FY 2011 Annual Reports for National Program 108 Food Safety

Executive Summary

Food Safety falls under Goal 4 of the Agency Strategic Plan:  Enhance Protection and Safety of the Nation’s Agriculture and Food Supply.  For the Nation to have safe and affordable food, the food system must be protected at each step from production to consumption.  The production and distribution system for food in the United States encompasses a diverse, extensive, and easily accessible system that is open to the introduction of pathogens (bacteria, viruses and parasites), bacterial toxins, fungal toxins (mycotoxins), and chemical contaminants through natural processes, global commerce, and intentional means.  In response to these threats, crop and livestock production systems must be protected during production, processing, and preparation from pathogens, toxins, and chemicals that cause disease in humans.

To ensure the security of production systems, Agricultural Research Service (ARS) conducts basic, applied, and developmental research resulting in new technologies, new and improved management practices, pest management strategies, sustainable production systems, and methods of controlling potential contaminants.  These ARS activities are key to providing a safe, plentiful, diverse, and affordable supply of food, fiber, and other agricultural products.

Mission Statement
To provide through research, the means to ensure that the food supply is safe for consumers and that food and feed meet foreign and domestic regulatory requirements.  Research seeks ways to assess, control or eliminate potentially harmful food contaminants, including both introduced and naturally occurring pathogenic bacteria, viruses and parasites, toxins and non-biological-based chemical contaminants, mycotoxins and plant toxins.  Food safety is a global issue; thus, the Program involves both national and international collaborations through formal and informal partnerships.  Accomplishments and outcomes are utilized in national and international strategies delivering research results to regulatory agencies, commodity organizations, industry and consumers for implementation.

Vision Statement
To increase public health through the development of technologies which protect food from pathogens, toxins, and chemical contaminants during production, processing, and preparation thus increasing the safety of the food supply.  

There is one research component and six problem statements in the current Action Plan for the program:

Component 1. Foodborne Contaminants

Problems Statements
1.A – Population Systems
·    This area identifies and characterizes the movement, structure, and dynamics of populations throughout food production, processing and storage; hence the entire safety continuum.  Major components of emphasis and interaction include epidemiology, ecology, host-pathogen relationships.  

1.B – Systems Biology
·    The concept of systems biology involves a unique integrative approach to understand the basic genetic components of pathogens, their expression, and directly relate this information to the microorganism’s biology.

1.C – Technologies for the Detection and Characterization of Contaminants
·    Challenges arise from either uncontrolled microbes entering through raw materials, contamination during processing, or from undesired chemical contaminants including chemical residues, and bacterial, fungal and plant toxins.  [Sensitive and specific] detection technologies are required at the earliest possible stage in the food chain, thus avoiding/preventing the need for processing interventions, or possible recall.
•    Development of technologies must yield method(s) that are faster and yield improved resolution.
•    In developing technologies decisions cannot be made in isolation.  There needs to be an integration of biology, epidemiology, and the physical sciences systems.

1.D – Intervention and Control Strategies
·    To ensure safe food and protect public health, intervention and control strategies must be identified, implemented, and then measured as to their impact on the reduction and control of food-borne pathogens or other zoonotic organisms, and chemical contaminants. This approach incorporates strategies in both pre- and post-harvest systems as the science dictates, to produce a complementary and efficient approach for food safety.

1.E – Predictive Microbiology
·    The behavior of any microorganism is deterministic and able to be predicted from knowledge of the microorganism itself, and the microorganism’s immediate environment. Behavioral predictions are an integral part of microbial risk assessment used to support food safety measures.

1.F – Chemical and Biological Contaminants: Methodology, Toxicology, and Toxinology
·    The regulation and control of veterinary drugs, residues, heavy metals, persistent organic pollutants, and biological toxins derived from bacteria, fungi and plants are an integral component of any food safety program to protect human health and the environment.

Selected Accomplishments for Agency Documents (2010-2011)

Stabilizer to improve sanitizing efficiency of chlorine.  The produce industry currently faces a major potential food safety problem, that chlorine levels needed to prevent pathogen survival in wash water are depleted during commercial operations.  Working closely with the produce industry, ARS scientists from Beltsville, Maryland, evaluated a novel chlorine stabilizer in maintaining free chlorine efficacy on pathogen survival and cross-contamination during commercial wash operating conditions.  In plant studies demonstrated that the patented compound (T128) significantly increases the efficacy of chlorine wash against bacterial cross contamination while maintaining the quality of leafy green vegetables under real world fresh-cut processing conditions.  This research is supported and conducted in collaboration with New Leaf Food Safety Solutions Inc. to optimize the application of T128 in the postharvest processing system.

Localization of lead in root crops.  Lead levels in produce have recently come under serious consumer and medical scrutiny particularly as nutritious snack foods for children.  Research conducted at Beltsville, Maryland, identified higher than normal levels of lead in carrots grown on old orchard soils where lead-arsenate insecticide had been used before 1950.   Peeled carrots were shown to have higher carrot lead, showing that the contamination pathway was not due to soil adherence to the roots.  Lead accumulates in the xylem portion of the root with very little lead in the rest of the storage root.  Additional root crops (beet, turnip, and radish) were similarly tested and lead accumulation was observed but considerably lower than that found in carrot.  This appears to result from the long xylem through the carrot compared to the wider diameter of the other root crops.  Potato had very low lead when grown on the same soils, showing that phloem-fed tissues such as tubers, fruits, and grains accumulate very low levels of lead even on high lead soils.  Overall, the findings support the Food and Drug Administration’s goal of understanding how carrots can be enriched in lead, and the industry’s need to limit production of crops with high lead levels.

Nanoparticles to inactivate foodborne pathogens.  Nanoparticles can be effective antimicrobial agents against foodborne pathogens.  ARS researchers at Wyndmoor, Pennsylvania, investigated the antimicrobial activities of two nanoparticles (magnesium oxide and zinc oxide) against three major foodborne pathogens: Escherichia coli O157, Salmonella sp, and Camplylobactor jejuni.  The results demonstrated that these nanoparticles dramatically killed those pathogens and, therefore, potentially can be added directly in foods or incorporated in packaging materials to improve microbiological safety.  This research explores a new application of nano-technology and inorganic antimicrobial compounds in the food safety area, and provides useful information to the food and packaging industries.  The impact of nanopaticles on environment and human health is not clear.  Currently nanotechnology is being evaluated in the Food and Drug Administration Critical Path Initiative.  Further toxicological studies are needed to determine the potential risks to humans which is a concern expressed by various international bodies.

Detection of veterinary drugs in animal tissues.  Currently, the USDA Food Safety Inspection Service (FSIS) uses a 7-plate microbial growth inhibition assay to screen for antimicrobial drug residues in beef samples from slaughter establishments throughout the U.S.  Drawbacks include that it takes 24 hours to yield a result, the responses do not identify the drug (only the antibiotic class), and it is unable to detect many common drugs of regulatory interest.  ARS researchers at Wyndmoor, Pennsylvania, developed, validated, and transferred to FSIS an improved screening method that also can identify individual drug residues in meat samples.  The technology targets 60 of the most important drugs of regulatory concern and is able to screen at concentrations below current regulatory tolerance levels.  A single analyst can perform preparation of 60 samples with the method in an 8-hour day for a series of sequential 10 minute analyses.  Implementation of the method in the USDA-FSIS National Residue Program will serve to improve the monitoring and enforcement of veterinary drug residues, and thereby assure better animal husbandry practices, reduce environmental contamination, decrease microbial antibiotic resistance, and increase food safety.


Detection and typing of non-O157 Shiga toxin-producing E. coli.  It has become evident that certain Shiga toxin-producing E. coli (STEC) serogroups, including E. coli O26, O45, O103, O111, O121, and O145 cause a similar illness in humans as E. coli O157:H7.  Since these “top six” non-O157 STEC serogroups can be as dangerous as E. coli O157:H7, the USDA Food Safety and Inspection Service (FSIS) has very recently declared these STEC as adulterants in beef like E. coli O157:H7.  At the request of the FSIS, ARS researchers at Wyndmoor, Pennsylvania, developed a method consisting of food enrichment, detection by the polymerase chain reaction targeting important genes involved in the disease process and serogroup-specific genes, and strain isolation protocols to detect and identify these non-O157 STEC pathogens in beef.  Further, ARS developed, evaluated, and transferred a latex agglutination tests (LATs) for detection and confirmation of the STECs.  The detection, isolation, and confirmation protocols will be useful for the food industry and in early 2012, will be employed by the FSIS to monitor for these important emerging pathogens in beef.

Detection of nivalenol and deoxynivalenol.  Nivalenol (NIV) is a trichothecene related to deoxynivalenol (DON, vomitoxin), a mycotoxin commonly found in cereal commodities in the U.S.  NIV has been reported to occur frequently in Asia and Europe, and a population of NIV-producing fungi was recently identified in the U.S.  This is of immediate concern because NIV may be as toxic (or more toxic) than DON.  For this reason rapid and sensitive methods for detecting NIV and DON are important.  ARS scientists from Peoria, Illinois, collaborated with Kirin Holdings Company (Gunma, Japan), the National Agricultural Research Center (Kumamoto, Japan), the Kobe Institute of Health (Kobe, Japan), and the National Institute of Health Sciences (Tokyo, Japan) in developing a novel antibody-based biosensor for the detection of NIV and DON in wheat.  Because the sensor can simultaneously detect both toxins it will find immediate utility in areas where these two toxins co-occur, assisting in the diversion of contaminated commodities from human food and animal feed supplies.

Neutralization of botulinum neurotoxin.  Clostridium botulinum neurotoxins (BoNTs), responsible for botulism food poisoning, are rapidly absorbed in small amounts.  Even though lethal they are concomitantly very difficult to detect.  Scientists in Albany, California, developed monoclonal antibodies specific for BoNTs and tested them for their ability to provide protection against botulism exposure in a mouse model system.  Following intravenous and oral exposures to lethal levels of toxin, the timing of antibody neutralization of the toxin was determined.  The results provided new information on the toxicity of BoNTs and revealed windows of opportunity for human therapeutic treatment with antibody.  A better understanding of the biology of toxins, the factors that affect their toxicity, and toxin neutralization are valuable tools for advancing food safety and defense.

Non-O157 Shiga toxin-producing E. coli in commercial ground beef.  Non-O157 Shiga toxin-producing E. coli are a collection of E. coli strains that produce various lethal Shiga toxins.  There are over 200 types of these E. coli strains and their ability to cause human foodborne illness ranges from harmless to those that can cause severe disease or death.  Recently, these strains have become an increasing concern to the beef industry, regulatory officials, and the public.  The USDA-FSIS has now classified some serotypes as adulterants, and thus new laws will come into effect in 2012.  ARS researchers at Clay Center, Nebraska, determined the prevalence and characterized non-O157 Shiga toxin-producing E. coli from over 4,000 commercial ground beef samples obtained from numerous manufacturers across the United States over a period of 24 months.  Markers of the bacteria were present in approximately one quarter of ground beef samples.  However, characterization of the specific bacterial strains obtained from the samples identified very few organisms that should be considered significant food safety threats.  The project provided the first large scale analysis of non-O157 in ground beef, and the results have been used by the beef industry and the FSIS to determine the best measures to take in regards to eliminating these pathogens from the beef supply.

Pharmacokinetics of perfluorooctanoic acid.  Perfluorooctanoic acid (PFOA) is a “nonstick” compound used in many industrial, commercial, and consumer products.  Due to its extensive use, PFOA is widely found in humans, wildlife, and the environment.  Cattle are exposed to PFOA while grazing in contaminated areas, but the extent to which PFOA accumulates in their meat is not known.  ARS researchers at Fargo, North Dakota, together with scientists at USDA-FSIS, conducted a study to determine to what degree PFOA concentrates in the edible tissues of beef cattle and whether this may be a concern for human exposure.  Beef cattle were fed a single dose of radiolabeled PFOA which could easily be tracked in the animals.  The PFOA was quickly excreted in animal’s urine and no detectable amounts were left in the animals after 8 days.  This study showed that PFOA was not likely to accumulate in beef and that consumption of beef should not be a significant source of exposure to PFOA.

Salmonella Enteritidis contamination of shell eggs.  Salmonella Enteritidis is the world’s leading cause of human salmonellosis.  It is unique among 2500 Salmonella serotypes, because it is able to colonize and survive in the internal contents of eggs produced by otherwise healthy appearing hens.  It does so with an efficiency and persistence that impacts epidemiology of human disease in a manner greater than all the other serotypes.  Salmonella Enteritidis presented a puzzle to the research and producer community, because numerous studies indicated that strains varied greatly in their ability to contaminate eggs.  However, genetic analysis repeatedly showed that the bacterium had very little genetic difference between strains in comparison to what is seen with other serotypes.  ARS scientists at Athens, Georgia, compared three whole genomes by high-density tiling arrays that generated a mutational map solved the puzzle of detecting genetic differences. Two of the strains were of the same phage type and were known to have no differences in gene content using DNA microarrays.  Application of 3 techniques found that 250 Single Nucleotide Polymorphisms (SNPs) differentiated these two strains that varied in the ability to contaminate eggs.  This information supports efforts to protect the food supply by improving epidemiological investigations and by providing new gene targets for improving vaccines.  Customers benefitting from this information are regulatory agencies such as the Food and Drug Administration, the Centers for Disease Control and the USDA-FSIS; in addition, producers of vaccines and field epidemiologists are benefitted.

Survival and virulence mechanisms in Salmonella.  Salmonella Typhimurium is a human foodborne pathogen and is one of the most prominent Salmonella serovars isolated from swine production farms.  Unfortunately, Salmonella Typhimurium can undetectably reside in pigs without causing noticeable infection.  These Salmonella-carrier pigs are a food safety problem for humans through contamination of penmates, the environment and slaughter plants that process pork for consumption.  In searching for improved intervention strategies against Salmonella on the farm, ARS researchers in Ames, Iowa, have identified a gene (poxA) in Salmonella Typhimurium that, when mutated, dramatically reduces the ability of the bacterium to survive numerous stress conditions as well as antibiotic and chemical exposures.  Furthermore, the gene mutation decreased the ability of Salmonella Typhimurium to colonize the pig.  As this genetic system is critical for the ability of the Salmonella to cause disease and resist antibiotics, it offers a novel target mechanism for intervention development against Salmonella.  

Organic acids reduce Salmonella in swine and poultry.  Salmonella bacteria are human pathogens that can reside in the gut of food animals such as swine, cattle, and poultry; these bacteria can contaminate meat products reaching the consumer and thus cause illness or even death.  Organic acids are a dietary additive that can improve animal growth efficiency and change the microbial population of the intestinal tract.  ARS researchers at College Station, Texas, demonstrated that including specific organic acids in the diets of pigs and chickens could reduce populations of Salmonella from 10 to 100 fold in the live animals.  This work has important food safety implications because it identifies another tool to help producers reduce the carriage of foodborne pathogens in meat-producing animals.  Reduced pathogen loads in animals at slaughter will result in microbiologically safer meat products reaching the consumer.

Irrigation pipelines as reservoirs.  In a primary study on the impact of biofilms, ARS scientists in Beltsville, Maryland, showed that pathogen and indicator microorganisms associated with biofilms on internal pipe walls could alter the microbial quality of irrigation water used to produce fresh produce.  This study showed that it is imperative to monitor water quality at fields, rather than just at the intake.  This study strongly suggests that back-flushing the irrigation system or disinfecting irrigation pipelines have to be considered as viable management practices by producers to improve the safety of irrigated produce.  Such decisions would have to be included within produce related good agricultural practices (GAPs).  

Faster analysis of pesticides in foods.  It is always desirable to use faster and better methods of analysis to monitor pesticide residues in foods.  ARS researchers at Wyndmoor, Pennsylvania, validated both qualitatively and quantitatively the updated QuEChERS (quick, easy, cheap, effective, rugged, and safe) version coupled to low-pressure gas chromatography-tandem mass spectrometry for fast analysis of 150 pesticides in cantaloupe, broccoli, lemon, sweet potato, and catfish.  The enhanced selectivity and sensitivity of this approach allowed the 150 pesticides to be separated in less than 7 minutes and to achieve less than 5ng/g limits of quantification for nearly all pesticides.  Qualitatively, no false positives or false negatives were observed for 100 randomly spiked and blinded extracts at contamination levels greater than 10ng/g in the different matrices.  This is a new state-of-the-art method for pesticide residue analysis that will be evaluated in an AOAC International collaborative study for enhanced technology transfer of the method to regulatory monitoring laboratories around the world.  

Sites of virus persistence in shellfish.  Virus contamination of shellfish has led to frequent outbreaks of hepatitis A and norovirus illnesses.  Once contaminated, shellfish retain high levels of these viruses for extended periods.  ARS researchers at Dover, Deleware, identified primitive blood cells of oysters, known as hemocytes, as the site of virus accumulation and persistence. Research developed simple and effective procedures to extract and test for viruses within the hemocytes.  This technology is a dramatic improvement over traditional testing methods for viruses in shellfish which rely on tedious dissection and testing of intestinal tissues that contain only a portion of the viral contaminants.  Improved assay methods will significantly enhance monitoring efforts and support regulatory agency such as the Food and Drug Administration, the Centers for Disease Control, and industry goals to reduce shellfish-associated enteric virus illness.

Dietary antibiotic effects on swine.  ARS researchers at Ames, Iowa, in collaboration with scientists at Michigan State University examined effects of feeding certain antibiotics to swine. They used a combination of phylotyping (bacterial identification by 16S rRNA gene sequences), metagenomic (sequencing and bioinformatic analysis of bacterial genes), quantitative PCR (qPCR), and culture-based approaches to investigate differences between swine fed a commercial diet containing aureomycin, sulfamethazine, and penicillin (ASP250) and siblings fed the same diet without antibiotics (control group).  By comparison with control animals, swine fed ASP250 diets for 2 weeks had higher Escherichia coli numbers in their feces; had elevated levels of bacterial genes for energy production and metabolism (consistent with the animal performance enhancing properties of the ASP250-diet); and had higher levels of genes encoding resistance both for the ASP250 antibiotics and other antibiotics in feces.  Investigations of dietary antibiotic effects on intestinal microbiomes are important for understanding how certain antibiotics enable animals to grow efficiently with less feed consumption and how antibiotic alternatives should behave.  Additionally, feeding subtherapeutic doses of antibiotics to farm animals will likely have collateral effects which should be considered by producers and commodity organizations in cost-benefit analyses of antibiotic use.  Data obtained from this study is currently being used in the selection of antibiotics and more importantly the design of antibiotic alternatives.

Aerosols from concentrated animal feeding operations.   Aerosols have been suspected as a means to transport food pathogens and contaminate fruits and vegetables grown in close proximity of concentrated animal feeding operations.  However, studies investigating such transport are scarce, and science-based guidelines for the produce industry regarding the safe distance between produce fields and processing plants, and animal operations are lacking.  ARS researchers in Albany, California, used gene sequencing methods to characterize bacterial communities in manure and air samples collected from dairies in the Central and Coastal valleys of California.  Whereas known enteric pathogens were not detected in aerosol samples, bacterial species originating from cows at the dairy operation in the vicinity were identified.  Thus, bacteria specific to each dairy may be used as tracers for detecting the source of pathogens that are transported to produce or fruit crops from feedlots or dairies.  These data suggest a new strategy and Good Agricultural Practice document needs to be developed for trace back epidemiological investigations by regulatory agencies and for quality testing by the produce industry.

E. coli O157:H7 and cattle confinement facilities.   There is growing interest in feeding beef cattle in enclosed deep-bedded confinement barns for improved cattle performance and ease of manure management.  However, there is no available information regarding the occurrence of pathogens in the manure/bedding material or in cattle that are housed in these barns.  ARS scientists in Clay Center, Nebraska, demonstrated that both E. coli O157:H7 prevalence and generic E. coli concentrations can occur at high levels in the manure/bedding material of these facilities, and can vary with differences in ambient seasonal temperatures.  Understanding microbial survival in the manure/bedding material is important because this material can be a source of pathogens for contamination of additional cattle, or of water, food, and feed crops when the material is applied to cropland.  Furthermore, this information is needed to develop recommendations and Good Agricultural Practices regarding the management of these facilities to reduce pathogens, thereby reducing the risk for contamination and human foodborne illness.

Antimicrobial interventions for Shiga toxin-producing E. coli.  Although the bacterium E. coli O157:H7 is currently most widely recognized, other E. coli types producing the same toxins have been implicated in cases of human diseases and are the subject of pending new regulatory policy.  ARS scientists in Clay Center, Nebraska, investigated whether antimicrobial compounds currently used by the meat industry to control E. coli O157:H7 are effective against these non-O157 Shiga toxin-producing groups (O26, O103, O111, and O145).  They determined that six antimicrobial compounds were equally effective against E. coli O157:H7 and non-O157 on fresh beef.  These results will assist the meat industry in developing effective antimicrobial intervention programs against these newly recognized and regulated pathogens.

Host response to Campylobacter in poultry.  Food-poisoning bacteria such as Campylobacter are significant causes of human disease; these pathogens can often be found as contaminants in poultry meat products.  New non-antibiotic therapeutics is needed to reduce or prevent colonization of poultry by these harmful bacteria; which will largely translate into pathogen-free meat products reaching the consumer.  ARS scientists at College Station, Texas, used modern molecular biological techniques (microarray analysis and real-time RT-PCR) to define gene expression changes in the avian spleen during an active Campylobacter infection.  This work is the first to develop a solid foundation for defining the complex mechanisms involved in innate immunity of poultry to Campylobacter infections, and in Campylobacter adaptation to the intracellular environment.  The work has broader impact in that it provides a valuable means for accessing unique information on host-pathogen interactions.  More definitive understanding of these molecular mechanisms will facilitate development of new and more effective intervention strategies for minimizing or even eliminating the occurrence of food-borne pathogens in commercial poultry.

Use of the agar diffusion assay to examine bactericidal activity of chelating agents.  Studies were conducted using the agar diffusion assay to examine the antibacterial activity of the chelators, ethylenediaminetetraacetic acid (EDTA) and ethylenediaminedisuccinic acid (EDDS).  EDTA is a synthetic, non-biodegradable chelator; and EDDS is a natural, biodegradable chelator.  Both chelators can bind minerals that produce water hardness thereby reducing water hardness and increasing the ability of soaps and detergents to remove dirt and debris during washing.  Results from experiments indicated that EDTA inhibited growth of all nine bacteria used in the study while EDDS inhibited the growth of two bacteria.  The addition of these chelating agents to formulations of sanitizers may improve the ability of the sanitizers to wash away microorganisms on chicken carcasses and reduce contamination by killing microorganisms.  Incorporating these chelators in sanitizers will reduce the number of bacteria on processed poultry.

Multi-drug resistance in Escherichia coli.  Characterization of bacterial plasmids (similar to small chromosomes) is necessary because genes encoding important traits such as antimicrobial resistance are frequently carried on plasmids.  ARS scientists at Athens, Georgia, analyzed the plasmid types, antimicrobial phenotypes, and genetic relationships of multi-drug resistant (MDR) Escherichia coli.  A total of 15 different plasmid types were detected, all isolates carried multiple plasmid types and extensive genotypic diversity was observed.  This study demonstrates that E. coli from animal sources are highly variable genotypically and are reservoirs of a diverse array of plasmids carrying antimicrobial resistance.  This work will significantly aid in the design of monitoring strategies by Federal agencies and researchers on the spread of MDR plasmids and determine their threat to human and animal health.