**National Program 108**

**Food Safety**

**2006-2010 Action Plan**

**Contents Page**

**Cover** 1

**Contents** 2

**Introduction** 3

**Mission and Vision** 4

**[Component]{.underline} 1**

-   1.1 Pathogens, Toxins and Chemical Contaminants Preharvest 5

1.1.1 Methodology 5

> 1.1.2 Epidemiology 7
>
> 1.1.3 Ecology, Host Pathogen and Chemical Contaminants
>
> Relationships 8
>
> 1.1.4 Intervention Strategies 10
>
> 1.1.5 Antibiotic Resistance 12

-   1.2 Pathogens, Toxins and Chemical Contaminants Postharvest 14

> 1.2.1 Detection and Validation 14
>
> 1.2.2 On-line Sensing Systems 16
>
> 1.2.3 Production and Processing Ecology 17
>
> 1.2.4 Processing Intervention Strategies 19
>
> 1.2.5 Omics 21
>
> 1.2.6 Safety and Health 22
>
> 1.2.7 Risk Assessment 24
>
> 1.2.8 Pathogenicity 25
>
> 1.2.9 Food Security 27

**[Component]{.underline} 2**

-   2.1 Mycotoxins and Plant Toxins 29

> 2.1.1 Toxin Methodology and Identification 30
>
> 2.1.2 Crop/Fungal/Toxin Relationships 31
>
> 2.1.3 Production Practices and Expert Systems 32
>
> 2.1.4 Breeding Resistant Crops 34
>
> 2.1.5 Biocontrol Technologies 36
>
> 2.1.6 Toxicity Evaluations and Mechanisms of Action 37

**Introduction**

Food Safety falls under Goal 3 of the Agency Strategic Plan: Enhance
Protection and Safety of the Nation's Agriculture and Food Supply. For
the Nation to have affordable and safe 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 has been a
diverse, extensive, and easily accessible system. This open system is
vulnerable to introduction of pathogens and toxins through natural
processes and global commerce and by intentional means. In response to
these threats, crop and livestock production systems must be protected
from the ravages of diseases whether domestic or exotic in origin. The
food supply must be protected during production, processing, and
preparation from pathogens, toxins, and chemical contamination that
cause disease in humans.

Food safety research seeks ways to assess and control potentially
harmful food contaminants. Research to ensure a secure agricultural
production system refers to work that reduces or eliminates factors that
threaten the ability of U.S. agriculture to produce enough food and
fiber, year to year, to meet the needs of American consumers. ARS will
conduct research designed to generate knowledge regarding new and
improved management practices, pest management strategies, sustainable
production systems, and control of potential contaminants for farms of
all sizes. These activities will ensure a secure production system able
to provide a safe, plentiful, diverse, and affordable supply of food,
fiber, and other agricultural products.

ARS will provide scientific information and technology to producers,
manufacturers, regulatory agencies, and consumers to support their
efforts to provide a secure, affordable, and safe supply of food, fiber,
and industrial products.

**Relationship of the NP 108 to the ARS Strategic Plan:** Outputs of NP
108 research support the 'Actionable Stratgeies" associated with the
performance measures shown below from the ARS Strategic Plan for
2003-2007, Objective 3.1: Provide Science-Based Knowledge on the Safe
Production, Storage, Processing, and Handling of Plant and Animal
Products and on the Detection and Control of Toxin-producing and/or
Pathogenic Bacteria and Fungi, Parasites, Chemical Contaminants,
Mycotoxins, and Plant Toxins so as to Assist Regulatory Agencies and the
Food Industry in Reducing the Incidence of Foodborne Illnesses.

**Performance Measures 3.1.1:** Develop new on-farm preharvest systems,
practices, and products to reduce pathogen and toxin contamination of
animal- and plant-derived foods**. Target:** Develop practices and/or
products that reduce preharvest contamination of major animal- and
plant-derived food products.

**Performance Measures 3.1.2:** Develop and transfer to Federal agencies
and the private sector systems that rapidly and accurately detect,
identify, and differentiate the most critical and economically important
foodborne microbial pathogens. **Target** Develop practices and/or
products that reduce postharvest contamination of major animal- and
plant-derived food products.

**Mission Statement:**

To provide through scientific research, the means to ensure that the
food supply is safe and secure for consumers and that food and feed meet
foreign and domestic regulatory requirements. Food safety 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.
Since food safety and food security are global issues, our research
program involves both national and international collaborations through
formal and informal partnerships. Our accomplishments and outcomes are
utilized in national and international strategies delivering research
results to regulatory agencies, commodity organizations and consumers.

Vision Statement:

For the Nation to have a safe, plentiful, diverse, affordable, quality
food supply, protected during production, processing, and preparation
from pathogens, toxins, and chemical contamination that cause disease in
humans.

The research components of this program include:

-   \[Microbial\] Pathogens, Toxins and \[non-biological-based\]
    Chemical Contaminants: subdivided in Pre-harvest and Post-harvest

-   Mycotoxins and Plant Toxins

**[Component 1.1.]{.underline} Pathogens, Toxins and Chemical
Contaminants - Preharvest**

The challenge to preharvest food safety research is to develop
information and products that will assure the safety of food products
from animal agriculture. Animal food products must be safe for the
consumer and produced with sensitivity to animal welfare concerns, and
they must be competitive in the national and international market place;
to make this possible research must develop interventions, that is
effective products and practices which will be used. Profit margins in
animal and poultry production are variable and often small, and
producers will not increase their cost of doing business, by using a new
vaccine or handling manure in a different manner, for instance, unless
they are convinced it will bring them a return in the health of their
animals, the price they will receive in the marketplace, or the
acceptability of their animals to meet regulatory agency standards.

The effectiveness of interventions during production must be considered
at the slaughterhouse level as well as at the farm itself. We need to
know both (1) if on farm interventions have really decreased the
pathogens in the GI tract of the food producing animal or bird at the
time of slaughter, and (2) has this made a difference in the pathogen
load of the animal products leaving the slaughter/packing plants.
Production of food products with the minimum of both pathogens and
chemical residues is important even if current regulatory guidelines are
being met. For example. the Food and Drug Administration has requested
that on farm practices be optimised to decrease the number of pathogens
in raw milk, even though pasteurization has been considered successful
for many years in providing safe milk for the American consumer. There
are certain cheese products, primarily soft cheeses that consumers
prefer to be made from unpasteurized milk, and they are available either
in imports or from ethnic markets. Some consumers also continue to
obtain raw milk for home consumption. Thus we must have the knowledge to
produce milk in the safest manner possible. Another reason to further
assure the microbiological safety of raw milk, or any other food product
of animal origin, is to provide a hedge against accidents or errors
occurring during the pasteurization, handling and transportation
processes which would provide an opportunity for pathogen growth.

**Problem Statement 1.1.1: Methodology**

Rapid, accurate, and sensitive methodology is needed for identification
and quantitation of epizootic pathogens and chemicals that may be
transmitted to humans, as well as for other bacteria that may affect
their occurrence in the animal and their environment. Previous standards
for the sensitivity of assays will not be acceptable in the future. For
example, acceptable precision and accuracy for all dioxin congeners can
now be achieved. This achievement has made a study with dioxins
conducted less than 10 years ago now obsolete.

State of the art methodology is needed for research that is conducted to
identify and quantify the pathogens and chemicals in both animal fluids
and tissues, and in their environment, including animal manure; to
establish the ecology and epidemiology of the pathogens; to delineate
the host-pathogen relationship of these zoonotic pathogens in food
producing animals; and to develop effective interventions that can be
easily instituted by the livestock and poultry producer.

**Research Needs**

Methods for accurate, timely and cost effective specific identification
and quantitative enumeration of pathogens and chemical contaminants with
appropriate speciation, are necessary not only for epidemiology,
ecological and intervention research, but also to support the risk
assessments which are carried out by the regulatory agencies, that is,
FDA and FSIS. New methods must provide a wide range of molecular
genetic, genomic and proteomic information on epizootic pathogens that
in turn enable the development, validation and standardization of new
rapid test methods, new drugs and vaccines, insights into key immune
responses, and improved intervention strategies.

**Outputs**

-   Methods to recover, identify and quantitate pathogens and chemical
    residues in the tissues and matrices, including animal manure, where
    they are expected to be found, or are needed for regulatory
    purposes.

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-   Methods to recover and enumerate \>non-culturable= pathogens from
    all environmental niches and to selectively enhance the growth of
    extremely slow growing organisms.

-   Methods that are sufficiently rapid and cost effective to be useful
    to both producers and regulatory agencies.

-   Genetic and serologic bases for new methods that will provide
    differentiation of bacterial clones to the extent needed for
    attribution of these sources.

-   Highly specific serological tests with the necessary sensitivity to
    detect animal carriers.

-   Development of microarrays to assess gene expression in various
    environments.

**Impact**

ARS will lead in developing and validating methodologies that have
regulatory, industry and research uses for solving preharvest pathogen
and chemical contamination problems. Fortunately there is a commonality
of interests among these groups which allow similar methods to be used
by each. Having reliable, cost effective identifications of pathogens
and chemicals in food animal tissues and environmental samples will
assist in carrying out risk assessments, in developing and validating
microbial models that predict the growth and virulence of pathogens, and
in identifying areas where interventions are most critically needed.
This information will assist FSIS, FDA and the food animal industry in
the production of safe food products.

# 

# Resources

ARS scientists assigned to projects under this Problem Statement are
located at Beltsville Agricultural Research Center, Beltsville, MD;
National Animal Disease Center, Ames, Iowa; Swine Odor and Manure
Management Research Laboratory Ames, Iowa; Meat Animal Research Center,
Clay Center, NE; Red River Valley Agricultural Research Center, Fargo,
ND; Western Regional Research Center, Albany, CA.; Southern Plains
Agricultural Research Center, College Station, TX; Livestock Issues
Research, Lubbock, TX; and Richard Russell Research Center, Athens, GA.

**Problem Statement 1.1.2: Epidemiology**

Epidemiology determines the origin and routes of transmission of
epizootic pathogens. The livestock industry continually incorporates
additional mobility, moving animals one or more times during the
production cycle, in order to benefit from large scale operations.
Animal mobility complicates the challenge to trace the flow of both
pathogens and specific identified genes, through the production cycle
from application of manure to crop or grazing lands, to the raising and
slaughter of meat animals, or to production of milk and eggs. For
instance, less than half of the dairy calves now grow and mature at the
premises where they are born, and thus whatever pathogens are associated
with them at birth or at common rearing facilities may be introduced to
the milking herds where they spend their productive lives. Also
cross-contamination occurs in multiple sites as animals are transported
to slaughter plants, at the plant through contact with workers and the
plant environment, and during fabrication in plant and in retail
supermarkets, particularly those that further grind or fabricate on
site. We need to be able to evaluate the contribution of each of these
sites.

**Research Needs**

Small scale, limited time frame and end point epidemiology studies can
be carried out by one research scientist with limited assistance, but
increasingly large scale research programs are necessary to meet the
needs of today=s agriculture which produces food for millions of
consumers. Large studies are needed in order to have the statistical
power to sample and analyze appropriate biological samples, recover the
isolates and determine the identity and numbers of pathogens, and
perform the necessary statistical analyses of the large volumes of data,
in order to reliably correlate laboratory data with herd information and
relevant production practices. A second vital consideration in
epidemiology is the need to conduct studies over an extended time frame.
Data from single time points do not reflect the large inherent variation
that is a part of bacterial reservoirs and amplification cycles, and do
not serve the needs of the animal industry. One program now in the
initial stages that addresses this deficiency is the Collaboration for
Animal Health and Food Safety Epidemiology (CAHFSE), a multiagency
program utilizing the resources of ARS, FSIS and APHIS in a single
coordinated framework to conduct epidemiology with repeat sampling over
an extended time frame. (See additional information under \>Antibiotic
Resistance'). Information-gathering projects that can detect early
trends and warning signals are also vital to biosecurity systems for
animal product food production.

**Outputs**

-   Development of coordinated food animal surveillance and epidemiology
    programs for food animal species which recognizes early warning
    signs of pathogen infection.

-   Estimates of the basic reproductive ratios (Ro=s) for epizootic
    pathogens in various food animal species for epidemiologic modeling
    and more accurate prediction of the effectiveness of interventions.

-   Epidemiological studies that elucidate chemical or pathogen presence
    and behavior in particularly important, suspected biological niches,
    including animal manure and other environmental niches.

**Impact**

Scientists will gain an additional understanding of the transmission and
dissemination of food safety pathogens from the farm through slaughter
environments from the epidemiology and surveillance data of food safety
pathogens in animal production. The contribution from all aspects of the
environment, including feed and water can also be evaluated. This
information will provide data needed to carry out risk assessments, to
develop and validate predictive microbial models, and assist in
determining critical directions for intervention strategies to reduce
the spread of pathogens both in food animals and poultry and to humans.
CAHFSE will enable USDA to identify and track emerging diseases and
identify and implement mitigation strategies in a timely manner thereby
averting negative effects on the economy, animal health and public
health. CAHFSE will also serve as a model for future surveillance
efforts on a national level, thus assisting FSIS, FDA and the food
animal industry in the production of safe food products.

# Resources

ARS scientists assigned to projects under this Problem Statement are
located at Beltsville Agricultural Research Center, Beltsville, MD;
National Animal Disease Center, Ames, Iowa; Meat Animal Research Center,
Clay Center, NE; and Richard Russell Research Center, Athens, GA.

**Problem Statement 1.1.3: Ecology, Host Pathogen and Chemical
Comtaminants Relationships**

The challenge of the future is to determine the attributes of the
ecological communities in which epizootic pathogens are found in animals
and their environments. It is not sufficient to know that food animals
may be carrying one or more specific epizootic pathogens; we need to
know their relationship to and the attributes of the microbial
communities in which pathogens live, particularly the gut of food
producing animals. To develop interventions that can control these
bacteria and protozoa we need to identify sites and mechanisms of
colonization in animals, to identify and characterize virulence
attributes and immunogenic responses, to learn how animals and microbes
affect one another and how this relationship is affected by animal well
being. We need to know where pathogens reside in the host, particularly
during the carrier state when they may be particularly difficult to
isolate. For instance, E. coli may be found in the gall bladder and
Salmonella in the protozoa of the rumen. Similarly we need to know the
metabolism, transport, sites of deposition and excretion of chemical
residues.

**Research Needs**

New methods are needed for describing the exceedingly large number of
very diverse organisms that inhabit the GI tract of animals in order to
gain an understanding of bacterial and fungal community composition, for
example, oligonucleotide fingerprinting of rRNA gene clones followed by
sorting into taxonomic clusters. We need to more clearly understand what
factors, including nutritional, allow animals to start shedding
epizootic pathogens in their feces when under stress, and particularly
when they are transported to slaughter. Similarly, information on diet
or other factors affecting the duration and fate of chemical residues is
needed. This basic information is necessary for development of
interventions that reduce both chemical residues and colonization and
shedding of pathogens in food animals.

**Outputs**

-   Use of molecular methods and genomics to assess the dynamics of the
    microbial intestinal flora and to more thoroughly understand the
    dynamics of various gut environments of food producing animals in
    order to elucidate effective means for improved control of food
    pathogens in the pre-harvest stage.

-   Use of such techniques as oligonucleotide fingerprinting of rDNA
    genes to assess species diversity and identify novel genes in order
    to identify opportunities for interventions to control pathogens.

-   Locating sequestration and deposition sites, and metabolic pathways
    to determine factors affecting both holding and release of chemical
    residues and pathogens.

-   Identification of SNP's (single nucleotide polymorphisms) that
    identify genes associated with host resistance and pathogen
    virulence and shedding.

-   Identification of environmental sites, both physical and biological,
    which provide pathogen reservoirs and sites of amplification.

-   Knowledge of the interrelationships among production practices,
    animal well- being and pathogen shedding.

**Impact**

Information gained from study of the ecology, genomic, host pathogen and
chemical residue relationships of food safety pathogens in animal
production will help scientists gain an additional understanding of the
transmission and dissemination of these pathogens and toxins in and
among food producing animals. An important underlying assumption for
this type of research is the "pre-harvest hypothesis" that is "live
animal" control efforts (implemented in or on the animal itself or in
its agricultural environment) will reduce downstream post-harvest
carcass and meat contamination and ultimately reduce human zoonotic and
food-borne disease risk. This information will provide critical data to
help reduce fecal shedding and transmission of pathogens to food, to
carry out risk assessments, to develop and validate predictive microbial
models, and assist in determining critical directions for intervention
strategies to reduce the spread of pathogens in food animals and poultry
and to humans. This research will also lead to both a core understanding
and improved methods to control stress and enhance well-being in
livestock resulting in improved and commercially viable handling,
feeding, therapeutic, and transportation practices which together will
help to reduce or eliminate food borne pathogen infection during
production and transportation stages in food producing animals. Thus it
will assist FSIS, FDA and the food animal industry in the production of
safe food products.

# 

# Resources

ARS scientists assigned to projects under this Problem Statement are
located at Beltsville Agricultural Research Center, Beltsville, MD;
National Animal Disease Center, Ames, Iowa; Swine Odor and Manure
Management Research Laboratory Ames, Iowa; Livestock Behavior Research
Unit, West Lafayette, IN; Meat Animal Research Center, Clay Center, NE;
Red River Valley Agricultural Research Center, Fargo, ND; Western
Regional Research Center, Albany, CA.; Southern Plains Agricultural
Research Center, College Station, TX; Livestock Issues Research,
Lubbock, TX; Poultry Production and Product Safety Research
Fayetteville, AR; and Richard Russell Research Center, Athens, GA.

**Problem Statement 1.1.4: Intervention Strategies**

Intervention strategies, including both products and practices, are
needed to reduce colonization and shedding of epizootic pathogens by
food producing animals. Successful interventions must cost the producer
almost nothing. Optimally an intervention could increase the
productivity of the animals in addition to decreasing the number of
pathogens, e.g., decreasing Salmonella while also increasing the rate of
gain and/or feed efficiency of the animals. Interventions that can be
used just prior to slaughter are particularly useful, as they can act
almost as a preharvest HAACP step with the animals having a very limited
opportunity to acquire additional infection prior to slaughter. Industry
has been reluctant to change rations and apply practices late in the
production cycle. The use of Optaflex, a new feed additive, by the beef
feeding industry starting 28 days before slaughter has now opened new
opportunities for other additives to be given to cattle during this
period just before slaughter.

With swine the present industry practice of holding animals in lairage
for at least 2 hours prior to slaughter provides an opportunity for
considerable reinfection and rapid systemic transit through the body
followed by excretion into the feces. This could limit the value of a
preharvest killing step unless it were combined with alternative methods
of transporting swine to slaughter. Chickens and cattle have the
opportunity to introduce pathogens into the slaughterhouse from their
hide, hair and feathers even when pathogens from their GI tract may have
been killed by chemical or biological intervention. Residues of any drug
used just prior to slaughter are also of great concern, thus drugs used
at this time must not absorbed from the GI tract, or must be otherwise
eliminated extremely rapidly, and/or must be extremely non toxic to the
animal.

Bacteriophage and bacteriocins both hold a great deal of theoretical
promise but these treatments, at least for food producing animals and
birds, have not yet been subjected to Food and Drug Administration
review. Past history for the use of competitive exclusion cultures (CEC)
and the bacteriocin nicin as methods of control, indicates there will be
significant regulatory hurdles before approval for the use of either
bacteriophage or other new bacteriocins. Although bacteriophage also
have a high level of specificity which may limit their broad
application, chemical or microbial treatments with narrow pathogen
targets, and specificity of action are sought after, as they are
associated with less antibacterial resistance and less disruption of
normal gut function.

**Research Needs**

The best interventions are simple, and changes in management practices
to control a pathogen would be ideal. However preventing exposure to
pathogens that are almost ubiquitous, as are most of the food safety
pathogens, is extremely difficult, and cleaning of the environment and
ration changes prior to slaughter have not yet been successful.
Production operations need products that are both effective and can be
approved by applicable regulatory agencies to successfully emphasize
food safety.

**Outputs**

-   Bacterial metabolic targets that allow interventions to be closely
    focused to specific metabolic pathways.

-   Interventions targeting specific metabolic endpoints.

-   Vaccines that decrease shedding of epizootic pathogens at the time
    of slaughter.

-   Effective interventions that also increase animal or bird
    productivity.

-   Effective modifications of production practices that particularly
    address animal welfare concerns and /or dietary components in
    reducing pathogens.

-   Interventions that prevent colonization or exclude pathogens from
    the gut.

**Impact**

The development of intervention strategies to control food safety
pathogens in animal production will provide technologies to help
producers clear these pathogens from food animals during on-farm
rearing. Again an important underlying assumption of intervention
strategies is the "pre-harvest hypothesis" that is, "live animal"
control efforts (implemented in or on the animal itself or in its
agricultural environment) will reduce downstream post-harvest carcass
and meat contamination and ultimately reduce human zoonotic and
food-borne disease risk. These intervention strategies will provide
environmentally compatible technologies to significantly decrease or
eliminate pathogens food animals during critical periods of on-farm
rearing to help reduce fecal shedding and transmission of pathogens to
food. The strategies can add critical information to predictive
microbial models, in order to reduce the spread of pathogens in food
animals and poultry and to humans. These interventions will assist
animal producers in meeting FSIS, FDA and the food animal industry goals
for the production of safe food products.

# Resources

ARS scientists assigned to projects under this Problem Statement are
located at Beltsville Agricultural Research Center, Beltsville MD;
National Animal Disease Center, Ames, Iowa; Swine Odor and Manure
Management Research Laboratory Ames, Iowa; Livestock Behavior Research
Unit, West Lafayette, IN; Meat Animal Research Center, Clay Center, NE;
Red River Valley Agricultural Research Center, Fargo, ND; Western
Regional Research Center, Albany, CA; Southern Plains Agricultural
Research Center, College Station, TX; Livestock Issues Research,
Lubbock,TX; Poultry Production and Product Safety Research Fayetteville,
AR; and Richard Russell Research Center, Athens, GA.

**Problem Statement 1.1.5: Antibiotic Resistance**

The emergence of antimicrobial resistance (AR) among food-borne and
commensal bacteria associated with food animal production has become an
important global issue. Despite growing concerns, information regarding
the development, prevalence, spread and persistence of AR in food-borne
and commensal bacteria is limited, and AR's impact on human health is
poorly understood.

Some facets of antimicrobial resistance (AR) cut across all the
components of preharvest food safety. AR is considered separately
because of its extreme visibility and importance to food safety, human
illness and the livestock industry. Both the CDC and the FDA believe
that the use of antibiotics in animal production, in particular
antibiotics used in feed, and for growth promotion, is a major cause of
antibiotic resistance in humans with resulting longer duration of
illness and/or untreatable illness with more severe sequelae. The result
is that it is more and more difficult for antibiotics for animals to
receive approval from the FDA, with the result that the livestock
industry is being deprived of valuable tools they have previously relied
upon to maintain healthy animals and flocks, and provide consumers with
safe and affordable animal food products.

Based on the precautionary principle, the EU has implemented a complete
ban on subtherapeutic use of most antimicrobials, although these drugs
are still widely used by prescription. Conversely, the US continues to
utilize subtherapeutic antimicrobials in animal production. Without
continued surveillance and accurate resistance data, trade negotiations
will be negatively impacted.

**Research Needs**

Throughout the farm-to-fork continuum, there are numerous processes that
affect the degree of microbial contamination of foods (with any
accompanying antimicrobial resistance), including the rearing,
transport, slaughter and processing of food animals, and the packaging
and preparation of foods prior to consumption. Research is necessary to
provide the necessary data to analyze specific resistance to antibiotics
in food animals as they are presented for slaughter, so that animals and
drugs that are not contributing to human resistance are not unfairly
penalized.

CAHFSE, first considered under 1.1.2 Epidemiology will enable ARS,
collaborating with other USDA agencies, to identify and track emerging
diseases and identify and implement mitigation strategies in a timely
manner which can avert economic, animal health and public health
consequences. Without continued surveillance, research and testing of
interventions, it would be impossible to determine on-farm practices
affecting food borne bacterial populations, including the antimicrobial
resistance associated with these bacteria. Such a surveillance program
will also be vital to the National Biosurveillance Integration System of
the DHS.

**Outputs**

-   Development of food animal surveillance and epidemiology programs,
    particularly the Collaboration for Animal Health and Food Safety
    Epidemiology (CAHFSE), together with other USDA agencies to assure
    early detection of epizootic pathogens and unique antibiotic
    resistance patterns.

-   Information about rates and patterns of spread of AR gene flow,
    among pathogenic and commensal microorganisms and in different
    environments of various food producing animals, including animal
    manure. This information can then be used for population genetic
    studies to map the spread of specific clones in populations across
    the U.S., and in risk assessments to help assess the effects of
    antibiotics in animal medicine.

-   Identifying linkages of bacterial AR genes to other genes affecting
    other aspects of bacterial physiology, particularly pathogenicity
    factors.

-   Combining information on the AR of isolates with genetic or other
    attributes to obtain specific identification and exclusion
    characteristics for isolates. .

**Impact**

Identification of factors responsible for mediating AR in food-borne and
commensal bacteria will provide vital information on AR mechanisms as
well as the incidence, persistence, and rates of dissemination of AR.
Scientists will gain an additional understanding of the transmission and
dissemination of these pathogens and their accompanying resistance from
the farm through slaughter environments. The contribution from all
aspects of the environment, including feed and water can also be
assessed. This information will provide data to carry out risk
assessments, to develop and validate predictive models of microbial and
antimicrobial resistance, and assist in determining critical directions
for intervention strategies to reduce the spread of AR in food animals
and poultry and to humans. Information gained from epidemiology and
surveillance of antibiotic resistance (AR) of food safety pathogens in
animal production using epidemiologic and molecular and population
genetic approaches will help. This will also help prevent physicians
from assuming that antibiotic resistance problems can be solved by
banning of certain antibiotics from animal use, thus postponing their
examination of the role of human antibiotic prescription and use
practices in AR. Thus it will assist FSIS, FDA and the food animal
industry in assuring production of safe food products.

# Resources

ARS scientists assigned to projects under this Problem Statement are
located at the National Animal Disease Center, Ames, Iowa; Southern
Plains Agricultural Research Center, College Station, TX; and Richard
Russell Research Center, Athens, GA.

**[Component 1.2]{.underline} Pathogens, Toxins and Chemical
Contaminants - Postharvest**

In order to direct the research program towards improving public health
the direction of the food chain must be reversed: plate-to-farm, not
farm-to-plate. This considers the consumer first, not the producer or
the processor, and is in-line with countries in the European Union and
Australasia. Additionally, the artificial barrier between pre- and
post-harvest is reconsidered, with food safety treated as a single
entity. NP108 Food Safety is a single integrated National Program,
however, for convenience and clarity, within the Action Plan the Program
is subdivided into various components and subcomponents, including pre
and post-harvest research issues.

Within the postharvest food safety program there are specific research
problems that have direct relevance for individual parts of the food
chain: these include, but should not be limited to the following:
detection; \[on-line\] sensing systems; production and processing
ecology; processing intervention strategies; omics; \[safety\] and
health; risk \[assessment\]; pathogenicity; and \[food\] security. Some
problems have a higher priority than others, while some, for example
health issues and food security are new, and require additional funding
to be implemented. The Action Plan is pragmatic, outlining the needs and
subsequent outputs that can likely be achieved during the next 5 year
cycle. The Action Plan is a working document, subject to realignment and
modification when and where necessary.

**Problem Statement 1.2.1: Detection and \[Validation\]**

Detection and quantitation of pathogens, toxins and chemical
contaminants are the central challenge to any food safety \[and food
security\] system. Diagnostic tools must be developed for the entire
food chain which allow the highest detection capability, and guarantee a
maximum degree of prevention. Where practical however, detection as
early as possible is best, thus avoiding or preventing the need for
significant processing interventions or even the recall of food products
from purchase endpoints. The term "diagnostic" implies analytical
methods, and denotes the method/mechanism to establish epidemiological
priorities.

**Research Needs**

Development of technologies and techniques is complicated, expensive and
time consuming. Therefore collaborations with other research entities,
both known, and those not previously considered, but who have the
technology or capacity to supply or assist innovative ideas must be
made. Most methods currently available were developed for a particular
target, and are not intended to screen for multiple agents. The research
program must be more forceful in having a systems approach, utilizing
all its expertise. Pragmatically, not every method developed has, or
will find utility. For regulatory use any developed methods must be
validated in various food matrices, and where necessary refined. There
is simply no point in developing any detection system or technique that
will have no use in the real world. Validation of utility may require
multidisciplinary, multi-location, and possibly multi-agency research
efforts, in particular direct interaction with FSIS, FDA and EPA. The
specific pathogens, toxins and chemical contaminants are not articulated
since food safety and food security priorities and needs shift with
time. Decisions have to be made about what is really critical,
especially in light of limited fiscal resources, time, rapidity etc.

**Outputs: (developing in various sub-components for both food safety
and security)**

[Sampling]{.underline}:

-   Best practice sample collection protocols for various food systems
    and matrices.

-   Innovative approaches to sample processing, for example universal
    separation and concentration steps.

-   Mathematical algorithms to maximize the probability of detection.

[Detection]{.underline}

-   Rapid systems for target amplification (genotypic/phenotypic) to
    maximize detection potential.

-   Multiple platform systems that will be combined with multiple target
    assays (multi-platform devices for universal contaminant detection).

-   Systems that function in real or near real-time, or have the
    potential to be automated for high-throughput.

[Validation]{.underline}

-   Determined under both laboratory, pilot-processing, and
    commercial-processing conditions, and where appropriate in
    association with the specific stakeholder end-user, and/or
    development partner.

-   The protocol for method validation for any specific pathogen, toxin
    or chemical contaminant detection will be different. However, the
    following generalities hold. The fundamental parameters for method
    validation will include evaluation of (1) sensitivity and
    selectivity; (2) accuracy, precision, recovery, and
    reproducibility; (3) stability and calibration curve (4) cost,
    robustness, and environmental impact.

-   Measurements for each (pathogen/toxin/chemical contaminant) in the
    matrix will be validated. In addition*,* the stability of the
    (pathogen/toxin/chemical contaminants) in spiked samples will be
    determined.

**Impact**

ARS will lead in developing and validating methodologies that have both
regulatory, industry and research use: a commonality of interests
between government and stakeholders. Having reliable, cost-effective
quantitative measurements of pathogen/toxicant in all food types can
provide data to carry out risk assessment, to develop and validate
predictive microbial models, and to identify areas where interventions
are most critically needed. This information will assist the
implementation of HACCP programs by FSIS, FDA, and their regulated
industries.

**Resources**

ARS scientists assigned to projects under this Problem Statement are
located at Beltsville Agricultural Research Center, Beltsville, MD;
Eastern Regional Research Center, Wyndmoor, PA; Meat Animal Research
Center, Clay Center, NE; and the Western Regional Research Center,
Albany, CA.

**Problem Statement 1.2.2: \[On-line\] Sensing Systems that Assist
Processing, and Have Application in Food Security**

With more focus on HACCP and HIMP (HACCP-based inspections models
project) both the FSIS and FDA have placed more of the burden of
inspection responsibility on the producers and processors. Plants are
also responsible for meeting other consumer protection (OCP) issues as
determined by regulatory agencies. In essence producers/ processors
assume the responsibility for inspection, and the regulatory agencies
perform oversight and verification to ensure standards are met. Under
HACCP, HIMP or GMP consumers demand safe, high quality food, however,
consumer demand for more food increases the need for, and pressure on
inspectors. Balancing consumer needs with the capabilities of the
inspection agencies and the producers/processors will not be easy.

**Research Needs**

During the past 5 year research cycle the Program has been pre-emptive
in devoting resources to developing automated, low cost, accurate,
on-line and hand-held, computerized inspection \[sensing\] systems for
poultry, beef and produce. These automated systems operate with minimum
human intervention and are able to function despite changes in physical
plant structure, and environmental conditions. The specific need is to
continue development and validation where appropriate with the goal of
commercial implementation asap. Future research with regulatory
agencies, security agencies, and industry collaboration and support will
focus on the following:

**Outputs**

-   Commercial implementation for detecting pathophysiological
    abnormalities, and feces/ingesta in poultry combined and integrated
    with on-line washing/ intervention systems developed by ARS.

-   Development, evaluation, validation, refinement, and commercial or
    near- commercial implementation of computerized, on-line
    detection/sensing systems for whole beef and pork carcasses; feces
    and defects on apples and other fresh fruits and vegetables;
    physical hazards (bones, glass, plastic, stones and metal) in foods;
    and physical defects in shell eggs.

-   Development, evaluation, validation, refinement and commercial
    implementation of head-gear systems; for use by small
    producer/processing operations. Expand their use to include other
    areas, for example: small and medium size processing facilities,
    cleaning and sanitation issues both in production and retail
    facilities.

-   Development of innovative sensing capabilities for use in military
    and food security applications. Any work should specifically be
    coordinated with needs in the Food \[Security\] problem 1.2.9 of the
    Action Plan.

**Impact**

On-line, computerized sensing-systems placed or used strategically in
food processing plants will assist and improve the regulatory and
in-house inspection system; minimizing the problems of human error and
variability; and increasing commercial processing productivity and
profitability.

**Resources**

ARS scientists assigned to projects under this Problem Statement are
located at Beltsville Agricultural Research Center, Beltsville, MD; and
the Richard Russell Research Center, Athens, GA.

**Problem Statement 1.2.3: Production and Processing Ecology**

Elucidating the ecology of pathogens was one of the major thrusts in the
previous Action Plan, and there is no doubt that understanding microbial
ecosystems is extremely useful when developing intervention strategies
to minimize contamination, to prevent the growth of pathogens, and kill
or remove pathogens at various stages of production processing,
transportation, marketing, and preparation for consumption. Such studies
are also a means utilize and validate any new, improved and innovative
methods to detect, differentiate, type, and quantify pathogens on some
foods, and within some production and processing environments. Research
also facilitates the identification of critical control points during
food production and processing; and allows development of alternative
HACCP/GMP systems.

Microbial ecology is perhaps however, the most contentious of the
problems in the post-harvest food safety program. Not unexpectedly, such
studies are often seen as collecting base-line data, which is not part
of ARS' mandate. However, these studies are appropriate if for example,
the data have a research purpose, and contributes to improved assessment
of the public health risk posed by the pathogen, or to form and/or
implement regulation(s).

**Research Needs**

The Program must build on previous ARS studies directed towards
understanding for example, biofilms especially in processing plants;
physiological status and survival in niches; attachment; quorum sensing;
influence of environmental stresses; or processing or packaging
technology. It is critical that such studies be integrated within other
problem areas of the research program, specifically, detection, omics,
pathogen modeling and food security. Collecting baseline data should be
undertaken with extreme caution; and only after specific regulatory
agency request. Studies would likely require direct collaboration with
FSIS/FDA laboratories. New priority areas for baseline studies could
include the following: plant environment sampling especially in blade or
pin tenderiser equipment (specific FSIS need); expanding the hide and
carcass mapping previously done in cattle to include swine and turkey
carcasses (specific FSIS need); foods produced with minimal processing
(RTE foods) or when prepared are subsequently associated with increased
public health risk (deli-meats) (specific FSIS and FDA need); foods
produced by new or alternate processing and/or packaging technologies;
exotic species (alligator, beefalo ratites: specific FSIS need) ethnic
food products (meats and produce); products produced and/or processed by
alternate methods (kosher, halal); and organic foods (USDA priority).

**Outputs**

-   Determine why certain, and often very specific pathogens
    (types/subtypes) persist not only on food but in the food processing
    environment.

-   Determine why certain types/subtypes are better adapted to
    production environments rather than the food processing environment,
    and vice versa.

-   Determine correlations between specific genetic types or clonal
    groups and virulence characteristics; and any relationships between
    the virulence characteristics and human infections.

-   Understand biofilm formation structure and composition both on foods
    and within processing, and transportation environments. Elucidate
    any differences between biofilms found on product, those found on
    equipment producing the product, and containers. Specifically
    addressing the formation and behavior of pathogens in biofilms
    associated with raw fruits and vegetables, both conventionally and
    organically produced: and elucidating the differences in biofilms on
    containers and equipment used in the produce industry.

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-   Determine the genes associated with altered physiological states,
    adaptation to extrinsic/intrinsic stresses, and quorum sensing.

-   Provide baseline data to regulatory agencies to write Performance
    Standards.

**Impact**

Number range CHAPTERExposure of animals, seafood and produce to
pathogens during production, slaughter and processing operations, and
transportation can be a significant source of contamination. Research
into the microbial ecology of pathogens on various surfaces is
critically important in order to understanding the phylogenetic
relationships between and within species, and how it may explain the
variability of types/strains encountered in food, the environment and in
humans, and the range of virulence characteristics expressed by the
different strains under varying conditions and stresses. Studies will
assist in understanding how biofilms are formed by elucidating the genes
and physical/chemical structures on the surface of pathogens responsible
for attachment and colonization. This will ultimately allow development
of improved food production, processing, handling, distribution, and
storage techniques, including identification of the factors that
contribute to the spread of pathogens and techniques for the elimination
of cross contamination

**Resources**

ARS scientists assigned to projects under this Problem Statement are
located at Beltsville Agricultural Research Center, Beltsville, MD;
Eastern Regional Research Center, Wyndmoor, PA; Meat Animal Research
Center, Clay Center, NE; Richard Russell Research Center, Athens, GA;
and the Western Regional Research Center, Albany, CA.

**Problem Statement 1.2.4: Processing Intervention Strategies**

New/alternative food processing technologies all have the ability to
inactivate microorganisms to varying degrees. However, the high
treatment intensities required for inactivation usually result in
adverse functional and/or sensory properties, significantly reducing
food quality. Quality and safety are intimately associated, especially
considering the change in consumer demands for more fresh (minimally
processed) and natural food products. Apart from irradiation, all of the
new technologies (used alone) are too costly, too energy expensive, and
cannot guarantee safety to be of practical use. Irradiation has high
potential, however, development and commercialization of irradiators
(including electron beam) has been hampered by unfavorable public
perception, despite endorsements by national and international
organizations, and increased industry interest.

**Research Needs**

A research strategy for the future needs to be based on future trends
(reverse food chain): consumer issues and concerns; traceability,
regulatory needs; and current/future fiscal and personnel resources.
Previously research has focused on commodities: poultry, produce,
ready-to-eat foods etc., future research must address very specific
needs with high impact. Therefore, the intervention program needs to be
re-defined, to focus on utilizing and integrating all aspects of ARS
food technology capabilities. Process engineering may play a larger role
than before. However, the caveat is that some lines of research have
limited applicability due to cost and practicality. Research involving
processing technologies that are unlikely to be implemented by industry
will have limited or no value and impact.

The need for more fresh (minimally processed) and natural food products
could for example be achieved in part by revisiting the (under-utilized)
hurdle effect concept which in and of itself is a minimal processing
technology that exploits interactions between preservation treatments.
Intelligent application of hurdle technology could readily be combined
with current (and alternate) processing technologies. There also needs
to be an increased effort to coordinate intervention(s) research with
other ARS Units/Centers viz-a-via: CEMMI to address modelling and risk
\[assessment\], and ERRC/RRRC food processing suites; and external to
ARS with the FDA-National Center for Food Safety and Technology,
Chicago, or University collaborators.

Specific needs articulated by both FSIS, FDA and commodity stakeholders
include developing, and validating intervention strategies for specific
foods and food types, and those useful for implementation in small and
very small processing plants. Other critical areas of focus include the
following: validating specific food cooking temperatures for food
handlers at retail, water and water reuse, air-decontamination in
processing facilities, shell egg processing, liquid egg processing,
juices (in particular orange-juice), carbonated and fermented beverages,
organic foods, and carcass dehairing. Specific processing technologies
to be utilized would include, but not be limited to: thermal and
non-thermal technologies such as radio-frequency (RF), electric field
(EF), microwaves, vacuum-steam-vacuum (where appropriate), irradiation,
high pressure, pulsed light, microfiltration, GRAS status chemicals, and
competitive exclusion using both bacteria and bacteriophage.

**Outputs**

-   Evaluate, develop, and validate through both laboratory, pilot-plant
    processing and commercial processing facilities the effect of single
    and combinations of intervention technologies (multi-target
    approach) on pathogen reduction. Ensure that lethality/intervention
    treatments do not negatively impact product quality.

-   Determine whether combinations of non-thermal technologies can be
    incorporated in the hurdle concept; and determine whether single or
    combinations of non-thermal technologies are more effective if used
    in combination with traditional interventions.

-   Increase fundamental understanding of the mechanisms, modes and
    sites of action at the cellular level of various intervention
    (inactivation) processes, and combination(s) thereof.

-   Develop methods to prevent the growth of pathogenic and spoilage
    microorganisms in minimally preserved, brined, and fresh-cut foods.
    Develop predictive models for growth survival and inactivation of
    pathogens critical to Problem Statement 1.2.7.

-   Utilize the inactivation data to model pathogen and non-pathogen
    behavior in complex food systems. These types of studies are
    fundamental to developing HACCP systems and regulations, and are
    critical to Problem Statement 1.2.7.

-   Develop of post-harvest interventions options for small and
    very-small FSIS regulated plants.

**Impact**

Postharvest operations of all sizes (large to very small) provide an
opportunity to remove or inactivate pathogens and their toxins acquired
during the production and processing phases. Pathogens may develop
resistance to antimicrobials from traditional measures used for pathogen
control. Successful technologies and strategies to eliminate, reduce, or
suppress human pathogens are needed for foods and food types associated
with foodborne illnesses, or at risk of becoming vehicles for human
pathogens. Development of individual or combinations for new or
innovative intervention technologies for (minimal) processing will be
developed based on an understanding their modes of action and effects on
the microbial ecology of a food product; inadequate suppression of
spoilage could create an opportunity for human pathogen growth and toxin
production.

**Resources**

ARS scientists assigned to projects under this Problem Statement are
located at Beltsville Agricultural Research Center, Beltsville, MD;
Eastern Regional Research Center, Wyndmoor, PA; Meat Animal Research
Center, Clay Center, NE; North Carolina State University, Raleigh, NC,
Richard Russell Research Center, Athens, GA; and the Western Regional
Research Center, Albany, CA.

**Problem Statement 1.2.5: Omics**

In 2001 the Program initiated an "omics" (genomics/proteomics) research
effort to sequence and annotate the genomes, construct microarrays,
conduct gene expression analysis, and construct data bases of several
critically important food-borne bacterial pathogens. Although this
research could be viewed in-part as a sub-component of Detection and
Validation 1.2.1, the rewards of carrying out this work are far greater
as are indicated in the Outputs. It is critically important that this
work be done collaboratively where possible and appropriate by combining
resources and expertise between different ARS Research Units, Federal
agencies, ARS and University partners and internationally. While ARS may
not have the capabilities to undertake complete genome sequencing,
outsourcing of this task has proven to be very cost effective. The
sequence data are directly provided to ARS for annotation, since we
understand the biology of the organism. Lack of understanding of the
organism's biology by the sequencers was a critical and severely
limiting problem with previous microbial genome analysis.

**Research Needs**

Initially the "omics' research focus was directed towards food safety:
the development of detection methods, phylogenetic analysis, and
elucidation of the biology of the organism at the molecular level under
advantageous and disadvantageous environments. However, future work will
also include microbial forensics as part of both food safety and food
security. [This is a direct request from various Federal
agencies.]{.underline} This specifically implies the directed use of
information for the development of new assays and physical analysis
methods, and for the identification of biocrime organisms.

Future initiatives progress from easier to more complex challenges and
should be directed towards undertaking the following: Complete genome
sequence analysis and annotation of 10-20 additional bacterial strains
from specific genera/species, and at least 3-5 close relatives. This
should also include the small viral genomes. [Finished
sequences]{.underline} should be the goal rather than draft. Developing
single (genera or species) and multiple (Pan-genera and species) arrays
for all critical food-borne pathogens. Developing protein microarrays:
where possible, allowing for loss of sensitivity and discrimination due
to conformation changes that occur during attachment. To meet this need
multiple toxin microarrays may initially be of the most utility and
value. Utilizing innovative (new technology) approaches to detect genera
and species, and differentiate both gross and subtle changes in
serotypes/strains. Establish a food-borne pathogen database based on
nucleic acid, DNA/RNA profiles, proteomics and other phenotypic
properties.

**Outputs**

-   Improved science-based risk assessment decisions to control
    foodborne pathogens. In particular, allowing comparisons (molecular
    systematics) of strains, providing information on genes that
    contribute to pathogenicity in human and/or animal hosts

-   Allow the study of expression of genes of interest, including, but
    not limited to genes involved in virulence and/or viability (growth
    and survival) in foods.

-   Increase understanding of population genetics and epidemiology, by
    assisting development of improved ways of molecular tracking of
    pathogens through the food chain.

-   Allow incorporation of sequence information into the design and
    optimization of nucleic acid arrays and chips.

-   Facilitate gene expression studies for identification of sequences
    expressed or repressed in response to external cues such as those
    likely to be encountered by the specific bacterium in the
    environment, foods, and/or humans.

-   Make possible more rapid and less costly diagnostic methods and
    vaccine development, and allowing analyses of resistance to
    antimicrobials, toxins, and disinfectants.

-   Reveal any novel genes associated with specific ecological niches,
    and allow for comparative genomic analyses to determine critical
    areas for targeting strategies for interventions and controls.

-   Identify regions of the genome that may have variations in the rate
    of nucleotide substitution or in the rate of intergenic
    recombination.

**Impact**

Number range CHAPTERGenome and proteome analysis are critical to the
development of rapid diagnostic tests/methods used by regulatory
agencies, industry and research agencies. Methods development
subsequently allows for the detection and enumeration of pathogens
during processing and storage (microbial ecology and epidemiology) which
leads to the development of predictive models and risk assessments,
critical in assisting both regulatory agencies in making food safety
decisions that impact public health, and industry for the development of
HACCP plans. Microbial models and risk assessment, combined with
genomics and proteomics will lead to the development of innovative
intervention strategies, and therapeutic agents. These in turn will
allow development of better HACCP plans which directly improve the
safety of the food supply. Genomics and proteomics will also allow a
greater understanding of beneficial genes and their products, which can
be utilized for the development of new agriculturally based products.
These products could be used for the development of alternate/innovative
intervention strategies, particularly in ready-to-eat foods.

**Resources**

ARS scientists assigned to projects under this Problem Statement are
located at Beltsville Agricultural Research Center, Beltsville, MD;
Eastern Regional Research Center, Wyndmoor, PA; Meat Animal Research
Center, Clay Center, NE; National Center Agricultural Utilization
Research, Peoria, IL; Richard Russell Research Center, Athens, GA; and
the Western Regional Research Center, Albany, CA.

**Problem Statement 1.2.6: \[Safety\] and Health**

Many health problems result from food safety events, and food borne
pathogens may have a role in 2-3% of chronic diseases, if not more.
Increasing consumer awareness of the link between diet and health has
promoted the introduction of components such as non-pathogenic
microorganisms used to produce fermented foods (probiotics) and certain
food compounds (prebiotics) to produce "functional foods". These foods
appear to benefit health through mitigating diseases. Countries within
the European Union, Asia and Australia have significant research
programs directed towards addressing health benefits of pre- and
pro-biotics. Two years ago NP107/108 (post-harvest) began an
inter-program collaboration on the role of probiotics in health using
pigs as a model system. Research has proceeded rapidly, and the
collaboration has developed into an excellent model of
Cross-Location/Cross-National-Program projects. While this research has
had a positive outcome and will continue with strong support from NPS,
it is unlikely that in the foreseeable future there will be any
significant new funds. Irrespective, of the funding issue, the problem
will remain a component of the Action Plan, and the NPL will endeavour
to obtain funds through external (international) collaborations.

**Research Needs**

Number range CHAPTERProbiotics are a class of microorganisms that can
establish and grow in a compartment in the host after consumption and
provide some positive health benefit. Benefits claimed include
protection against pathogens, stimulation of the gut immune system,
correcting some bowel diseases, reduced allergic disease and protection
against carcinogens. However, the basis of such claims is often
confounded by a lack of demonstrable growth and function of the
probiotic in the gut. Scientific validation of many of the claims of
probiotics activity is missing. Consumer concern about probiotic
reliability, efficacy and safety can be addressed by hypothesis-based
and statistically validated testing of probiotics under controlled
experimental conditions. Therefore, the need of this research is to test
if dietary probiotics can enhance immune function against infectious
agents, prevent the onset of allergenic disease, and improve the
function of the GI tract.

**Outputs**

-   Develop animal model(s) to test the unique nature of various
    probiotic strains and their efficacy against particular disease
    conditions will benefit the functional food industry.

-   Provide information on the prophylactic use of dietary probiotics to
    enhance the mucosal immune system and to provide a more robust
    response to infection especially for neonates and the elderly.

-   Provide a description of biomarkers that associate probiotic
    consumption with improved immune function would benefit human
    health.

-   Provide practical recommendations for dietary changes to improve
    health status and reduce the cost of health care.

-   Determining the efficacy of probiotic treatment of swine on
    resistance to infection will have inherent value to the swine
    industry for control of these infections.

**Impact**

Number range CHAPTERStudies will provide information on the relationship
between probiotics, diet, and immune function and identify biomarkers of
nutritional status. Sound scientific evidence for probiotic efficacy
will provide the food industry and regulatory agencies with relevant
information for concerned consumers. Those at risk of chronic
nutrition-related diseases will directly benefit. Research will also
impact development of value-added food products, such as those
containing probiotics or plant sterols. Other areas impacted will be by
increasing opportunities for initiating joint (international) programs
in omics to develop useful bacterial stains for food use; and future
development of probiotic strains to produce innovative foods with
increased bioactive capabilities useful for improved food safety and
\[food\] security.

**Resources**

ARS scientists assigned to projects under this Problem Statement are
located at Beltsville Agricultural Research Center, Beltsville, MD; and
the Eastern Regional Research Center, Wyndmoor, PA.

**Problem Statement 1.2.7: Risk \[Assessment\]**

ARS does not conduct risk assessment (RA) per se. The Program provides
data, models, and expertise in order for the regulatory/action agencies
to conduct their RA. ARS is recognized as a world leader in the
development of food-related predictive models, and these models are an
integral part of microbial risk assessment used to support food safety
measures adopted by member countries of the WTO. Also, these models
assist in identifying specific food processing steps, and associated
Critical Limits that can serve as Critical Control Points in HACCP
systems, as well as to facilitate food safety decisions when process
deviations occur.

**Research Needs**

There are a number of diverse critical issues in RA that must be
addressed in future ARS research. Broadly, they include research that
expands exposure assessment, dose response, risk characterization, and
overall Quantitative Microbial Risk Assessment (QMRA), including the
development of more robust predictive and process risk models that
specifically address hazards in complex food matrices. Specific outputs
include, but are not limited to those listed below:

**Outputs**

-   Determine the influence of the food matrix on the infective dose of
    a specific pathogen, and the subsequent biological response. Examine
    the uncertainties that surround dose-response models derived from
    animal studies, and determine which models more accurately function
    as surrogates for humans. Determine if dose-response data from
    epidemiological investigations are ultimately better.

-   Develop and validate models that consider the inherent complexities
    if intrinsic and extrinsic parameters; or the macro- and/or
    micro-environment in food matrices. Determine the strategies needed
    to extrapolate model predictions to more complex foods.

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-   Determine and elucidate objective measures to assess model
    performance versus observation. Develop alternate responses or
    options, for example iterative approaches for specific food
    applications.

-   Determine what strategies should be used to predict the distribution
    of lag times, and the worst-case scenario, since lag time has the
    highest uncertainty in predictive modelling.

-   Develop data, and use these data for the development of predictive
    models for growth survival and inactivation of pathogens critical in
    minimally processed, brined, and fresh-cut produce.

-   Determine the strategies for converting and integrating ARS models
    into fully functional process risk models.

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-   Compile quantitative food microbiology data into a database so that
    they can be exploited for model development and validation,
    especially when resources are limited.

**Impact**

Dose-response models underpin the establishment of Food Safety
Objectives, and will assist public health by determining host responses
to specific challenges. Research will address realistic processing and
handling conditions that provide greater value to food industries, risk
assessors, and risk managers. The validation of current models; and
development and validation (including accuracy) of new models is
critical since there are no universally accepted criteria for
determining safe prediction zones. Models with the greatest value must
couple quantitative microbial ecology with a better understanding of the
physiological responses of microorganisms to stressors (interventions)
used in food processing. We will address through creative approaches the
behaviour of Select Agents in foods since they may not follow
conventional pathogen growth and survival patterns. A quantitative
database can be achieved through further expansion of Combase through
CEMMI.

**Resources**

ARS scientists assigned to projects under this Problem Statement are
located at Eastern Regional Research Center, Wyndmoor, PA; and the North
Carolina State University, Raleigh, NC.

**Problem Statement 1.2.8: Pathogenicity**

Microbial genomic efforts, for example, the availability of genome (and
gene) sequences and microarrays have allowed us to obtain an increased
appreciation and understanding of pathogenicity and virulence. We have
already begun to compare different strains and species to determine
phylogenetic relationships, especially in relation to ecological niche;
understanding the role of intrinsic and extrinsic stress responses; and
understanding the role of quorum sensing. To achieve the "Outputs" for
these research needs will require all of the omics and ecology
capability and expertise that both currently exists, is made available
in the future. Being able to identify the "bad bugs" will require also
the ability to do functional studies in models that are relevant to
human disease. This will be a particularly difficult but exciting area
for research and will require researchers and collaborators both
nationally and internationally with special expertise in many aspects of
microbiology related to pathogenesis and virulence (attachment,
invasion, cell culture, biochemistry, molecular biology), not just
expertise in food microbiology.

**Research Needs**

Pathogens have the capacity to readily and rapidly adapt and evolve. We
must increase our understanding of the genetic mechanisms that make one
strain of a microorganism pathogenic, while others within the same
species are not; in essence, how do microorganisms become pathogenic?
Virulence is the degree of pathogenicity, so why are some pathogens
highly virulent while others are less virulent? How or is virulence
directly related to the infective dose? What other factors impact
virulence? Understanding pathogenicity and virulence are basic and
critical issues, however, data are implicitly needed for pathogen
control and risk assessment. Control strategies that were once effective
may not remain so, hence they force us to change or develop new
production processes and products to maintain and improve both food
safety and food security. Good risk assessment(s) are predicated on
understanding the pathogen, dose response and the behavior in foods

**Outputs**

-   Isolation and characterization of virulence factors such as toxins,
    adhesions and invasions, and determination of how they interact, for
    example, do various factors behave synergistically or
    antagonistically?

-   Utilize genomics and bioinformatics to classify pathogens based on
    specific virulence factors, rather than by name or by non-virulent
    associated phenotypic traits. This will improve the evaluation of
    safety currently focussed on organisms that have differential
    pathogenicity.

-   Determine the ecological basis for predominance, persistence and
    succession along the food chain to prioritize points for
    intervention application. (See Problem Statement 1.2.3)

-   Utilize genomics and bioinformatics information to understand why
    evolutionary shifts occur, and identify the selection pressures
    responsible for the shifts.

-   Utilizing bioinformatics to make predictions, for example on protein
    structure and function.

-   Determine whether quorum sensing occur in foods, or on surfaces
    (biofilms), and if so, whether it is factor in determining or
    regulating virulence in foodborne pathogens

-   Determine through analysis of gene/protein expression profiles the
    responses to intrinsic and extrinsic stressors imposed during
    production, processing and storage.

-   Determine if stress-regulatory-response-systems may also be targets
    for the design of innovative antimicrobial agents.

-   Though understanding virulence, develop improved detection and
    identification methods that enable differentiation of virulent from
    non-virulent.

-   Initiate studies through collaborations with other Federal agencies,
    affiliated research establishments and Universities to determine the
    infectious dose of various pathogens and their toxins, in various
    food matrices, both pre and post stress.

**Impact**

Sequencing and annotating pathogen genomes will provide the ability to
understand the nature and differences between pathogens and
non-pathogens, and identify factors that encode for variations in
virulence. Physiological characteristics of pathogens are known to be
affected by stress. This work will determine the effect on virulence
mechanisms which directly impacts pathogenicity and the emergence of new
pathogen types. If virulence is correlated with quorum sensing this
could potentially be a mechanism to disrupt or negate pathogenicity by
targeting communication pathways with novel antimicrobials. Research
will allow better risk assessment and allow more efficient allocation of
risk management resources focussing on only the critical pathogens in
the food supply.

**Resources**

ARS scientists assigned to projects under this Problem Statement are
located at Beltsville Agricultural Research Center, Beltsville, MD;
Eastern Regional Research Center, Wyndmoor, PA; Meat Animal Research
Center, Clay Center, NE; National Center Agricultural Utilization
Research, Peoria, IL; Richard Russell Research Center, Athens, GA; and
the Western Regional Research Center, Albany, CA.

**Problem Statement 1.2.9: \[Food\] Security**

To enhance the effectiveness of food security a layered approach must be
taken. Three areas need to be addressed: detection (and validation);
prevention, and response and recovery, not all of which the NP108
Program can assist with. At present the Program can and will only focus
on detection and prevention, with detection technologies and their
validation in food systems being the principal directive. Detection of
intentionally added biological and/or chemical agents is central to
\[food\] security. ARS's contribution with its partners/collaborators
(both government and industry) will be very high. Currently we have
technologies that can detect many pathogens, toxins and other food
(residue) contaminants. However, the breadth of these technologies must
be expanded both in scope and limits of detection. Prevention is a
proactive response, relying upon a high degree of intuition and
awareness within food safety practices and procedures to prevent an
event. The current evaluation of high risk foods (Food Shields) is a
first-start, however, all individual HACCP programs, and critical points
within the various food production, processing and transportation
pathways must be re-evaluated, and points of concern identified and made
invulnerable.

**Research Needs**

Currently, there are no validated methods for the detection of Select
Agents in food; and there are no deployable [and]{.underline} rapid
biological detection capabilities for food and water. [These needs were
directed to ARS from several Federal Departments/Agencies and Food
Security Committees,]{.underline} and include but are not limited to the
following:

-   New approaches to sampling protocols to maximize the probability of
    detecting intentional contamination.

-   Innovative approaches to sample processing such as universal
    separation and concentration steps; automating systems for
    high-throughput.

-   Rapid systems for target amplification to maximize detection
    potential allied to multi-platform devices for universal contaminant
    detection, and where possible develop robust systems that function
    in real or near real-time.

-   Validating technologies to detect Select Agents in various food
    matrices in both in pilot plant and real world situations; conduct
    simulation and validation tests. Although assays for many Select
    Agents exist for clinical or environmental samples, few exist that
    are validated for use in the food matrices of concern.

-   Elucidating if and how Select Agents are affected by intervention
    strategies in various food matrices, including their viability
    after-processing.

-   Technologies to deactivate/neutralize of Select Agent hazards in
    food, and studies to determine the effect of these processes on food
    quality.

-   Decontamination technologies to safely clean food processing,
    distribution, and storage operations contaminated with select
    agents.

[Other potential research areas]{.underline} (based on needs articulated
by Stakeholders) that could be incorporated into the Action Plan at a
later date.

-   With other government agencies and industry produce a best practices
    handbook which establishes standards and requirements for a secure
    food supply chain to reduce the probability, severity and extent of
    an event.

-   Biosensors (tagging methods) for real-time integrated tracking of
    product that can be monitored through geospacial information systems
    (GIS).

-   Infective dose studies of biological toxins in various food
    matrices. What is effect of pathogen and toxin combinations;
    examining synergy, and changes in lethal dose level? Examine the
    effects and markers of toxicological responses in model systems.

**Outputs**

-   Develop best practice sample collection and analysis system for
    foods of concern.

-   Number range CHAPTERValidate methods and diagnostics (nucleic acid
    based, serological, analytical assays) specifically developed for
    the detection of select agents (biological agents, toxins, chemical
    residues and pesticides) in various food matrices.

-   Develop systems and models that validate the behavior of pathogen in
    foods, and the effect of various intervention strategies on select
    agents.

-   Identify, develop and validate decontamination systems for various
    food commodities, with particular reference to quantity, level and
    type of contamination. Develop simulation models for strategies of
    containment, decontamination and disposal of both of the affected
    food; the production and processing facilities, within the
    distribution system, and environmental fate, persistence and impact
    of the select agent.

**Impact**

Agencies within the Federal government have begun to implement strategic
action plans to further ensure the safety of the nations food supply. It
is apparent that even if a small number of contaminants were
intentionally introduced into some part of the food chain, such an
action would have the potential to seriously damage public confidence.
Apart from the impact on health and safety, bioterrorism against the
food supply would also directly harm the nation's economy. U.S.
agriculture employs nearly one quarter of the workforce, and annually
contributes over one trillion dollars to the gross domestic product. The
food production industry annually exceeds two hundred billion, with
exports over fifty-five billion.

There are several critical needs in order to implement a comprehensive
food security strategy. The ARS Food Safety program's role is to
establish methods to protect food that has been identified as at-risk;
strengthen and expand laboratory preparedness; and to develop rapid and
confirmatory laboratory methods to analyze suspect foods for Select
Agents, toxins and chemical contaminants.

**Resources**

ARS scientists assigned to projects under this Problem Statement are
located at Beltsville Agricultural Research Center, Beltsville, MD;
Eastern Regional Research Center, Wyndmoor, PA; Meat Animal Research
Center, Clay Center, NE; National Center Agricultural Utilization
Research, Peoria, IL; North Carolina State University, Raleigh, NC;
Richard Russell Research Center, Athens, GA; and the Western Regional
Research Center, Albany, CA.

**[Component 2.]{.underline} Mycotoxins and Plant Toxins**

Food products of plant origin may contain mycotoxins as a result of
fungal growth/toxin production in/on the plant prior to harvest, as well
as during transportation and storage. Intrinsic plant metabolites that
are toxic to mammals and heavy metals from soil uptake can also be
transmitted to food products. Contamination may make the plant commodity
itself unacceptable for human and animal food, or it may result in
residues of the toxins in food products of animal origin, primarily
milk. The potential for the presence of mycotoxins in commodities is a
threat to competitiveness of US agriculture in the world export market,
where limits of acceptability are generally more stringent. Furthermore,
the potential presence of mycotoxins in the human food supply is
detrimental to the public perception of a safe and healthy food supply.

Mycotoxins which are diverse chemically, are considered to be secondary
fungal metabolites: that is having a role other than for primary energy
and cell structure purposes. Mycotoxins have been thought to perhaps act
as defence mechanisms, but their precise function(s) have never been
elucidated. Evidence is now beginning to accumulate that at least one
mycotoxin may affect the cellular redox homeostasis through action on
the mitochondrial antioxidative stress system as a metabolic target.
Other physiological targets will undoubtedly be pinpointed for other
toxins (they are not toxins as far as the producing fungi are
concerned). Each fungus is adapted to grow well in certain crops and in
certain ecological niches thus they have particular climatic optima,
resulting in explosive growth and fungal production with favorable
weather conditions.

The mycotoxins of most concern are aflatoxin, fumonisins, vomitoxin and
ochratoxin, one or more of which may contaminate peanuts, corn,
cottonseed, tree nuts and/or wheat. Fungi, particularly endophytic fungi
with a very close relationship to the host plant, may produce
metabolites that are essential to plant survival under stressed
conditions, and at the same time are toxic to insect or mammalian
herbivores. The mutualistic symbiosis of endophyte and grasses is
beneficial to the host plant through insect and nematode resistance,
drought tolerance, and improved competition with other plant species.
Examples are the lolines and ergopeptine alkaloids in tall fescue.

Direct translocation of unsafe levels of chemicals from the soil on
which they are grown, e.g., heavy metals and nitrates, or deposition by
air currents, are other mechanisms that can result in toxins in edible
plants. Toxic compounds may also be formed by the plant itself for
fulfilling some physiological need, or as a defense mechanism, for
example, against herbivores, both insect and mammalian. When plants
cannot make their own necessary protective chemicals the need may be met
by endophytic fungi as explained above. In either case the compounds can
be toxic to insects, nematodes and/or mammals consuming the plant. In
developing genetic solutions we must be careful to not inadvertently
enhance factors which may be responsible for pest resistance in plants,
and which simultaneously may have unanticipated consequences in animals
and humans. (Residues of pesticides are not considered in the Food
Safety National Program.)

**Problem Statement 2.1.1: Toxin Methodology and Identification**

Rapid and affordable chemical toxin identification and quantitation are
the basis of both industry and regulatory food safety assurance
activities, and they are now of vital importance to homeland security.
In addition because mycotoxins are unevenly distributed in contaminated
plant commodities, both regulatory agencies and the producing industries
need methodologies to accurately determine the contamination of the
entire tested lot, which may be thousands of tons, as in a loaded river
barge.

**Research Needs**

Non-destructive, rapid, accurate, sensitive and affordable detection
methods are needed. The most useful and effective methods are non
invasive or require only a minimum of sample preparation, and preferably
provide a quantitative assessment of the amounts of mycotoxins or plant
toxins present on the food commodity. In addition, they must be
compatible with today=s high speed, high volume commerce. Possible
methods include near infra red and multi spectral analyses, as well as
the more traditional screening methods, that are rapid, sensitive, easy
to perform and quickly learned, e.g., fluorescence polarization
immunoassays, ELISAs, etc. To develop the necessary reagents for these
new assays we may need to use genetic components, computational
chemistry approaches and novel synthetic chemistries.

To be truly effective in a high volume industrial operation sorting of a
commodity with a spectral analysis for mycotoxin contamination must be
combined with recognition and removal, such as a high volume optical
sorter. Such a machine would in a single pass, simultaneously sort and
reject mycotoxin-containing kernels from contaminated grain. Use of this
technology to examine all product could overcome the very serious
problem of sampling commodities with unequal distribution of toxins.
Although more work will be necessary to improve the accuracy and
specificity of NIR to detect very low amounts of mycotoxin
contamination, it is an important first step in developing a useful
online technology.

**Outputs**

-   Rapid, accurate, sensitive and potentially affordable detection
    methods that are compatible with high speed, high volume commerce,
    that can be linked to optical sorters, and that are preferably
    non-destructive.

-   Spectral analysis of contaminants to identify key optical signals
    that can be used for identification.

-   Expand the use of genetic based determinants in PCR based tests
    including microarrays to rapidly identify mycotoxigenic fungi in
    contaminated food products.

-   Novel mycotoxin-binding materials such as molecularly imprinted
    polymers, novel carbohydrates, and improved antibodies that bind
    mycotoxins.

# 

# Impact

Protection of both the human food supply and animal feeds requires the
rapid detection of toxins, and in particular mycotoxins, in commodities,
foods, and feeds. This includes both the commodities with which they are
usually associated as well as those in which they have not traditionally
been found, the latter as a tool in support of enhanced food security.
ARS will lead in developing and validating methodologies and rapid
screening assays including on-line methods that have regulatory,
industry and research uses for solving mycotoxin contamination problems.
Detection of toxins is the essential first step in the proper
identification of contaminated materials, and ARS methods will aid,
where necessary, the proper diversion of such materials to alternative
uses. Rapid ARS methods will aid both today's high speed high volume
commerce and the regulatory agencies. Fortunately, there is a
commonality of interests among these groups which allow similar methods
to be used by each. Reliable, cost effective identification methods for
toxins in food animal tissues and environmental samples will provide
data to help resolve food security concerns, to carry out risk
assessments, to develop and validate predictive microbial models, and to
identify areas where interventions are most critically needed. This
information will assist FSIS, FDA and the food crops industries in
continually assuring production and delivery of safe food products

# Resources

ARS scientists assigned to projects under this Problem Statement are
located at National Center for Agricultural Utilization Research,
Peoria, IL; Western Regional Research Center, Albany, CA.; Southern
Regional Research Center, New Orleans, LA; and the Richard Russell
Research Center, Athens, GA.

**Problem Statement 2.1.2: Crop/Fungal/Insect/Toxin Relationships**

A thorough understanding of the very complex
fungal/insect/crop/environment interactions during both fungal and plant
growth and maturation is necessary to develop effective strategies to
prevent toxin accumulation, whether of fungal or plant origin, in crop
plants. We need to elucidate factors affecting, either positively or
negatively, the interaction between soil and crops to identify
interventions, such as, avoidance of certain soil series, use of soil
amendments, crop production practices and/or development of crop
varieties that do not absorb the toxic trace elements

**Research Needs**

In many cases mycotoxin producing fungi are introduced to the food crop
by insects. Control of these insects can offer a practical avenue for
controlling fungi on the crop - unless of course the weather pattern is
so favorable for fungal growth and toxin formation that no method can be
successful. As an example, Bt-resistant corn has significantly reduced
levels of mycotoxins compared to non-Bt corn; but the degree of benefit
is highly dependent on the timing and makeup of the insect pest complex
in the particular crop year. Response to specific pheromones and
kairomones by insect pests of tree nuts can also provide additional
means for controlling specific fungus carrying insects.

**Outputs**

-   Determining the effects of weather on toxin accumulation.

-   Identify and develop commercially practical synthesis for specific
    pheromones and kairomones that can help control insect pests of tree
    nuts

-   Developing mechanisms and products for preventing fungal
    introduction by insects.

-   Determination of the sources and mechanisms of plant accumulation of
    toxic amounts of heavy metals or other toxins in food crop plants.

**Impact**

The thorough understanding gained through this research of the very
complex fungal/insect/crop/environment interactions present during both
fungal and plant growth and maturation will result in effective new
strategies to prevent toxin accumulation, whether of fungal or plant
origin, in crop plants. Knowledge of the biology of the production
systems will provide data to help construct predictive systems, and to
determine under what conditions is it economical to implement specific
management strategies, particularly for insect control. Crops will be
able to be produced in areas where it is not otherwise economical or
even possible to do so. Having this thorough understanding of
crop/insect/ toxin/environmental relationships will help prevent food
security concerns, carry out risk assessments, develop and validate
predictive microbial models, and identify areas where interventions are
most critically needed. This information will assist FSIS, FDA and the
food crops industries in the continued production and delivery of safe
food products

# Resources

ARS scientists assigned to projects under this Problem Statement are
located at Beltsville Agricultural Research Center, Beltsville, MD;
National Center for Agricultural Utilization Research, Peoria, IL;
Western Regional Research Center, Albany, CA.; Southern Regional
Research Center, New Orleans, LA; National Peanut Research Laboratory,
Dawson, GA; and Richard Russell Research Center, Athens, GA

**Problem Statement 2.1.3: Production Practices and Expert Systems**

Optimal cultural and crop handling practices facilitated by expert
systems, where they are available, can help decrease mycotoxin
accumulation. Optimized practices are generally the least expensive
methods for controlling aflatoxins. For instance, ARS research has
demonstrated that the rehydration procedure used to facilitate cracking
of closed-shell pistachios results in exceptionally high aflatoxin
levels. In another crop ARS showed that the second phase of
contamination of cottonseed, that is, between boll opening and harvest,
is the most important factor predisposing the crop to aflatoxin
contamination, rather than the earlier phase as was traditionally
emphasized. This knowledge helps cotton breeders target events in plant
growth and development cycle for reducing aflatoxin susceptibility of
their crop. For corn ARS developed data and formulated a computer
program to give useful predictions for mycotoxin occurrence in most corn
hybrids in most years in the Midwest.

**Research Needs**

We need various good agricultural and handling practices to provide
valuable comprehensive management information and tools for producers in
order to help assure the lowest possible toxin levels in food crops. We
also need to gain an understanding of the role of crop management
practices, including use of rotation crops, on the ecology of
mycotoxigenic fungi contamination in order to optimize the application
of competitive exclusion products.

**Outputs**

-   Identification of cultural crop production and handling practices
    that can assist in the reduction of mycotoxins in crops. This
    includes effects of various management strategies, e.g., rotations,
    tillage, and cover crops, and herbicide-resistant crops under
    different weather conditions, and insect control

-   Formulation of user-friendly computer programs to provide useful
    predictions for mycotoxin occurrence.

**Impact**

We will be able to predict when mycotoxin problems may occur, and to
determine under what conditions it is economical to implement specific
management strategies, particularly for insect control. We will have
effective strategies to prevent toxin accumulation, whether of fungal or
plant origin, in crop plants. Corn will be less likely to exceed FDA
tolerances and guidance levels for mycotoxin levels each year, and we
will know what corn lines and treatments have insect resistance and thus
tend to have reduced mycotoxin levels. This will result in corn more
often being a viable economic crop for the south, especially in rotation
with cotton and soybeans. This information regarding optimum production
practices and the ability to predict when contamination may occur in the
field will help develop and validate predictive microbial models and
risk assessments, and will help prevent food security concerns. The
information will assist FSIS, FDA and the food animal industry in the
continued secure production and delivery of safe food products

# Resources

ARS scientists assigned to projects under this Problem Statement are
located at National Center for Agricultural Utilization Research,
Peoria, IL; Western Regional Research Center, Albany, CA.; and Crop
Genetics and Production Research, Stoneville, MS.

**Problem Statement 2.1.4: Breeding Resistant Crops**

Mycotoxin contamination of food crops cannot be completely prevented by
improved cultural practices, insect control, and even competititve
exclusion products. To economically produce crops that meet regulatory
guidelines we need intrinsic crop resistance to colonization by the
fungus and subsequent production of toxins. A thorough understanding and
utilization of fungal genomics, including functional genomics offers an
unprecedented opportunity to affect mycotoxin control through resistant
crops. This genomic information can facilitate identification/
characterization of the complex set of genes involved in fungal
virulence, and the signalling pathways between the fungus and the
environment, and fungal reproduction/survival, all of which must be
understood if fungal infection and mycotoxin production in crops is to
be prevented.

A significant early development in this area is the determination that
the effective aflatoxin resistance in the Tulare variety of walnut is
due to high levels of hydrolyzable tannins in the seed coat that persist
throughout the growing season, in contrast to other varieties. Fungal
tannase hydrolyzes tannins to form gallic acid, which affects fungal
response to oxidative stress and is a potent inhibitor of aflatoxin
synthesis. This information explains the basis of a naturally occurring
antifungal/antimycotoxin mechanism and points to the possibility of
prevention of aflatoxin biosynthesis by disruption of oxidative stress
response pathways. In addition proteomics has been used to identify
several fungal resistance-related characteristics and stress-responsive
proteins/genes in resistant lines of corn.

**Research Needs**

Both genomic and proteomic information are needed to guide traditional
breeding, marker assisted selection and/or genetic engineering to
develop aflatoxin-resistant varieties of crops. We particularly need
molecular biological approaches to discover, introduce and evaluate
plant-derived selectable markers for transgenic plant production.

**Outputs**

-   Identification of unique fungal genes for specific biological and
    physiological functions for use in highly sensitive PCR based tests
    (including microarrays).

-   Completion of the identity of the gene clusters and biochemical
    pathways required for the production of common mycotoxins.

-   An understanding of how environmental factors affect the fungus,
    which genes are turned on during the plant-fungus interaction and
    mycotoxin production, and which are essential to fungal survival in
    the field environment.

-   An understanding of the effect of the plant on the growth and
    sporulation of the fungi and on mycotoxin production under various
    crop production and stress conditions.

-   Use of identified genes for marker assisted selection of corn
    resistant to mycotoxins.

**Impact**

Knowledge of fungal genomics will provide the basis to bring about
mycotoxin control through resistant crops. Fungal genomics can help
elucidate the dynamics of the fungal-crop relationship, including the
role and mechanism of action of secondary metabolites in pathogenesis to
the host plant, and the mechanism of transmission from one plant to the
next. Fungal genomic information can help identify both the genes in the
biochemical pathways that lead to mycotoxin formation, and the
regulatory elements of mycotoxin production. Genomic information will
also provide identification/ characterization of the complex set of
genes involved in fungal virulence, and the signalling pathways between
the fungus and the environment, and fungal reproduction/survival which
will help prevent fungal infection and mycotoxin production in crops.
This genetic information in turn will identify effective control
strategies for mycotoxins during crop production, and, set the stage for
providing farmers with crop varieties that will not support fungal
growth and toxin production. A lower amount of crop will need to be
discarded because it does not meet regulatory guidance levels. Thus, the
results of this research will assist the food commodity industries in
the secure production and delivery of safe food products with lower
mycotoxin levels.

# Resources

ARS scientists assigned to projects under this Problem Statement are
located at National Center for Agricultural Utilization Research,
Peoria, IL; Western Regional Research Center, Albany, CA.; Southern
Regional Research Center, New Orleans, LA; Crop Genetics and Production
Research, Stoneville, MS; and Richard Russell Research Center, Athens,
GA

**Problem Statement 2.1.5: Biocontrol Technologies**

Biocontrol is the use of living organisms to control pests. The correct
selection of organisms can provide a highly selective tool for
controlling the pest of concern with minimal adverse effects on the
environment. The simplest systems are where the organisms are amplified
in the laboratory and then applied to the field; just as effective, and
certainly more elegant and less expensive, are biocontrol organisms that
amplify in the field to the necessary numbers to provide effective crop
protection. Research to date has been highly successful in developing
biocontrol strain technologies that use competitive exclusion to prevent
aflatoxin in cotton and peanuts, and some promising trials have been
carried out with corn and tree nuts. Biocontrol strains of organisms may
be either bacteria or fungi, and they may be endophytic or confined to
the outside of the plant.

**Research Needs**

New and improved biocontrol agents must be identified and developed for
continually recognized new crop/fungal control opportunities. We need to
understand the ecological relationship of the biocontrol fungi to other
plant microorganisms, soil microorganisms, and the treated crops and the
environment to help assure optimal delivery methods and timing. It is
particularly important that ARS partner with commodity-producing
industries, state boards or other development authorities, and/or crop
protection industries to complete the development of promising
biocontrol products. ARS must provide substantial assistance in
interpreting the data and making it available to EPA in an
understandable form in order to obtain the necessary, but often tedious,
regulatory approvals. It may also be necessary to develop commercial
scale production systems, including simple strain identification,
starter culture procedures, scale-up procedures, quality control
procedures and methods to assess efficacy for fungal biological control
agents. Only following a prodigious amount of often frustrating work can
products be made available to commodity producers.

**Outputs**

-   New or modified effective biocontrol organisms and delivery systems
    that do not introduce toxic factors.

-   Establishment of baseline levels of the atoxigenic biological
    control organisms to provide a basis for determining the influence
    of applied biocontrol products on natural microbial communities,
    particularly when applied over large crop acreages.

-   Further delineation of the role of endophytic fungi in regulating
    plant metabolism and in providing effective defense against
    predators and stresses.

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-   Determination of the effect of control strain distribution on the
    ecology of the producing area.

-   Safety, efficacy, and stability data necessary for maintenance and
    expansion of biopesticide registrations with EPA.

**Impact**

Effective and economical biocontrol strains that are approved by
regulatory agencies and readily available to the producing industries,
i.e., peanuts, corn, cottonseed and tree nuts will provide control of
aflatoxin in most years under most environmental conditions. Although
there is occasionally a year with environmental conditions favourable to
toxin production such that no single intervention can reduce losses from
mycotoxin contamination, long-term use of competitive exclusion products
will assist producers sufficiently that there is financial incentive to
continue crop production and stay in business. The results of this
research will thus assist the food commodity industries in the continued
secure production and delivery of safe food products with lower
mycotoxin levels.

# Resources

ARS scientists assigned to projects under this Problem Statement are
located at Western Regional Research Center, Albany, CA.; Southern
Regional Research Center, New Orleans, LA; Crop Genetics and Production
Research, Stoneville, MS; National Peanut Research Laboratory, Dawson,
GA; and Richard Russell Research Center, Athens, GA

**Problem Statement 2.1.6: Toxicity Evaluations and Mechanism of
Action**

FDA has established guidelines for several mycotoxins in the US, but
their toxicity continues to be assessed by the world community. Moreover
new research including human epidemiology studies continually raises new
questions of cause and effect links to human disease. There are also
questions about the effectiveness of certain industrial crop handling
and food product preparation procedures for irreversibly reducing
mycotoxins. In addition the presence of heavy metals and plant toxins
may be strictly regulated by the FDA, and also by the EPA, when there is
evidence that they may occur in drinking water.

**Research Needs**

Critical data for science-based risk assessments world-wide are needed
as the basis for regulatory decisions to protect consumers; and thus
continued assessment of certain aspects of mammalian toxicity is
necessary. Production of crops in developing countries is faced with
constraints, e.g., severe drought without water for irrigation, that are
of lesser importance in the U.S. Information is needed for the FDA and
WHO to make solid recommendations regarding safe levels of mycotoxins in
food. Toxicity of chemicals may be strongly affected by dietary
constituents, and specific relationships need to be elucidated and
recognized. We need to correlate the bases of identification tests for
certain regulated chemicals with the active moiety of these chemicals as
confirmed by tests of mammalian toxicity.

**Outputs**

-   Chemical speciation of toxins following ingestion and elucidation of
    their specific biological effects with relevant dietary components.

-   Mechanism of action based bioassays for \>mycotoxin - like= activity
    in foods and other matrices.

-   Chemical analyses that accurately predict the biological activity of
    toxins as they are found in prepared food products, and assistance
    in the development of production procedures to achieve safe levels.

-   Specific molecular endpoints of toxins in mammalian systems and
    downstream mechanisms of action using *in vivo* systems, plant
    models, and whole animal test systems; and validation of
    toxicological endpoints in whole animal feeding studies.

**Impact**

This research will provide information for the FDA and WHO to make solid
recommendations with a scientific basis regarding safe levels of
mycotoxins in food. The producing industries and the public will more
likely accept regulatory decisions when they know and understand the
basis of the regulatory guidance. Having a solid basis for
identification tests for the active moiety of mycotoxins and plant
toxins will assist in the identification and confirmation by the
regulatory agencies of suspect food contamination, either naturally
occurring or by bioterrorists. This will help assure food security, help
consumers accept regulator's statement and believe in the safety of
their food supply, and thus maintain healthy eating habits.

# Resources

ARS scientists assigned to projects under this Problem Statement are
located at Beltsville Agricultural Research Center, Beltsville, MD; and
Richard Russell Research Center, Athens, GA.