Project Number: s-295 Title: Enhancing Food Safety Through Control of Food-borne Disease Agents Duration: October 1, 2000 to September 30, 2005




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НазваниеProject Number: s-295 Title: Enhancing Food Safety Through Control of Food-borne Disease Agents Duration: October 1, 2000 to September 30, 2005
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Project Number: S-295


Title: Enhancing Food Safety Through Control of Food-borne Disease Agents

Duration: October 1, 2000 to September 30, 2005

STATEMENT OF THE PROBLEM


In 1999, the Centers for Disease Control and Prevention (CDC) reported new, more accurate estimates of food-borne illnesses that occur annually. There are an estimated 76 million cases of food-borne illness, 325,000 hospitalizations, and 5,000 deaths each year from food-borne diseases (Mead et al., 1999). The new surveillance system, FoodNet, indicated that there were more cases of food-borne illness occurring, but fewer deaths were caused by food-borne disease agents than previously reported. Campylobacter spp. was responsible for the most cases of food-borne illness while Salmonella (nontyphoidal) caused the most deaths with Listeria monocytogenes also causing a significant number of deaths. Overall, the report indicates that food-borne pathogens have a significant impact on human health and the food industry in the United States.

In addition to human suffering, food-borne illnesses also have a substantial economic impact in the United States. It is estimated that the annual cost of food-borne illness in the U.S. is $5-$6 billion for loss of productivity and medical expenses (Marks and Roberts, 1993). The most costly food-borne illnesses are caused by Toxoplasma gondii, Salmonella spp., Campylobacter spp., and enterohemorrhagic Escherichia coli.

New methods to prevent, reduce or eliminate food-borne disease agents at all points of the food chain, from “farm to fork”, need to be investigated to improve the safety of the food supply to prevent illnesses and deaths and to prevent economic losses to the food industry.

JUSTIFICATION


Across the nation there are numerous scientists who study food safety. Some study the behavior of pathogens at the pre-harvest stage of the food chain while others focus on preventing food-borne pathogen occurrence in the food product itself. A cooperative effort among these scientists can result in a reduction of the number of pathogens in the food supply and in the number of deaths and food-borne illnesses that occur annually. Live animal research, the pre-harvest segment of the food chain, is the first step in reducing pathogens in the environment.

A pathogen commonly associated with production agriculture, especially cattle, is E. coli O157:H7. E. coli O157:H7 is a member of the enterohemorrhagic group of pathogenic E. coli (EHEC). This agent has emerged as a food-borne and waterborne pathogen of humans that causes hemorrhagic colitis, hemolytic uremic syndrome, and thrombotic thrombocytopenic purpura. Outbreaks involving undercooked ground beef (Bell et al., 1994) and a variety of other foods including salami (CDC, 1995) and apple cider (Besser et al., 1993) have been documented.

Most human disease outbreaks caused by this organism have been linked to bovine fecal contamination of food or water. E. coli O157:H7 inhabits the intestinal tract of cattle, but it is unclear whether it actually colonizes the bovine intestinal tract (Shere et al., 1998). Reducing this organism in cattle prior to slaughter may reduce contamination of waters subject to runoff and of food produced in the processing plant.

Scientists participating in this project will form a multi-state team to study the impact of bacteriophage on E. coli O157:H7 occurrence and survival in live cattle. Bacteriophage are viruses which infect bacteria, and use their host to make multiple copies of themselves. Because they target specific receptors on the bacterial surface, phage are often strain-specific. Scientists at Auburn have identified several bacteriophage that inhibit E. coli O157:H7, but have no effect on other strains of E. coli. This limited host specificity is the basis for phage typing of bacterial strains. It also provides an advantage in targeting a species of pathogenic bacterium in an animal while leaving commensal flora unaffected. Thus, selected phages could be used prophylactically to protect an animal against infection with a specific pathogen, or therapeutically to remove an infectious agent from an animal. Phage can be grown to high titer inexpensively, facilitating their use in animals to reduce E. coli O157:H7.

One major deficiency preventing pathogen removal is the lack of knowledge of the epidemiology of O157 in nature, including definitive data regarding what triggers shedding of the organism from infected cattle. Antibiotic usage in cattle is widespread, both to treat infections (therapeutic) and to promote growth (sub-therapeutic). We hypothesize that therapeutic and sub-therapeutic antimicrobial treatment of cattle colonized with E. coli O157:H7 perturbs the normal bovine gut flora, resulting in a modulation in shedding frequency and quantity of the pathogen, followed potentially by transmission to new hosts, and contamination of food and water. We suspect that some antimicrobial drugs will enhance E. coli O157 shedding from calves, while others will not. The application of this research could help veterinarians more carefully choose both the type of antimicrobial drug to use to treat common bovine infections or to promote growth, and the timing of administration of these drugs, thus providing a pre-harvest food safety critical control point for reducing O157 in meat and uncooked foods such as fruits and vegetables contaminated by bovine feces.

In addition to E. coli O157:H7, there is a consensus among the scientists that we must work toward the control of salmonella and other pathogens at the pre-harvest stage. The occurrence of the pathogen is related to a number of factors including the innate resistance of the animal, the infectious dose, the virulence and the serotype. Certain measures can and have been used to successfully lower or abolish the incidence of salmonellosis. These efforts are very much dependent on improvements in management. Environmental stresses, poor feed or nutrition, improper sanitation and animal density all play a vital role in salmonella control. Because of the great diversity in the manner in which pathogens spread no one single solution will solve the problem. As in cattle, broad-spectrum antibiotics are commonly used in most species, but with variable results. Prophylactic use of antibiotics in animal feeds has also come under fire by many consumer groups with concerns over the increasing number of antibiotic resistant strains. Other options available to the producer for control of potential pathogens include: early weaning to remove the young animal from possible contamination from the adult, higher pelleting temperatures, acidification of the feed or water with propionic or other organic acids, identification and removal of carrier animals, improved sanitation in the facilities and nutritional manipulation of the gastrointestinal tract through the use of oligosaccharides, probiotics or competitive exclusion products. A multi-state project will allow several scientists to study different factors under the same conditions to determine which ones are most effective in reducing the pathogen in the live animal.

Although a large majority of publicity and research has focused on pre-harvest food safety in animal foods, fresh produce has been implicated in major food-borne illness outbreaks (Rasmussen et al., 1993; Besser et al., 1993; Beuchat, 1996; Griffin & Tauxe, 1991; Neill et al., 1994). An increasing number of these outbreaks have been directly linked to fecal contamination of fresh and minimally processed produce. USDA/FDA guidelines for fruit and vegetable production dedicate an entire section of the proposal to the proper handling of treated and untreated manure. The use of untreated manure on produce crops carries a higher risk of contamination as compared to treated manure which has markedly reduced levels of pathogens. The contamination with fecal pathogens poses a potential threat to cause food-borne illness if sufficient levels of viable pathogens are contained on the produce. In an attempt to reduce this risk, the guidelines suggest incorporating raw manure into the soil prior to planting as well as maximizing the time between manure application and harvest. Additionally, holding the manure for several days may result in a natural reduction of pathogens. Initially, the draft USDA/FDA guidelines recommended holding times of 60 days. This recommendation was taken from the Organic Food Production Act of 1990, which was based on pathogen survival in feces. Subsequent research has shown that E. coli O157:H7 is capable of surviving for more than 70 days in feces (Wang et al., 1996). The research performed thus far has investigated the survival and growth of pathogens in feces or composting manure which do not accurately depict the survival of pathogens in untreated manure when applied on cropland and exposed to various environmental stress factors such as UV, moisture and temperature fluctuations and a mixed, competitive, microbial flora.

Both organic and nonorganic produce growers utilize untreated manure as a source of crop fertilizer. This widespread use of untreated manure is deemed "essential" by the produce growers and "potentially hazardous" by food safety experts. The response of produce growers across the country was strong opposition to the implementation of guidelines for untreated manure because of the lack of research that was based on the recommended holding time for untreated manure on crops. Both growers and government officials admit to the need for additional research regarding untreated manure before implementation of these guidelines. Thus far, food safety research has been directed more towards inhibiting food-borne pathogens after the farm rather than at the farm, where a major portal exists for the entry of pathogens into the food system. Much of the founding work for pathogen survival in manure was performed prior to the emergence of present day pathogens that plague the food industry. E. coli O157:H7 is an example of a new and virulent strain, mutated from a once nonpathogenic and commonly isolated bacterium (Riley et al., 1983). This new virulent strain is also known to have physical characteristics such as enhanced acid tolerance that has changed it's potential in causing food-borne illness in foods that before were thought as safe with adequate protective measures due to the acidic nature of the product. It is not known whether E. coli O157:H7 also has an increased resistance level to the conditions found in untreated manure and the environmental factors that exist when spread onto cropland. There is a critical need for the establishment of minimum holding times for untreated manure when applied to cropland that will be used to produce minimally processed and fresh fruit and vegetables. The results of this work will provide viable data to support guidelines for untreated manure holding time standards that will ensure inactivation of key food-borne disease causing pathogens that may be introduced onto fruit and vegetable crops during manure application. With the increasing concern of the safety of imported produce, these results could also be extended to support recommendations for countries exporting produce to the United States.

While E. coli and Salmonella are major concerns in pre-harvest food-safety, Campylobacter jejuni also causes a spectrum of diseases including gastroenteritis, proctitis, septicemia, meningitis, abortion, arthritis and Guillain-Barré syndrome (GBS) (On, 1996) and originates in the pre-harvest environment. Campylobacter was targeted as one of the four most important food-borne pathogens in the United States based on the number of reported cases and their severity (CDC, 1994; FSIS, 1995; Buzby and Roberts, 1996; ASM, 1997). The number of cases of Campylobacter enteritis is estimated at 1.1 to 7 million per year making C. jejuni and C. coli the most commonly isolated enterobacterial pathogens (Buzby and Roberts, 1996; ASM, 1997; Mead et al., 1999). Campylobacters are estimated to cause nearly 100 deaths annually in the US (Mead et al., 1999). The combined annual costs of human illness due to Campylobacter infection was reported in the range of $1.3 to $6.2 billion (Buzby and Roberts, 1996, 1997).

Contaminated poultry is the most common route of transmission from animals to humans (Altekruse et al. 1997; Konkel and Cieplak, 1996; Harris et al., 1986). Smith et al. (1999) observed an increase in fluoroquinolone (FQ) resistant C. jejuni poultry isolates following approval for veterinary use in the US in 1995. Nearly 90% (80/91) of the market chickens contained Campylobacter and 20% of these chickens (18/91) contained Campylobacter isolates resistant to at least 32 ug/ml of Cip. Resistant isolates were detected in birds from 15 processing plants located in 9 different states suggesting that FQ resistance is becoming widespread. A more comprehensive surveillance can be conducted through collaborations developed in the regional project. C. jejuni infections in immuno compromised human patients and other susceptible individuals which result in severe gastroenteritis or infections of deep tissues, require antibiotic treatment for successful resolution (Adler-Mosca et al., 1991). Failure of antibiotic therapy due to development or transfer of antibiotic resistance is extremely important because these severe infections are the largest contributors to the health and economic impact of this foodborne disease agent. Erm and Cip, are the antibiotics of choice for treatment of severe C. jejuni infections (Konkel and Cieplak 1996; Smith et al., 1999). The 1998 report of the National Antimicrobial Resistance Monitoring System (NARMS) reported that of the 332 C. jejuni isolates submitted, 181 (54%) were resistant to antibiotics surveyed including Cip (MIC 4 ug/ml; 13.3%) and Erm (MIC 8 ug/ml; 2.2%). Little is known about the ability of Campylobacter to transfer chromosomally mediated Cip or Erm resistance to susceptible strains. Trieber and Taylor (1999) reported transfer of ErmR from resistant to susceptible isolates via natural transformation using naked genomic DNA but transfer efficiencies were not reported. The ability of C. jejuni and C. coli to develop or acquire Erm and Cip resistance via spontaneous mutation or transfer of resistance genes can result in failed antibiotic therapy. Since FQs are among the antibiotics of choice for treatment of infections due to Salmonella, Shigella, and Vibrio (Dryden et al.,1996) antibiotic resistant Campylobacters have the potential to serve as reservoirs for acquired resistance for these important enteric pathogens.

CipR in C. jejuni. In C. jejuni, the primary target for nalidxic acid (Nal) and the FQ antibiotics appears to be DNA gyrase (Heisig and Tschorny, 1994; Wiedemann and Heisig, 1994; Gaunt and Piddock, 1996; Piddock, 1995b; Piddock, 1995a). Mutations in codon thr86 in the quinolone resistance-determining region (QRDR) in gyrA in C. jejuni are strongly associated with high-level CipR (HLCR; MIC 16 ug/ml) in the limited number of strains (30 out of 30 isolates) described to date (Charvalos et al., 1996; Husmann et al., 1997; Gaunt and Piddock, 1996; Gibreel et al., 1998; Ruiz et al., 1998). CipS isolates did not contain this mutation. However, a direct causal relationship between codon 86 mutations and HLCR has not been conclusively established.

Collaboration among the scientists in this group could establish this relationship by examining numerous isolates from pre-harvest and processing environments.

Recent food-borne disease outbreaks have also brought about increased consumer demand and subsequent regulatory pressure to improve the microbiological safety of raw food commodities from in food processing facilities. It has been well established that raw meat and poultry are prevalent sources of food-borne pathogens and vehicles of food-borne disease. To address the safety of meats and poultry, the USDA-FSIS has implemented new regulations (USDA, 1996). While not mandating the use of specific antimicrobial treatments, these new regulations call for pathogen reduction measures and set microbiological performance standards that all meat and poultry processors must meet. In addition to food safety issues attributed to raw meats and poultry, food-borne disease attributed to fresh fruits and vegetables has doubled over the last ten years. Increased consumption, greater geographical variety, and consolidation of agricultural production areas have, in part, contributed to this increase in produce-borne illness. While the prevalence of food-borne pathogens on fresh produce is typically lower than that encountered on raw meat and poultry, many fruits and vegetables, in contrast to meats and poultry, are consumed raw. The occurrence of human pathogens on raw food commodities can represent a substantial food safety risk to the consumer. Therefore, consumer demands and regulatory pressure have and will continue to challenge food producers and processors to take steps to further reduce or eliminate pathogens in their commodities.

Recently, there has been renewed and keen interest to develop antimicrobial treatments, most in the form of dips, washes, sprays, etc., that can be applied to raw foods to reduce or eliminate the pathogen load. For antimicrobial treatments to be commercially feasible, and therefore have impact on food safety, their efficacy must be clearly established. Unfortunately, it has been difficult to compare research findings to fully establish the efficacy of potential treatments. The difficulty in comparing results arises primarily due to differences in experimental procedures used from one researcher to the next, which often accounts for apparent variation in results from study to study. This lack of consistent results and difficulty in comparing results from different studies has proven to be an obstacle in the adoption of novel treatments in commercial processing. That is, few novel antimicrobial treatments have been adopted by food producers and processors. Thus, there is a need to standardize methods of evaluating antimicrobial treatments. A standardized tested method would be an invaluable tool in the development of efficacious and feasible antimicrobial treatments that can be applied to improve the microbiological safety of foods (Conner and Bilgili, 1994).

To standardize methodologies, inter-laboratory studies are required. A multi-state project provides an ideal structure under which this needed collaborative approach can be implemented effectively. Moreover, the current participants have been actively involved in evaluation of potential antimicrobial treatments, which provides a critical mass of researchers to conduct these inter-laboratory studies. Therefore, an objective of this project is to generate and compare data needed to develop (and recommend) a fundamental testing protocol for assessing the antimicrobial activity of potential treatments. Development of such a protocol would allow for evaluation of a wide range of treatments in a standard format in any laboratory, which in turn will allow for better comparison of results from one study to the next. Subsequently, a means of comparing results will facilitate the adoption of novel treatments that can be used by producers and processors to maintain and improve the microbiological safety of their products.

Another aspect of the new USDA/FSIS regulatory requirements with regard to pathogen reduction is the implementation of HACCP plans in meat and poultry processing facilities. Traditional approaches to food safety have focused on prevention of pathogen growth. Today, the low infectious dose required of some pathogens dictates that successful prevention must focus on reducing, controlling or eliminating the microorganisms with a Hazard Analysis and Critical Control Points (HACCP) plan. HACCP was initially developed by NASA and Pillsbury to provide the safest food possible for the space program (Bauman, 1992). The National Advisory Committee for the Microbiological Criteria for Foods have standardized the original HACCP concept into 7 step by step principles that can be used by the food processing industry to reduce, prevent or eliminate biological, chemical, and physical hazard from occurring in the final food product (NACMCF, 1998).

All meat and Poultry processors are required to implement HACCP plans by January, 2000 (USDA, 1996). A properly written, implemented plan should reduce hazards if the appropriate hazards were identified and the hazard analysis, critical limits, and corrective actions are based on sound, scientific data. However, much of the scientific information needed to develop scientifically sound HACCP plans is not available. Simple, easy to understand information needs to be developed to assist processors in setting critical limits and to understand the relationships between temperatures in the processing room and the temperature of the meat products and/or on the surfaces on which the products are processed. Greater understanding and control of effects of processing environmental conditions on meat safety during primal cut and ground product preparation and use will result from use of such visual aids.

Additionally, it is imperative to the success of HACCP to assess and validate the effectiveness of implemented HACCP plans by utilizing objective measurements of biological hazards to determine if the plan reduces the risk of food-borne pathogens such as Salmonella, E. coli O157:H7, Campylobacter, and Listeria monocytogenes. Processors may be operating under a HACCP system, but there is not scientific evidence that the plan is actually controlling hazards.

A multi-state approach to HACCP validation is ideal to develop the information needed for HACCP validation. Currently HACCP plans are based on data generated in controlled laboratory conditions. In-plant research provides actual measurements in the plant environment to determine if the HACCP plan is resulting in a safer product. Measurements are needed in a variety of different processing plant environments to determine if there is variation in data collected in different processing plants. Many of the scientists participating in this project have extension appointments and have a good working relationship with the food industry. This relationship provides an avenue into the plants to collect data.

The states participating are Alabama, Arkansas, Iowa, Kentucky, Michigan, Minnesota, Mississippi, Nebraska, New York, North Carolina, South Carolina, Tennessee and Virginia.

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