Introduction

The ARS food safety research program significantly expanded to more than $82 million in 2000, with a budget increase of  $5.9 million for preharvest research and $5.4 million for postharvest research. The funds were used for research on manure handling and distribution, risk assessment, antibiotic resistance, fungal toxins, zoonotic disease risk, aflatoxins, pathogen control in fruits and vegetables, pathogen control during slaughter and processing, antimicrobial resistance, food safety engineering, and research specifically directed to Listeria monocytogenes and E. coli O157:H7.

 During fiscal year 2000, several new research projects were initiated: a program in association with the National Alliance for Food Safety to specifically address issues on Listeria monocytogenes and E. coli O157:H7; a molecular systematics program to develop molecular characterization methods; and a program to address listeriosis, in Pullman, Washington and Dubois, Idaho. A new risk assessment program was implemented in association with FSIS and UMES.  The Food Safety Research Information Office based at the National Agricultural Library, Beltsville, Maryland, was officially created with the appointment of a coordinator/director.

In fiscal year 2000, ARS held several workshops to help relate research progress to customer and stakeholder needs.  ARS in conjunction with CSREES, held the Congressionally mandated National Conference on Food Safety Research, in September in St. Louis, Missouri.  The purpose of the meeting was to assess the research needs of a variety of audiences, including federal regulatory agencies, producers, processing and handling companies, retailers, consumers and consumer agencies.  The 20th Annual ARS-FSIS Research Program Planning Meeting held in December in Washington, DC, brought together ARS scientists and FSIS staff for a research progress update and discussion of FSIS needs for meeting their regulatory responsibilities.  Representatives from other Federal agencies with food safety programs, for example, the Food and Drug Administration’s Center for Food Safety and Applied Nutrition (FDA-CFSAN) also participated.  The 12th Annual Aflatoxin Elimination Workshop held December 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 2^nd Annual Fresh Fruit and Vegetable Research Planning Workshop held in November in Sonoma, California, brought together ARS scientists, FDA-CFSAN staff, and university collaborator researchers for a research progress update and discussion of FDA needs to meet regulatory responsibilities.

The National Food Safety Team conducted a joint ARS-IFR (Institute of Food Research-BBSRC, Norwich, United Kingdom) Food Safety Pathogen Workshop in July at Wyndmoor, Pennsylvania, to identify specific program areas for collaboration between ARS and scientists within the European Union (EU).  Six areas of collaboration were identified, several of which have been implemented.  The National Food Safety Team conducted a Dairy-Milk Pathogen Workshop in association with the AMS and FDA-CFSAN in March, at Beltsville, Maryland, to provide a forum for research and regulatory food-safety agencies to review and reach consensus on research priorities to assure the safety of milk and milk products.  The National Food Safety Team and ARS scientists, in association with staff from the FDA-CFSAN, represented the United States in developing Bilateral Cooperative Food Safety Research Agreements with Mexico and the EU.

 Program Components 

 In fiscal year 2000 approximately 75 percent of the research (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).  Three separate peer review panels were convened to provide in-depth critiques of 5-year research project plans.  Panels were comprised of scientific peers with expert knowledge in 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 O157:H7 and related EHEC, Shigella, Listeria, Yersinia,  Aeromonas, Arcobacter, Vibrio, and Clostridium; the parasites Cryptosporidium and Toxoplasma; and various viruses, hepatitis A Norwalk, and Norwalk-like.  New research programs were initiated in manure handling and distribution, risk assessment, antibiotic resistance, zoonotic disease risk, pathogen control in fruits and vegetables, pathogen control during slaughter and processing, antimicrobial resistance, food-safety engineering, and research specifically directed to Listeria monocytogenes and E. coli O157:H7.

 Certification program to prevent trichinella in swine.  A pre-harvest certification program for Trichinella is needed to assure consumers that the pork they buy and bring into their homes is free of this parasite.  ARS scientists, together with the pork industry, developed the certification system based on knowledge of risk factors, detection methods, and good management practices.  Certification requires pork producers to meet certain management criteria that eliminate risk of exposure of pigs to the Trichinella parasite.  This certification program has been adopted by the pork industry, and APHIS and FSIS are currently developing regulations for management of the program.  Certification should also help USDA establish equivalency agreements with foreign markets regarding the safety of US pork.

Preventing Salmonella in pork.  ARS scientists evaluated the practice of holding swine at the abattoir prior to slaughter as a source of Salmonella infection of market pigs.  Salmonella isolation rates were significantly higher for penmates necropsied after transport and held at the abattoir, compared to cultures collected at the farm.  In addition, a greater number of different Salmonella serotypes were isolated from samples collected at the slaughterhouse compared to the farm.  To confirm the possibility of such rapid infection with Salmonella, 40 pigs were exposed to feces containing a marked strain of Salmonella.  Within 2 to 3 hours, 80 percent of the exposed animals were positive for the marked strain, and after 6 hours all of the animals had at least one tissue sample test positive.  Together, these studies with swine show that control of pathogens in the preharvest environment can significantly affect carcass contamination and improve the safety of the food supply.

Incidence of E.coli O157:H7 in feedlot cattle.  Foodborne disease from enteric pathogens such as E. coli O157:H7 is a major public health concern in the U.S. today.  ARS scientists completed a survey of E. coli O157:H7 infection in feedlot cattle presented for slaughter.  The prevalence of infection was found to be significantly higher than previously reported, with 28 percent of all animals tested found to be infected.  Additionally, infection was also widespread, with 72 percent of slaughter groups testing positive for E. coli O157:H7.  This is the first comprehensive study of prevalence in fed cattle at slaughter and is also the first demonstration of a significant association between prevalence of infection in the live animal and carcass contamination.  Thus, control of foodborne pathogens such as E. coli O157:H7 in the preharvest phase of production will have significant effects on carcass contamination and public health.

Multiple antibiotic resistant pathogens in poultry.  ARS scientists challenged broiler chicks on the day of hatch with either a sensitive or penta-resistant Salmonella typhnimurium DT104 to assess the effect on virulence and/or colonization.  They determined that penta-resistant bacteria, previously reported to be highly pathogenic, did not cause clinical illness in broiler chicks; however there was a significant increase in the number of birds that were colonized in the penta-resistant group.  In contrast to in vitro studies, these data indicate that acquisition of multiple resistance can affect colonization rates and may affect the number of bacteria that may reach the food chain.  These data increase our understanding of the transmission and significance of antibiotic resistant pathogens in animals which can be used to develop recommendations to decrease the incidence of antibiotic resistance in food producing animals.

Recognition of innate immunological responsiveness in poultry.  There is a need for one or more genetic lines of poultry that are resistant to pathogen colonization.  In collaboration with a commercial partner, ARS scientists determined the innate immunological responsiveness of three commercially available lines of chickens.  Evaluation of the general functional activities of cells in the birds, known as heterophils and monocytes, identified one commercial line that was significantly more efficient in responding effectively to pathogen challenge shortly after hatch.  Further testing and selection of this line resistant to pathogens will benefit the poultry production industry and help make poultry food products safe.

Rapid detection of viruses in aquaculture products.  Rapid methods are needed for the detection of enteric viruses such as hepatitis A, Norwalk, and rotavirus in food and water.  ARS succeeded in developing a much safer and more rapid analytical method for the cell-culture-based enumeration of hepatitis A virus, and human and simian rotavirus using enhanced chemiluminescence technology.  The new method reduces by 5 days the previous assay procedure, and eliminates the use of  radioactive isotopes in the detection protocol.  The new method will have broad-based appeal for regulatory and action agency monitoring of aquaculture products such as oysters; and will be particularly useful in determining the effectiveness of processing strategies for the inactivation of viruses.

Ensuring the safety of apple cider.  There is a critical need to assure the safety of fresh, unpasteurized fruit juices, such as apple cider.  Removal of pathogens from the surface of fruit before processing is considered a critical control point in the processing of apple juice.  Previous ARS studies demonstrated limitations in the efficiency of washing apples as a means of reducing microbial populations, even when fruit was washed with 5 percent hydrogen peroxide. ARS has now demonstrated the conditions for improving the efficiency of hydrogen peroxide treatments by mechanical detachment of adhering bacteria and by improving contact of the attached bacteria and wash solution.  This improvement brings the FDA’s 99.999 percent (5-log) populations reduction target for unpasteurized apple cider within reach.

Decontamination of sprouts.  Naturally contaminated seeds must be decontaminated before being used to grow sprouts for human consumption. ARS had previously shown that laboratory contaminated seeds could be decontaminated with 20,000 ppm of free chlorine providing a 99.99 percent (4-log) reduction in pathogens. ARS has now demonstrated that this method was effective in sanitizing naturally contaminated seed. As a result, sprout growers, consumers, and regulatory agencies can have a greater confidence in the safety of sprouts grown from seeds treated in this manner.

Cooling of retail foods  Inadequate.  Cooling of foods in retail food operations may allow the bacterium Clostridium perfringens to grow to potentially hazardous, infective dose levels.  ARS has now established the safe cooling rate for cured beef, pork, chicken, and uncured chicken by defining the time and temperature needed to control C. pefringens.  A predictive model has been developed to predict growth from spores at temperatures applicable to the cooling of cooked meats.  The information will enable regulatory agencies and the food industry to evaluate the safety of cooked products.

Irradiation treatment to inactivate pathogens on hot-dogs.  ARS evaluated the use of irradiation to eliminate the bacterial pathogen Listeria monocytogenes from hot-dogs.  Studies concluded that a 99.999 percent (5-log) reduction of this pathogen was achieved with a radiation dose of 3.6kGy, thus meeting the regulatory goal of the Food and Drug Administration for this pathogen. Differences in radiation sensitivity were discovered that depended on product formulation, therfore, the irradiation processing step would require product dependent adjustment.

Intervention strategies for ground meats.  In the normal processes of breaking down the animal carcass into smaller meat cuts and trim, there are additional opportunities to spread or increase bacterial contamination.  ARS designed a combination treatment process for the microbiological decontamination of pork trim prior to grinding.  The processes were shown to reduce and control populations of fecal bacteria on pork trim and in the resultant ground pork.  The work provides industry with a process to improve both the microbiological safety and shelf life of ground pork products, and assist processors in meeting the proposed Salmonella performance standards for fresh pork sausages.

Levels of E. coli in beef.  ARS determined the relationship between cattle contamination and subsequent carcass contamination.  Results showed an unexpectedly high number of animals per lot entered the slaughter plant carrying E. coli O157:H7/NM; however, very few carcasses were still contaminated after processing.  These data have contributed to the food safety and policy debates regarding the commonness of E. coli O157:H7/NM, the usefulness of sampling procedures, and strategies to eliminate E. coli 0157:H7/NM contamination of the beef supply.

Chemical Residues

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 a reliable, effective, accurate, user-friendly, cost-effective residue detection methodology that requires minimal amounts of organic solvents to detect these residues.

Significance of the endogenous hormones 17-estradiol and testosterone in animal manure.  The endogenous hormones, 17-estradiol and testosterone, are potent estrogenic and androgenic substances that are mainly eliminated in animal manure.  ARS scientists analyzed samples related to manure handling practices for the levels of these native hormones using a commercial enzyme immunoassay kit.  The results showed that 1) hormones are present in water adjacent to manure pits, 2) some degradation of these hormones occurs when allowed to stand at 20 degrees C, and 3) the hormones are water soluble and residues can be transported in the environment.  These results identify an area for evaluation of the significance of residues of environmentally bioactive substances.

Rapid analytical method for detection of pesticide residues in eggs.  ARS developed and evaluated a novel approach of rapid analysis for approximately 40 pesticides in eggs, using direct sample introduction/GC/MS-MS.  The rapid, sensitive, quantitative, confirmatory, simple, inexpensive, safe, and rugged procedure was developed to monitor pesticide levels as low as 1 ng/g.  The approach also avoids the costly, time-consuming, and labor-intensive clean-up and solvent evaporation steps associated with traditional methods.  This approach has the potential to make a strong impact in the analysis of many types of pesticides and other semi-volatile chemicals in a variety of matrices, including food.

Rapid method for the determination of thyreostats a banned growth promotants.  A novel, multiresidue, analytical, screening method was developed for the isolation and detection of thyreostatic agents in meat tissue without the need for a mercury column cleanup step.  The total amount of solvent used for the analysis is less than 30 mL, and recoveries from fortified meat tissue were consistent and greater than 85 percent for thiouracil, tapazole, 6-methyl-2-thiouracil, and 6-n-propyl-2-thiouracil.  GC/MS-MS conditions were also devised for reliable confirmation of the presence of the drugs in the samples.  This method provides a cheaper and safer alternative to the current method adopted by FSIS and the EU. 

Mycotoxins

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 plant-fungus interactions and toxin production in the field.

Preventing aflatoxin in peanuts.  ARS has developed the information that is necessary to obtain an experimental use permit(EUP) for testing a product that utilizes competitive strains of A. flavus and A. parasiticus to prevent contamination of peanuts with aflatoxin.  A Georgia company is interested in the technology and is discussing the licensing of the technology from ARS.  An experimental use permit will allow the product to be field tested in more than 10 acres of peanuts per season in order to determine if it will effectively exclude toxin-producing fungi from peanuts grown under commercial conditions, and keep the aflatoxin content less than the tolerance required by European markets.

Competitive exclusion to prevent aflatoxin production in cottonseed.  Aflatoxins are extremely toxic substances produced by some strains of Aspergillus flavus and most strains of Aspergillus parasiticus.  These fungi are ubiquitous and infect many crops including peanuts, corn, cottonseed and tree nuts, and even wild plants growing in noncultivated areas.  Both the health risks and the reduced profitability of contaminated crops create a need to prevent the formation of aflatoxins.  Strains of A. flavus, which do not produce aflatoxin, may be used to competitively exclude the toxin producing strains prior to cotton boll development.  ARS developed the information necessary for EPA to allow the treatment of up to 20,000 acres of cotton to further assess and confirm the value of this product in preventing aflatoxin in cottonseed in a wide variety of environmental conditions and production practices.  Use of this product to prevent aflatoxin in cottonseed will help assure its safety for animal feed purposes, in particular cottonseed in dairy cattle feed, and will help to maintain continued profitability of the crop.

Targeting genes to the corn kernel pedicel.  To decrease mycotoxins as a health and economic concern we need to breed crop species that are resistant to mycotoxins, in particular corn.  ARS scientists develop a biotechnological tool, a gene promotor with the ability to express genes added to corn plants in a precise tissue specific manner in kernel pedicels, a common site for infection of corn plants by mycotoxin-producing fungi.  ARS has characterized and refined this tissue specific promoter to make it a user-friendly with a versatile gene expression cassette system for plant breeders to add novel genes for evaluation in corn.  A patent application has been submitted.  This is a critical advance in the development of new corn varieties that are more resistant to mycotoxins.

Assaying pistachios cost effectively.  Analysis of pistachios currently requires a very large nut sample to reduce sampling errors.  ARS developed a calibrated 3-step sampling plan for pistachios that reduces the amount of nuts and testing effort required to characterize a lot for aflatoxin.  Use of this plan with an average sample of 23 lbs. results in the same precision in aflatoxin determination as the usual 1-step test using 110 lbs. of nuts.  Adoption of this plan will save the industry approximately $1 M/ year in sample and test costs.

New plant odor attractive to codling female moths can reduce aflatoxin contamination.  Aflatoxin is produced by the fungus Aspergillus flavus, which is spread by tree nut insect pests.  A new plant odor was found to be specifically attractive to female moths, which lay eggs that hatch into larvae that cause the feeding damage and provide an entry for the fungus.  This odor can be combined with a pesticide to lure only codling moths, and thus eliminate the need for widespread application of pesticides and at the same time control aflatoxin contamination of tree nuts.

Toxic Plants

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

Decreasing the risk of toxic cadmium in foods.  In order to decrease the levels of the toxic element cadmium, we need to understand how other elements affect its bioavailability in the human diet.  In cooperation with an ARS Human Nutrition Laboratory a feeding test was conducted using labeled sunflower kernels to quantitate the role of crop zinc, iron, and calcium on absorption of crop cadmium.  Based on kidney and liver cadmium from isotope, kernel iron and calcium contributed to lower bioavailability of crop cadmium, but zinc had less effect.  This information will help in assessing the significance of amounts of cadmium in specific foods.

Isocupressic acid conjugates pave the way to prevent pine needle abortion.  Pine needle abortion causes significant losses to cattle producers in the west.  ARS scientists prepared isocupressic acid-protein conjugates using ovalbumin for investigation of potential immune-based assays ELISA, and vaccines to better diagnose and reduce or prevent abortion in cattle from ponderosa pine needles.  Since isocupressic acid is a small molecule and not naturally immunogenic, it was conjugated to ovalbumin to elicit an immune response.  This is a significant first step toward development of assays or vaccines that can effectively decrease abortion losses.