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

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.

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 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.

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).

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.

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.