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					Title: Biosecurity and Diversity of Foodborne Pathogen Populations on Poultry Farms.

Project Summary:
Campylobacter jejuni, Listeria monocytogenes, and Salmonella spp. are among the leading
foodborne bacterial pathogens in the USA . Poultry products are a major source of these
organisms. Using cross-sectional and longitudinal study designs, we will assess the prevalence of
these foodborne pathogens in commercial chicken and turkey production. For each pathogen we
will determine the population diversity on farms, after transportation to the abattoir, and at the
end of the processing line. Sampling will also occur on selected breeder farms and hatcheries
in an effort to assess the relative importance of these sources of transmission. In addition, we
will sample vectors such as arthropods, rodents, and feed; as well as other poultry houses, and
non-poultry farms (swine and cattle) found in proximity to the study farms. We will survey each
farm in order to determine management and environmental risk factors that may be associated
with foodborne pathogens. The longitudinal study will allow us to assess the impact of a
biosecurity extension program on the level of foodborne pathogens at the farm. Results from
these studies will provide us with key information to improve our biosecurity and sanitation
extension programs. We will also integrate the research findings into our educational programs,
using multimedia technology, and assess their value as learning tools. Finally, a communication
network will be established for the purpose of providing access to the information by all
stakeholders, in particular, those directly involved in poultry production, since they are on the
front line of any foodborne pathogen reduction efforts.
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Introduction
         The Centers for Disease Control and Prevention estimates that about 5,000 people die and
75 million get sick each year from foodborne illness. Outbreaks of foodborne illness are
associated with many different foods, yet contaminated meat, poultry, and egg products are the
most frequently reported vehicles (around 37% of all outbreaks). Campylobacter jejuni, Listeria
monocytogenes, and Salmonella spp. are among the leading foodborne bacterial pathogens in the
US and worldwide (Mead et al., 1999, Hubbert W et al, 1996). Consumption of poultry products
has often been among the major risk factors implicated in foodborne diseases due to these agents.
Salmonella and Campylobacter, in particular, have been shown to be important both at pre- and
post-harvest levels, whereas L. monocytogenes is particularly important as a post-harvest
contaminant. Among Campylobacter spp, C. jejuni is known to be the most important species
for two major reasons: first, this species is the foremost cause of human bacterial gastroenteritis
in the world (Nachamkin, I, 2000, Hubbert W et al, 1996). Second, the recent emergence of
fluoroquinolone resistant C. jejuni, mainly among isolates collected from poultry and humans
(Smith, K et al., 1999), presents a growing public health risk. So far, there has been little work on
the molecular epidemiology of Campylobacter spp.. The genomic diversity of this agent on
farms, as well as for the other two foodborne pathogens mentioned above is not well known. The
distribution of these agents in the poultry environment also needs to be studied as part of an
effort to objectively determine environmental, management, and even bird factors that might be
associated with their presence or higher prevalence.
         Arthropods, especially the housefly (Musca domestica), very likely contribute to the
transmission and maintenance of bacteria associated with foodborne illnesses in the preharvest
interval. Housefies have been shown to transmit Campylobacter experimentally and half of all
flies captured near poultry houses containing naturally infected chickens were positive for the
bacterium (Rosef and Kapperud, 1983; Shane et al., 1985). The housefly is the predominant pest
in the southeastern US. Specifically, these highly mobile insects (flights up to 12 km within 24
hours; Greenberg, 1973) are capable of disseminating foodborne bacteria within and between
poultry houses (Lysyk and Axtell 1986).
         The three major research components of this proposed study are 1) characterization of
foodborne pathogens at phenotypic and genotypic levels; 2) determination of risk factors; and 3)
assessment of the impact of biosecurity measures (on-farm interventions) on these pathogens.
Our long-term goal is to increase our understanding of the epidemiology of foodborne pathogens
in order to develop, implement, and evaluate extension and education programs targeted at
people involved in the poultry industry, with the ultimate goal of contributing to a significant
reduction of foodborne pathogens.
         The poultry industry is showing interest in this project because new diseases affecting
poultry continue to emerge (an average of almost one per year over the past 20 years). The global
movement of poultry, poultry products, equipment and people facilitate the spread of pathogenic
agents. In addition, the intensive nature of modern poultry production aids in the development
and spread of infectious diseases. Many of these diseases have immunosuppressive effects. There
is growing evidence that they may have a substantial impact on poultry production. Furthermore,
immunosuppressive diseases may also have an impact on the incidence and diversity of
foodborne pathogens. For example, Campylobacter has been associated with enteric diseases in
turkeys (Barnes et al, 2000). Unfortunately, there is still a paucity of information on the possible
role of these poultry diseases relative to foodborne pathogens, and about the possible value that
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biosecurity measures could contribute to the reduction of both categories of infectious agents
(i.e. poultry and foodborne pathogens).
         Biosecurity is defined as “health plan or measures designed to protect a population from
transmissible infectious agents”. This includes sanitation, pest control, and any measures
intended to break the chain of infection (Toma and Vaillancourt, 1999). Our proposal aims at
assessing an integrated biosecurity extension training program (e.g., sanitation, pest control,
traffic control, etc.) under commercial field conditions. It has the merit of integrating newly
developed multimedia extension material with on-farm and laboratory research activities
designed to assess this extension effort, to determine whether it has a significant impact on
foodborne pathogens, and lead to improved extension and educational material (Figure 1).
     Many critical pre-harvest foodborne pathogen issues have not yet been addressed because of
the challenges associated with such research. Our research and extension teams have several
unique advantages that will allow us to meet these challenges:
     1. Because of important epidemics that have affected the North Carolina poultry industry
   over the past 7 years (poult enteritis mortality syndrome; Mycoplasma infections; turkey
   coronavirus enteritis; infectious laryngotracheitis), members of our group have developed
   privileged relationships with poultry companies and the NC Department of Agriculture. This
   unique partnership between state, industry, and academia was fostered by a USDA Fund for
   Rural America grant on emerging and reemerging infectious poultry diseases. We now have an
   extensive database that includes Geographical Information System coordinates for all farms,
   and environmental and management data for a several hundred chicken and turkey farms. We
   also have a weekly interaction via e-mail with all the poultry companies east of Raleigh, which
   represent an annual turkey production of about 40 million birds and an annual chicken
   production of about 350 million broilers. The main purpose of these contacts is data sharing
   among all members of this state-industry-academia alliance.
     2. The Poultry Coordinating Committee (a group of faculty from the Departments of Poultry
   Science, Agriculture and Resource Economics, Entomology, Food Science, and Veterinary
   Medicine) has been involved with developing and offering a workshop that introduces the
   concepts of on-farm HACCP (hazard analysis critical control point) to the poultry industry. To
   date 4 workshops have been held throughout the state. These workshops have been well
   attended by representatives from major integrators, and participants have expressed an interest
   in having options to use the Internet for the second phase of training. Thus, the members of
   this committee, some of whom are also key personnel in this project, are in the process of
   developing an on-line on-farm food safety course, which incorporates HACCP principles.
     3. Members of our extension team have received two grants that are directly relevant to the
   proposed project (US Poultry & Egg grant to design a multimedia biosecurity training
   program, $89,000); and a Delta grant entitled “Distance Learning for Educating Students in the
   Concepts of On-farm Production Practices Associated with Poultry Product Safety” ($15,000).
     4. The NCSU-CVM teaching animal unit (TAU) is a commercial facility on the veterinary
   campus that can house up to 8000 broiler chickens or 2800 turkeys. It is also within 20 meters
   of a swine production facility. This facility will be used to test the hypothesis that organisms
   may be shared between poultry and swine production units.
     5. We have the ability to produce extension material in Spanish, the most commonly spoken
   language among poultry farm workers, as well as English. We also have the ability to develop
   survey instruments in both languages. This has proven very useful when we investigated a
   major Mycoplasma gallisepticum epidemic last year.
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    6. The multidisciplinary composition of our team, from clinicians, extension experts, to
  basic scientists.
    Knowledge will be obtained about the phenotypic and genotypic diversity of foodborne
pathogens in poultry flocks, relative to their environment (including neighboring farms) over
time. This will provide us with key information to evaluate existing extension and educational
programs to supportthe poultry industry in its effort to reduce the risk of contamination with
foodborne pathogens.
        Our proposal is relevant to the first three priorities as outlined in the Federal Register: 1.
analysis, assessment, and communication of risk (objectives 1 to 4 and 6 of this grant); 2. control
measures for foodborne microbial pathogens (objective 5); 3. sources and incidence of microbial
pathogens (objectives 1 to 3).

Objectives:
   1. Assess the prevalence of leading foodborne bacterial pathogens (Campylobacter,
      Salmonella,and Listeria) for two major commodities: chicken and turkey production.
   2. Determine for each pathogen the diversity of its population on poultry farms, after
      transportation to the processing plant, and at post-chill (i.e., assessment of the different
      types of Salmonella, Listeria, and Campylobacter on farms and at the slaughter plant)
   3. Assess the relative importance of sources of transmission of these foodborne pathogens
      including arthropods, rodents, feed, as well as establishing the significance of close
      proximity of poultry and non-poultry farms (i.e., do they share the same populations of
      bacteria?).
   4. Determine risk factors associated with foodborne pathogens.
   5. Determine the impact of a biosecurity (including sanitation) extension program on
      foodborne pathogens at the farm level.
   6. Adjust our biosecurity and sanitation extension program, integrate the research findings
      in our educational programs, assess their value as learning tools and establish a
      communication network for the purpose of providing access to the information to all
      stakeholders.

Methods:
1. Assess the prevalence of leading foodborne bacterial pathogens (Campylobacter, Salmonella,
and Listeria) for two major commodities: chicken and turkey production.

Farm selection: As part of the farm selection process for a cross-sectional study, we will have
access to data on Salmonella monitoring from two turkey companies and one chicken company.
These companies have birds on about 1200 farms. A subset of 90 farms (45 turkey and 45
chicken farms) will be sampled to confirm the Salmonella status of the current resident flocks,
which will allow us to also establish the prevalence of Campylobacter and Listeria. The oldest
flock on each farm will be sampled if more than one flock is present on any given farm (on-farm
and processing plant sampling: see objective 2). This will provide us with a point prevalence
assessment of the three foodborne pathogens. This sampling will also contribute to the final farm
selection for a longitudinal (prospective) study (objective 5). This objective is essentially
focusing on grow-out facilities. However, results from breeder farms and hatcheries will also be
included in the analysis (comparison of pathogens between breeder farms, hatcheries, grow-out,
and abattoir).
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        Using farms selected for the cross-sectional and the longitudinal studies, the NC
Agricultural and Technical State University members of our team (Drs Willis and Worku) will
assess the relative importance and significance of used vs unused litter in the colonization of
chickens and turkeys with Campylobacter jejuni. Turkey and chicken farms (flock and
environment) will be sampled at time of placement and at 3 and 6 weeks of age (end of grow-out
for chickens). Samples for turkey flocks will also be taken at 10 and 15 weeks of age. Twenty
fresh feces cecal droppings will be collected in the early morning. Sterile rayon-tipped
applicators will be used to gather a part of each dropping. Caryr-Blair transport medium will be
used to transport samples to the laboratory. In the lab, broth will be placed and spread on BBL-
Campy plates. The plates will be incubated in controlled atmosphere of 5% 02, 10% C02, and
85% N2 for 24 hours at 42º C. Confirmation will be done by using the serological latex
agglutination test. Bacterial isolates from these sources will be strain characterized by high-
resolution genotyping. The relative importance of live haul transmission (crate sanitation) will
also be determined. Crates will be sampled prior to bird pick up and after unloading at the
processing plant. The isolates obtained from transport crates will be subjected to strain
characterization for farm trace back. The effectiveness of pre-and post-sanitation intervention on
Campylobacter jejuni survivability in transport crates will be assessed. Isolation and genomic
fingerprinting will follow the same procedures as mentioned under objective 2.

2. Determine for each pathogen the diversity of its population on poultry farms, after
transportation to the processing plant, and at post-chill (i.e., assessment of the different types of
Salmonella, Listeria, and Campylobacter within farms and at the slaughter plant)

On-farm and slaughter plant sampling strategy
    For each selected grow-out flock, five pooled samples of feces will be collected (five
birds/pool) in one poultry house. Sampling will be performed at the following times: a) two
weeks prior to processing; b) at arrival at the processing plant; c) at shackle time; d) post-chill
carcasses (no feces available; five pooled carcass rinses).
    In order to identify biosecurity measures that will be effective in preventing food
contamination, we must determine whether events that occur throughout the production process
impact bacterial contamination. It is possible that the effectiveness of any measure implemented
on the farm will be overwhelmed by increased bacterial shedding or by the introduction of new
agents during transport. To study this, we will culture samples taken before transportation, from
trucks, and pre-shackle, and post-chill. By performing phenotypic and genotypic analysis on
these strains, we will determine whether the biosecurity measures we test are likely to have an
important impact on foodborne pathogen contamination at different stages of production.

Identification of Salmonella from fecal and environmental samples
        We will culture Salmonella using methods shown previously to be effective for bacterial
isolation from animal and environmental sources (Bager et al., 1991). One gram of feces (or
bacteria from pooled carcass rinses) will be pre-enriched in 10 ml buffer peptone water and
incubated over night at 37ºC. Then, 100 l of this will then be enriched overnight in Rappaport
Vassilliadas medium at a 1:100 ratio. Samples will then be plated on xylose lysine tryptone
(XLT4) agar and suspected colonies will be identified as Salmonellae by fluorescence using
MUCAP (Ref). Identification will be confirmed using triple sugar iron (TSI) and Urea media.
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Isolates found to be positive will be sent to the National Veterinary Services Laboratory (NVSL)
for serotyping.

Phenotypic Characterization
        We will characterize strains at both the phenotypic and genotypic level. Phenotypic
characterization includes two steps: antimicrobial resistance patterns (antibiograms) for all
organisms, and phage resistance patterns for a subset of our isolates. Antimicrobial
susceptibility testing will include both a semi-automated method (Vitek; Biomerieux) and Kirby-
Bauer disc diffusion. In this way we can test for resistance to 14 antimicrobial agents used in
veterinary and human medicine. Antimicrobial resistance testing has several advantages for this
study. It both provides an initial discrimination among isolates and, in combination with
genotypic testing, will provide a more complete characterization of the cultured strains. Further,
information about resistance may be, in and of itself, valuable information. Data from
antimicrobial susceptibility testing will thus be compiled, and strains will be classified based on
their respective resistance patterns. These data will be combined with further genotypic
characterization, as well as to distinguish isolates collected from different sources.
    The second phenotypic characterization method will be phage typing. This method can be
performed only on two serovars of Salmonella; Typhimurium and Enteritidis. Based on the
findings of our study, we will perform phage typing on representative isolates with different
antibiograms to identify the presence of phage types with known public health significance.

Genotypic Characterization
        In order to study the relatedness of Salmonella strains obtained from animal and
environmental samples, it is essential to have a reproducible method to distinguish
phenotypically identical strains from one another. Such a method must also be rapid and easy to
employ, since this work requires analysis of a large number of strains. We have chosen a
relatively new method; amplified fragment length polymorphism analysis (AFLP). AFLP is
particularly sensitive in that it can detect genomic polymorphisms of as little as one base pair
difference (Vos et al., 1995). With this technique, genomic DNA is cut with two restriction
enzymes, EcoRI and MseI, then linkers specific to each end are ligated to the resulting
fragments. PCR primers complementary to the linkers are then used to amplify the fragments.
To limit the number of amplicons produced, a single adenine is included at the 3’ end of one
primer, requiring that the genomic DNA have a complementary thymine adjacent to its EcoRI
site. One primer is also infrared labeled so that resulting bands can be separated with great
sensitivity using an automated infrared-reading sequencing apparatus. This technique produces
up to 200 distinct bands, ranging in size from 50 to 700 bp. Different strains produce unique
banding patterns, identified using computer-assisted analysis, allowing us to distinguish closely
related strains from those that are more distantly related. The value of this technique is that it
does not rely on a single locus to differentiate strains, but instead samples multiple loci from the
bacterial genome. It has been successfully used to establish phylogenetic relationships among
and between bacterial species, including Salmonella (Jackson et al, 1999; Janssen et al, 1996;
Linstedt et al., 2000, De Sai et al., 2001). It has also been shown to be reproducible and to be at
least comparable in differentiating strains to other typing techniques, such as PCR-based
ribotyping and pulse-field gel electrophoresis (Janssen et al, 1996; Koeleman et al, 1998;
Lindstedt et al., 2000). We have thus far completed AFLP profiles on over 1000 Salmonella
strains for another project. We have found that we can differentiate strains obtained from
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different sources and can find polymorphisms that characterize specific strains and antimicrobial
resistance patterns.
         One disadvantage of AFLP is that, although it samples multiple loci for polymorphisms,
it produces fragments that make up no more than 1-2% of the genome. It is obvious, therefore,
that sequence differences among strains can be missed. Therefore, in order to ensure that strains
with identical antibiograms and patterns on initial AFLP analysis are indeed identical (or at least
very closely related), we will perform additional AFLP analyses on those strains using different
primer extensions. We will substitute a cytosine, guanine, or thymine residue at the end of the
EcoRI amplification primer, and thus create up to three additional fingerprints for each strain in
question. In this way, we should be able to differentiate strains even when they are closely
related.

Data Analysis and Interpretation of Results
        We can analyze AFLP gels to produce implied phylogenetic trees with the software
Quantity One 4.1 (Bio-Rad) and using the neighbor-joining method of this same program to
create phylogenetic trees (Saitou and Nei, 1987). Our strategy will be to first establish general
relationships among strains by scoring only a few of the polymorphisms present. These
relationships will guide us in making comparisons among strains. We will then analyze all
polymorphisms to determine which isolates are clonal, and which are closely related, but
different by AFLP. Once we have created complete phylogenetic trees, we can make
comparisons that will allow us to address the questions raised for the following objectives.
    Baseline data will provide information about bacterial prevalence. Phenotypic and genotypic
analysis of isolates will additionally show the diversity of the cultured isolates, both in terms of
important characteristics, such as antibiotic resistance, and at the genetic level. We will be able
to determine whether strains isolated from different sources and over time (longitudinal study)
are identical, suggesting the expansion of a clonal population, or whether they represent distinct
strains of the same bacterial species.

Identification and characterization of Campylobacter
         Fecal dropping sample collection will follow the same scheme as described above for
Salmonella. Samples will be transported on ice to the laboratory where they will be processed
with minimum delay. For isolation of Campylobacter, serial dilutions will be plated directly
onto selective media (Campy-Cefex; Stern et al., 1992) and the plates will be incubated in a
microaerobic atmosphere at 42C utilizing the Campy-Pac system. Plates will be examined
following 24 hours incubation. Bacteria from colonies with appearance suggestive of
Campylobacter will be examined by gram stain and by phase-contrast microscopy using wet
mounts, to determine whether the darting motility and cell shape typical of Campylobacter are
present. Campylobacter colony forming units (CFUs) in the original fecal sample will be
enumerated, and 2 putative Campylobacter colonies will be subcultured at 42C
microaerobically. Following 36 hours of growth, the subcultures will be catalogued, and the cell
mass will be swabbed off the plates and preserved in freezing medium (Brucella broth with 15%
sterile glycerol) at –70C.
         Campylobacter will be isolated from broiler carcasses (chill-tank) originating from the
farms being monitored, following enrichments of carcass rinses in Bolton Broth (4 h 37C
followed by 20h at 42C) and subsequent growth in direct plating on Campy-Cefex agar media
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and incubation in a microaerobic atmosphere at 42C. Bacterial isolates will be processed as
described above for fecal dropping isolates.
        Strain characterization will focus on the following: differentiation of C. jejuni from C.
coli; high-resolution genotyping; and determination of antibiotic resistance.

Differentiation of C. jejuni from C. coli.
        C. jejuni and C. coli have been estimated to cause 80-90% and 5-10% , respectively, of
human Campylobacter infections (Nachamkin et al., 2000). The majority of Campylobacter
isolates from broilers appears to be C. jejuni, whereas C. coli has been found to be predominant
in swine (Aarestrup et al., 1997;Saenz et al., 2000). Although both species are potential human
pathogens, their differentiation is meaningful, as they appear to vary in the occurrence of
resistance to different antibiotics (Aarestrup et al., 1997; Saenz et al., 2000). The two species
may have additional, but currently poorly understood, differences in their physiology and
pathogenesis.
        C. jejuni differs from C. coli in having hippuricase activity, and the corresponding genes
(hip). To differentiate C. jejuni from C. coli, PCR utilizing the published hippuricase gene
primers (Chan et al., 2000 ) will be used. In our own experience with isolates from broilers, such
hip-based PCR has been reliable and reproducible, yielding clear results for all isolates,
including those that gave ambiguous results with the enzymatic assay. The PCR products will be
identified by agarose gel electrophoresis, and known C. jejuni and C. coli isolates will be
included periodically as positive and negative controls, respectively.

High-resolution genotyping
       AFLP has recently been shown to be an excellent high-resolution method for genotyping
of Campylobacter. The AFLP protocols will be basically the same as those described above for
Salmonella with modifications as described.

Antimicrobial resistance of the Campylobacter isolates.
        Campylobacter strains will be tested for antibiotic resistance to nalidixic acid,
ciprofloxacin, erythromycin, azithromycin, and tetracycline by using the E-test method (AB
Biodisk™ AB Biodisk of North America, Inc., Piscataway, NJ). Four E-test strips will be placed
at ninety-degree angles onto a plate and incubation will be for 48 hours at 37C
microaerobically. These conditions were chosen to conform with those in use by the National
Antimicrobial Resistance Monitoring System: Enteric Bacteria (NARMS), specifically for
Campylobacter (Tollefson et al., 1999). Our choice to focus on the five antibiotics of most
clinical interest was also guided by the need to contain costs, considering that E-strips are
expensive and a large number of isolates will be involved in the project.
        There are currently no recommended antibiotic breakpoint concentrations for
Campylobacter, and results will be recorded in terms of MIC data. To allow for comparison of
our data with those of others who have investigated antibiotic resistance in Campylobacter
(Aarestrup et al., 1997; Saenz et al., 2000), we will use the National Committee for Clinical
Laboratory Standards (NCCLS) breakpoints for resistance (NCCLS, 1994).

Identification and characterization of Listeria monocytogenes
        Samples for Listeria analysis (fecal droppings and carcass rinses) will be sent by
overnight delivery on ice to the laboratory of Dr. Elliott Ryser at Michigan State University and
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analyzed within 24 hr of receipt. In the procedure, 25-g fecal and 25-ml rinse water samples will
be enriched in 225 ml of UVM according to the VIDASTM L. monocytogenes enrichment
procedure. After 40-48 h of incubation at 30oC, a portion of the sample will be boiled for 10
minutes and screened for the presence of L. monocytogenes using the mini-VIDAS System that
is available and operational in the laboratory of Dr. Ryser. The VIDASTM LMO Assay is an
automated enzyme-linked immunofluorescent assay that specifically detects the presence of L.
monocytogenes. Unboiled enrichment cultures yielding positive results with VIDASTM LMO
Assay will be streaked to Modified Oxford Medium (OXOID) for isolation of Listeria species,
including L. monocytogenes. Since this plating medium will support the growth of other Listeria
spp. in addition to L. monocytogenes, up to five Listeria colonies per plate will be subjected to a
battery of morphological and biochemical tests (e.g. Gram stain, catalase, CAMP hemolysis,
fermentation of rhamnose and xylose) for positive identification of L. monocytogenes. In
addition, all isolates identified as L. monocytogenes will be serotyped using commercially
available antisera. While of little threat to human health, Listeria spp. other than L.
monocytogenes would still be of relevance as indicators in terms of evaluating the possible
impact of biosecurity measures.
        High-resolution genotyping will be done by Pulsed-field Gel Electrophoresis (PFGE).
The PFGE subtyping data will be valuable for future incorporation into the PulseNet database.
Currently the L. monocytogenes PulseNet database consists primarily of human clinical isolates,
but typing data from animal and food-derived strains are becoming increasingly desirable for
epidemiologic monitoring of the sources and reservoirs of the pathogen in nature and in the food
chain.

3. Assess the relative importance of sources of transmission of these foodborne pathogens
including arthropods, rodents, and feed, as well as establishing the significance of close
proximity of poultry and non-poultry farms (i.e., do they share the same populations of
bacteria?).
        Because we expect a high prevalence of Salmonella-positive flocks, we will seek a 2
positive to 1 negative ratio for the cross-sectional study, resulting in the selection of 40 positive
farms and 20 negative ones. Additional testing will be conducted, if necessary, until these
numbers are attained. Salmonella status was selected as the determinant factor for cohort
selection because, of the three pathogens, it is the one that can be isolated with the most
consistency.
    The 40 Salmonella-positive farms will be separated in two cohorts of 20 farms (10 turkey
and 10 chicken units per cohort). One cohort will comprise farms whose growers and employees
will participate in the multimedia biosecurity extension program (intervention group). The other
cohort will also have 20 farms. However, the growers and their employees will not participate in
the program (non-intervention group). For each chicken farm, we will follow 6 flocks; for each
turkey farm, we will follow 3 flocks. This follow-up will take between 12 to 16 months,
depending on the farm type. Therefore, we should gather data on 60 chicken flocks and 30 turkey
flocks for each cohort.
    In this study, we will use other poultry houses on the same farm and swine and beef cattle
farms proximal to poultry farms as a model to study the significance of other host animals as
risks to poultry. The participating turkey companies are also involved in pig production. This
will facilitate the process of getting authorization to sample these herds. We will consider vectors
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(vehicles) that may transmit these organisms between different host species. These include
arthropods such as flies, rodents, and fomites including transportation equipment.

3.1 Rodent monitoring:
Mice will be the primary target species for the study. Twelve Victor Tin Cat Repeating
MouseTraps will be placed in selected study houses in both intervention and non-intervention
groups. Monitoring will be conducted over a 24-hour period at intervals of every 2-4 weeks.
Trapped rodents will be tabulated and submitted for pathogen culture.
         Breeders: It will not be possible to monitor all of the breeder flocks producing the
grow-out flocks included in this study. Hence, we will only select the breeder flocks that are at
the origin of one turkey and one chicken grow-out flock, to a maximum of 8 breeder flocks
(e.g., 3 turkey breeder flocks providing all the birds for a turkey grow-out flock included in the
study and 5 chicken breeder flocks providing all the birds for one chicken grow-out flock).
Each breeder house will receive 10 traps placed beneath slats along walls and 10 on top of
slats distributed between walls and adjacent to nest boxes, and 1 each in the egg room and
cooler.
         Chicken and turkey grow-outs: As for the breeders, it will not be possible to do pest
monitoring for all the flocks. Five turkey and 5 chicken farms will be monitored (including the
ones originating from the breeder farms selected for the study) Ten traps will be placed on
floors along walls (wire enclosures if significant bird interference occurs), and 10 distributed
on building exteriors along walls, feed bins and service buildings. Another type of trapping
device (glue board) may also be used depending on the structure of each building.

3.2 Insect monitoring:
        Our efforts will concentrate on the housefly. Two fly monitoring devices (spot cards
and sticky cards) will be used. A full complement of monitoring devices (20) will be placed in
study houses. Half of the cards in each house will be distributed at random locations at 2-3
meters above the litter or manure surface (height A). The remaining half of the devices will be
randomly distributed approximately 1 meter above the litter or manure surface (height B).
Devices will be replaced at maximum intervals of 7 days for the duration of the study.
Retrieved devices will be counted (spots or flies per card) and recorded for analysis. Trapped
flies will be submitted for pathogen culture.
3.2.1. Determine the potential of houseflies to support and harbor Salmonella, Listeria, and
Campylobacter (SLC).
        3.2.1a. Evaluate time course survival of bacteria in the crop and gut of the housefly: We
will evaluate the potential of SLC bacteria to be supported and survive in the crop or gut of the
fly. We will provide adult houseflies with foods inoculated with one of four treatments including
a control and either Salmonella spp., L. monocytogenes, or C. jejuni selected for study by Altier
and Kathariou labs. Housefies will be provided with the selected treatment at a single feeding.
Flies will be sacrificed at 0.5, 1, 3, 6, 9, 12 and 24 hr intervals and subsequent days. Insects will
be surface sterilized and the crops removed aseptically and placed intominimal medium. Crop
tissues will be homogenized and serially passed on selective medium. Cultures will be
confirmed by methodology developed presented under objective 2.
        3.2.1b. Evaluate the potential of housefies to transmit bacteria to eggs: Housefies will be
provided with foods inoculated with pure cultures of SLC. Flies will be held in cages. Fresh
eggs will be collected from laying hens while moist. The moist egg will be placed in an
                                                                                                      13

inoculation cup in the fly cage to allow the fly to come to the surface of the moist egg. Eggs will
be removed after 5 minutes in the cage. A group of exposed eggs will also be treated with
quaternary ammonia to determine the effect of disinfectants on the passage of bacteria into the
egg. Treatments will be compared to positive and negative controls. Eggs will be incubated for
7 days to allow the embryo to develop at which time it will be sacrificed for culturing and
identification of bacteria.

3.2 .2. Evaluate the inter-relationship between Salmonella spp., Listeria monocytogenes, and
Campylobacter jejuni fecal shedding, and muscoid fly prevalence in meat bird production.
         3.2.2a. Monitor the movement and dispersal potential of muscoid flies in the
transmission of selected strains of bacteria within and between farms: Housefly associated
bacterial cultures will be identified using phenotypic/genotypic characterization as described
previously. Adult flies will be collected from five broiler farms and five turkey farms (10 total)
monthly using sweep-net or sticky cards. Freeze killed flies will be surface sterilized and the
crops aseptically removed. Fly crops will be stored in minimal medium until homogenized and
tissues serially passed and plated on selective medium. Cultures of bacteria will be identified
according to the methods previously described.
         3.2.2b. Monitoring of the density dependent relationship of flies to bacterial prevalence
in adjacent poultry houses on the 10 farms above (a): The relationship of fly, bird, and bacteria
will be directly compared using phenotype/genotype characterizations described previously.
Muscoid fly densities will be monitored using spot cards and sticky cards (Lysyk and Axtell
1986, and Hogsette et al. 1993). Spot cards will be placed for 7 days and sticky cards for 24
hours. Fecal samples (cloacal swabs) will be taken from 10 birds randomly selected from the
flock. Fly and fecal samples will be stored in whirl-pac bags and transported on dry ice to the
laboratory for culturing and characterization.
         3.2.2c. Seasonal prevalence of foodborne and antibiotic resistant foodborne bacteria
within poultry houses. The role of the housefly in the transmission of foodborne bacteria is
limited by the seasonal abundance of pathogens and the flies themselves. During this monitoring
period we will obtain data on the seasonal abundance, prevalence and transmission potential of
flies for these important bacteria. Using the aforementioned methodology we will monitor flies
and the presence of characterized bacteria throughout the longitudinal study.

4. Determine risk factors associated with foodborne pathogens.
        Each of the 90 farms included in the cross-sectional study will be surveyed using a
standardized pre-tested questionnaire designed to obtain information such as: pets, breeder flocks
and age of breeder flocks, litter condition, treatment of litter, farm traffic, mortality disposal, etc.
Water properties and water activity of the litter will also be assessed at the time of the survey
visit (Hayes et al., 2000). The dependent variables will be the foodborne pathogen status of each
farm based on the sampling performed for objective 2.

Field trial as part of the assessment of the role of breeder flocks on foodborne pathogen
contamination:
        The NCSU-CVM Teaching Animal Unit (TAU) raises 5000 broilers per year in an
isolated unit using all-in-all-out production. This facility is unique, in that it is well isolated and
is managed under optimal biosecurity standards. It is located at least 20 miles from other
domestic poultry.
                                                                                                 14

        A breeder flock will be identified and sampled for the presence of Salmonella spp. and
Campylobacter spp. Progeny of this breeder flock will be placed at the TAU (no other breeder
flock contributing to this grow-out flock). Likewise, 20,000 chicks from the same breeder flock
will be placed in one commercial house in eastern North Carolina to serve as a paired flock from
the original breeders. Flock and environmental sampling will be performed as previously
described at time of placement and weekly thereafter until processing and cultured for
Salmonella spp., Campylobacter spp., and Listeria. These isolates will also be characterized as
described above.
        All isolates will be saved and compared to determine 1) the relationship between
pathogens isolated from the breeder flock and the paired grow-out flocks; 2) when positive
samples are first obtained for both broiler flocks; and 3) duration of the positive status for
specific pathogens. Finally, we will test pigs housed near the TAU poultry unit for the presence
of the same organisms.

5. Determine the impact of a biosecurity extension program on foodborne pathogens at the farm
level.
        In this study, we will assess the effectiveness of the biosecurity program by
characterizing isolates collected pre- and post-intervention. We will also conduct pre/post-
intervention assessment of pest status on selected farms. Finally, we will compare the two
cohorts intervention versus non-intervention groups: a sample size of 40 positive versus 20
negative farms will allow the detection of a significant factor given a risk ratio of 3 (based on a
95% confidence and 80% power) (Epi-info 2000, Center for Disease Control and Prevention).
This would be for farm level factors. However, we will have data on 120 chicken flocks and 60
turkey flocks. The analysis at the flock level will use adjustment for clustering (farm) as
described by McDermott et al., 1994)
        The biosecurity program may be immediately recognized as effective if the numbers of
pathogenic bacterial present in the birds or the environment are greatly reduced. However,
stronger conclusions about these measures may be possible based upon genotypic analysis of
cultured organisms. For example, pre-intervention flocks may carry Salmonella of a number of
serovars representing many strains. A successful intervention program might not only reduce the
total number of bacteria, but also decrease flock contamination from an outside source, a fact
that could be shown by the clonal nature of remaining strains by genomic fingerprinting.

6. Adjust our biosecurity and sanitation extension program, integrate the research findings in our
educational programs, assess their value as learning tools and establish a communication
network for the purpose of providing access to the information to all stakeholders.
         Findings of these studies will be developed into recommendations and made available to
growers, extension agents, and integrators in a timely fashion and a variety of formats (written,
electronic, presentation, etc.) to facilitate their inclusion in pathogen reduction activities.
Selected forms of information will be provided in both English and Spanish. The information
will become part of a media asset management system (Cumulus, Canto Inc.) and database
currently being implemented by D. Ley at NCSU-CVM. Target audiences for this and other
forms of information transfer will be: growers and their employees; company farm service
people; county extension agents; other relevant integrator representatives (e.g., feed delivery and
farm maintenance personnel); poultry industry associations (e.g. National Turkey Federation,
etc.), allied poultry industries (e.g. biologics and pharmaceutical companies), State and Federal
                                                                                                  15

animal health and diagnostic officials, and clinical and research experts. Modular instructional
material is being developed through cooperation with NCSU Faculty in Food Science, Poultry
Science, Entomology, Agriculture and Resource Economics, and the CVM. The material will be
delivered as a formal on-line and classroom course for students in Poultry Science, Food
Science, Entomology, Veterinary Medicine, or for on-farm HACCP certification of industry
personnel (growers, integrator service/management personnel, extension). Individual modules
may also be utilized as stand alone training packages for training growers, integrators, agents,
etc. and can easily be modified based on research findings.
         A recent survey of the North Carolina poultry industry revealed that most integrators
realize the need for pre-harvest pathogen reduction training. Approximately half of those
surveyed requested that if offered, they would prefer to receive training via the Internet.
Additionally, the industry expressed a need for graduates who are certified in HACCP training (9
CFR Part 304, et al. Pathogen Reduction; Hazard Analysis and Critical Control Point Systems,
Final Rule).
         Where appropriate evaluation is practical, a pre-test/post-test retention assessment will be
conducted and tabulated for each module and according to each educational delivery method and
student background. Smaller focus groups will be organized to test short term and long term
retention (written questionnaire and interviews). We will also develop a questionnaire to gather
user opinions of the material (Web based information; printed materials; guide sheets; manuals;
multimedia).
         A continually updated directory of stakeholders and resources will be established and
maintained at the lead institution (NCSU) with input from all partners. This information will be
compiled into computer-based information management systems or databases. The primary
storage devices will be NCSU-CVM servers. The software programs for these purposes are
already available to the investigators and/or central service facilities (e.g. Biomedical
Communications at the NCSU-CVM). These databases and associated software will facilitate
effective development and dissemination of information by traditional media (e.g. fact sheet,
newsletters, 35mm slide sets and video tapes) and Internet-based electronic media (e-mail lists,
list servers, Web pages, CDs etc.).

Data entry and analysis:
        All data will be entered into a database file (STATISTIX, version 7; Analytical Software,
Tallahassee, Fla.). Simple descriptive statistics (frequency distribution, mean, standard deviation,
minimum, median, maximum) will be done using STATISTIX. The data will then be exported to
a formatted ASCII file. The Statistical Analysis System (SAS Institute Inc., Cary, NC) will be
used for multivariate analyses. Given dichotomous outcomes [presence versus absence of a
pathogen], logistic regression will be used. This will include a two-step procedure: first,
univariate analyses will be conducted to screen independent variables. Pearson’s chi-square test
will be used to screen categorical variables. Continuous variables will be regressed on the
outcome using unconditional logistic regression. A p-value of < 0.15 will be considered to enter
the multivariate analysis (backward stepwise logistic regression). Flock level analyses (with up
to 6 flocks per farm) will be adjusted for clustering (farm).

Potential problems and limitations:
        Although farms will be divided into two cohorts for the longitudinal study, it is possible
that growers from the intervention cohort (biosecurity training) will communicate with growers
                                                                                                16

from the non-intervention (status quo) group. Thus, there could be transfer of intervention
strategies to this cohort. This could effectively minimize the opportunity to detect a significant
difference between the two groups. We should be able to determine whether or not this occurred
when we survey both groups at the end of the project.
        Identification of stakeholders and compiling information into appropriate computer
databases are primarily clerical efforts requiring time but are otherwise easily achievable. The
challenge is to keep the information up to date and inclusive, which also requires constant
attention and time. Collating and developing biosecurity/foodborne pathogen-related
information for development as educational material will require coordination, and contributions
from the research partners. Dr. Ley (NCSU-CVM) has agreed to coordinate all of the activities
under this specific aim. The limitation of what can be accomplished will be imposed primarily
by time and money. Therefore, realistic priorities have been set to include newsletters and
informational Web pages. Electronic communications and dissemination of educational
materials is likely to have the most cost effective bottom line, and be readily achievable by the
project partners. However, some stakeholders do not have the Internet access necessary to take
advantage of electronic communications. This limitation may be partially overcome by
encouraging stakeholders to ‘buy in’ to the necessary technology, or by making such technology
accessible. For example, a poultry company could make a computer terminal with access to the
internet (at its main office) available to growers.

Cooperation and Institutional Units Involved:
        Three institutions are involved in the proposed project. North Carolina State University is
the lead institution where researchers and extension specialists from 5 different departments are
collaborating. This group has field and laboratory expertise in all key disciplines (epidemiology,
microbiology, molecular biology and epidemiology, pathology, entomology, poultry science,
communication, and extension). In addition to this core group, two members of the North
Carolina Agricultural and Technical (NC A&T) State University (Worku and Willis) and one
from Michigan State University (Ryser) complete the project team. The NCSU group requested
the participation of Drs Worku, Willis, and Ryser. Dr Ryser has collaborated with Dr Kathariou
on other Listeria projects. His laboratory can perform analyses not available in Dr. Kathariou’s
laboratory. Drs Willis and Worku are from a historically black institution located in Greensboro,
North Carolina. This institution is located close to farms that will be included in the study. But
more importantly, and like the NCSU group, they have a well-established relationship with the
poultry industry. Dr. Willis’ research on litter and sanitation complements our field work. Dr
Worku will characterize at the molecular level the Campylobacter isolates collected by Dr
Willis. All data generated by the three institutions will be merged to cover our research
objectives. Finally, since the NC A&T State University is part of the cooperative extension
system, the extension and educational programs developed during this project can be
implemented there as well.

Equipment and Facilities:
All equipment and facilities needed for this project are already in place in the 5 departments
associated with this proposal.
Laboratory facilities: The microbiologists participating in this study each have laboratories
equipped fully for bacterial culturing and identification, and for molecular biology. One of them
(C. Altier) is also the director of the Clinical Microbiology Laboratory at the NCSU College of
                                                                                                17

Veterinary Medicine where bacterial identification and susceptibility testing will be performed
using a Vitek computerized bacterial identification and susceptibility system (bioMerieux Vitek,
Inc.). AFLP analysis will be performed using a Li-Cor 4000 sequencer maintained by the
College of Veterinary Medicine. All of the software required for gel analysis is maintained on
site, and is available to us.
Computer hardware and software: The department of Farm Animal Health and Resource
Management already has all the computers and software packages to enter the data (Excel
spreadsheet) and complete the statistical analysis (SAS; Statistix).
Communication equipment and facilities: The College of Veterinary Medicine at North Carolina
State University houses the Biomedical Communication Group. This group has two professional
photographers, four multimedia specialists with expertise in web design, one professional video
specialist, and two additional technicians. These professionals already have the network of
computers (including an in-house server dedicated to the storage of images) and the necessary
authorwares required to produce the extension and educational training programs. In addition,
the Learning Technology Service Department at NCSU provides extensive support for on-line
course development and improvement. Mr. Gene Lambert from Paradigm Media in California is
the communication specialist leading the technical production of the biosecurity and sanitation
material funded by the United States Poultry and Egg Association, and his company also has all
the needed equipment and facilities for multimedia production and post-production.
Transportation: North Carolina State University has an extensive motorpool available for farm
visits. The state of North Carolina motorpool will also be available if needed.
Access to chicken, turkey, and swine farms: We have a long-standing relationship with poultry
companies in the state. They have all participated in field research with our institutions for well
over a decade. More recently, they were involved in epidemiological investigation on respiratory
and enteric diseases that included a survey of all farm employees; sampling (blood, feces,
tissues) of affected and non-affected birds; and the communication of all laboratory results
(including farm names and locations) to all other poultry companies in the region. All turkey
companies are also involved in swine production. A few also own slaughter plants. Therefore,
we do not anticipate any restrictions for farm and processing plant access.

Project Timetable:
Development of biosecurity multimedia training program: Ongoing; completion date: May 2002
Selection of farms based on company data: December 2001 – January 2002
Testing of flocks on selected farms to confirm status: November 2001 – February 2002
Survey of farms (cross-sectional study): March 2002 – September 2002
Selection of Salmonella-positive farms for prospective study: September 2002 – October 2002
Application/evaluation of biosecurity training program (extension): October 2002 – March 2003
Sampling and surveys for prospective study: November 2002 – March 2004
Data entry (including validation): November 2002 – May 2004
Analysis: May 2004 – July 2004
Clinical trials: February 2002 – February 2004
Production of next generation of extension and educational programs based on cross-sectional
and prospective study: May 2004 – September 2004
Evaluation of extension and educational material: September 2004 – December 2004
                                                                                                         18

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                                                                                                    20


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Key personnel:
Jean-Pierre Vaillancourt (Epidemiologist): Principal investigator. Responsible for the design of
the biosecurity extension program. Responsible with others for the development, pre-testing, and
administration of surveys and the conduct of the cross-sectional and longitudinal studies. (Time
commitment: 30%).
Jesse Grimes (Extension Poultry Specialist) Dr Grimes will be involved with the teams
involved in the epidemiology/survey work directed at the turkey, the turkey hatchery and turkey
breeder segments. He will also be a member of the team that has responsibility for taking the
research findings and utilizing them in development of the educational components directed at
reducing the incidence of microbiological food safety hazards from contributing to consumer
illness. This will involve development of published bulletins, multimedia formatted information
and incorporation of the information into a distance leaning course emphasizing on farm HACCP
principles. (Time commitment: 9%)
Michael Wineland (Extension Poultry Specialist): Dr Wineland will be involved with the teams
involved in the epidemiology/survey work directed at the broiler, the broiler hatchery and broiler
breeder segments. He will also be a member of the team that has responsibility for taking the
research findings and utilizing them in development of the educational components directed at
reducing the incidence of microbiological food safety hazards from contributing to consumer
illness. This will involve development of published bulletins, multimedia formatted information
and incorporation of the information into a distance leaning course emphasizing on farm HACCP
principles. (Time commitment: 9%)
Wes Watson (Entomologist): Research and Extension. Dr. Watson is responsible for the
entomological component of the study, pest surveillance, preliminary isolation and culturing of
bacteria, coordination of the graduate students program. Results of this study will be utilized in
Medical Veterinary Entomology courses taught by Dr.Watson, and extension programs
coordinated and developed by Livestock and Poultry Extension Entomologist, Mike Stringham.
(Time commitment: 15%)
                                                                                                 21

Michael Stringham (Entomologist): Co-PI. Responsible for arthropod and rodent sampling
studies with W. Watson. Responsible with others for the developoment and pre-testing of
surveys and development and delivery of Extension training programs and assessment (Time
commitment: 10%)
Craig Altier (Microbiologist): Responsible for identification and characterization of Salmonella
obtained in this study, including both phenotypic analysis and molecular fingerprinting of
isolates (Time commitment: 10%).
Wondwossen Gebreyes (Molecular epidemiologist): Co-principal investigator. Responsible for
isolation, identification, phenotypic characterization and genotypic fingerprinting of Salmonellae
and Campylobacter, analysis of strains to study diversity of the pathogens and identification of
risk factors, and study the impact of biosecurity measures on the selection of strains. (Time
committment: 20%)
David Ley (Microbiologist and media asset management): Co-investigator. Responsible for
information and media asset management. Responsible with others for communications with
stakeholders and development of extension and distance learning content, particularly via
electronic media (internet, WWW). Responsible with others for implementation of DNA
fingerprinting gel analysis system and database, and molecular epidemiology (Time
commitment: 10%)
John Barnes (Pathologist): responsible to determine flock health status (pathology lab) for
selected farms; also involved in the educational and extension programs (Time commitment 5%).
Andrea Miles (Poultry veterinarian and outreach professional): Involved in development of on-
line pre-harvest food safety course, member of NCSU Poultry Coordinating Committee. Will act
as liaison between extension team, research team and poultry companies in delivery and revision
of educational programs (Time commitment: 5%).
Dennis Wages (Poultry veterinarian, director of the Teaching Animal Unit, NCSU): Co-
Investigator: Responsible for sample collection in and use of Teaching Animal Unit flocks for
bacterial monitoring and isolation (Time commitment: 5%).
Sophia Kathariou (Microbiologist): Responsible for the isolation and characterization
(including molecular subtyping) of Campylobacter from poultry fecal dropping and carcass
samples. (Time commitment: 10%)
Elliot Ryser (Microbiologist, Michigan State University): Collaborator. Responsible for Listeria
isolation, identification and characterization by serotyping and pulsed-field gel electrophoresis in
conjunction with the Michigan Department of Community Health (Time commitment: 5%).
Willie Willis (Poultry scientist, A&T): Co- investigator. Responsible for conducting litter and
live haul studies at the various production sites. Data will be collected on the prevalence of
Campylobacter jejuni in chicken and turkeys reared on used and unused litter. Seasonal
varability will be observed. Additionally, sanitation in the live haul division will be assessed.
(Time commitment: 10%).
Mulumebet Worku (Biotechnologist, A&T): Dr Worku will work with Dr Willis for
Campylobacter studies (molecular characterization of strains). (Time commitment: 5%)
                                                                                              22



Collaborative and/or Subcontractual Arrangements

The collaboration between the three institutions is described in the text of the grant and under
the “Cooperation and Institutional Units Involved” and the “Key Personnel” sections. Please see
the letters from Drs. Willis, Worku and Ryser (attached).

				
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