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During fiscal year 1999, four new research programs were initiated: a molecular systematics and diagnostics program for parasites in foods and food animals; a food safety-engineering program at Purdue University, for the development of new technology and systems to detect and prevent chemical and microbial food contaminants; a food safety-remote sensing program with the Institute for Technology Development, Stennis Space Centre, specifically focused on the development of imaging technology on-line for the inspection of poultry products during processing; and a Food Safety Research Information Office at the USDA National Agricultural Library.

In fiscal year 1999, ARS held several workshops to help relate the research progress in the program to customer and stakeholder needs. The 11th Annual Aflatoxin Elimination Workshop held October, in St. Louis, Missouri updated the peanut, corn, cottonseed and tree nut industries on ARS supported research progress made to control aflatoxin in these commodities. ARS in conjunction with CSREES, held the Congressionally mandated National Conference on Food Safety Research, in November in Alexandria, Virginia. 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 19th Annual ARS-FSIS Research Program Planning Meeting held in December in Athens, Georgia, brought together ARS scientists and FSIS staff for a research progress update, and discussion of FSIS needs for meeting their regulatory responsibilities.

The National Program Staff conducted the ARS Food Safety National Program Program Planning Workshop in February 1999, at Wyndmoor, Pennsylvania, to identify specific program research priorities and objectives. A second National Planning Workshop to address the research needs of the Produce and Imported Foods Safety Initiative (PFSI) was held in May 1999, in Monterey, California. A 5-year Action Plan was developed from the Workshops and is available on the World Wide Web: www.nps.ars.usda.gov/programs/108/action plan . Research progress with regards to three of the National Program components,microbial pathogens, residues and toxic plants was documented in the 1999Progress Report on Food Safety Research Conducted by ARS, provided bothin hardcover form, and on the World Wide Web: www.nps.ars.usda.gov/programs/108/progress report .

Early exposure of beef calves to E. coliO157:H7. ARS scientists demonstrated that most beef calves are exposed to E. coliO157:H7 even in relatively isolated range environments. This exposureoccurs prior to weaning, shipping, and mixing with calves from diversesources, circumstances which commonly contribute to exposure to infectiousagents. Thus, in order to prevent calf infections with E. coliO157:H7, interventions must be implemented early in the life of the calf.This information will help target the timing of procedures to prevent infectionof calves with this dangerous zoonotic pathogen.

Protection against Salmonellainfections in broiler birds. During the first 7-14 days after hatch, poultry has an immune system that is functionally immature, with only limited capability to respond to pathogenic infections. Thus newly hatched chicks are extremely susceptible to infection with zoonotic pathogens such as Salmonella. ARS scientists demonstrated that administration of Salmonella enteritidis-immune lymphokines (SILK) to day-old chickens and turkeys, by routesroutinely used by the industry for vaccines, conferred protection to thebirds against localized enteric Salmonellainfections, and hastened immune system development. Prophylactic administrationof SILK can reduce the incidence of Salmonellain broiler birds and thus help to protect the public health.

Detection of Salmonella typhimuriumDT-104 using a unique antibiotic resistance gene. Salmonella typhimuriumDT-104 is a highly pathogenic, foodborne bacteria that is difficult andtime-consuming to identify. ARS scientists discovered that DT-104 containsa unique gene, which they named St-flo, that confers resistance to theantibiotics chloramphenicol and florfenical. They developed a DNA-baseddetection system with nearly 100% sensitivity and specificity for detectionof the St-flo gene, which allows identification of the pathogen in 2 days.Rapid detection of DT-104 is important to identify outbreaks, to identifyindividuals for treatment, and to protect the public health.

Color of cooked beef patties and the food safety risk. Previous USDA advice to consumers on cooking hamburgers was lack of pink/red color equaled done and safe. This advice however, may have been oversimplistic, since patties subjected to grilling may lose their red color (premature browning), but not yet have reached a temperature high enough to destroy pathogenic bacteria, such as E. coliO157:H7. To determine when color gives false information, ARS collaboratedwith the Food Safety Inspection Service to determine when premature browningoccurs, by using meat stored frozen or nonfrozen, and thawed either inthe refrigerator, at room temperature, or by microwave. The studies showedthat thawed ground beef produced more brown color when cooked than pattiescooked either fresh or rapidly thawed. The USDA now advises that cookedbeef patty color is not a good indicator of safe food and that consumersshould not eat ground beef patties that are pink or red in the middle unlessa food thermometer has been used to verify cooked temperature.

Treating surfaces to resist pathogens. New methods are being devised to make inanimate surfaces in the processing areas more resistant to bacterial contamination. ARS scientists tested various methods of treating stainless steel, including sanding, grinding, polishing, and electrochemical, to determine which method best inhibited bacterial attachment. Stainless steel samples that had been electropolished showed the most significant reduction in bacterial attachment. This will aid manufacturers in designing and producing equipment that is not conducive to bacterial growth.

Rapid Test for E. coliO157:H7. The presence of pathogenic bacteria at any stage of food production, processing, and distribution must be determined quickly in order to allow proper treatment of the food before consumption by the general public. ARS scientists in cooperation with IGEN of Gaithersburg, Maryland, developed an immunomagnetic-electrochemiluminescent test for the detection of as few as a single E. coliO157:H7 bacterium in 25 grams of ground or unground beef. The inexpensive,user-friendly test can be conducted in less than 8 hours and is 100 timesmore sensitive than tests currently available. This test will allow detectionand quantification of this important pathogen, thus ensuring a safe foodsupply and protecting public health.

Epidemiology of Salmonellaand Campylobacterin broilers. Although the incidence of Salmonellaand Campylobacterin broilers has been decreasing, further measures are needed to assurea safe food supply. ARS conducted a nationwide epidemiology study of Salmonellaand Campylobacterin broilers to: identify the most importance sources of Campylobacterand Salmonellain the production environment, and determine intervention strategies thateffectively reduce transmission and limit consumer exposure to Campylobacterand Salmonella. The results of this study has allowed development of a commercial scaletrial that targets four control points to further reduce the levels ofpathogens on poultry; these are hatching cabinet disinfection, consistentuse of new paper pads for newly hatched chickens, competition exclusion,and litter treatment in the production house.

Rapid detection of antibiotic resistant Salmonella. Rapid methods are needed to identify multi-drug antibiotic resistant bacteria, such as, Salmonella typhimuriumDT104 in food producing animals and their environment. ARS determinedthat the resistance to the antibiotics florfenical and chloramphincol areconferred by the flogene. Presumptive identification of this multiresistance can now be maderapidly based on the presence of the gene and its resulting phenotype.This information will enhance the development of a rapid test kit to identifyDT104 in a timely manner, and slow its spread to food products.

Using competitive exclusion for pathogens in swine. A competitive exclusion culture was developed to control Salmonellaand other enteropathogens in swine. This culture significantly reducedmortality in baby and weaned pigs, and in addition significantly reducedmortality associated with pathogenic E. coliin very early-weaned pigs. The individual bacteria have been isolatedand a recombined culture has been developed that has the same efficacyas the original culture. Specific identification of the bacterial componentsof such cultures is necessary to allow reproducibility of results and forFDA approvals.

Contamination of the Chesapeake Bay. ARS research to characterize the extent and distribution of Cryptosporidium parvumoocysts in Chesapeake Bay indicates that oysters from all monitoring sitesare contaminated and contain viable oocysts. A real-time fluorescent-probebased PCR assay was developed for the detection of oocysts in less thantwo hours, with a detection limit of 5 to 100 oocysts depending on whetherthe water is clear or turbid. By detecting C. parvumARS has shown that shellfish can be good indicators of fecal pollutionof surface waters and that oysters pose a public health risk if eaten raw.Sequencing of the PCR products from some oysters indicates that the oocystsare C. parvum, bovine genotype. Utilizing PCR testing for detection of Cryptosporidiumincreases our ability to detect lighter infections and to differentiatespecies infectious for humans versus those noninfectious for humans andin some cases to determine the source of the fecal contamination.

Broiler transportation. Studies compared conventional solid to elevated wire mesh flooring during transport and holding of poultry broilers prior to slaughter. Results indicated that although broilers transported and held on solid flooring had noticeably dirtier breast feathers and higher coliform and E. colicounts prior to scalding and defeathering, bacteria recovery from externalcarcass rinses did not differ between the solid and wire flooring treatmentsafter defeathering. The processing steps of immersion scalding and defeatheringconsistently lowered carcass bacteria counts and the incidence of Campylobacter-positive, but not Salmonella-positive carcasses.

Cold shock sensitizes pathogens in fruit juices. Research showed that a sensitizing cold treatment prior to thermal processing increased the effectiveness of the thermal process for the destruction of L. monocytogenes, E. coliO157:H7 and S. typhimuriumin juices. The inducible and enhanced vulnerability to heat in model andfood systems shows the potential for this approach to be used by the foodindustry as a practical and efficacious post-processing intervention strategyto eliminate these bacterial pathogens. Cold shock prior to pasteurizationof fruit juices could provide an extra measure of safety and would alsoallow juice processors to reduce thermal processing requirements for pathogencontrol, while maintaining the fresh qualities of the juice.

Heat resistance of Listeria monocytogenes. Studies were performed to determine the heat treatment required to kill L. monocytogenes. The data was used to develop a mathematical model for determining theeffects of environmental parameters on thermal resistance of the bacterium.Using this predictive model, food processors should be able to design thermalprocesses for the production of a safe food with extended shelf life withoutsubstantially adversely affecting the quality of the product.

New method for detecting E. coliin ground beef. A procedure to rapidly detect the E. coliin beef hamburger patties was developed. Streptavidin coated magneticbeads and biotin-labeled anti E. coliO157 antibodies were used to capture the bacteria, which were furtherimmobilized on biotinylated nitrocellulose membrane sticks. The bacteriawere further treated with fluorescein-labeled anti E. coliO157 antibodies and urease conjugated anti fluorescein antibody. Ureasecatalyzed production rate of NH3 from urea was then determined. The approachallows detection of E. coliO157:H7 at a level of ~1 CFU/g after a six-hour enrichment. The approachmay be applied for field use, that is, sample collection from remote locationsaway from central laboratories, since the immobilized bacterial samplesare stable for 48 h at 4C without significant changes in signal.

Color as an indicator of hamburger safety. ARS research resulted in the FSIS issuing a message to consumers that "consumers should not eat ground beef patties that are pink or red in the middle unless a food thermometer has been used to verify cooked temperature". Subsequent surveys indicate that most consumers either do not own a meat thermometer or if they own one, do not use it in cooking meat products. Thus, additional joint ARS-FSIS studies on premature brown color in cooked beef patties were conducted, focusing on:

(1) the use of outdoor gas grills as the cooking equipment and,

(2) temperature and color changes post-cooking and before consumption. Results indicated that internal color of patties prepared and cooked under these conditions is even less reliable as an indicator of food safety. A workshop held in Washington, DC in November 1998 resulted in the formation of a Food Temperature Indicator Association to conduct research and perform consumer affairs regarding temperature devices. Also several major retailers, for example, Giant food stores have initiated ground beef thermometer usage programs employing ARS-FSIS research findings on safe cooking of beef patties.

Apple decontamination. The efficacy of commercial brush washing equipment, used in conjunction with conventional and experimental washing and sanitizing agents, in decontaminating apples was investigated in a commercial cider mill operated in Placerville, California. Results indicated that brush washing in the presence or absence of washing and sanitizing agents did not significantly reduce the bacterial population on apples or in cider. Cider contamination probably resulted from the inability of such washing systems to inactivate or remove microorganisms in inaccessible calyx and stem areas of apples.

E. coliO157:H7 contamination of apples used for cider. Studies were conducted to determine whether areas of decay in grounder apples ("drops") may be a potential source of E. coliO157:H7 in apple cider made from such fruit. Wounds in fresh apples inoculatedwith this human pathogen and one of two decay fungi, Penicillium expansumor Glomerella cingulataindicated that the P. exapansumfungus was antagonistic to the bacteria. However, apple wounds containing G. cingulataand E. coliO157:H7, showed extensive bacterial growth. Since the extent of growthof E. coliO157:H7 in the presence of Glomerellawould be sufficient to contaminate a large quantity of cider, these resultsclearly demonstrate the potential risk of using decayed apples for productionof unpasteurized cider.

Decontamination of sprouts. Research on novel combination treatments for decontamination of alfalfa seeds was conducted, and several alternative treatments were realized. A combination of treatments using gamma irradiation followed by chemical sanitizing with 2% calcium hypochlorite yielded a 5 log reduction in the population of Salmonellaon inoculated seeds, a reduction level that could not be achieved withthe individual treatments alone. The efficacy of 20,000 ppm chlorine insanitizing seed contaminated with E. coliO157:H7 was confirmed. Data generated on the use of 2% calcium hypochloritewas used in support of a successful joint USDA/industry petition to theEPA to allow use of this agent for sanitizing seed destined for sprouting.This technology is now widely used in the U.S. sprouting industry. Thecombination treatment strategy should enable sprout growers to achieveFDA goals with current technology if there are no unforeseen regulatoryrestrictions or economic constraints.

Detection of dioxins. ARS has developed a rapid, inexpensive immunoaffinity column (IAC) clean-up method for the detection of polychlorinated dibenzo-p-dioxins (PCDDs) and related compounds, from serum samples. Studies showedthat several of the most toxic PCDDs bound to the IAC, and the congenerscould be readily eluted before analysis by high resolution gas chromatography-highresolution mass spectrometry. Addition of a prewashing step before applyingthe sample, dramatically increased the recovery of 2,3,7,8-TCDD from 16%to 83%. Verification studies of samples provided by the CDC indicated thatsix dioxin and furan congeners were quantitated within the 95% confidencelimits of the method at the part-per-quadrillion (ppq) level. This simplertechnology will be of importance to regulatory agencies and other scientistsfor reducing the time and costs associated with dioxin analysis, presently$1000/sample.

Automated system for detection and removal of pistachios contaminated with aflatoxin. Aflatoxins are a family of structurally related toxins produced by molds in the genus Aspergillus. The toxins are usually associated with tree nuts, peanuts, corn, andcottonseed and affect a wide variety of different animals, including humans.ARS scientists developed an automated system that identifies and removesaflatoxin containing pistachio nuts from packing house production lines.The high-speed processing software and hardware is linked to sorters equippedwith digital image cameras, instead of conventional color sensors. Thesystem allows highly accurate, real-time sorting of the nuts, reduces lossesfrom sorting errors, ensures edibility, and increases marketability ofthe pistachio nuts especially in countries with stringent aflatoxin standards.Prototypes of the imaging system are currently being tested at a majorpistachio processing plant in central California.

Toxicity studies of fumonisins help to establish regulatory guidance. Several diseases of farm animals are caused by toxic metabolites produced by Fusarium moniliformeassociated with major crops, particularly corn. The Food and Drug Administration(FDA) is concerned about levels of the toxin, fumonisin B1, in grain andmay issue regulatory guidance. ARS scientists directly contributed to thedesign and successful completion of chronic toxicity and carcinogenicitystudies of fumonisin B1 in rats and mice, as well as teratology studiesof the mycotoxin in rabbits and rats. These studies showed that fumonisinB1 is carcinogenic in rodents, provided target organ and dose-responsedata, and also showed that fumonisin is not teratogenic. The data is criticalfor completion of FDA risk assessments to protect the public health.

Marker strains of Aspergillus flavusfacilitate crop resistance determination. Aflatoxins are natural poisons produced by two common fungi, Aspergillus flavusand Aspergillus parasiticus. These fungi invade crops such as, corn, peanuts, cotton and treenuts,and produce aflatoxins, thus making the crops unfit for sale under theexisting regulatory guidelines. ARS scientists have developed marker strainsof A. parasiticusto monitor the progression of aflatoxigenic fungi during invasion of cornand cotton and their ability to make toxin. These marker strains of thefungus allow the rapid screening of germplasm of the affected crops, bothin the laboratory and in the field, to determine its resistance to funalinvasion and toxin formation.

Disrupted fungal gene will help determine toxin biosynthesis pathway. The fungus Fusarium graminearumthat causes Fusarium head scab also produces the toxin deoxynivalenol(DON), that is harmful to humans and animals that eat toxin contaminatedfood or feed. ARS scientists have cloned the TRI101 gene from Fusariumgraminearum, and by gene disruption, identified its role in DON biosynthesisand toxin resistance. Mutant strains with a disrupted TRI101 gene willbe useful as tools to identify additional toxin resistance genes in Fusariumand to determine the DON biosynthesis pathway, thus leading to effectivecontrol strategies.

Natural resistance to aflatoxin formation found in Tulare walnuts. Contamination of tree nuts by aflatoxins produced by the fungi, Aspergillus flavusand Aspergillus parasiticus, is a serious problem because of the potential threat to human healthand the resulting stringent regulatory levels imposed for these toxins.The current US guideline set by the FDA is 20 ppb total aflatoxins, however,the European Community has instituted a standard of 2 ppb aflatoxin B1and 4 ppb total aflatoxins. ARS research has demonstrated a significantdifference in the ability of almonds and walnuts to support aflatoxin production,and there is further significant varietal difference within each crop.In these studies the Tulare variety of walnut completely suppressed aflatoxinproduction, which is the first example of a commercial variety of any susceptiblecrop plant that shows complete resistance to aflatoxin. This informationsuggests that selections of tree nut breeding lines for aflatoxin resistancecan be effectively made.

Spatial reference of Aspergillus flavusfungal communities aids assessment of aflatoxin control strategy. Because an aflatoxin management strategy to modify the Aspergillus flavuscommunity structure is being implemented over thousands of acres in Arizona,it is important to understand the spatial characteristics of communitiesof this fungal. Soil samples were collected statewide, and spatially referencedusing GPS. These samples were used to characterize A. flavuscommunities statewide by both incidences of A. flavusS strain and by regional shifts in S strain incidence over time. Thiswork provides the framework that to assist both the assessment and implementationof aflatoxin management programs based on reducing toxigenic A. flavuscommunities.

Utilizing tall larkspur without toxic effects Larkspur (Delphinium spp.) is toxic to grazing cattle and causes serious losses to livestock producers in western ranges. ARS research established that toxic alkaloid levels in tall larkspur vary between species, locations and over the growing season, however, the alkaloids appear to be synthesized in the first 3-4 weeks of growth followed by dilution of concentrations as the plants continue to grow throughout the season. Understanding such patterns will help predict the potential toxicity of larkspur populations to susceptible grazing livestock.

Identification of low dose effects of locoweed will aid decisions on range management Locoweed (species of Astragalusand Oxytropis) is a major management problem for cattle ranchers since it causes abortion,weak calves at birth, and weight loss in the stocker-yearling herd. ARSresearch demonstrated that alkaline phosphatase and serum iron levels changevery quickly (within 24 hours of initial exposure to swainsonine) and thesechanges occur prior to changes in other cellular damage markers. This indicatesthat swainsonine alters cellular metabolism and nutrient utilization, andthat this may occur before tissue damage or clinical signs are present.This information will help ranchers make more informed management decisionson utilization of ranges.