, heritable, natural resistance to aflatoxin.

New genus of plant friendly bacteria found which are antagonistic to fungi. Some fungi which grow on food crops produce mycotoxins which may harm people and food producing animals. ARS scientists established the DNA fingerprint of the endophytic bacterium Bacillus mojavensis and compared its molecular profile with other isolates and species within the large group of related species. All B. mojavensis isolates were endophytic and antagonistic to fungi, but other species within the group were not. This newly established Genus of bacteria forms natural associations with plants, and is both plant friendly and antagonistic to fungi. Members of the Genus have potential uses for exploitation to provide plant disease protection and to prevent toxin production.

Chemical structural rearrangement reforms toxic molecule. The occurrence of fumonisins in corn is a potential human and animal health hazard, and it decreases the value of corn to producers. ARS scientists previously determined that a particular structural modification of fumonisin destroyed its ability to cause the fundamental adverse event that leads to toxicity, but other scientists then had contradictory results. In further studies ARS scientists found that the modified nontoxic fumonisin is unstable and that it can spontaneously rearrange with time at either room temperature or freezing temperatures to the form that is toxic. This finding provides insight into the chemical and biological properties of fumonisns, and is important as efforts to develop methods to decontaminate fumonisin contaminated corn products are explored.

Research information on which to base regulatory guidelines for fumonisins. Fumonisins which may contaminate U.S. corn were included among the mycotoxins recently reviewed by Food and Agricultural Organization (FAO)/World Health Organization (WHO) Joint Committee on Food Additives (JEFCA), February 2001. ARS research played a major part in defining the acute and subchronic toxicity of fumonisins, the mechanism of action and the pharmacokinetics. This provided the basic work for both the longterm carcinogenicity studies conducted by the FDA, the JEFCA recommendations, and the recent FDA guidelines on fumonisins in human food and animal feed. ARS research thus provided a solid information foundation on which to make recommendations for fumonisn exposure levels that both protect the consumer and drastically reduce losses in income to corn growers, processors, and exporters.

This program component seeks to minimize the exposure of animals and humans to natural toxins from poisonous plants.

Natural levels of cadmium in sunflower are not toxic. Sale of U.S. sunflower seeds was prevented in some countries based on natural cadmium concentration in soil. (The cadmium level present in sunflower was believed harmful due to toxicities reported with high soil associated cadmium in rice.) A feeding test was conducted in rats using purified diets containing polished rice or sunflower kernel containing various levels of ¹⁰⁹Cd. The study showed that because polished rice supplied little Zn, Fe, or Ca, the marginal supply of these elements caused a much larger increase of absorbed Cd in the kidney and liver than found for sunflower kernels that supplied adequate amounts of these elements. Thus, the difference in bioavailability of grain Cd among crops may have been the most important factor where riceCd caused human disease attributed to soil Cd. The results should allow the U.S. to request that regulations under consideration be the FAO/WHO for the Codex Alimentarius should be different for sunflower and other grains than for rice.

Fungal endophyte was found in locoweed that produces the toxin swainsonine. Locoweed on western grazing areas is associated with unthrifty, unprofitable animals resulting in high livestock losses. Endophytic Alternaria fungi were found in five of sixteen different populations of locoweed tested. There was a high correlation between endophyte infected plants and plants with high swainsonine levels. This finding provides crucial information to allow scientists to determine the origin (plant, fungus, or combination) and environmental factors affecting the presence of the toxin found in locoweed.