**FY 2008 Annual Reports for National Program 108, Food Safety**

**Executive Summary**

Food Safety falls under Goal 4 of the Agency Strategic Plan**, to
Enhance Protection and Safety of the Nation's Agriculture and Food
Supply**. The program's mission is 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. To ensure a secure agricultural system, ARS
works to reduce or eliminate factors that threaten the ability of U.S.
agriculture to produce enough food, year-to-year, to meet the needs of
American consumers. Research is 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. In particular, 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 to support further
efforts to provide a secure, affordable, and safe food supply.

The program's vision is to increase public health through the
development of technologies that **protect food from pathogens, toxins,
and chemical contamination during production, processing, and
preparation, thus** increasing the safety of the food supply. The
research components of the program during 2008 were:

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

-   Mycotoxins and Plant Toxins

**Program Highlights**

During 2008, there were several significant events in the Food Safety
National Program. Dr. Mary Torrence joined the Office of National
Programs to jointly oversee the program after her previous position as
National Program Leader for Food Safety at the USDA-Cooperative State
Research, Education, and Extension Service, where she oversaw the Food
Safety Epidemiology Program. Also, draft outlines were developed for the
NP 108 2011-2015 Strategic Plan and NP 108 2011-2015 Strategic Action
Plan, which will form the basis for food safety research in the next
Office of Scientific Quality Review (OSQR) cycle. In 2009, the National
Program will undergo an external Retrospective Review and a Stakeholder
Workshop prior to preparation of the next OSQR research cycle.

**Selected Research Highlights**

[Detection of melamine]{.underline}. The detection of
melamine-contaminated imported food products is a critical issue for the
Food and Drug Administration (FDA). ARS scientists developed a rapid,
nondestructive detection/identification method for melamine and its
derivatives in pet foods, based on Raman spectroscopic techniques. A
patent disclosure was approved for the method, and a Cooperative
Research and Development Agreement (CRADA) was initiated, resulting in
the development of two prototype hand-held devices currently undergoing
testing and validation in commercial settings. This work will have a
direct impact on the FDA's ability to detect melamine and related
contaminants in foods.

[Quantitative reduction of *Listeria monocytogenes* using
bacteriophage]{.underline}. New classes of GRAS (generally recognized as
safe) antimicrobials are in strong demand as mitigation strategies
against *L. monocytogenes* in various ready-to-eat food products. The
FDA has recently approved two lytic bacteriophage preparations as GRAS
for the protection of ready-to-eat food products against the bacteria.
In collaboration with scientists at Mississippi State University, ARS
scientists have developed models to determine the efficacy of the
bacteriophage preparations against *L. monocytogenes* in aquaculture
products. This research validates new methods for controlling Listeria
in ready-to-eat foods, and will allow the FDA to develop better risk
assessments.\
\
[Persistence of *Escherichia coli* on spinach leaves
evaluated]{.underline}. The contamination of leafy-green, fresh cut
produce is a major food safety concern. ARS scientists examined how long
*E. coli O157:H7* could survive under field conditions on spinach
leaves, comparing the bacteria's survival on spinach plants and in
organic soil in a growth chamber that simulated field conditions.
Populations of *E. coli O157:H7* survived for a shorter duration on
spinach shoots than in soil, with non-pathogenic *E. coli* detected
intermittently on spinach up to 28 days after inoculation. The results
suggest that *E. coli* survives for extended periods of time both on
leaves and in the soil. This work is critically important for producers
and regulatory agencies in developing effective agricultural practices
for fresh cut produce.

[Radiation sensitivity of fresh vegetables tested]{.underline}. The
produce industry is requesting a "kill" step to ensure the microbial
safety of fresh produce and to gain the consumer confidence. ARS
scientists demonstrated that a moderate (?---kGy isn't very
reader-friendly) dose of 1 radiation can achieve at least a 99.999
percent reduction of *E. coli O157:H7* inoculated onto the surface of
fresh produce. ARS further examined the effects of irradiation on the
quality of 13 common fresh-cut vegetables (Iceberg, Romaine, red and
green leaf lettuce, spinach, tomato, cilantro, parsley, green onion,
carrot, broccoli, red cabbage, and celery) and found that the
appearance, texture, and aroma of most of the vegetables were not
negatively affected, even after 14 days storage, although the vitamin C
content was reduced in a few vegetables. Further, no detectable amount
of furan (a possible carcinogen) was produced from irradiation. This
information is critical to the real-world application and implementation
of irradiation as a food safety intervention for fresh produce.

[Blade tenderization evaluated as food safety concern]{.underline}.
Blade tenderization is a process whereby needles are used to tenderize
whole muscle pieces of meat that are then cut into steaks, but may force
cells of pathogenic bacteria residing on the outside of the meat into
its center. The question remained, however, whether cooking would be
adequate to kill cells that are inside in addition to those on the
surface of the steaks. ARS scientists evaluated whether cooking
blade-tenderized steaks on a commercial gas grill would eliminate *E.
coli O157:H7*. In cooking the steaks on an open-flame gas grill to
internal temperatures ranging from 120° to 140°F, scientists discovered
that regardless of steak temperature or thickness, 1,000 cells of the
pathogen could be readily killed. These results validate that mechanical
blade tenderization transfers *E. coli O157:H7* into the interior of
steaks, with the majority of the cells remaining in the top 1 cm of the
meat, but that by cooking the blade-tenderized steak on a
commercial-style gas grill, any pathogen cells distributed throughout
the meat can be effectively eliminated. This information is critical for
regulatory agencies such as the USDA-Food Safety Inspection Service
Program, industry, and consumers.

[Detection of anthelmintic drug residues enhanced]{.underline}. The
monitoring of veterinary drug residues in meat and milk products is a
critical issue for regulatory agencies world-wide. ARS scientists
developed and validated a new liquid chromatographic-tandem mass
spectrometric (LC-MS/MS) multi-residue method to simultaneously quantify
and identify 38 of the most widely used anthelmintic veterinary drugs
(including benzimidazoles, macrocyclic lactones, and flukicides) in milk
and liver. The procedure utilizes a simple modification of the
ARS-developed QuEChERS method, which was initially developed for
pesticide residue analysis. The new method is capable of detecting and
quantifying sufficiently low levels of all targeted drug residues, and
was successfully validated for implementation in regulatory monitoring
labs in the United States, European Union, and other countries.

[Antibiotic resistance in Salmonella studied]{.underline}. Antibiotic
resistant bacterialpathogens pose a serious threat to human and animal
health. The bacterial strains that are resistant to multiple antibiotics
(MR) frequently harbor plasmids (extra-chromosomal circular DNA) or
other mobile genetic elements carrying resistance genes that can be
spread to non-resistant bacteria, and it is necessary to assess the
prevalence of such elements and understand the mechanisms of
transmission. ARS scientists examined 4 small plasmids conferring
kanamycin resistance in *Salmonella* strains that cause human illness,
determining that these plasmids are closely related to each other and to
other plasmids from *E. coli* and other pathogens that cause
gastrointestinal illness and that they exist in moderately high
abundance within bacterial cells. Moreover, since their presence is not
routinely assessed by typing methods used to characterize the MR
plasmids, the prevalence of this plasmid group may be underestimated.
This study underscores the need for regulatory and public health
agencies to continue to survey the emergence and spread of MR strains
and to monitor the presence of this group of plasmids conferring
resistance to kanamycin in *Salmonella*.

[Detection of other pathogenic *Escherichia coli*]{.underline}.
Serogroups of *E. coli* such as O2, O63, O28ac are of serious public
health concern, and improved methods for their detection and
identification are needed. ARS scientists developed multiplex PCR
(polymerase chain reaction) assays that target genes specific to each
serogroup based on DNA sequence information. Research results showed
that these genes could be used as diagnostic markers to rapidly identify
these serogroups in food and other types of samples.

[Shiga toxin-producing *E. coli* (STEC) from swine evaluated for
pathogenicity]{.underline}. STEC from cattle are known to cause disease
in humans; however, it was unknown whether swine harbored similar
pathogens. ARS scientists isolated and examined over 200 different STEC
strains from swine feces to determine the presence of genes involved in
causing disease (virulence), resistance to antibiotics, and acid
tolerance. Results indicated that the strains possessed 22 different
combinations of virulence genes, including genes found in *E. coli*
strains that cause disease in humans and animals, displayed resistance
to a number of antibiotics (with one strain resistant to 15), and were
also found to be resistant to acids; acid resistance is particularly
critical, given its vital role in the survival of bacteria in acidic
foods and in determining the dose needed to infect humans. The study
clearly demonstrated that swine STEC are a heterogeneous group of
organisms possessing important virulence genes that can potentially
cause human illness, information that is critical for producers and
regulatory agencies to implement safe and effective production
practices.

[Optical pathogen detection method created]{.underline}. Rapid methods
for the detection of harmful bacterial pathogens in food are necessary
to prevent the distribution of contaminated foods, thus protecting
consumers from bacterial food poisoning. In collaboration with Purdue
University, ARS developed a semi-automated Bacteria Rapid Detection
using Optical scattering Technology, or BARDOT system, to identify
bacteria from colonies on a plate without the need for expensive and
time consuming biochemical or microbiological tests. The simple system
involves growing the pathogens from the food in specialized liquid
culture media, subsequent plating on a solid media, and then shining a
red laser light through a bacterial colony and collecting the image
generated using a digital camera. Under test conditions, the actual
BARDOT analysis took 5-10 minutes to identify all bacterial colonies,
illustrating the system's utility as a simple and rapid method with
direct application to the food industry and regulatory agencies. The
BARDOT system (now patented) has been validated for the identification
of *L. monocytogenes*, *E. coli*, *Salmonella,* and *Vibrio* isolated
from food and for *L. monocytogenes* isolated from clinical samples.
Three BARDOT systems are currently being built for collaborative
evaluation by an academic, a government, and a food testing reference
lab.

[On-farm risk factors for *Yersinia enterocolitica*
assessed]{.underline}. Swine are the major animal reservoir for *Y.
enterocolitica* strains, a major human foodborne pathogen and one of the
eight bacterial foodborne pathogens under FoodNet surveillance. ARS
scientists examined critical factors for *Y. enterocolitica* infection
based on their screening of feces and tonsilar swabs of hogs on 122
premises. Four risk factors were identified (location, E. coli
vaccination, deaths due to scours \[diarrhea\], and diet components) and
the odds ratio for infection determined. This study was the first
attempt to identify risk factors for *Y. enterocolitica* in the United
States hog population, and its results are critical for swine producers
and regulatory agencies such as the USDA Animal and Plant Health
Inspection Service (APHIS).

[Aflatoxin contamination of almonds prevented]{.underline}. Aflatoxin
contamination is a critical issue for the California almond industry,
valued at over \$2 billion annually. ARS scientists found that the navel
orange worm (NOW), an insect pest, is a major contributor to alflatoxin
contamination of almonds, carrying spores of the *Aspergillus* fungus to
the almond kernel through feeding wounds produced by the insect, where
the spores subsequently germinate and grow. This finding will have an
immediate impact on the almond industry by facilitating the
implementation of insect control measures using host-plant volatiles
(HPVs), which trap, confuse, or distract the insects from locating the
host-plant.

[Bioavailability of botulinum neurotoxins in mice poison
evaluated]{.underline}. Along with potent neurotoxins (BoNT), the crude
food poisoning mixture produced by *Clostridium botulinum* bacteria
includes other non-toxic proteins. ARS scientists compared the toxicity
of crude versus purified BoNT preparations presented to mice in food and
showed that these accessory proteins protect BoNT from destruction and
deactivation in the digestive tract. These results will help scientists
understand how these potent biothreat toxins work and develop strategies
for maintaining a safe and secure food supply.

[Risk factors for produce contamination studied]{.underline}. Little is
known about specific plant factors that affect the colonization of
lettuce by the human pathogens *E. coli O157:H7* and *Salmonella
enterica*, and such information is necessary in order to develop
adequate good agricultural practices (GAPs) and Hazard analysis critical
control point (HACCP) guidelines for the produce industry. ARS
scientists demonstrated that enteric pathogens had higher growth rates
and grew to greater population sizes on the young inner leaves than on
the older middle leaves of Romaine lettuce, a difference partly
attributable to differences in nitrogen levels available to bacterial
cells on the surface of the leaves. These observations are valuable for
risk assessment of produce contamination, the development of efficient
sampling strategies to detect contamination of produce, and the design
of a control strategy by modulating nitrogen levels in lettuce fields.

[*Campylobacter* outbreak from milk contamination assessed]{.underline}.
The second largest outbreak of *Campylobacter* illness in U.S. history
occurred as a result of contaminated milk processed at a California
correctional facility dairy. At about the same time, a small outbreak of
*Campylobacter jejuni* illnesses occurred due to the consumption of raw
milk from a small organic dairy. A study of both dairies by ARS
scientists in Albany, California, in coordination with the California
Department of Public Health, involved sampling cattle feces, water,
milk, and other samples and isolating and screening *C. jejuni* using a
novel method. Suspect outbreak-related strains were genotyped and
compared to the human outbreak strains. The successful isolation of
outbreak-related strains from both dairies provided crucial
epidemiological information for explaining the outbreaks, and the
results obtained were critical for ongoing negotiations between
producers and State of California representatives concerning coliform
levels in raw milk deemed acceptable for human consumption.

[Genetic markers identified to assess fungal population
biology]{.underline}. The use of atoxigenic strains of *Aspergillus
flavus* as biocontrol agents has been discussed due to the potential
development of "super" toxin producers. To assess this possibility,
genetic markers are required to measure the potential for genetic
exchange. In order to determine genetic relationships among various
atoxigenic and toxigenic strains of *A. flavus* and to determine the
rate at which genes are exchanged among the various strains, ARS
scientists developed molecular markers and identified 68 microsatellite
loci from the genome sequence of *A. flavus NRRL3357.* Among these
microsatellite loci, 24 sites were identified as useful for population
biology studies on genetic distribution across strains, variability
within locations, and lack of interfering variation in adjacent DNA
locations. This work will allow through careful genetic manipulation the
design and development of safer biocontrol atoxigenic strains of *A.
flavus* that cannot become super toxin producers.

[*Escherichia coli O157:H7* higher in cattle fed wet distillers
grains]{.underline}. Demand for corn has driven cattle producers to rely
on other available feedstuffs, such as wet distillers grains with
solubles (WDGS). Small studies on the prevalence of *E. coli O157:H7* in
WDGS used in cattle diets have produced mixed results. In the first
long-term study on this topic with a large animal group, ARS scientists,
utilizing 600 calf-fed steers in the feedlot environment, examined the
level and prevalence of *E. coli O157:H7* on hides and in feces for 245
days through the growing and finishing phases of production. They found
that feeding 14 percent WDGS (on dry matter basis) in the growing phase
was associated with a slightly higher prevalence of *E. coli O157:H7* in
the feces compared to animals not fed WDGS, and that in the finishing
phase, animals fed 40 percent WDGS had greater prevalence of the
pathogen on hides and in feces compared to those not fed WDGS. Thus, the
higher prevalence of *E. coli O157:H7* in cattle fed high levels of WDGS
could result in a greater pathogen load at time of slaughter.

[Heat stress, tolerance, and *E. coli* prevalence studied]{.underline}.
Numerous environmental stressors have been identified that may impact
infection, prevalence, or levels of pathogens such as *Escherichia coli
O157:H7* that are shed by livestock. Over a 2-year period, ARS
scientists individually examined feedlot cattle for signs of heat stress
on days when the temperature/humidity index was in the "high danger" or
"emergency" categories. In addition, the scientists assessed the scoring
temperament of individual animals during the normal 28-day weighing
schedule to determine stress tolerance. Scientists examined any
correlations between heat stress and stress tolerance and *generic* or
*E.coli O157* levels and found no relationship between these factors.
This information is critical for industry and regulatory agencies in
providing healthy cattle at slaughter.

[Chemicals identified for destruction of estrogens in
water]{.underline}. Worldwide, surface waters are being made more
estrogenic as food animals in concentrated confinement secrete natural
estrogens and humans secrete synthetic versions of the hormone. Further,
both synthetic and natural estrogens may be incompletely removed by
municipal sewage treatment plants and agricultural waste handling
systems, posing unknown health implications to consumers of the water.
In this study, ARS scientists demonstrated that trace levels of Fe-TAML
(a commercially available chemical) makes hydrogen peroxide more
efficient at degrading natural and synthetic reproductive hormones
present in water, demonstrating the chemicals' potential utility for
removing the hormones from agricultural and municipal waste streams. The
work will have both national (EPA) and international impact on the
bioremediation of water sources.

[Drug-resistant *E. coli* in swine]{.underline}. Bacteria have become
increasingly resistant to antibiotics, raising concern for health
caregivers and the medical community that this resistance may be
transferred from animals to humans. Definitive evidence of whether or
not this transfer occurs is lacking, however, largely due to the lack of
controlled study populations. ARS scientists, in collaboration with
Texas A&M University, studied a semi-closed, fully integrated swine
production operation and discovered that certain production groups of
swine, such as boars and nursery piglets, and certain worker
populations, such as slaughter plant workers, were at higher risk of
carrying multidrug-resistant *E. coli* than were other populations
within the operation. The researchers thus found that certain production
practices and occupational exposures may indeed increase the risk of
antibiotic resistance transfer in pathogenic microorganisms. The
research also identified potential critical control points that may be
targeted to reduce the transmission of antimicrobial-resistant bacteria
from farm animals to humans demonstrating important food safety
implications.

[Mechanical vector discovered for *Salmonella* in poultry]{.underline}.
The lesser mealworm is a common insect that infests commercial poultry
houses. While the mealworm does not overtly affect the birds, it is not
known what role the insect might play in the spread of pathogenic or
food-poisoning microorganisms within the poultry environment,
particularly its capability to infect birds with these microbes. After
externally disinfecting the beetle, ARS scientists showed that
relatively short exposures of the beetles to low levels of *Salmonella*
result in the rapid acquisition of viable bacteria into its alimentary
canal, illustrating the beetle's has potentially a signifcant role as a
vector for the pathogen. This work will be extended by project
scientists to develop biosecurity procedures against the beetle, which
should ultimately result in less pathogen colonization of the birds and,
ultimately, in microbiologically safer poultry meat products reaching
the consumer. This insect is also being evaluated as a model system for
understanding the mechanisms of antimicrobial resistance.

[Non-aflatoxigenic *Aspergillus flavus* strains patented]{.underline}.
Aflatoxins are a group of toxins produced by the fungus *A. flavus,*
which frequently contaminate corn in the mid south area, especially in
the Mississippi Delta. Reducing mycotoxin contamination in corn will
produce a safer feed/food supply that will be readily acceptable on the
world market. ARS scientists have patented a non-toxigenic *A. flavus*
strain (K49) that was shown to reduce aflatoxin in corn by up to 93
percent under field conditions. This technology provides the groundwork
for industry to test the use of non-toxigenic strains and develop a
commercial product that controls aflatoxin contamination in corn.

[Antioxidants shown to inhibit aflatoxin production]{.underline}. ARS
scientists in Albany, California, and New Orleans, Louisiana, have
discovered how to prevent aflatoxin production with safe, common natural
chemicals. Caffeic acid, an antioxidant, was found to reduce more than
95 percent of aflatoxin production by *Aspergillus flavus* without
affecting fungal growth. In conducting a microarray analysis of *A.
flavus* treated with caffeic acid, scientists found decreased expression
of almost all genes in the aflatoxin biosynthetic cluster, suggesting
that the caffeic acid triggers the induction of alkyl hydroperoxide
reductases (enzymes), which prevent the fungus from synthesizing
aflatoxin. In addition, the research has elucidated the mechanism by
which these compounds cause the fungus to turn off the machinery that
synthesizes aflatoxin, which is produced by the fungi to protect it from
chemical attacks by plants. This information provides insight into
controlling the genes that trigger aflatoxin biosynthesis and should
help in devising crop breeding methods to prevent aflatoxin
contamination.

[Serotyping *Salmonella*]{.underline}. Serotyping *Salmonella* is an
expensive, time consuming task; therefore, development of a rapid
high-throughput molecular technique is needed by regulatory agencies.
ARS scientists adapted a previously developed multiplex PCR *Salmonella*
serotyping technique to perform on a high-throughput platform. The new
technology can identify the top 31 serotypes, representing 75 percent of
all clinically isolated *Salmonella* from humans and animals. This
technology significantly improves the efficiency and effectiveness of
analysis, allowing the determination of up to 90 isolates in one day at
just \$1.50/sample, as compared to several days and \~\$40.00 for
traditional serotyping. Moreover, the technology requires little
training, no specific anti-sera, and only standard DNA sequencing
instruments. This technique could replace traditional serotyping for
most *Salmonella* isolates implicated in foodborne outbreaks. It is
currently being tested by several Federal and State public health
laboratories in the United States and also by the Public Health Agency
of Canada.

[Multiplication of *Salmonella enteritidis* studied in
eggs]{.underline}. Although chickens infected with *Salmonella* do not
deposit this pathogen inside egg yolks very often, there are occasions
where bacteria from the surrounding albumen might penetrate through the
membrane surrounding the yolk, resulting in rapid and extensive
*Salmonella* growth in the nutrient-rich interior contents of the yolk
prior to egg refrigeration. ARS scientists used a laboratory egg
contamination model to assess the ability of *S. enteritidis* strains to
multiply on the vitelline membrane or to penetrate this membrane and
multiply inside yolks during incubation at warm temperatures. Studies
determined that *S. enteritidis* were all able to penetrate from the
exterior of the yolk (vitelline) membrane into the yolk contents during
as little as 12 hours of incubation at 30°C and that the concentration
of *S. enteritidis* after incubation was significantly higher in whole
yolks than in yolk contents at both 12 hours and 36 hours. These results
demonstrate that extensive bacterial multiplication on the yolk membrane
may occur in addition to (and before) penetration into the yolk
contents, further supporting regulatory rules that emphasize rapid
refrigeration of eggs for protecting consumers from egg-borne illnesses
by *Salmonella*.

[Micro-crack detection method developed for table eggs]{.underline}. The
USDA Agricultural Marketing Service (AMS) asked ARS to develop a method
to help graders identify hairline micro-cracks in table eggs. ARS
scientists developed a 20-egg batch-process imaging system to visually
enhance and enable detection of these small cracks, resulting in an
extremely accurate method to detect the cracks. Further enhancements to
the system include a user-friendly, touch-screen database method for
recording the number of egg cracks and other egg features that cause
downgrades, which the AMS graders are currently documenting. The system
will help the graders by increasing their accuracy, removing
subjectivity, reducing data transfer errors, increasing their
productivity, and dramatically changing the way eggs are currently
graded.