SURVIVAL AND DISTRIBUTION OF Escherichia coli O157 IN BOVINE MANURE by ALEJANDRO ECHEVERRY, B.S.

A THESIS IN FOOD TECHNOLOGY Submitted to the Graduate Faculty of Texas Tech University in Partial Fulfillment of the Requirements for the Degree of MASTER OF SCIENCE

Approved

Mindy M. Brashears Chairperson of the Committee

John R. Blanton

Accepted

John Borrelli Dean of the Graduate School December, 2004

ACKNOWLEDGEMENTS

I would like to acknowledge and thank Dr. Mindy Brashears for helping me to initiate this research. My decision to pursue a Master’s degree was facilitated on the assistantship awarded by her that allowed me to travel from Colombia and work in her laboratory at Texas Tech University. Her advice, continuous support and encouragement while letting me focus on the research and work at my own pace was extremely important to me. She has been a close advisor, mentor and friend, and I grew both personally and academically under her supervision. I also want to show my appreciation to the other members of my committee, Dr. Guy Loneragan and Dr. John Blanton, for their time and contribution to this work. I wish to thank all colleagues and friends at TTU, specially Jason Mann and Karen Killinger-Mann for helping me with lab work and being close friends. I specifically want to thank Amy Hoyle for her valuable time reviewing and correcting this thesis and being a close confident and friend. Thanks to the sample collection team at WTAMU and student workers at the food microbiology lab at TTU for their assistance during sampling and microbiological processing. Lastly, I want to thank my family for their support, patience, and specially, for trying to understand the goals of my work and studies while abroad.

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TABLE OF CONTENTS

ACKNOWLEDGEMENTS………………………………………………………………ii LIST OF TABLES….............................….............…………………………………viii LIST OF FIGURES…..…………………………………………………………….......ix LIST OF ABBREVIATIONS……...……………………………………………….........x ABSTRACT…………………………………………………………………………......xi CHAPTER I. INTRODUCTION/LITERATURE REVIEW……............................……………1 1.1. Escherichia coli….……........………………………………………………1 1.2. Enterohemorrhagic Escherichia coli (EHEC) and Escherichia coli O157:H7 ..................................................................3 1.2.1. Characteristics............................................................................5 1.2.2. Pathogenicity..............................................................................6 1.2.2.1. Acid Tolerance...............................................................6 1.2.2.2. Thermal inactivation.......................................................9 1.2.2.3. Antibiotic resistance.....................................................10 1.2.3. Human Disease........................................................................12 1.2.3.1. Hemorrhagic Colitis (HC).............................................13 1.2.3.2. Hemolytic Uremic Syndrome (HUS)............................15 1.2.3.3. Thrombotic Thrombocytopenic Purpura (TTP)............17 1.2.3.4. Treatment and Outcome..............................................18

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1.2.4. Reservoirs of Escherichia coli O157:H7...................................20 1.2.4.1. Cattle............................................................................21 1.2.4.2. Domestic Animals........................................................22 1.2.4.3. Birds and Other Animal Sources..................................23 1.2.4.4. Humans........................................................................24 1.2.5. Survival of Escherichia coli O157:H7........................................26 1.2.5.1 Survival in Foods.........................................................26 1.2.5.2 Survival in Water...........................................................30 1.2.5.3 Survival in the Environment, Soil and Other Sources...32 1.2.5.4. Survival in Feces and Slurry........................................34 1.3. Disease Outbreaks.............................................................................38 1.3.1. Geographic Distribution............................................................40 1.3.2. Seasonality...............................................................................41 1.3.3. Susceptible Populations...........................................................43 Infective Dose..................................................................44 1.4. Escherichia coli O157:H7 Detection Methods....................................44 1.4.1. Enrichment Methods.......................................................46 1.4.2. Immunomagnetic Separation (IMS)................................48 1.4.3. Culture Methods..............................................................51 1.4.4. Latex Agglutination Test..................................................55 1.5. Preharvest Food Safety.....................................................................56 1.5.1. Importance................................................................................56

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1.5.2. Prevalence of Escherichia coli O157:H7 in feedlots and cattle....................................................................57 1.5.2.1. Prevalence in Dairy Cattle...........................................57 1.5.2.2. Prevalence in Beef Cattle............................................62 II. SURVIVAL OF ESCHERICHIA COLI O157:H7 IN BOVINE FECES UNDER VARIOUS STORAGE CONDITIONS.................66 2.1. Abstract..............................................................................................66 2.2. Introduction........................................................................................67 2.3. Materials and Methods.......................................................................71 2.3.1. Study Design ...........................................................................71 2.3.2. Sample Collection.....................................................................72 2.3.3. Strains Conditions.....................................................................73 2.3.4. Inoculated Sample Preparation................................................73 2.3.5. Control Sample Preparation.....................................................74 2.3.6. Microbial Analysis for Detection of E. coli O157:H7.................74 2.3.6.1. Detection of E. coli O157:H7 using immunomagnetic separation....................................................................74 Isolation of E. coli O157:H7..........................................75 2.3.6.2. Quantification of E. coli O157:H7 by direct plating.......76 2.3.7. Sampling Times and Storage Conditions.................................77 2.3.8. Statistical Analysis....................................................................78 2.4. Results...............................................................................................79 2.4.1. Cooler Results..........................................................................79

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2.4.2. Refrigeration Results................................................................80 2.4.3. Room Temperature Results......................................................80 2.4.4. 37°C Results.............................................................................82 2.5. Discussion..........................................................................................82 III. NON UNIFORM DISTRIBUTION OF ESCHERICHIA COLI O157:H7 IN BOVINE FECES AND UNDERESTIMATION OF PREVALENCE...........................................................................................89 3.1. Abstract..............................................................................................89 3.2. Introduction........................................................................................90 3.3. Materials and Methods.......................................................................95 3.3.1. Study Design ...........................................................................95 3.3.2. Sample Collection.....................................................................95 3.3.3. Microbial Analysis for Detection of E. coli O157:H7.................96 3.3.4. Statistical Analysis....................................................................97 3.4. Results...............................................................................................98 3.4.1. Overall Results.........................................................................98 3.4.2. Feedlot Results.........................................................................99 3.4.3. Final Results for Fecal Pats....................................................102 3.4.3.1. Final Results by Sampling Position............................102 3.4.3.2. Final results for sub-samples within fecal pats..........105 IV. GENERAL DISCUSSION/SUMMARY OF FINDINGS...........................112 Overview...............................................................................................112

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BIBLIOGRAPHY.........................................................................................119 APPENDIX..................................................................................................158

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LIST OF TABLES

1.1.

Examples of reported outbreaks and the number of cases of Escherichia coli O157:H7 infections worldwide.........................................39

2.1.

Least square means values for the population of E. coli O157:H7 (log10 cfu/g) in inoculated bovine feces held at different temperature conditions at each of the sampling times..................................................81

2.2.

Final IMS results for E. coli O157:H7 obtained from inoculated bovine fecal samples incubated at 37°C...............................................................83

3.1.

Distribution and prevalence of fecal pats positive for E. coli O157 in each feedlot involved in the study.......................................................100

3.2.

Distribution and frequency of the total number of sub-samples that returned positive E. coli O157:H7 results for each of the participating feedlots....................................................................................................101

3.3.

Estimated E. coli O157:H7 prevalence for any given number of sub-samples (positions) collected...........................................................107

A. 1

Probability of difference between Least Square Means (LSM) (Log10 CFU/g) for E. coli O157:H7 populations when comparing cooler results to the rest of the treatments during time.....................................................159

A. 2

Probability of difference between Least Square Means (LSM) (Log10 CFU/g) for E. coli O157:H7populations when comparing 4.4°C results to the rest of the treatments during time.....................................................160

A. 3

Probability of difference between Least Square Means (LSM) (Log10 CFU/g) for E. coli O157:H7 populations when comparing 37°C results to the rest of the treatments during time. ...................................................161

A. 4

Probability of difference between Least Square Means (LSM) (Log10 CFU/g) for E. coli O157:H7 populations results at 23°C during time.........................................................................................................162

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LIST OF FIGURES

3.1.

Total number of positive Escherichia coli O157:H7 sub-samples (By their respective position).................................................................................103

3.2.

Respective Escherichia coli O157:H7 prevalence for each of the positions sampled...................................................................................................104

3.3.

Cumulative prevalence of E. coli O157 in positive fecal pats according to the number of samples within the pat.....................................................105

3.4.

Variation in E. coli O157:H7 prevalence in bovine feces with number of samples collected....................................................................................108

A. 5

Sampling Methodology............................................................................163

A. 6

Change of E. coli O157:H7 population in inoculated bovine manure held under cooler conditions.....................................................165

A. 7

Change of E. coli O157:H7 population in inoculated bovine manure held under refrigeration at 4.4°C................................................166

A. 8

Trend of E. coli O157:H7 population in inoculated bovine manure held at room temperature conditions (23°C).......................................................167

A. 9

Viable E. coli O157:H7 counts in inoculated bovine manure after incubation at 37°C...................................................................................168

A. 10 Escherichia coli O157:H7 behavior in inoculated bovine feces at different temperature conditions (Superposition of Appendix 6 to Appendix 9 results)..........................................................................169

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LIST OF ABBREVIATIONS

CDC.................................................... Center for Disease Control and Prevention DAEC ...............................................................Diffusely Adherent Escherichia coli EAggEC........................................................... Enteroaggregative Escherichia coli EHEC............................................................. Enterohemorrhagic Escherichia coli EIEC...................................................................... Enteroinvasive Escherichia coli ELISA.......................................... Enzyme-linked-immunosorbent serologic assay EPEC ................................................................Enteropathogenic Escherichia coli ETEC.................................................................... Enterotoxigenic Escherichia coli FDA..........................................................................Food and Drug Administration FSIS ...............................................................Food Safety and Inspection Service HC ............................................................................................Hemorrhagic colitis HUS............................................................................ Hemolytic uremic syndrome IMS ............................................................................Immunomagnetic separation MAC............................................................................................. MacConkey agar SMAC .............................................................................Sorbitol MacConkey agar STEC..................................................................... Shigatoxigenic Escherichia coli Stx .........................................................................................................Shiga toxin TTP............................................................ Thrombotic thrombocytopenic purpura VBNC............................................................................... Viable but nonculturable VTEC ............................................................Verotoxin-producing Escherichia coli VTs .........................................................................................................Verotoxins

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ABSTRACT

Escherichia coli O157:H7 has become one of the most important emerging foodborne pathogens with many recent outbreaks being associated with cattle. Although estimates of E. coli O157:H7 prevalence in cattle have increased over time likely due improvements in detection methodologies, fecal collection methodologies and sample transport conditions from farm to microbiological laboratories for further analysis may be factors for underestimation of prevalence of this pathogen. In this study, a new sampling methodology was analyzed and comparison of survival of E. coli O157:H7 in feces of cattle under various experimental conditions was also determined. For the first part of the study, bovine fecal samples were inoculated with a cocktail of four different antibiotic resistant E. coli O157:H7 strains. Each inoculated sample was subdivided and subjected to each of the four following conditions: 37°C, room temperature (23°C), refrigeration temperature (4.4°C) and in plastic coolers with refrigerant packs in order to simulate transportation conditions. Samples from each of the temperature conditions were taken at 0 h, 24 h, 48 h, 120 h, and 144 h and subjected to detection and quantification of E. coli O157:H7. Overall, holding samples at temperatures equal to or below to 23° C resulted in detectable populations for up to 168 h. At 37oC, samples were not recovered by any of the methods used after 48 hours. These results indicate that

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holding samples at room temperature or lower for up to 168 h prior to processing will not reduce the pathogen’s population. For the second part of the study, the objective was to evaluate fecal pat sampling strategies to improve accuracy of E. coli O157:H7 prevalence estimates. A total of 120 fresh fecal pats from cattle were used in this study. From each fecal pat five samples were collected systematically going from West to East (positions 1 to 5 respectively) in north to south lines direction to avoid cross contamination and cultured for E. coli O157:H7 within two hours using IMS separation. Of the 120 fecal pats, 96 (80%) had no positive samples in any of the 5 samples. One sample was positive in 13 of the pats, 2 in 4 of the pats, 3 in 2 of the pats, 4 in 3 of the pats and only 2 of the pats had all 5 samples positive. Of the 600 total sub-samples analyzed, 49 were positive with 14, 9, 8, 8, and 10 on position 1, 2, 3, 4, and 5 respectively. An increase in the prevalence of 2.45-fold (from 8.17% to 20%) was observed when sampling 5 positions per fecal pat as compared to the estimated prevalence obtained when just 1 sub-sample was obtained. Prevalence estimates may be underestimated as a result of an uneven distribution in fecal material; therefore sampling procedure plays a critical role in E. coli O157:H7 detection in bovine fecal pats.

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CHAPTER I INTRODUCTION/LITERATURE REVIEW

1.1. Escherichia coli Escherichia coli is a microorganism described and characterized for the first time by Dr. Theodor Escherisch in 1885. It belongs to the Enterobacteriaceae family, with other microorganisms including Salmonella, Yersinia, Shigella, Citrobacter, Klebsiella, Enterobacter, and Proteus genera. The term Escherichia refers to the genus that is composed of gram-negative, aerobic, facultative anaerobic, non sporeforming rods, and the term coli to the species within the family. The food industry has been using non-pathogenic E. coli since the earliest 1900’s as an indicator of fecal contamination in water and milk (Bell et al., 1998), as well as an indicator of the sanitary conditions in the food processing environment. Non-pathogenic strains of E. coli are part of the normal enteric flora of humans and warm-blooded animals’ intestines, living as commensals in the bowel and being the predominant facultative anaerobe organism in the human gastrointestinal tract (Doyle et al., 1997). However, presence in the human bowel is believed to be less than 1% of the natural flora, a very small proportion of the total bacterial number in the bowel, with quantities around 108 cells/g of feces. Most strains are harmless to their host and their role in the body remains uncertain. Some studies suggest that E. coli serves a beneficial function in the body by synthesizing

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vitamins and by outcompeting other pathogenic bacteria that may be ingested with food or water (Fratamico, 2002). While most strains of E. coli are non-pathogenic, some strains can cause several different forms of gastroenteritis, each with its own symptoms and epidemiology. Pathogenic strains are divided into six different categories according to the mechanisms by which diarrhea is produced, virulence properties, pathogencity mechanisms, clinical syndromes and manifestations, and serological subgroups (Donnenberg et al., 2000; Doyle et al., 1997). The categories of the pathogenic strains E. coli are enteropathogenic E. coli (EPEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC), enteroaggregative E. coli (EAggEC), diffusely adherent E. coli (DAEC), and enterohemorrhagic E. coli (EHEC). The mechanisms by which diarrhea is produced vary for each type of E. coli and include attachment of the bacteria to the intestinal cells, invasion, and production of enterotoxins (Fratamico et al., 2002). Pathogenic E. coli isolates are classically differentiated on the basis of three surface antigens: the somatic lipopolysaccharide or cell – wall antigens (O), the flagellar antigens (H), and the capsular antigens (K). To date approximately 174 O antigens, 56 H antigens, and 103 K antigens have been identified (Doyle et al., 1997; Fratamico et al., 2002), with the combination of the different antigens defining the E. coli serotype. Nowadays, the use of serogroups together with other characteristics like biotype or enterotoxin production help in the differentiation of

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those strains that cause infections and illness in both humans and animals (Bell et al., 1998).

1.2. Enterohemorrhagic Escherichia coli (EHEC) and Escherichia coli O157:H7 Among the hundreds of strains that cause disease, those in the enterohemorrhagic E. coli (EHEC) group are the most notorious of all due the severity of the disease and number of cases and outbreaks related to it. Although relatively rare, the number of reported clinical cases from non-O157:H7 EHEC are increasing (Chapman et al., 1996; Hennessy et al., 2004; Karch et al., 1996; Paton et al., 1998). The EHEC group was identified for the first time as a cause of human disease when two consecutive outbreaks of hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS) in the United States were linked to the serotype O157:H7 in 1982 after consumption of undercooked hamburger patties in a chain of fast food restaurant (Riley et al., 1983). During the past twenty years, E. coli O157:H7 has emerged as a major disease causing pathogen, capable of causing high morbidity and mortality numbers among humans that become infected (Altekruse et al., 1997). Other serotypes that cause disease in the EHEC group include O157:NM (nonmotile), O103, O111:NM, O111:H8, O145, and O26:H11. Illnesses caused by these serotypes are similar to those caused by E. coli O157:H7.

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The EHEC group is also referred to in the literature as verocytotoxigenic E. coli (VTEC) or shiga toxin-producing E. coli (STEC) due the production of toxins that are closely homologous to the Shiga toxin (Stx) produced by Shigella dysenteriae type 1 (Renter et al., 2002). These toxins are also called verotoxins (VTs) or Shigalike toxin (SLT) due their ability to cause cytopathic effects, a block in protein synthesis causing cellular death in Vero cells from the African green monkey kidney cells (Doyle et al., 1997). Some reports estimate that more than 100 serogroups of E. coli produce these kind of toxins (Renter et al., 2002; Verweyen et al., 2000), while others studies report that it could be as high as 200 O:H VTEC serotypes, all of them isolated from animals, food, and the environment (Lindqvist et al., 1998). However, not all STEC are considered pathogenic and do not attach to epithelial cells. Other virulence factors are required to cause infection and disease in humans. Many verocytotoxigenic E. coli also have been found in healthy adult feces, but they haven’t been implicated in outbreaks (Donnenberg et al., 2000). Escherichia coli O157:H7 was first described in 1975 in California after it was isolated from a woman with bloody diarrhea, but its identification as an enteropathogen was not until two, nearly simultaneous, U.S. outbreaks during 1982 (Neill et al., 2001; Terajima et al., 2000). It is considered a serious threat to public health in developed countries. In the United States alone, the single greatest cause of hemorrhagic colitis and Hemolytic Uremic Syndrome (HUS) is E. coli O157:H7 (Altekruse et al., 1997; Peacock et al., 2001; Terajima et al., 2000; Williams et al.,

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1997) and more than 100 outbreaks of E. coli O157:H7 have occurred since 1982 (Varade et al., 2000).

1.2.1. Characteristics In the EHEC group, E. coli O157:H7 and E. coli O157:NM cause the majority and most severe outbreaks of gastrointestinal illnesses related to E. coli (Fratamico et al., 2002) from infections that range from asymptomatic conditions to mild bloody diarrhea or even severe hemorrhagic colitis. Severity of symptoms usually depends on the immune status of the person infected with the pathogen. Those who are immunocompromised suffer the most severe symptoms. Most of the strains of the EHEC group possess several unique characteristics not found in other E. coli strains: inability to grow well at temperatures ≥ 44.5°C; inability to ferment sorbitol within 24 hours; possession of virulence determinant factors that allow an intimate attachment or adherence to the intestinal cells within the host; and genes that encode for the production of very powerful verotoxins, which are immunologically and genetically related to those produced by Shigella dysenteriae (Doyle et al., 1997; Fratamico et al. 2002). These cytotoxins inhibit protein synthesis, causing damage to vascular endothelial cells in certain organs and even cellular death. Cytotoxins also contribute to the extraintestinal complications during infection with E. coli O157:H7. Another physiological difference important in isolation of E. coli O157 is the fact that it does not produce the enzyme

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β-glucuronidase. In typical E. coli isolation methods, this enzyme will cleave MUG into a fluorescent compound, but O157 isolates will not cleave this compound.

1.2.2. Pathogenicity Production of Shiga toxins is believed to be required for the occurrence of severe complications that are present during an EHEC infection (Donnenberg et al., 2000), but the precise mechanisms of pathogenicity are not completely clearly understood. It is believed that E. coli O157:H7 causes disease by adhering to the host cell membrane, where Stx 1 is produced after invasion of the host cells (Doyle et al., 1997).

1.2.2.1. Acid tolerance A critical factor that accounts for the pathogencity of E. coli O157:H7 is its ability to survive for a defined period of time at a pH that is lethal to others. This is considered an advantage and allows the bacteria to survive in foods with low pH or in the environment outside the host’s gastrointestinal tract. Foods that in the past were considered intrinsically safe in terms of pathogenic transmission due their low pH and acidity such as cheese, yoghurt, orange juices, and mayonnaise have been recently associated with outbreaks of E. coli O157:H7. In addition, research has shown the pathogen’s ability to survive on these acidic food products (Doyle et al., 1998; Meng et al., 1998a; Zhao et al., 1994).

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Apple cider, a beverage that normally has a pH between 3.0 to 3.8, was implicated in an outbreak of E. coli O157:H7 in southeastern Massachusetts in the fall of 1991, where twenty-three cases of infection were identified as hemorrhagic colitis (HC) and four children were diagnosed with hemorrhagic uremic syndrome (HUS) (Besser et al., 1993). At that time, apple cider differed from apple juice in the way it was processed. Apple cider remained unpasteurized while apple juice was pasteurized. In 1996, outbreaks in Connecticut and New York were also associated with the consumption of unpasteurized apple cider, affecting people with ages that ranged from 2 to 73 years. In that outbreak, E. coli O157:H7 caused illness in 66 persons and at least 1 death was a result of HUS, resulting in the largest outbreak caused by this pathogen through juice consumption (CDC, 1997). Although the apples were brushed and washed with potable municipal water and the cider was added with 0.1% of potassium sorbate, the preservative in the unpasteurized cider didn’t kill the microorganism which was later confirmed by an independent research study (Zhao et al., 1993). Survival studies have been conducted in various foods to address the acid tolerance properties of E. coli O157:H7. Inoculation studies have revealed survival under laboratory conditions for 35 days in buttermilk (pH 4.1), 12 days in yoghurt (pH 4.17 to 4.39), and 28 days in sour cream (pH 4.3) (Dineen et al., 1998). E. coli O157:H7 was recovered following fermentation, drying and storage of sausages (pH 4.5) for up to 2 months at 4°C, and in mayonnaise (pH 3.6 to 3.9) for 5 to 7 weeks at 5°C and for 1 to 3 weeks at 20°C (Doyle et al., 1997). Escherichia coli O157:H7 was

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found to survive in inoculated apple cider with pH values in the range of 3.6 to 4.0 from 10 to 31 days at 8°C, and from 2 to 3 days at 25°C (Zhao et al., 1993). Low pH values have shown little effect by themselves to inhibit E. coli O157:H7, so other processes must be used together with the acidic conditions of the food to achieve death of the pathogen. As a result of the E. coli O157:H7 apple juice outbreaks, the Food and Drug Administration (FDA) recommended juice processors adopt a hazard analysis and critical control (HACCP) program in order to identify potential hazards during processing and implement controls to reduce the likelihood of foodborne illness among consumers. Since September 8, 1998, the FDA requires that any container of apple juice, and apple cider that hasn’t been pasteurized or treated to bear a warning label that reads, “Warning: this product has not been pasteurized and, therefore, may contain harmful bacteria that can cause serious illness in children, the elderly, and persons with weakened immune systems.” in order to inform consumers of the risk associated with the consumption of untreated products. The same warning label was also required for all other juices after November 5, 1998 (FDA, 2004). However, there is still a risk associated to consumption of juice since small apple cider producers, who are unable to purchase a pasteurizer, are still allowed to produce unpasteurized cider as long as they comply with the warning label (Uljas et al., 1999).

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1.2.2.2. Thermal Inactivation Escherichia coli can grow at a temperature ranging from 7 to 46°C, with an optimum of 37°C. Several different undercooked and unpasteurized foods have been linked to E. coli O157:H7 outbreaks. One of the best known, most widespread outbreaks occurred in the U.S. between December 1992 and January 1993, involving Washington, Idaho, California, and Nevada. This outbreak affected 731 persons between 4 months to 88 years of age and resulted in 56 persons developing HUS and the death of 4 children. After an epidemiologic investigation, it was found that hamburgers contaminated with E. coli O157:H7 served at a fast food chain restaurant were the vehicle of infection. When reviewing the restaurant’s cooking procedures it was found that the internal temperature of the patties were below 60°C and the restaurant was not achieving the 68.3°C internal temperature required by Washington State (Bell et al., 1994; CDC, 1993). Presently, the required internal temperature when cooking ground beef is 71.1°C (160°F) as established by the Food Safety and Inspection Service (FSIS). Studies on the thermal sensitivity of E. coli O157:H7 in foods have revealed that the pathogen does not have a high resistance to heat. Pasteurization, or the process of treating liquid foods with heat for a specific period of time at a specific temperature, is an effective method to destroy pathogens. Thermal pasteurization conditions vary according to the beverage’s properties (Al-Taher et al., 2004) such as viscosity, solids content, texture, and heat transmission. Other conditions that may affect the time/temperature relationship in the pasteurization process are the

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desired final result such as retention of nutritional value, flavor, color, and destruction of the pertinent microorganism. Pasteurization of milk at 71.7°C (161°F) for 15 seconds is an effective treatment to achieve a 5 log reduction of E. coli O157:H7 (Al-Taher et al., 2004). In apple, orange, and white grape juices at pH 3.9, a 5 log reduction is achieved after a thermal process of 3 seconds at 71.1°C (160°F) (FDA, 2004).

1.2.2.3. Antibiotic Resistance Antibiotics are sometimes used in food-producing animals that can harbor pathogens such as cattle, horses, and other livestock for three main purposes: 1) growth promotion, 2) disease prophylaxes at subtherapeutic concentrations for prevention of infection by pathogenic bacteria, even if no symptoms of illness are shown by the animals, and 3) as therapeutics when disease truly occurs in the animals (Galland et al., 2001; Schroeder et al., 2002). However, the use of antibiotics in animals creates a selective pressure that contributes to the survival of strains strong enough to resist the agent (Altekruse et al. 1997). Streptomycin, sulfasoxazole, tetracycline, and tetracycline derivatives are drugs rarely used to treat diarrhea in humans, but their use is common in animals for the economic benefits. There has been an increased trend of E. coli O157:H7 resistance to antibiotics approved for feedlot animals like ampicillin, tetracycline, amoxicillin/clavulanic acid, and rifampin; as well as oxacillin and vancomycin, although these last two antibiotics are not approved for use in cattle (Galland et al., 2001; APHIS, 1999). In one study,

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it was observed that E. coli O157:H7 isolates and STEC strains were resistant to kanamycin and ampicillin and showed multiple resistance to streptomycin, sulfamethoxazole, and tetracycline (Zhao et al., 2001). In another study, it was observed that all tested E. coli O157:H7 and STEC strains were resistant to at least one antibiotic. Of 29 E. coli O157:H7 and 21 STEC colonies tested, 14% and 33% were resistant to Ampicillin and 21% and 19% were resistant to Kanamycin, respectively. Some of the isolates also showed resistance to multiple antimicrobials with streptomycin, sulfamethoxazole, and tetracycline the most characteristic arrangement triad (Zhao et al., 2001). As part of the microbial adaptation and change, other factors such as transformation, transduction and conjugation allow the transfer of genetic material among bacteria, producing serotypes that are resistant to antibiotics or that produce disease-causing toxins (Morse et al., 1995; Feng et al., 1995). A prudent use of antibiotics in cattle is recommended in order to prevent resistance and keep the drug’s efficacy, because of the potential adverse effects, clinical implications, and acceleration in the development of antimicrobial resistant bacteria that could colonize the human gastrointestinal tract through the food chain (Schroeder et al., 2002; Zhao et al., 2001). It is also possible that antibiotic resistant E. coli O157:H7 can spread resistance factors to other microorganisms found either in the animal tract or in the environment (Galland et al., 2001). By prudently using therapeutic antibiotics when needed and limiting or reducing their availability, feedlot

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management could become a key factor to reduce antimicrobial resistance in the animal pre-harvest environment (APHIS, 1999).

1.2.3. Human Disease Microorganisms adapt according to the changes in their environment, while increasing their survival chances. By doing so, sometimes these changes resulted in the ability of some bacteria to produce illness in the host. Microorganisms that were not expected to be found on foods or that were not recognized previously as nonpathogenic have caused outbreaks of foodborne illness. Escherichia coli O157:H7 is considered an emerging pathogen, meaning that the disease produced by this organism was either previously unknown, or that the bacteria was not recognized as a cause of illness until very recently. According to Morse et al. (1995), an emerging pathogen is a microorganism that just appeared very recently in the population, or which has existed but its incidence at its normal geographic location is increasing very rapidly. Escherichia coli O157:H7 is a major cause of HC and hemolytic uremic syndrome (HUS), the most common cause of acute renal failure in children (CDC, 1993). Hemolytic uremic syndrome was first described in 1955, but its cause was not identified then. Another severe manifestation of infection with E. coli O157:H7 also includes thrombotic thrombocytopenic purpura (TTP), a life-threatening renal disease (Doyle, 1991). The emergence of pathogenic bacteria may be occurring due the contribution of different factors. In the case of Escherichia coli O157:H7, changes in food

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processing technology may be an underlying cause for its recent appearance and broad clinical spectrum of disease in the human population (Morse et al., 1995). For example, food-mass production methods used in the making of ground beef products allow frozen patties to be made from meat of several different carcasses. If one of the carcasses is contaminated with E. coli O157:H7, even in very small numbers, the blending and grinding process can distribute the pathogen in the entire batch contaminating several pounds of meat, as some epidemiological studies shown in previous outbreaks (CDC, 1993; Riley et al., 1983). According to the United States Department of Agriculture (USDA), one infected animal can contaminate as much as 16 tons of ground meat used in the making of hamburger patties, and a single batch can contain meat from as many as one hundred animals (Juska et al., 2000).

1.2.3.1. Hemorrhagic Colitis (HC) Infection with E. coli O157:H7 can cause a wide variety of outcomes (Fratamico et al., 2002; Paton et al., 1998; Tschäpe et al., 2001), with cases being reported worldwide. Bloody diarrhea caused by E. coli O157:H7, where infection of the large intestine occurs, is clinically different from that produced by other gastrointestinal pathogens. It is likely that many cases of watery, non-bloody diarrhea are not reported to health departments. Patients that experience mild to moderate abdominal pain and watery diarrhea and who don’t present themselves for examination and/or medical help may account for the underestimation of the

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illness caused by E. coli O157:H7 (Hennessy et al., 2004; Mead et al., 1999; Peacock et al., 2001; Talan et al., 2001). After ingestion of E. coli O157:H7, the clinical symptoms range from one to eight days, with an average incubation period of 3 days (Fratamico et al., 2002; Peacock et al., 2002). Initially, patients develop abdominal cramps and watery diarrhea, with a varying percentage of these patients’ diarrhea resolving without further complications. The cramps can be very severe, with the cecum and the ascending colon as the most affected areas that can mimic an acute abdomen inflammation and lead to exploratory laparotomy. Fever is usually absent or mild but occasionally can exceed 102° F (38.9°C). In mild disease without bloody diarrhea, patients have less abdominal cramps, vomiting, and fever and are less likely to develop systemic sequelae, HUS, or to die. When watery diarrhea becomes bloody, occurring in one-quarter to threequarters of patients, it can range from occult positive or just a few visible quantities to the entire stool being compose of blood, usually without clots. This outcome usually occurs due to an infection with E. coli O157:H7 or Shigella; however, in developed countries, Salmonella and Campylobacter are also a major cause for this symptom (Talan et al., 2001). Infections with E. coli O157:H7 rarely produce fever as occurs in infections with Shigella, a predominant pathogen among adults with bloody stools that require hospitalization. The occurrence of bloody diarrhea can happen as often as 15 to 30 minutes (Feng et al., 2001). Vomiting is also reported in about 30% to 50% of cases. Other

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health problems that occur with HC are elevation in the blood leukocytes, edema, and hemorrhage of the lamina propia, superficial ulceration, tenesmus, pseudomembrane formation, and necrosis of the superficial colonic mucosa (Fratamico et al., 2002; Varade et al, 2000). Approximately 95% of the cases of HC resolve completely without further complication, however, the remaining 5% develop hemolytic uremic syndrome.

1.2.3.2. Hemolytic Uremic Syndrome (HUS) Hemolytic uremic syndrome, a term used for the first time in 1955, is defined as a disorder where kidney failure, hemolytic anemia, and thrombocytopenia (platelet deficiency) develops, usually after 7 days of the onset of diarrhea (Shapiro et al., 2002; Varade et al, 2000). These symptoms are also accompanied by coagulation defects and variable nervous system signs. When these symptoms occur in adults, the syndrome can be diagnosed as thrombotic thrombocytopenic purpura (TTP). Hemorrhagic colitis usually occurs before hemolytic uremic syndrome, but “atypical” HUS can occur without diarrhea in the prodrome. The risk of developing HUS after an EHEC infection is between 2% to 7% (Verweyen et al., 2000). The toxicity mechanisms used by E. coli O157:H7 to cause illness in the host are very complex and not completely understood. After ingestion of E. coli O157:H7 the bacteria colonize the mucosal site of the intestine by attaching to the epithelial cells of the gut where water and electrolyte loss is induced causing diarrhea; as

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mentioned before, and dehydration. The virulence of E. coli O157:H7 is determined by properties encoded on the bacterial chromosome such as acid resistance, expression of shiga toxins (Stx), and adherence factors like the effacing lesion phenotype (A/E lesion), pedestal formation, aggregation of polarized actin, enterohemolysin and serine protease, translocated intimin receptor, and host inflammatory mediators, which allow the pathogen to adhere and avoid expulsion (Trabulsi et al., 2002; Varade et al, 2000; Verweyen et al., 2000). This close attachment induces the transfer of a verotoxin to the mucosa where it is transported by the epithelial cells, causing microvascular injury and producing thrombosis, hemorrhage and necrosis which is expressed as bloody diarrhea. Toxin absorption is facilitated by the damage of the gut wall; and it can spread systematically through circulation to the kidney and other organs. Renal injury is caused mainly by the expression of high levels of Gb3, the receptor of the verotoxin. Some studies have shown that the binding of Stx to Gb3 receptor decreases after 2 years of age, accounting for the difference in renal injury and HUS between children and adults after an E. coli O157:H7 infection. Patients younger than 5 years of age tend to shed E. coli O157:H7 for longer periods than the adults, which explain the confirmed transmission of infection among preschool children (Peacock et al., 2001; Terajima et al., 2000). Some studies suggest that E. coli O157:H7 is the major cause of postdiarrheal HUS among adults and children in developed countries (Banatvala et al., 2001). E. coli O157:H7 accounts for 70% of cases, but other serotypes including

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O111:H-, O111:H8, O26:H-, O113:H21, O130:H11 are responsible for the remaining 20% to 30% of EHEC infections (Elliot et al., 2001; Neill et al., 2001; Talan et al., 2001). Although rare, bloody diarrhea and HUS have also been associated with other pathogenic infections by Campylobacter, Salmonella, Citrobacter, Aeromonas, Shigella dysenteriae type 1, and even Streptococcus pneumoniae. In addition, viruses like varicella, echovirus, Coxsackie’s A and B, and human immunodeficiency virus 1 (HIV) have also been linked to HUS. Finally, noninfectious development of HUS has also been associated with cancer, oral contraceptives, pregnancy, organ (kidney, liver, heart, bone marrow) transplantation, and/or the administration of mitomycin C, cyclosporine A, tacrolimus, and other therapeutics used in cancer patients during chemotherapy (Shapiro et al., 2002; Varade et al., 2000).

1.2.3.3. Thrombotic Thrombocytopenic Purpura (TTP) Thrombotic Thrombocytopenic Purpura (TTP) is characterized by damage to the endothelium, fragmentation and destruction of red blood cells, consumption of platelets, and an inadequate supply of blood to the tissues. The kidneys are especially susceptible to such vascular damage, but the brain, intestines and other organs can also be affected (Donnenberg et al., 2000; Fratamico et al., 2002). The differentiation between TTP and HUS is not very clear and sometimes TTP diagnosis is given to patients with E. coli O157:H7 infection when the prodromal diarrhea is absent and the patient is afflicted with and a less pronounced renal failure (Besser et al., 1999, Talan et al., 2001). In TTP, the deficiency of a protein in

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the plasma called von Willebrand factor (vWF) also cause a condition characterized by excessive bleeding, which tends to affect adults rather than children.

1.2.3.4. Treatment and Outcome Most people recover from the HC without the use of antibiotics or any other specific treatment in 5 to 10 days with the exception of the individuals who develop HUS (CDC, 2003). However, HUS has no specific treatment once the illness started. Supportive care is usually necessary to prevent further complications, including careful fluid administration in order to prevent overhydration, hyponatremia, and seizures. Careful blood-product transfusion, treatment of electrolyte disturbances, control of seizures, hypertension, intake, output, and weight should be monitored closely; medication may also be administered to control the symptoms (Peacock et al., 2001; Varade et al., 2000). Steroids may be prescribed to reduce inflammation is some cases. Dialysis is necessary when renal failure is progressive or prolonged. The use of antibiotics for treatment of HC caused by an E. coli O157:H7 infection is not recommended since some studies have found that antimicrobials are a risk factor and patients under antibiotic treatment are more likely to develop HUS (Paton et al., 1998; Verweyen et al., 2000). It is speculated that antibiotics increase the amount of toxin produced by E. coli O157:H7, which is released in the gut after lysis of the bacteria, altering and reducing the normal gut flora and prolonging the curse of the disease (Shapiro et al., 2002; Varade et al., 2000).

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Persons with only diarrhea usually recover completely without any further complications. Early supportive care at the beginning of EC infections may decrease the risk of progression to HUS. Children under 10 years are more likely to develop hemorrhagic uremic syndrome, with kidney dysfunction due to necrosis. The annual incidence of HUS in the United States varies from 0.2 to 3.4 cases per 100,000 children, but the average is estimated to be 3 cases per 100,000 children less than five years old (Dunn et al., 2003). It has been estimated that 2-7% of persons with E. coli O157:H7 infection will develop HUS, and even as high as 20% as some outbreaks have shown (Besser at al., 1993; Dunn, 2003). All people are susceptible to hemorrhagic colitis, but the younger (those between 6 months and 4 years of age) are especially at risk being the most susceptible to the disease caused by E. coli O157:H7 with a mortality rate between 3% - 15% due to renal failure (CDC, 2003; Salyers et al., 2001). Most adult patients are women who have been taking oral contraceptives, are postpartum, or are having obstetric complications such as preeclampsia. The elderly and the immunocompromised are at increased risk of a fatal outcome, with a mortality rate for the elderly as high as 50% (CDC, 2003). Approximately 5% to 33% of HUS patients develop chronic renal failure or permanent kidney damage often requiring blood transfusions, long term dialysis, and even kidney transplantation (Peacock et al., 2001; Peacock et al., 2001), and 30% may experience acute neurological complications such as stroke, seizure, lethargy, hemiparesis, decerebrate posturing, and coma (Varade et al., 2000). In some cases, surgery is required to remove part of the bowel. About 8% of those who develop

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HUS will have life long complications such as hypertension. Other outcomes like pancreatitis, diabetes mellitus, and pleural effusions may occur as very rare complications of the disease. Approximately 85% of HUS cases recover with supportive care and regain normal renal function, but long term follow-up may be required (Dunn et al., 2003; Peacock et al., 2001).

1.2.4. Reservoirs of Escherichia coli O157:H7. The emerging nature of E. coli O157:H7 demands that the ecological niches and routes of infection used by the pathogen be studied and characterized. As a result several different kinds of animals which may harbor pathogens, including E. coli O157:H7, that can be transmitted to foods by fecal contamination have been studied in order to assess the magnitude of the problem posed by the microorganism. Enterohaemorrhagic E. coli can be isolated frequently from the gastrointestinal tract of many herbivorous animal species used in food production, and host infected animals don’t show symptoms of illness or infection. Manure is a public health concern because during slaughter and processing, fecal material from the gastrointestinal tract and soiled hides containing E. coli O157:H7 and other pathogens can contaminate the carcasses and may enter the food supply. According to the National Agricultural Statistics Service (2004), it is estimated that every year about 1.6 billion tons of manure are produce in the U. S, with cattle accounting for approximately 90% of that amount. Manure can also come in direct contact with

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other foods when used as slurry to fertilize crops and can contaminate water sources used either for irrigation or animal and human consumption (Fukushima et al., 1999; IFT, 2002; Juska et al., 2000). On the farm, inactivation of manure compost to reduce pathogen loads before using it as a crop fertilizer is recommended in order to reduce the risk of transmission of E. coli O157:H7 and other pathogens (Duffy, 2003; Jiang et al., 2003).

1.2.4.1. Cattle. After consumption of undercooked ground beef in two almost simultaneous outbreaks in 1982, E. coli O157:H7 was recognized for the first time as a pathogen (Riley et al., 1993). Other outbreaks of bovine origin have occurred since then, which indicates that cattle and their products are a primary source and vehicle for this pathogen. Due the strong epidemiological evidence linking E. coli O157:H7 infections with the consumption of meat and meat products, several surveys in the United States have been established in order to estimate the true incidence and prevalence of the pathogen at feedlots and slaughterhouses (Parry et al., 2002). In The United States, beef and dairy cattle have been established to date as the most important natural reservoirs of E. coli O157:H7. Worldwide, many other countries have also reported high rates of carriage of the pathogen in the same animals (Feng, 2001; Kehl, 2002; Kobayashi et al., 2001). According to the National Agricultural Statistics Service (2004) there is approximately 94,882,000 cattle (nondairy) head in the U.S., being E. coli O157:H7 highly prevalent and widely distributed

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in groups of animals at the feedlot (Khaitsa et al., 2003). The lower gastrointestinal tract of mature ruminants has been found to be the predominant location for the proliferation of E. coli O157:H7, with the cecum and the colon as the likely sites for colonization. Despite infection, no disease or clinical manifestations are caused by the pathogen (Callaway et al., 2003; Grauke et al., 2002; Naylor et al., 2003). Escherichia coli O157:H7 has been proven to be very versatile and can adapt and survive the acidic conditions in the rumen and hindgut, while propagating within the gastrointestinal tract, indicating the probability that the bacteria is part of the normal stomach flora in cattle. The average duration of individual animals to test positive for E. coli O157:H7 is 30 days; however, cattle can shed the pathogen for up to 1 year. Young animals tend to carry E. coli O157:H7 more frequently than older animals (Besser et al., 1997; Zhao et al., 1995).

1.2.4.2. Domestic Animals. Several different reservoirs have been identified for E. coli O157:H7. In general, ruminants appear to be colonized by STEC more often than are other animals. Studies in sheep have revealed that sheep and cattle also harbor other serotypes other than EHEC, usually in higher levels and with more frequency than E. coli O157:H7 (Cornick et al., 2000). Pigs, horses, goats, rabbits, and poultry have also tested positive for E. coli O157:H7 (Chapman et al., 1997; Hancock et al., 1998). When infected, these animals could contribute to spread the pathogen between herds or within farms/feedlots (Duffy, 2003; Fratamico et al., 2002; Synge

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et al., 2000). Animals that are in much closer contact with people, such as cats and dogs, may also pose a potential risk for spreading the pathogen. Escherichia coli O157:H7 has been isolated from feces of these animals which have a frequent contact with children (Duffy, 2003; Hancock et al., 1998; Synge et al., 2000). Since the presence of these two domestic animals in family households are considered normal, good hygiene practices are necessary to reduce the risk of illness after petting and handling the animals and before cooking and consuming food. Petting zoos and open farms that are tourist attractions also have been reported as places where visitors have suffered from E. coli O157:H7 infections. In at least two different episodes, contact with the farm animals and consumption of foods from outdoor stalls under poor hygiene conditions may account for outbreaks (Parry et al., 2002; Payne et al., 2003).

1.2.4.3. Birds and Other Animal Sources. Undomesticated animals are a potential route of E. coli O157:H7 transmission in large areas of the environment. Escherichia coli O157:H7 has isolated from a diverse range of wild, asymptomatic animals. Ruminants like deer, elks and caribou (Donnenberg et al., 2000; Hancock et al., 1994) have tested positive for the pathogen. Undercooked deer meat and jerky were involved in two different outbreaks with E. coli O157:H7 in 1988 and 1995; increasing awareness of the role of products from different animals as a source for this pathogen (Fischer et al., 2001, Rabatsky-Ehr et al., 2002). Homemade food products by hunters pose a potential

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risk when animal carcasses are not handled and processed rapidly and properly, and when contaminated meat is not cooked thoroughly. Enterohaemorrhagic E. coli also has been isolated from bird droppings including ravens, doves, and seagulls (Doyle, 1997; Wallace et al., 1997; Whittam et al., 2000), indicating that these animals and their feces are a potential vector for the spread of the pathogen in the environment. The presence of birds and pigeons in open areas in the cities where people converge may account for a potential transmission risk of EHEC. Finally, insects such as flies have tested positive for E. coli O157:H7 and may be a vector to spread the pathogen onto the food surface by carrying the pathogen in their intestine and/or body parts. Despite the wide variety of animals that harbor E. coli O157:H7, cattle and their products seems to be the main reservoir and vectors in the environment for causing human infection (Duffy, 2003; Hancock et al., 1998).

1.2.4.4. Humans. The reservoirs for E. coli O157:H7 are animals, primarily cattle, from which contaminated food products or water spread the pathogen to humans. Humans are not considered reservoirs but host, since any asymptomatic E. coli O157:H7 carrier has yet to be identified. However, humans can be considered vectors in person-to person transmission of the pathogen. Serological test have indicated that farmers and people residing in rural areas have antibodies indicating a previous exposure and/or infection to EHEC. In one study in Wisconsin, E. coli O157:H7 antibodies were present in higher numbers among rural children when compared to non-farm

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resident children (Belongia et al., 2003). A continuous exposure to E. coli O157:H7 on the farm causing subclinical or mild infections may induce immunity and reduce the severity of illness among residents of those areas when infected with the pathogen. The use of enzyme-linked immunoabsorbent assay (ELISA) test in patients to screen for E. coli O157:H7 antibodies have been effective to detect infection with the pathogen even weeks after the onset of disease (Chart et al., 1991). Person-to-person transmission of E. coli O157:H7 by the fecal-oral route has also been reported, making its control difficult in child day-care, geriatric centers and institutions for those with physical and/or mental disabilities (CDC, 2003; Parry et al., 1998). In a reported outbreak, this path of transmission was the cause of illness among people in contact with the original patients, especially between siblings. Failure to follow good hygiene practices when nursing young toddlers, not disinfecting areas where diapers are changed, lack of toilet training for the youngest children, frequent hand to mouth contact, and not washing the hands of the children before eating account for transmission in this specific outbreak as the microorganism was shed in the patients feces up to 2 weeks after infection (Williams et al., 1997). Person-to-person transmission may also occur in food-related outbreaks. Secondary transmission was also associated with approximately 11% of all infected people in a large outbreak involving more than 700 cases after consumption of undercooked hamburgers in 1993 (Bell et al., 1994; CDC, 1993).

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1.2.5. Survival of Escherichia coli O157:H7 The majority of studies that have been done to determine the temperature effects on the growth of E. coli have used cocktail mixtures of EPEC, ETEC, and EIEC. After recognition of E. coli O157:H7 as a causation of food-borne illness, studies have focused specifically in the growth, survival, and inactivation characteristics of this pathogen (Bell et al., 1998; McClure et al., 2000). Studies have been performed to understand the behavior of E. coli O157:H7 in different substrates and foods for varying periods of time, as well as the effect that different intrinsic and extrinsic factors such as hot and cold temperatures, pH, organic acids, water activity (Aw), salt, control of reduction-oxidation potential (RO), fat content, irradiation, and preservatives will have on this specific pathogen.

1.2.5.1 Survival in foods As mentioned before, E. coli O157:H7 possesses very characteristic factors that allow it to survive in acidic foods; however, it has no heat resistant attributes which allows for control of the organism by proper cooking of the food product to a specific temperature and time. Escherichia coli O157:H7 is a cause for concern especially if present in foods that do not go through a treatment process to eliminate the pathogen, or that could be contaminated after such process and before packaging as in the case of ready-to-eat (RTE) products. After the outbreak in 1993, (Bell et al., 1994; CDC, 1993), the USDA considered E. coli O157:H7 an adulterant if

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present in ground beef, setting for the first time in the United States’ history a zero tolerance policy for presence of a microorganism in raw meat product. Microorganisms are not killed instantly when exposed to a lethal agent, but rather, the population decreases exponentially. The D value, or “decimal reduction time”, is used in food microbiology to describe at any given temperature the time required in minutes to reduce 90% (or 1 log) a specific microbial population in a specific food (Doyle et al., 1997). The D value is also affected by other food factors such as pH, water activity (Aw), content of preservatives, product composition, and the size of the microbial population, among others. The presence of fat in ground beef also increases E. coli O157:H7 tolerance to heat. Studies have revealed that cooking ground beef with 17-20% fat at 57.2°C and 62.8°C have D values of 4.5 and 0.40, respectively. In addition, these studies found that when ground beef patties have 30.5% fat and are cooked at 57.2°C and 62.8°C, D values of 5.3 and 0.47 are observed (Doyle et al., 1997). Cooking hamburgers to an internal temperature of 71.1°C (160°F) for 15 seconds is required to assure adequate cooking and prevent outbreaks. Pasteurization is also an accepted heating method to destroy this pathogen in milk, fruit juices and ciders. As mentioned before, treatment of milk for 15 seconds at 71.7°C (161°F) allows a 5-log reduction of E. coli O157:H7. (Al-Taher et al., 2004; FDA, 2004). The consumption of unpasteurized apple ciders, especially among children and immunocompromised is not recommended since the pathogen can survive in foods with low pH (Besser et al., 1993; CDC, 1997). However, some

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studies have been performed to examine the effect of treatment combinations to reduce E. coli O157:H7 by 5-log in unpasteurized apple cider. The use of multiplehurdle technologies allow small mill processors, which may not have the economic resources to purchase and install a pasteurizer or ultraviolet disinfection equipment, to process safer juice products. In one study, apple cider (pH 3.3) was frozen at -20°C for 48 hours and then thawed at 4°C for 4 hours to reduce the pathogen by at least 5 log units. A 5 log reduction of E. coli O157:H7 in apple cider (pH 4.1) was also achieved when the cider was tempered for 6 hours at 35°C, frozen for 48 h at 20°C and thawed at 4°C for 4 hours. Other successful treatments include addition of 0.1% of sorbic acid to the cider before tempering at 25°C for 12 hours or temper it at 35°C for 4 hours followed by freezing and thawing (Uljas et al., 1999). Low temperatures are used to reduce metabolic and enzymatic activities of microorganisms, thus reducing or preventing their growth. Storage of foods at low temperature conditions can reduce the population of microorganisms but not as completely when compared to a heat treatment. The use of low temperatures in food systems, normally below -2°C, results in dehydration and the formation of ice crystals and concentration of solutes within the cell, causing denaturation and destabilization of the structural and functional molecules. However, if the freezing process occurs very rapidly, cells do not suffer large, irreversible structural damage, and injured cells can recover and grow when conditions are favorable (Ray, 2001). Studies have shown recovery of E. coli O157:H7 in artificially inoculated foods after frozen storage. In one study, E. coli O157:H7 was recovered from inoculated

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strawberries, radishes, and cabbage after 2 and 4 weeks of storage at -20°C (HaraKudo et al., 2000). Ground beef used in the manufacturing of hamburger patties is often produced in a central location and distributed under frozen conditions to fast food restaurants in different locations. In the 1993 E. coli O157:H7 multistate outbreak involving undercooked hamburgers, contaminated frozen patties were involved with illness 6 weeks after the production date (Bell et al., 1994; Tuttle et al., 1999). Studies performed after that outbreak in inoculated ground beef patties (20% fat) revealed that E. coli O157:H7 can survive for up to 4 weeks after storage at -2°C with a 1.5 log reduction in the population. Storage of ground beef at -20°C for 12 months established recovery of the pathogen with an approximate reduction of 1.0 log (Ansay et al., 1999), demonstrating the ability of E. coli O157:H7 to survive in hamburgers for long periods of time at frozen temperatures with little decline in numbers of viable cells. Survival studies conducted in pepperoni, a meat product that is typically heated after fermentation, have shown the ability of E. coli O157:H7 to survive this production process. These types of products are commonly believed to be intrinsically safe due their low water activity (Aw=0.87-0.89), pH (4.68-4.85) and high content of salt (4.58-5.13%). Fermentation and drying alone in one particular study resulted in a 1 to 2 log unit reduction in the numbers of the pathogen; however, the reduction was not enough to comply with an USDA FSIS requirement to eliminate 5 log of E. coli O157:H7 per g of meat. Increased fat content in the product was also found to contribute in a lower reduction in the numbers of the pathogen (Faith et al.,

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1998). As seen in the examples above, E. coli O157:H7 displays a unique ability to survive in a wide variety of food products subjected to different process conditions for long periods of time, allowing them to serve as vehicles in the transmission of infections.

1.2.5.2. Survival in water Many of the outbreaks caused by E. coli O157:H7 have been associated with consumption of water from different environmental sources. Waterborne outbreaks have been caused through unintentional contamination of drinking and recreational water with manure harboring the pathogen (Samadpour et al., 2002). Application of manure to the land to fertilize crops also can result in runoffs that can contaminate aquifers, supplies and distribution systems of drinking water (Guan et al., 2003; Maule, 2000). In the summer of 1998, a large outbreak infected approximately 157 persons in a small town in Wyoming (Olsen et al., 2002). Epidemiological studies associated the illness among residents with the consumption of contaminated, unchlorinated water from a municipal water supply. However, microbiological studies performed on cold water from storage tanks did not detect the pathogen. It is believed that the microorganism was in a viable but nonculturable state (VNCS) which did not allow detection of the pathogen by traditional plating methods (Wang et al., 1998). Studies of the survival of a mixture of 5 strains of E. coli O157:H7 in water, performed by Wang et al. (1998), showed the greatest survival of the pathogen in filtered, autoclaved municipal water and lowest

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survival rate in lake water. Despite of the source of the water, survival of the pathogen was also high when the temperature was 8°C compared to 25°C. The study also demonstrated that there was a reduction of E. coli O157:H7 by 1.0 to 2.0 log units after 91 days of holding the water at 8°C. Some studies revealed that the water offered to livestock at different farms has a high microbial load (Faith et al., 1996; Hancock et al., 1998); accounting for a possible source of infection with this pathogen. Some studies revealed that water troughs used in feedlots can serve as both an important environmental reservoir and long term source of E. coli O157:H7 (LeJeune et al., 2001; Van Donkersgoed et al., 2001). Infected cattle may contaminate water troughs with their own feces and saliva, resulting in a niche for the microorganism and allowing for infection of healthy animals after a period of time. In one study in particular, an association between the prevalence of E. coli O157:H7 in water troughs and factors such as season, climatic temperatures, precipitation the week before sampling, and coliform and E. coli counts in the water trough was found (Van Donkersgoed et al., 2001). Studies have also been performed at the feedlot level to detect the prevalence of E. coli O157 in water sources. In a study performed in four major feeder cattle states, over 60% of feedlots and 13% of pens had at least one positive result for the pathogen in water samples collected from tanks in the pens (Sargeant et al., 2003). In another study involving large cattle environments, a prevalence of 0.51%, 0.25%, and 0.41% of E. coli O157 was found in water samples taken from cattle tanks, lakes, and creeks, respectively (Renter et al., 2003). Although the prevalence is low, positive results

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indicate a wide distribution and survival of the pathogen in water environments available to cattle at the feedlots, accounting for the infection and persistence of this pathogen in the animals. The incoming water sources, when contaminated with E. coli O157:H7 are also an important, continuous form of introduction of the pathogen in the farm (Van Donkersgoed et al., 2001).

1.2.5.3. Survival in environment, soil, and other sources A recurring persistence of pathogens in the environment may be a factor in their presence in animal reservoirs (Maule, 2000). Preharvest studies performed at several farms and feedlots have found that E. coli O157:H7 can be present in environments outside production areas. Studies on dairy and beef cattle demonstrated an extended geographic distribution on farms, and the pathogen was isolated from birds, flies, raccoons, wild animals feces, pen floors, water, water troughs, sediments, and feed (Renter et al., 2003; Shere et al., 1998; Smith et al., 2001; Van Donkersgoed et al., 2001). Wang et al. (1998) demonstrated that E. coli O157:H7 could exist and multiply in water sources, and other studies have shown its continued persistence in water sources such as troughs, tanks, ponds, and creeks, which accounts for dissemination of the microorganisms across wide areas (LeJeune et al., 2001; Renter et al., 2002). Sources such as cattle feeds and forages, which can be contaminated by manure and feces at the farm, have been postulated as a source of E. coli O157:H7. Wind, dust, and insects may be key factors in propagating the microorganism from feces over wide geographic areas.

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Survival of E. coli O157:H7 in soils may be affected by factors such as pH, texture, water content and bacterial competition, which could account for the differences found in the survival of the pathogen in this substratum (Maule 2000; Ogden et al., 2001). In many studies, presence in soil is a concern due the potential to spread the pathogen to the food chain, especially through produce consumed without further processing. Infections with E. coli O157:H7 have been linked to the consumption of raw products like alfalfa sprouts (Breuer et al., 2001), radish sprouts (Michino et al, 1999; Watanabe et al., 1999), pea salad, lettuce, cantaloupe, and other vegetables (Feng, 2001; Fratamico et al., 2002; Parry et al., 2002). One study was performed to determine the persistence of E. coli O157:H7 in different substrates. The pathogen was recovered in fallow soils, rye roots, and alfalfa roots after 25-41, 47-96, and 92 days, respectively (Gagliardi et al., 2002), showing the ability of the microorganism to persist for long periods of time. Finally, studies have been performed on different food contact surfaces used during the manufacturing and processing of foods, due to their potential to contaminate products if not cleaned and sanitized properly during working shifts. Studies revealed that bowl cutters and grinders, used in size reduction and blending of meat, ingredients and spices, are a vehicle in the contamination of meat products (Flores, 2004; Flores et al., 2002). In one study after the processing of contaminated meat, it was found that the knife and knife guard of the cutter were likely to be contaminated, spreading the pathogen to the subsequent batch and equipment surroundings. It was also determined that a contaminated knife works as

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a vehicle in distributing the pathogen into meat. Maule (1999) reported survival of 107 CFU inoculum of E. coli O157 on stainless steel surfaces held at 4°C, with a decrease less than 10-fold over a 60 day study. However, a similar inoculum of the pathogen on copper surfaces was undetectable after 10 hours. Other studies (Maule, 2000; Wachtel et al., 2003) revealed that in addition to survival on stainless steel, E. coli O157:H7 was able to survive at room and chill temperatures on domestic cutting boards made of plastic, showing the potential role of these utensils in the crosscontamination of foods. Rinsing plastic tables for 15 seconds with warm water (35°C) was not enough to decrease the number of disease-causing bacteria. Proper washing and sanitizing of hands and cooking utensils; the use of different cutting boards according to the type of product that is been handled to prevent crosscontamination; and strict enforcement of food safety practices in cooking/processing areas is recommended to avoid the spread of potential infections. The use of porous surfaces such as cutting boards made of wood is not recommended by the USDA due the higher potential for microorganisms to be trapped; the probability of microorganisms to increase in number when nutrients are present, and the difficulty of cleaning and sanitizing when compared to plastic and other inert surfaces (Ak et al., 1994; Welker et al., 1997).

1.2.5.4. Survival in feces and slurry Because cattle are recognized as the principal reservoir of E. coli O157:H7 and their feces are responsible for transmission of the pathogen at the farm, studies

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have been performed on the survival and growth characteristics of the pathogen in feces. The large amount of waste produced each year by these animals indicates the high potential of this route in shedding, transmission and infection of farm animals (Besser et al., 1997; Dargatz et al., 1997; Khaitsa et al., 2003). Studies have been conducted in feces from naturally infected and inoculated animals, including cattle and sheep, and growth characteristics have been established in manure and slurries used to fertilize crops. Application of slurry, a biomass that could contain a combination of animal and vegetable waste such as feces, urine, animal byproducts, grass clippings, leaves, feed, and sewage sludge is a normal practice in agricultural production, often without treatment to reduce their bacterial content (Gagliardi et al., 2002). Before use, slurries should be composted and matured until they have minimal biological activity since humans can be exposed to enteric pathogens after ingestion of food contaminated with this fertilizer. One study revealed the ability of loam and clay soils to retain E. coli O157 for more than 20 weeks after application of slurry containing the pathogen (Fenton et al., 2000). However, E. coli O157 was detected only in the upper layers of the soil and grass for the first week after application, implying that grass and silage fed to animals could be a vehicle in the transmission and infection with the pathogen. To prevent and reduce the risk of pathogens when fertilizing crops, composting is recommended to decline the microbiological load in animal waste (Jiang et al., 2003). In this process, instead of spreading animal feces directly to the

35

crops, effluents from farming operations and manure are normally collected in a bioreactor together with nutrients such as water, urine, and bulking agents like spilt feed and sulfates (Jiang et al., 2003; Kudva et al., 1998) for the anaerobic, metabolic processes of the bacteria and fungi. Heat is generated, and after diverse change in the compost conditions, there is a final inactivation of pathogens, larvae, and seeds in the organic mixture. At that time, the slurry can be used as a fertilizer. In one study, it was shown that under very controlled conditions, E. coli O157:H7 can be inactivated at different rates. Very small populations of the pathogen were detected in the compost mixture after 7 days when enrichment was used in the recovery of the bacteria. If the temperature in the bioreactor is set at a minimum of 50°C, it is recommended to compost manure for at least 2 weeks before using it as a crop fertilizer (Jiang et al., 2003). However, when manure is subjected to fluctuating environmental conditions, other studies have revealed a longer survival time of E. coli O157:H7. Kudva et al. (1998) reported that the pathogen survived in nonaerated ovine manure from inoculated sheep for more than a year and in bovine manure from inoculated cattle for 47 days. In the afore mentioned study, it was possible to recover the bacteria by a selective enrichment technique when samples in the middle and at the bottom of an ovine manure pile were collected. However, analysis of the dry fecal matter taken from the top of the pile found that the bacteria did not survive. In a study in cow manure-amended soil it was found that the pathogen was able to survive for up to 77, 226, and 231 days when held at 5°C, 15°C, and 21°C, respectively; however, the long survival rates in the study were performed on

36

autoclaved soil (Jiang et al., 2002). The authors believed that microorganisms in manure and soil had an antagonistic effect on the survival characteristics of E. coli O157:H7 since the pathogen in unautoclaved samples had a lower survival rate. Although the above studies show an increased survival rate of the pathogen in animal waste, the environmental conditions, such as the use of slurry as fertilizers on the same farm where cattle are held do not typically occur in American feedlots. Beef cattle are usually held in small pens where frequent contact between animals and their own feces occur. This may account for a possible source of reinfection of the animals and long prevalence of the microorganism in the farm environment. Representative studies on the growth characteristics of E. coli O157:H7 in pure bovine feces indicate that the pathogen can survive in feces for long periods of time while maintaining their ability to produce verotoxins, which confirm feces as a vehicle for transmitting bacteria. In one particular study, a cocktail of five E. coli O157:H7 strains were inoculated in bovine feces at a high level (2.7 x 107 CFU/g) and subjected to 3 different environmental conditions (Wang et al., 1996). It was reported that feces stored at 37°C and 22°C resulted in a positive recovery of E. coli O157:H7 using an enrichment procedure after 7 and 8 weeks, respectively, but the longest recovery was found in samples held at 5°C, where the inoculum survived for up to 70 days with no substantial decrease in numbers of viable bacteria.

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1.3. Disease Outbreaks. Escherichia coli O157:H7 outbreaks have been linked to the consumption of several different foods with the majority of outbreaks involving ground beef. Fecal contamination of products and water sources along with a failure to comply with good manufacturing practices have led to the presence of the pathogen in food products and the causation of numerous outbreaks. Table 1.1. lists some of the outbreaks that have been linked to E. coli O157:H7. Eventually the number of illnesses and deaths caused by the microorganism and the lessons learned during the epidemiological studies of the outbreaks prompted a strengthening of manufacturing and traceability control of foods in the industry and an increase of cooking temperatures in fast food restaurants in order to prevent of E. coli O157:H7 infections. The first outbreak which recognized E. coli O157:H7 as a foodborne pathogen was in 1982, where contaminated hamburgers not cooked to the specified temperature in a fast food chain restaurant were to blame (Riley et al., 1983). Since then, many other outbreaks have occurred due the presence of the pathogen; however, it did not gain national attention until another multistate outbreak in 1993 caused the death of four young children and more than 700 cases of illness among persons of 0 to 74 years of age (Bell et al., 1994). After this highly publicized outbreak, the demand for a safer food supplied increased among consumers, causing the need for a pathogen reduction in foods. The Food Safety and Inspection Service (FSIS) established new provisions for all meat and

38

Table 1.1 Selected examples of reported outbreaks of Escherichia coli O157:H7 worldwide

39

Year

Location

Source/Cause

Cases (Deaths)

Reference

1982

USA/Restaurant

Hamburgers

>47 (0)

Riley et al., 1983

1993

USA/Mill

Fresh apple cider

23 (0)

Besser et al., 1993

1994

USA/Restaurant

Hamburgers

>501 (4)

Bell et al., 1994

1995

USA

Beach associated

12

Mudgett et al., 1998

1995

USA/Daycare center

Unknown

24 (0)

Williams et al., 1997

1996

USA

Apple cider

66 (1)

CDC, 1997

1996

Scotland

Cooked meats

490 (16)

Dundas et al., 2001

1996

Japan/School

Radish sprouts

>9451 (12)

Michino et al., 1999

1996

Japan/Factory

Radish sprouts

74 (1)

Watanabe et al., 1999

1997

USA

Alfalfa sprouts

64 (0)

Breuer et al., 2001

1998

USA

Municipal Water

157 (0)

Olsen et al., 2002

1999

USA/Lake

Swimming related

36 (0)

Samadpour et al., 2002

poultry plants; requiring the mandatory implementation of a hazard analysis and critical control point (HACCP) system in those plants to identify risky and potentially hazardous practices that account for microbial contamination. Despite the improvements in processing techniques and often voluntary implementation of HACCP in areas of food processing other than meat and poultry, the pathogen continues to cause outbreaks through a novel wide range of products. Among the unusual outbreaks have been those which have occurred after consumption of unpasteurized apple cider (Besser et al., 1993; CDC 1997), alfalfa sprouts (Breuer et al., 2001), white radish sprouts (Michino et al., 1999; Watanabe et al., 1999) and other food products including cantaloupes, lettuce, potatoes, pea salad, and other fresh produce, raw milk, mayonnaise, salmon roe, cheddar cheese, yogurt and hard salami (Altekruse et al., 1997; Fratamico et al., 2002; McClure et al., 2000). Outbreaks have also been linked to the use of recreational water and the consumption of water from municipal systems, as well as person to person transmissions in nursing homes and daycare centers (Mudgett et al., 1998; Williams et al., 1997; Olsen et al., 2002; Samadpour et al., 2002).

1.3.1. Geographic Distribution Reports describing the occurrence of infections with E. coli O157:H7 have been worldwide. Most of the studies that have been published addressing infections with this pathogen occur in the United States and Canada (Doyle et al., 1997);

40

however, sporadic illnesses associated with E. coli O157:H7 and other EHEC strains have also been reported in different countries. Escherichia coli O157:H7 was found to be the major cause of outbreaks in Japan (Michino et al., 1999; Watanabe et al., 1999) and in Scotland in 1996 (Dundas et al., 2001). Countries that have reported both outbreaks and the presence of E. coli O157:H7 and EHEC include Australia, Argentina, Brazil, Germany, the Netherlands, Chile, France, Italy, Finland, and Hungary, among others (Caprioli et al., 1998; Dundas et al., 2001; Heuvelink et al., 1998; López et al., 1998). The disparity in the number of illness caused by this pathogen as well as its distribution worldwide may be due several factors including a true variation in the geographical prevalence of the microorganism, cooking and eating habits in each country, better surveillance for the detection and reporting of human illness, and differences in the microbial methodology used in the isolation of the pathogen (Tschäpe et al., 2001).

1.3.2. Seasonality. Many studies have shown a seasonal variation of E. coli O157:H7. Escherichia coli O157:H7 infections in humans and animals peak during the warmest months of the year, with 83.4 % of the United States outbreaks occurring between May and late October (Doyle et al., 1997; Dunn, 2003). Interestingly, a similar trend with summertime seasonality has also been described for cases of HUS and foodborne illnesses (FDA, 2003; Neill et al., 2001). Some hypotheses for this trend could include an increase in the prevalence of E. coli O157:H7 in livestock, together with

41

an increase of outdoor cooking activities where ground beef is a major ingredient. Improper handling and cooking procedures, food temperature abuse, and eating habits (raw or pink ground meat) might be other causes of the increasing numbers. Temperature changes in the environment can also stimulate changes in bacteria pathogenesis and survival factors, aggravating the risk of foodborne illness (Doyle et al., 1997). Several authors have reported an increase in the prevalence of the pathogen during the summer. Hancock et al. (1994) were the first to report a tendency in the seasonal variation of E. coli O157:H7, with the highest number of colonies isolated in September. After that, other studies performed in cattle have found similar trends in the prevalence and shedding of the pathogen in cattle. In a one-year-study conducted in Washington State, fecal samples were collected monthly from different animals immediately after slaughter in order to detect changes in the prevalence of E. coli O157:H7. The persistence of the pathogen in cattle was at its lowest with 4.8% in December, varying to the highest point in May, with 36.8% prevalence (Chapman et al., 1997). In the Netherlands, a study in dairy cattle analyzed at various ages, also found that the fecal excretion of E. coli O157 in the animals was higher during the July-September quarter than at any other period of the year (Heuvelink et al., 1998). It is believed that warm temperatures together with moist conditions which occur during the summer months and changes in the diet can promote the survival and outgrowth of bacterial population in feedlots and cattle environments.

42

1.3.3. Susceptible Populations. All age groups are susceptible to infections with E. coli O157:H7, but the younger are the most affected with hemorrhagic colitis. Those between 6 months and 4 years of age are especially at risk, with a mortality rate between 3% - 15% due to renal failure (CDC, 2003; Salyers et al., 2001, Shapiro, 2002). In a multistate outbreak in 1993, the age range of the infected was between 4 months and 88 years, and fatalities due to HUS complications included 4 children (Bell et al., 1994; CDC, 1993). In another example of the susceptibility of younger to infection with E. coli O157:H7, an outbreak in Japanese elementary schools resulted in more than 6000 young students infected with the pathogen after consumption of contaminated radish sprouts (Fukushima et al., 1999; Michino et al., 1999; Watanabe et al., 1999). In adult HUS patients, most are women who have been taking oral contraceptives, are postpartum, or are having obstetric complications such as preeclampsia. The elderly and the immunocompromised are also at increased risk of a fatal outcome as one outbreak in Scotland proved in 1996. It was very remarkable that of the total number of patients, with an age range among 18 months and 94 years, admitted to hospitals in that outbreak, all those who died were older than 65 years of age. Of the 16 total deaths, 11 had a complete HUS diagnosis, and of those, 7 had neurological complications due the infection. This illustrates the risk of complications and high mortality rates that these patients have after infection with E. coli O157:H7 (Dundas et al., 2001).

43

1.3.3.1. Infective Dose. According to the FDA (2001), the infectious dose for E. coli O157:H7 is unknown; however, data obtained from several different outbreaks suggest that this pathogen has a very low infectious dose, most likely a result of its ability to resist acid environments with very low pH like the ones found in the rumen or stomach (Diez-Gonzalez et al., 1998; Doyle et al., 1997). Some reports suggest that less than 1000 microorganisms are required for symptoms of the illness to develop and to cause disease (Donnenberg et al., 2000), but recovery and quantification of E. coli O157:H7 from foods in other studies involved with outbreaks indicate a dose as low as ≤ 100 or even ≤ 10 microorganisms can cause illness in young children, the elderly and immunocompromised people (Neill et al., 2001; Peacock et al., 2001; Varade, 2000). Studies performed on ground beef patties involved in a large outbreak revealed a median most probable number (MPN) of 1.5 E. coli O157:H7 organisms/gram of ground beef, or about 67.5 organisms/ hamburger (Tuttle et al., 1999). The evidence showing such a small infectious dose to get infected with this microorganism reinforces the zero tolerance the USDA has for its presence in ground beef.

1.4. Escherichia coli O157:H7 Detection Methods During an outbreak, early detection and recognition of the causative agent is required to allow for proper treatment of the patients (De Boer et al., 2000). This is

44

especially true in the case of infection with E. coli O157:H7, since some studies suggest that the use of antibiotics increases the risk of developing HUS (Neill et al., 2001). Isolation of the causative agent is also required not only to administrate the proper medication, but also to promptly identify other possible cases and routes of transmission, and limit the number of deaths that may occur (Bell et al., 1994). Sensitive detections methods for E. coli O157:H7 allows surveillance nets to be activated at health department levels and initiate procedures to recall food products if necessary. The food industry also requires and demands rapid and accurate identification methods to verify the processes and the environmental sanitation conditions of the plant in order to provide a safe food product. Isolation of E. coli O157:H7 from animal, food, environmental, and clinical cases is based on the unique phenotypic and genotypic characteristics of this microorganism that allows its detection and confirmation with the use of serological and biochemical tests. Escherichia coli O157:H7 differs from generic E. coli in some unique aspects, including the absence or slow sorbitol fermentation (March et al., 1986), no haemolysis (or disintegration of red cells with the release of hemoglobin) on sheep and rabbit blood agar (Wells et al., 1983), and no manifestation of βglucuronidase activity (Reinders et al., 2004). A wide variety of methods and protocols describing the culture techniques for the isolation of the pathogen using these properties have been reported (Chapman et al., 1997; Faith et al., 1996; McDonough et al.,. 2000; Meyer-Broseta et al., 2001). Three general steps are required: enrichment of the sample, isolation of the presumptive colonies, and final

45

confirmation of the bacteria. Most of the studies conducted follow these accepted steps, but may differ due to the media used or the supplements added to the media (Tortorello, 2000).

1.4.1 Enrichment methods Enrichment (also known as pre-enrichment) is a step in which samples are added to a specific medium for a determined period of time, allowing injured cells to recover, increase and multiply. Enrichment, followed by plating in selective media, allows the recovery of bacteria when the number of cells in a sample is very low. This is very true in the case of E. coli, which accounts for just a small percentage of the flora found in the gastro intestinal tract (De Boer et al., 2000; Zimbro et al., 2003). The use of different media, supplements, and incubation time in this essential step has returned different results in various studies (Barkocy-Gallagher et al., 2002). Buffered peptone water (BPW) is a medium that has been used to improve the recovery of E. coli O157:H7 from different sources. Fenlon et al. (2000) successfully used BPW supplemented with vancomycin for the detection of E. coli O157:H7 from soil and water after an incubation period of 6 h at 37°C. Isolation of E. coli O157:H7 from fecal samples taken from cattle, sheep, and pigs using the same procedure have been reported (Chapman et al., 1994; Chapman et al., 1997). Kerr et al. (2001) used BPW to detect the pathogen in fecal samples, but they used a two-step method. In this method, 1 g of feces was added to 9 ml of BPW and

46

incubated for 2 h at 37°C. After that, 1 ml was removed and added to 9 ml of BPW containing vancomycin and cefsulodin and incubated overnight at 37°C. Restaino et al. (2001) also used BPW supplemented with vancomycin, cefsulodin, and cefixime (BPW-VCC) in a study of selective broths for the recovery of heat and freeze injured E. coli O157:H7 cells from ground beef. Gram-negative (GN) broth, a selective medium normally used for the microbiological examination of foods, is used often in the detection of E. coli O157:H7. The sample is added to the media, allowing the growth of coliforms while inhibiting competitive flora such as Gram-positive bacteria (Zimbro et al., 2003). GN broth has been proven to be successful in the isolation of E. coli O157:H7 from stools of patients with HUS (Karch et al., 1996). However, GN broth is usually supplemented with vancomycin, cefixime and cefsulodin (GN-VCC broth) and used in the detection of E. coli O157:H7 from fecal samples, feed and water (Brashears et al., 2003; Dodd et al., 2003; LeJeune et al., 2001). Another medium commonly used for preenrichment is tryptic soy broth (TSB), a solution that supports and promotes the growth of different microorganisms and has been used in several studies (Fukushima et al., 1999; Kudva et al., 1998; Zimbro et al., 2003). Tryptic soy broth supplemented with cefixime and vancomycin (TSBcv) was used by Dargatz et al. (1997) as enrichment in the isolation of E. coli O157:H7 from cattle feces in a study involving 100 feedlots. Hancock et al. (1998) also used the same enrichment broth for the detection of E. coli O157:H7 from feces of cattle and other animal species, by incubating the samples at 37°C for 16-24 h

47

before plating. In another study, where different types of enrichment media were compared, it was found that the use of TSB supplemented with cefixime and vancomycin (TSBcv) was more sensitive in the detection of E. coli O157:H7 from bovine feces than when a direct plating method was used (Sanderson et al., 1995). In another study, where feeds were artificially inoculated with E. coli O157:H7, the use of TSBcv as enrichment yielded more sensitive results than the sole use of TSB in the recovery of the pathogen (Dodd et al., 2003). Escherichia coli (EC) broth was also used successfully in the preenrichment step for the isolation of E. coli O157:H7. In two different studies in the Netherlands, samples from bovine and ovine feces, milk, water, silage and insects were inoculated in EC broth yielding positive results in the detection of this pathogen. Modification to this media also includes the addition of an antibiotic called novobiocin (mEC+n) that is used to inhibit Gram-positive bacteria (Heuvelink et al., 1998; Restaino et al., 2001).

1.4.2. Immunomagnetic Separation (IMS) Immunoassays refer to a method that allows the detection and identification of a protein based on the affinity and formation of an antigen-antibody complex. Once a specific target is formed, the unwanted material on a sample can be washed away, allowing the desirable target to be separated and recovered from other components (Anonymous, 2003). The use of this system allows the isolation and detection of E. coli O157:H7 by concentrating the pathogen from samples where its

48

numbers are minimal compared to the total flora (De Boer et al., 2000; Fratamico, 1999), as is the case of samples containing fecal material. Immunomagnetic separation (IMS) is considered as part of the pre-enrichment step, contributing to increase retrieval of microorganisms, such as E. coli O157:H7, by the use of polystyrene beads coated with pathogen specific antibodies. The beads are superparamagnetic, i.e., they exhibit magnetic properties only when they are placed under a magnetic field; however, once the magnetic field is removed, no residual magnetism remains on them allowing distribution of the particles in a suspension and discharge of the undesirable material (Anonymous, 2003; Fratamico et al., 1999; Strockbine et al., 1998). In general, the tubes containing the beads and the samples (which have been subjected to a preenrichment step) are placed at room temperature under continuous rotation to allow formation of the beads-cells complex, after which the tubes are placed in a magnetic field, allowing concentration of the target material on one side of the tube. The supernatant containing contaminants, small pieces of the sample (i.e. food or organic material) and undesirable microorganisms can be removed by aspiration, and only the bead-cell complex remains in the tube. Washing the complex with a detergent such as phosphate-buffered-saline (PBS) and repeating the mixing and exposure to the magnetic field several times, allows an increase in the sensitivity of the method. However, despite the specificity of the antibodies and the use of detergents to wash the sample, interaction between the beads and undesirable cells cannot be eliminated, resulting in a non-pure sample at the end of the cycle (Wright

49

et al., 1994). In one study, non-sorbitol fermenting (NSF) organisms that were recovered after using IMS and which apparently also adhered to the E. coli O157 magnetic beads were categorized. In that occasion, the two most common groups were E. coli non-O157 and Proteus spp.. Other microorganisms such as Hafnia alvei, Klebsiella ozaenae, and Providencia alcalifaciens were also isolated with beads coated specifically for E. coli O157 (Chapman et al., 1996). After the IMS protocol has been accomplished, a suspension containing the beads-cells complex is plated onto agar followed by incubation, allowing the detection and isolation of the presumptive colonies. The use of IMS for the recovery of E. coli O157:H7 has been proved successfully in numerous studies involving food, environmental, and fecal samples. In one of the earliest studies assessing the use of IMS, it was found that the method was a sensitive procedure to recover the pathogen from inoculated, minced beef samples in a range limit of 2-20 E. coli O157:H7 CFU/10 g of beef. Inclusion of this step before plating was believed to increase sensitivity around 100-fold in the recovery of the pathogen (Wright et al., 1994). During the same study, the use of IMS also allowed milk samples from a dairy herd to be linked to a small outbreak of infection with E. coli O157:H7. A similar increase in the sensitivity of IMS was reported by Chapman et al. (1994), when comparing the use of IMS and direct culture for the isolation of E. coli O157:H7 from inoculated bovine feces. The use of IMS has also been used in the recovery of E. coli O157:H7 from humans presenting bloody diarrhea, acute non-bloody diarrhea, and even from

50

asymptomatic persons who have been in contact with infected people. In one study, the use of IMS allowed detection of the pathogen in 173 samples of 690 submitted by patients presenting diarrhea symptoms, whereas direct plating method resulted in detection of only 52 samples (Chapman et al., 1996). Karch et al. (1996) also reported isolation of E. coli O157 strains from 90% of HUS patients after using IMS. Several studies had used IMS successfully in the recovery of E. coli O157:H7 from naturally infected and artificially inoculated feces and hides (Elder et al., 2000; Parham et al., 2003; Ransom et al., 2002; Sanderson et al., 1995; Tutenel et al., 2003). In one study, Barkocy-Gallagher et al. (2002) reported the use of IMS followed by plating on selective media, achieving high specificity in the recovery of E. coli O157:H7 from bovine carcasses, hides, and fecal samples. Other studies where IMS has been a fast, reliable tool in the recovery of this pathogen include ground beef (Guan et al., 2002; Lindqvist et al., 1998; Tu et al., 2001), bovine carcasses (Kang et al., 2001), and naturally infected and artificially inoculated foods and water (Ogden et al., 2001; Pyle et al., 1999).

1.4.3. Culture methods Since earlier studies involving specimens from HUS patients, E. coli O157:H7 showed typical reactions for generic E. coli except one characteristic result: the slow or lack of sorbitol fermentation within 24 h. MacConkey agar (MAC), a medium used as a differential plating media for the detection and isolation of gram-negative microorganisms from different sources was used in earlier studies. Its use is based

51

on fermentative reactions, allowing in this mode the isolation and differentiation of enteric rods (Zimbro et al., 2003). When plated on MAC, E. coli O157:H7 ferments lactose rapidly and cannot be differentiated from other generic E. coli strains and enteric microorganisms that may also grow in the agar (Strockbine et al., 1998). This finding was followed with the introduction of sorbitol-MacConkey agar (SMAC), where lactose was replaced with sorbitol as a carbohydrate source (March et al., 1986; Zimbro et al., 2003). The use of SMAC as a differential medium to detect E. coli O157:H7 has been proved to be very efficient, since non-sorbitol fermenting (NSF) cultures appear either colorless or pale yellow as compared with sorbitolfermenting microorganisms which appear pink. This media allows a fast, easier and more reliable recognition of presumptive E. coli O157:H7 colonies (Bettelheim et al., 2003; De Boer et al., 2000; March et al., 1986). Because of the serious illness and complications associated to E. coli O157:H7 infections, the CDC recommends clinical laboratories to screen all stools, or at least bloody stools, with SMAC to test for the presence of this pathogen. When patients have had a history of bloody diarrhea, it is also suggested to use SMAC to test for the presence of E. coli O157:H7 (Boyce et al., 1995; CDC, 1993; Kehl, 2002). However, SMAC plates are not 100% effective for the detection of this pathogen, since it has been reported that some E. coli O157:H7 isolates can ferment sorbitol (Neill et al., 2001), giving false negative results on this test. Despite the advantage of using SMAC for diagnosis and detection of E. coli O157:H7, serogroups other than O157 as well as microorganisms of other genera are also NSF, growing too as colorless colonies on

52

SMAC, forcing investigators to conduct further tests to confirm the colonies on the plates. In order to avoid errors during investigations and the diagnoses of falsepositive samples, several other studies have been performed to enhance the SMAC agar for the detection of E. coli O157:H7. The use of antibiotics in the media improved the detection methods as seen when cefixime, a potent antimicrobial with a broad in-vitro activity against pathogens, especially enteric gram-negative bacteria, was added to SMAC. The use of cefixime on SMAC agar enhanced the detection of E. coli O157:H7 while inhibiting the growth of both generic E. coli and Proteus spp., another NSF bacteria that may also be found in the samples, saving consequently time and money during investigations (Chapman et al., 1991). A subsequent study found that the use of potassium tellurite increased the detection and selectivity of SMAC to E. coli O157:H7. Tellurite, a rare substance in the environment with oxidative properties to gram-negative microorganisms such as generic E. coli and Aeromonas spp., favored the growth of E. coli O157:H7 (Strockbine et al., 1998; Zadik et al., 1993; Zimbro et al., 2003). Since then, several studies have used both cefixime and tellurite together with SMAC (CT-SMAC) agar and their use has became a regular protocol method to inhibit background flora in fecal (BarkocyGallagher et al., 2002; Brashears et al., 2003; Chapman et al., 1994; Hu et al., 1999) and environmental samples (Fujisawa et al., 1996; Renter et al., 2003; Sargeant et al., 2003; Zhao et al., 1995). In one study, three different media were compared for the recovery of E. coli O157:H7. Feces from inoculated calves were plated on

53

different agars, and it was found that CT-SMAC was a more sensitive medium for the recovery of E. coli O157:H7 colonies when contrasted to the results of samples plated on solely SMAC or cefixime-SMAC agar (C-SMAC), confirming CT-SMAC as an excellent tool in the detection and differentiation of E. coli O157:H7 (Sanderson et al., 1995). Another commonly used differential method for the detection of E. coli O157:H7 is based on the biochemical use of chromogenic compounds, substances that remain colorless until acted upon by an enzyme. Fluorocult E. coli O157 agar, a medium that contains 4-methylumbelliferyl-b-D-glucuronide (MUG), is used to take advantage of this characteristic. When MUG is cleaved by β-D-glucuronidase (GUD), an enzyme produce by E. coli strains, a compound called 4methylumbelliferone (MU) is produced (Manafi, 2000). The presence of MU in the media can be confirmed with long-wave ultraviolet light (366nm), yielding a blue fluorescence color. Most E. coli strains, between 94-96%, exhibit GUD activity; however, E. coli O157:H7 do not possess the enzyme and cannot hydrolyze the substrate. Since no fluorescence is produced under UV light, identification of presumptive E. coli O157:H7 colonies is facilitated with the use of this differential agar (Manafi, 2000; Strockbine et al., 1998). To present, only one isolated variant of E. coli O157:H7 has shown GUD activity in the United States; however, atypical variants had also shown positive GUD activity in Europe (Hayes et al., 1995; Strockbine et al., 1998). 4-methylumbelliferyl-b-D-glucuronide can also be added to other media to facilitate the detection of E. coli O157:H7, as proved one specific

54

study in Japan where MUG was added to CT-SMAC and used successfully in the isolation and identification of this pathogen from inoculated radish sprouts (Fujisawa et al., 2000).

1.4.4. Latex Agglutination Test Isolated colonies must be tested in order to confirm their identity. Colorless, NSF, MUG-negative colonies are selected from plates and subjected to an specific agglutination test to confirm the presumptive isolates as E. coli O157. These latex tests, based on serological and somatic properties, are designated to establish the display of the O157, H7, or both antigens in the culture through the formation of an agglutinate or precipitate when positive (Mach et al., 1989; Strockbine et al., 1998). The test reagent consists of latex particles covered with rabbit antibodies specific and reactive to the O157 somatic antigen in the pathogen. Once the presumptive positive sample and the reagent are mixed together and put under rotation, usually for a minute or two, the formation of particles visible to the naked eye confirms the presence of the microorganism (Chapman et al., 1989). The use of this serological test is at its best when used together with SMAC agar for the isolation of NSF. In one study, where three different commercial latex tests were compared against a method using a specific antiserum in a tube agglutination assay, 159 strains of E. coli O157:H7 previously isolated from human stools and other sources were tested. All the strains tested yielded 100 % sensitivity and 100% specificity with each one of the agglutination tests when compared to the CDC reference antisera (Sowers et al.,

55

1996). Agglutination tests have been proved to be an excellent and sensitive alternative to identify the O157 antigens when used as directed by the manufacturers, allowing a rapid confirmation when compared to standard serological methods.

1.5. Preharvest Food Safety 1.5.1. Importance The implementation of HACCP systems in the food industry had improved the quality and safety of food products; however, despite the efforts to lower the levels of pathogens in food, they still occur causing numerous illness and deaths. The concept behind preharvest food safety is to prevent and/or decrease the levels of pathogens in the animals and in the preharvest environment in the first place, in order for the occurrence of these contaminants and pathogens in beef and meat products to also be reduced (Childers et al., 1996). A decrease in the shedding levels could also translates as a reduction in the contamination of other foods involved in outbreaks such as fresh produce, fruits and water. By understanding the complex ecology, epidemiology, interaction, and dissemination of E. coli O157:H7 within cattle, animals, and the environment, new strategies and intervention methods can be implemented to prevent the risk of serious foodborne diseases among consumers.

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1.5.2. Prevalence of Escherichia coli O157:H7 in feedlots and cattle. Several studies have been conducted in the preharvest area in order to determine the ecology of E. coli O157:H7 in cattle. These studies assessing the numbers of E. coli O157:H7 can be divided in different sets such as conducted on farm, prior or just after slaughter, and on carcasses after hide removal and evisceration (Meyer-Broseta et al., 2001). Reports of the prevalence and distribution of this pathogen has increased through the years, in good part because of the use of improved technologies that are faster and more reliable in detecting low numbers of the pathogen in the samples. Despite the use of better microbiological assays, prevalence estimates have varied due factors including, but not limited to, type of cattle (beef or dairy), age of the animals, sampling and sensitivity of culturing methods, seasonality, and the number and size of the animals and locations involved in the studies.

1.5.2.1. Prevalence in dairy cattle There are numerous studies that examine the prevalence of E. coli O157:H7 in dairy cattle, varying in scale, location and microbiological methods used to detect the pathogen. In one of the earliest epidemiological studies, feces were obtained with the aid of swabs from the rectum of individual animals and plated directly onto SMAC in order to isolate the pathogen. Final confirmation of the isolates was achieved with the use of a latex agglutination test specific for the O157 antigen. In

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that occasion, it was established that dairy cattle at farms in Washington State presented an overall E. coli O157:H7 prevalence of 0.28% in individual animals and an overall 8.3% prevalence in herds (Hancock et al., 1994). Despite the low numbers in the recovery of the pathogen, the authors suggested a tendency towards a seasonal variation in E. coli O157:H7 shedding among herds, which peaks during the summer and fall. In a subsequent survey conducted on dairy farms in Wisconsin, fecal samples obtained by digital rectal retrieval were examined. After preenrichment in EC-broth and plating on CT-SMAC, it was found a prevalence in individual animals of 1.8% and an overall herd prevalence of 7.1%; very similar data when compared to the previous study (Faith et al., 1996). A subsequent longitudinal study in the same state (Wisconsin) was also performed by Shere et al., (1998). Samples were obtained, preenriched and plated as described previously (Faith et al., 1996) and confirmed with a latex agglutination test. Although this study did not report a seasonal variation in the shedding of this pathogen, two interesting remarks were observed: first, an intermittent E. coli O157:H7 transmission among cattle; second, the indication that the use of antibiotics in the animals was a risk factor in the shedding of the pathogen in cattle. In a study performed in three Northwestern states (Idaho, Oregon, and Washington), the E. coli O157 prevalence in dairy cattle was investigated through collection of fecal material with the aid of cotton swabs. After enrichment in cv-TSB for 16-24 h, samples were plated on SMAC for detection of NSF bacteria. This

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examination showed a prevalence ranging from 1.1% to 6.1% within herds (Hancock et al., 1998). In all farms involved in the study, at least one E. coli O157 colony was isolated, indicating a low but wide distribution of the pathogen in those areas. Studies performed in places other than the USA have resulted in different estimations on the frequency of E. coli O157:H7 and STEC in dairy cattle. In a 4month study period in Sheffield (UK), from late May until late August, an increase in the prevalence among dairy cattle (8.3%) was reported (Chapman et al., 1994). In this study, fecal samples, collected by rectal swabs, were subjected to preenrichment in VCC-BPW for 6 h, followed by IMS and plating on CT-SMAC. When the authors compared the results obtained following this procedure to those obtained from direct plating on CT-SMAC, a 100-fold increase in the recovery sensitivity of E. coli O157 was observed. In another study in Japan, it was reported that the prevalence of STEC was 46% in calves, 66% in heifers, and 69% in cows as determined with the use of PCR when screening rectal stool samples; however, when characterizing the microorganisms, only 1 colony out of 92 isolates belonged to the O157 serogroup (Kobayashi et al., 2001). These results may suggest that although E. coli O157 is present in other countries, other serotypes may account for the majority of STEC infections in places other than North America. In a characterization study conducted in the Netherlands, cattle and calves fecal samples were collected for examination (Heuvelink et al., 1998a). Isolation of the pathogen was conducted by two methods: IMS after preenrichment in EC+novobiocin broth for 6 h and selective plating of dilutions onto CT-SMAC after

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18-20 h of incubation in the same broth. Colonies were confirmed by the use of an agglutination test. Overall, E. coli O157 was isolated in 11.1% of adult cattle and 0.5% of veal calves examined, showing the IMS technique to be more sensitive in detecting the pathogen and significantly more sensitive than direct plating by seven fold in this particular study. In the next study, samples obtained by digital rectal retrieval were collected at different farms and examined using the same procedure of the previous study. Microbiological examination resulted in a prevalence among cattle varying from 0.8% to 22.4%. A seasonal variation was also observed, with the highest rate of positive E. coli O157 isolates recovered in September and October, and the lowest recovered during the winter (Heuvelink et al., 1998b). A disparity in the pathogen’s prevalence was also observed by age group, with calves between 4 to 12 months exhibiting the highest excretion rate (21.7%) as compared to calves younger than 4 months (6.7%), heifers ages 1 to 2 years (3.7%), cows ages 2 to 3 years (2.2%), and cattle older than 3 years (10.7%). A study conducted in Canada during a 1-year period determined the prevalence of E. coli O157:H7 in cattle at slaughter. Fecal and rumen samples were analyzed both by 1) direct plating on CT-SMAC and 2) enrichment in TSB for 6 h followed by IMS and isolation on CT-SMAC. Recovery of E. coli O157:H7 was higher when IMS was used, resulting in an overall 7.5% prevalence on the samples. A seasonal variation in prevalence was also observed, with the highest (19.7%) in the summer, and the smallest (0.7%) in the winter (Van Donkersgoed et al., 1999), supporting results from previous studies.

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In a longitudinal study of calves conducted in Finland, rectal fecal samples were obtained from individual animals with the use of plastic gloves. Samples were enriched in TSB and subjected to IMS; followed by plating on both SMAC and CTSMAC for further isolation of the pathogen. Upon arrival at the farm, all animals were negative for E. coli O157; however, positive animals were detected one day later. During the fattening period, the infection among animals ranged from 0-38.5% and at slaughter, positive fecal samples varied from 9.7%-38.9% (Lahti et al., 2003). This study found that the animals were shedders of the pathogen in colder months (temperatures between 0-3°C) and suggested that infection of animals was probably acquired at the finishing farm. In one of the earliest E. coli O157:H7 prevalence studies, which was conducted in New York State during the periods of August to September of 1995, a comparison of culture methods for the recovery of the pathogen was also performed. Fecal samples were obtained from animals (primarily Holstein cattle) just hours prior to slaughter and examined by different methods. The authors established that the use of a broth enrichment rather than direct plating was more successful in the recovery of the pathogen. By plating the samples directly onto SMAC, the prevalence was 0.96%; however, by incorporating TSBcv in the enrichment step and plating on CT-SMAC, the E. coli O157:H7 prevalence results were 1.3% (McDonough et al., 2000). Although an increase of just 0.34% in the prevalence was observed, the use of preenrichment followed by IMS proved again to be more sensitive in the recovery of the pathogen.

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1.5.2.2. Prevalence in beef cattle Along with results obtained from dairy cattle studies, beef cattle also have shown differences in the occurrence of E. coli O157:H7. Numerous microbiological methods have been used to detect the pathogen, and studies differ in several factors. In a large study in Kansas and Nebraska, performed at the level of range production environments, the frequency of E. coli O157 was determined from different sources (Renter et al., 2003). Fresh beef fecal samples were obtained after animals were observed defecating. After arrival to the lab, 1 g of the sample was incubated in universal preenrichment broth for 16-18 h, plated on CT-SMAC, and finally confirmed with latex agglutination resulting in a low prevalence of the pathogen (0.90%) in the 9122 total examined samples. In contrast, another study resulted in a higher prevalence of the pathogen, with an overall 15.7% recurrence among the examined cattle (Chapman et al., 1997). In this study, fecal samples were obtained with swabs from the rectum of cattle immediately after slaughter, preenriched in BPW-VCC for 6 h, subjected to IMS and inoculated on CT-SMAC. The authors reported a seasonal variation in the recovery of E. coli O157, with a monthly prevalence in individual animals ranging from 4.8% in December to 36.8% in May, the largest to date reported for individual animals. In a study performed in Midwestern United States feedyards, recovery of E. coli O157:H7 was achieved after collection of feces from the rectum of animals. Samples were subjected to enrichment in VCC-GN broth, IMS, and isolation on CTSMAC. The investigation revealed a prevalence of cattle shedding the pathogen

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ranging from 0.7% to 79.8%, and resulted in 100% of the feedlots testing positive for this pathogen (Smith et al., 2001). In a longitudinal observational study, the prevalence and duration of shedding by feedlot cattle was conducted during 19 weeks. Animals during the finishing period were tested by analyzing feces collected from the rectum. During the course of the study, the prevalence of E. coli O157:H7 shedding varied from 1% to 80% (Khaitsa et al., 2003). This investigation revealed three characteristic prevalence intervals for the pathogen in that particular feedlot. The first interval, or pre-epidemic, was characterized by a low incidence in the fecal excretion of E. coli O157:H7 for the first eight weeks; the second period, or epidemic, revealed a dramatically increase in the number of animals infected after week 9; and the third phase, or post-epidemic, where a substantial decrease in shedding was observed during the last 5 weeks of the study. The importance of this study was the suggestion that the variation of the prevalence of E. coli O157:H7 in the observed animals was not only affected by the incidence or occurrence of new cases of infection, but also a function of the interval or period of time in which animals are infected. The observation made by Khaitsa et al. (2003) coincides to those concluded earlier by Besser et al., (1997), in which excretion of the pathogen by dairy cattle was temporary and of short duration, normally for a period of four weeks. In another survey performed in Midwestern United States processing plants, the frequency of E. coli O157:H7 and EHEC O157 was estimated (Elder et al., 2000). The samples were collected during late summer (July) and early fall (August)

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of 1999. Fecal, hide, and carcasses (preevisceration) samples presented a prevalence of 28%, 11%, and 43%, respectively. An overall 46.3% of all carcasses sampled positive for EHEC O157 in at least one site. The most notorious finding was that the pathogen was isolated from at least one animal sample in 72% of the lots involved in the study, confirming a wide distribution of the microorganism. A similar observation was noticed later in a large study among cattle within one month of market (feeding periods) conducted at feedlots located in Kansas, Nebraska, Texas, and Oklahoma; states representing approximately 70% of the cattle on feed in the United States (Sargeant et al., 2003). Sampling was conducted during May to August 2001. One gram of fecal material was collected after animals were observed defecating, and enriched in VCC-GN broth for 6 h. Immunomagnetic separation was conducted on all samples, followed by plating and isolation on CT-SMAC. Final testing was conducted with a latex agglutination test. Overall, 10.2% of fecal samples were positive for the pathogen; and over 95% of the participating feedlots had at least one positive E. coli O157:H7 fecal sample, supporting the theory of a large distribution of the bacteria in the environment. More recently, Barkocy-Gallagher et al., (2003) confirmed a seasonal variation in the recovery of E. coli O157:H7, this time in a research performed at three beef processing plants located in the Midwestern USA. After animals were slaughtered, samples were obtained from the hide, feces, and the carcasses prior to the preevisceration wash. Hide and carcasses samples were obtained by swabbing the sample area, while fecal samples were obtained from the colon after

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evisceration. Samples were subjected to enrichment in TSB, detection with IMS, and isolation on CT-SMAC. Overall, the fecal, hides, and carcasses samples resulted in 5.9%, 60.6%, and 26.7% prevalence, respectively. For all the types of samples examined, the highest E. coli O157:H7 prevalence peaked in the summer with 12.9%, 73.5%, and 40.8%; and decreased to 0.3%, 29.4%, and 1.2% during the winter for feces, hides, and preevisceration carcasses, respectively. The primary source for E. coli O157:H7 recovery was the hides, imputing them as the major contributing factor in carcass contamination. Despite the difference in the scale and types of production systems evaluated, a trend towards an increase in the number of animals infected with E. coli O157:H7 can be seen due an increment in the sensitivity of the reliable microbiological methods used to detect the pathogen. More research is needed concerning the occurrence of infection and shedding of E. coli O157:H7 among food animals to better understand its prevalence and ecology. This research will benefit the design and implementation of control methods to reduce the presence of the pathogen.

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CHAPTER II SURVIVAL OF ESCHERICHIA COLI O157:H7 IN BOVINE FECES UNDER VARIOUS STORAGE CONDITIONS

2.1. Abstract Although E. coli O157:H7 prevalence estimates in cattle have increased over time due improvements in detection methodologies, fecal sample transport conditions from farm to microbiological laboratories for their further analysis may be a factor for prevalence underestimation of this pathogen, because the pathogen may not survive under certain storage conditions. The objective of this study was to compare the survival of E. coli O157:H7 in bovine feces under various experimental conditions to ensure maximum recovery of the pathogen after collection. Bovine fecal samples were inoculated with an inoculum (1x105 cfu/g) of a cocktail containing 4 different E. coli O157:H7 strains. Each inoculated sample was subdivided and placed in each of the 4 following conditions: 37°C, room temperature (23°C), refrigeration temperature (4.4°C) and in plastic coolers with refrigerant packs to simulate transportation conditions. Samples from each of the temperature conditions were taken at 0 h, 24 h, 48 h, 120 h, and 144 h and subjected to both detection and quantification of E. coli O157:H7. A statistical decrease in the population of the pathogen was observable after 120 h in the samples held at 37°C (P < 0.0001); and by time 168 h at 4.4°C (P = 0.033). No

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decrease was observable at 23°C after 168 h (P = 0.3348). Under the cooler conditions the samples showed an increase in the population numbers (P = 0.0133) after 48 h. At 37oC, detection of the pathogen with IMS was negative after 48 h, either by direct plating or by IMS. Holding samples in an ice cooler resulted in detectable populations in inoculated and control treatments for up to 120 h. At room temperature, control samples were positive for the pathogen for up to 48 hours with the use of IMS; while inoculated samples were positive through the entire study. Overall, at all temperatures less than 23°C, the E. coli O157:H7 survived in the samples for 168 h indicating that shipment and storage under these conditions prior to microbiological analysis would be acceptable and may not be affecting obtained results.

2.2. Introduction Escherichia coli O157:H7 is an important human pathogen, which accounts for nearly 74,000 infections and approximately 61 deaths yearly (CDC, 2003; Mead et al., 1999). Several routes of transmission have been identified (Renter et al., 2002; Griffin et al., 1991), including environmental and water sources in addition to animal sources (Ibekwe et al., 2003; Van Donkersgoed et al., 2001; LeJeune et al., 2001; Rasmussen et al., 2001). The majority of the outbreaks have been linked to the consumption of a wide variety of foods (Michino et al., 1999; Griffin et al., 1991; Altekruse et al., 1997; Watanabe et al., 1999), with the greatest percentage involving consumption of undercooked

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ground beef products (Cassin et al., 1998; Eisel et al., 1997; Tuttle et al., 1999; Riley et al., 1983). Different animals can harbor E. coli O157:H7 (Beutin et al., 1996; Beutin, 1999), but cattle, both beef and dairy, are believed to be the major reservoirs of this pathogen (Maule, 2000; Hancock et al., 1994; Wells et al., 1991). Numerous scientist have studied the prevalence of this pathogen among cattle at various ages of the animal using a wide variety of both sampling and detection methods (Hancock et al., 1994; Hancock et al, 1997; Renter et al., 2003; Renter et al., 2004; Chapman et al., 1989; Chapman et al., 1992). In recent studies, detection of E. coli O157:H7 in different environmental samples has been possible because of sensitive methods that allow detection of the pathogen even when present in very small numbers. In a study involving different animals on a farm, the persistence of E. coli O157:H7 in cattle was observed during a 1-year period. The persistence of this pathogen in these animals varied from 4.8% at the lowest prevalence in December to 36.8% of individual animals shedding the pathogen at its highest prevalence in May, for an overall 15.7% prevalence over the year (Chapman et al., 1997). In another study, cattle from different lots presented for slaughter at a meat processing plant were tested for E. coli O157. In that study, 21 out of 29 lots (72%) presented at least one positive fecal sample; and 26 out of 30 lots (87%) had at least one E. coli O157 positive carcass sample at pre-evisceration (Elder et al., 2000). Smith et al. (2001) reported that at least one positive colony was recovered in all the pens from 100% of the feedlots involved in that study;

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suggesting these results that E. coli O157 is more common in group of animals and feedyards than previously reported. As the methods used in different studies improve, the prevalence of the pathogen could increase, yielding a higher, more accurate reporting of the prevalence of the pathogen. Pre-harvest food safety is part of the farm-to-table continuum, in which public health can be improved by making all the segments of the food chain responsible of reducing the hazards associated with the consumption of food products (Childers et al., 1996; WHO, 2001). One of the main objectives of preharvest food safety is to decrease the numbers of pathogens at the farm-animal production level, in order to reduce their presence in the final product while retaining high quality and nutritional value. Consequently, the occurrence of a lower number of pathogens in the food may translate in a reduction and prevention of several numbers of cases of foodborne illness and/or deaths after consumption of contaminated foods. Several different management strategies and interventions have been suggested, tested, and implemented in order to reduce E. coli O157:H7 both in live animals and in the carcasses which could become contaminated after slaughter (Dorsa et al., 1997; Gill et al., 2004; Kang et al., 2001). Some of the strategies and methods have been proven effective while others have not. Changes in management practices such as withholding feed, chlorination of water, scraping pens and changing the types of feed, either in the days prior to slaughter or during the fattening period, are not considered to be an effective

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means of controlling E. coli O157:H7 in the pre-harvest environment (Van Baale et al., 2004; Diez-Gonzalez et al., 1998; Berg et al., 2004; Russell et al., 2000; Braden et al., 2004; Grauke et al., 2003; Buchko et al., 2000; Callaway et al., 2003; Van Baale et al., 2004). While some of the studies report reduction of the pathogen, others report no change or an increase in pathogens loads. Because data are conflicting, no management practice can be pinpointed as a control measure for pathogens in the feedlot. Interventions such as the use of competitive microflora and direct feed microbials (DFM) have been proven effective in reducing E. coli O157 in the live animal, but not eliminating it completely (Brashears et al., 2003; Tkalcic et al., 2003; Younts-Dahl et al., 2004; Zhao et al., 1998; Zhao et al., 2003). Use of other interventions such as bacteriophages (O’ Flynn et al., 2004; Kudva et al., 1999; Tanji et al., 2004); sodium chlorate (Edrington et al., 2003; Rice et al., 1999; Zhao et al., 2001; Callaway et al., 2002; Callaway et al., 2004), neomycin sulfate (Alali et al., 2004), and vaccination of animals (Potter et al., 2004) have also been proven effective, but are still in the investigative stages and need regulatory approval before they can be implemented (Castillo et al., 1998; Byrne et al., 2000; Gill, 2004; Bosilevac et al., 2004; Longstreeth et al., 1997; Nou et al., 2003). In order to determine if the efforts made in using interventions are effectively reducing and controlling the numbers of E. coli O157:H7 in cattle, the status of the animal, i.e., positive or negative for E. coli O157:H7 by

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microbiological exams, has to be established. In most of the cases, the locations of the different feedlots, feedyards, and slaughterhouses where the use of these interventions are studied and performed are in a different site to those where the samples are analyzed microbiologically. Despite the use of overnight shipping services, sometimes samples can take several days to reach the laboratory, possibly affecting in this way the survival of the pathogen, and therefore, the outcome of the investigation and the validity of the results. Additionally, laboratories may receive a large number of samples in a day that can not all be processed immediately and must be held. The objectives of this study were to determine simultaneously the survival characteristics of E. coli O157:H7 in bovine feces under different storage conditions. By simulating the transportation and handling conditions that fecal samples encounter, the effects of factors such as temperature and length of storage on the pathogen’s population during shipping can be studied and analyzed. Bovine fecal samples inoculated with a cocktail of antibiotic resistant E. coli O157:H7 strains were held at different temperature conditions for up to 7 days and analyzed microbiologically for numbers of and presence of the pathogen at three different times.

2.3. Materials And Methods 2.3.1. Study Design This study had two treatments with four storage conditions analyzed over time, and may be analyzed by a 2 x 4 factorial with repeated measurements

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during time design. Three replications were conducted for both treatments during the entire experiment. Sampling was performed at separate occasions and no attempt was made to ensure that the fecal samples were exactly from the same animals or pens; however, all fecal samples were initially standardized by testing to determine if they were positive for E. coli O157 naturally. Randomization was performed at two different times: when collecting the fecal material, randomization was performed to assign the manure either to the control or to the inoculated treatment and the second randomization was performed after inoculation of the fecal material with E. coli O157:H7 in the treatment and addition of buffered peptone water to the control. Samples were mixed, subdivided, and randomly assigned to each of the temperature conditions to which they were held for the course of the study.

2.3.2. Sample Collection Fresh fecal samples were aseptically obtained from cattle housed at the Texas Tech University Burnett center facilities in New Deal, Texas. At each replication, a total of two samples, each one with approximately 1000 g of fresh feces, were obtained directly from the pen and placed in sterile, labeled plastic bags. Each collected sample was taken by the use of disposable gloves. After collection bags were stored in a plastic cooler with ice packs, transported directly from the farm to Texas Tech University food microbiology lab, and examined within 30-60 minutes after collection.

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2.3.3. Microbiological Culture Selection and Conditions A cocktail of 4 different streptomycin-resistant Escherichia coli O157:H7 strains (920, 922, 944, and 966) from the Texas Tech University Food Microbiology laboratory was prepared prior to use. All E. coli O157:H7 isolates were originally isolated from cattle. Stock cultures maintained in tryptic soy broth (TSB, Difco) plus 15% wt/vol glycerol (BHD Inc., Toronto, Canada) that were previously frozen in cryovials at -80°C were grown separately and subcultured twice in TSB for 24 h at 37°C prior to experimental use. Equal volumes of the four different strains were combined after culturing to obtain the inoculation mixture.

2.3.4. Inoculated Sample Preparation Immediately after arrival, 1000 g of feces were inoculated with a mixture of 99 ml of buffered peptone water (BPW, Difco) with 11 ml of the cocktail previously described. Feces inside the bag were mixed manually for two minutes to homogenously distribute the E. coli O157:H7 cocktail in the entire sample. After mixing the inoculated sample was divided into 4 sub-samples. Each subsample of the inoculated feces, approximately 250 g, was taken with the use of sterile spatulas and placed in sterile Whirl-Pak bags labeled for each temperature treatment: 1) 37°C, 2) RT (room temperature at 23°C), 3) Cooler, and 4) refrigeration temperature at 4.4°C.

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2.3.5. Control Sample Preparation 110 ml of sterile BPW was added to 1000 g of feces and mixed manually for two minutes. This process was done simultaneously as the inoculated sample preparation. Special attention was placed in order to avoid crosscontamination with the inoculated samples. Non-inoculated manure was also divided in four sub-samples, randomly assigned to the same temperature holding conditions, and placed into sterile, labeled Whirl-Pak bags as described previously.

2.3.6. Microbial Analysis for Detection of E. coli O157:H7 Immediately after subdividing the fecal samples, 10 g of feces for each treatment and control were transferred for enrichment directly into dilution bottles containing 90 ml of GN broth, Hajna (Difco laboratories, Becton Dickinson and Co., Sparks, MD.) supplemented with 10 µg/L of vancomycin (Sigma – Aldrich Co., St Louis, MO), 8 µg/L of cefsulodin (Sigma), and 50 µg/L of cefixime (Dynal Inc., Lake Success, NY); (VCC - GNBroth) and incubated at 37°C for 6 h (Fenlon et al., 2000; Foster et al., 2003; Sanderson et al., 1995; Chapman et al., 1994; Chapman et al., 1997; Restaino et al., 2001; Zhao et al., 2001; Taormina et al., 1998; MacRae et al., 1997; Fujisawa et al., 2000; Fujisawa et al., 2002)..

2.3.6.1. Detection of E. coli O157:H7 using Immunomagnetic Separation After pre-enrichment, 1 ml of each sub-sample, control and inoculated treatment, was analyzed by a sensitive essay using immunomagnetic separation

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(IMS) with 20 µl of dynabeads anti – E. coli O157 (Dynal Biotech ASA, Oslo, Norway). The sample was washed three times in phosphate buffered saline with tween 20 (PBST; Sigma) while subjected to a magnetic field via a Dynal magnetic concentrator in order to separate and capture the bacteria (LekowskaKochaniak et al., 2002; Wright et al., 1994; Johnsen et al., 2001; Safarikova et al., 2001; Drysdale et al., 2004; Cubbon et al., 1996; Parham et al., 2003; Kerr et al., 2001; Chapman et al., 1994; Chapman et al., 1996; Tu et al., 2001; Tomoyasu, 1998). After completion of the procedure, the beads were resuspended in 0.05 ml of the phosphate buffer. The bead-bacteria suspension was vortexed and spread onto sorbitol MacConkey agar plates (SMAC; Difco laboratories, Becton Dickinson and Co., Sparks, MD.) supplemented with cefixime (0.05 µl/L) and potassium tellurite (50 µl/L) (CT-SMAC; Dynal) in order to inhibit Proteus spp. and gram-positive microorganisms, and incubated at 37°C for 18 h to 24 h in an aerobic chamber (Renter et al., 2003; Duffy et al., 1999; De Boer et al., 2000; Barkocy-Gallagher et al., 2002; Paton et al., 1998; Sargeant et al., 2003; Sanderson et al., 1995; Chapman et al., 1991; Zadik et al., 1993; Hu et al., 1999).

2.3.6.1.1. Isolation of E. coli O157:H7 After incubation two typical colorless sorbitol-negative colonies per plate were picked and streaked for isolation on CT-SMAC and placed overnight at 37°C (Karch et al., 2001; Pearce et al., 1994; Bettelheim et al., 2003; March et

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al., 1986; Fujisawa et al., 2000; Fujisawa et al., 2002; Hammack et al., 1997). A single sorbitol-non-fermenting colony was selected and subcultured onto MacConkey (MAC, Difco) and Fluorocult E. coli O157:H7 agars (Fluorocult, EM Science, Gibbstown, NJ) and incubated overnight at 37°C. After incubation pink lactose-positive, 4-methylumbelliferyl-β-D-glucuronide (MUG) negative colonies were transferred from the MAC plates into tryptic soy broth (TSB, Sigma), triple sugar iron agar tubes (TSI, Difco), and MacConkey broth (MACb, Difco) for further testing and incubated overnight in aerobic conditions at 37°C (De Boer, 1998; Restaino et al., 1999; Manafi, 1996; Manafi, 2000; Manafi et al., 2001). Colonies that were indole-positive on TSB, dextrose, lactose and/or sucrose positive fermenting plus gas production on TSI, and gram negative lactose fermenting on MACb were considered as presumptive positive E. coli O157:H7. For final confirmation results, colonies from the MAC plates described before and that were stored under refrigeration temperatures at 4.4°C for less than 24 h were subjected to a commercial latex agglutination test specific for the O157:H7 antigen (Remel, Lenexa, Kans.); (Sowers et al., 1996; Chapman et al., 1989; March et al., 1989). For this study, only colonies that were confirmed with the agglutination test were considered positive.

2.3.6.2. Quantification of E. coli O157:H7 by direct plating Immediately after subdividing both inoculated and control samples, 11 g of feces were weighed and placed into stomacher filter bags with 99 ml of BPW. All

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sub-samples were mixed well for 2 min at norm speed (Stomacher 400 Laboratory blender, Seward Medical, London). E. coli O157:H7 counts were determined by duplicate plating by direct culture of the mixed dilution with the use of an autoplater (Autoplate 4000, Spiral Biotech, Inc., Norwood, MA). 50 µl of the suspension was spread onto EC medium with MUG agar plates (ECMUG, Difco) supplemented with streptomycin and incubated at 37°C for 18 h to 24 h in an aerobic chamber. After incubation duplicate plates were counted with the Q – count (Spiral Biotech, Inc.). After enumeration, plates were examined under UV light to detect 4-methylumbelliferyl-β-D-glucuronide negative colonies. Up to 10 MUG negative colonies were selected per plate, streaked for isolation on MAC agar plates, and placed overnight at 37°C. After incubation pink, lactose-positive colonies from each plate were subjected to a latex agglutination test for final confirmation (Sowers et al., 1996; Chapman et al., 1989; March et al., 1989). Final counts in this step were corrected 1) by taking into account the exact weight of each fecal sample before addition of BPW, and 2) the percentage of colonies that resulted positive after the agglutination test before transforming results into log10 data

2.3.7. Sampling times and storage conditions Detection with IMS and direct plating for quantification of E. coli O157:H7 were conducted for each sub-sample in every replication at times 0 h (immediately after subdividing samples), 24 h, 48 h, 120 h, and 168 h. Inoculated

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and control sub-samples were stored in the following conditions: Incubator (37°C), room temperature (23°C), refrigeration temperature (4.4°C) and inside a insulated container (Rubbermaid, model 9482, 21 ¾ in x 13 5/8 in x 15 ½ in). The plastic cooler was filled with refrigerant packs (Utek, Polyfoam Packers Corporation, Wheeling, IL) that were not replaced during the curse of the replication to mimic shipping conditions. Temperatures inside the cooler were measured at each sampling time.

2.3.8. Statistical Analysis All the experiments were performed in triplicate for each of the temperature conditions. Once counts were obtained for each of the sub-samples, the cfu/g was transformed into log10 units. To analyze statistical significant differences in the survival of E. coli O157:H7 among the different treatments (the effect of each temperature on bacterial growth), the data was analyzed by using the Proc Mixed Procedure of the SAS system (SAS, 2001). In this model the dependent variable or characteristic evaluated was the population of bacteria at a given time (Log10 CFU). The different temperatures and the treatments were the fixed effects in the models; the treatments (either inoculated or control) also were assigned randomly during the experiment. Each one of the three replications was considered as blocks, since no differences in the population of E. coli O157:H7 were expected due to the source of feces that were used in the experiment.

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2.4. Results Sub-samples were analyzed to detect simultaneously the presence of E. coli O157:H7 with IMS and to quantify the survival of the pathogen at different temperature conditions. For each of the treatments, the least squares mean (LSM) estimates of average E. coli O157:H7 fecal population (log10 cfu/g) obtained by using the quantification method can be observed in Table 2.1. No E. coli O157:H7 colonies were recovered from the control samples using this method. In general, the average initial amount of inoculated E. coli O157:H7 in bovine feces was 5.01 log10 cfu/g, with a range starting from 4.91 to 5.06 log10 cfu/g at the time of inoculation. There were not significant differences among the populations at time 0 h.

2.4.1. Cooler Results In the cooler, the E. coli O157:H7 survived during the entire course of the study with very few differences observed. In addition to the quantification data, the pathogen was also detected in the samples held in the cooler. While the numbers of E. coli O157:H7 detected from time 0 h to 168 h are very similar, the E. coli O157:H7 showed a statistical significant increase in the population by 0.09 log10 cfu/g (P = 0.0349), after 24 h, reaching its maximum at 48 h (P = 0.0133) and decreasing after 168 h to 5.02 log10 cfu/g (P = 0.264). Because there was very little variation in the data, significant differences were observed, but they are likely not practical.

79

2.4.2. Refrigeration Results At 4.4°C, there was a slight decrease in the population of E. coli O157:H7 by the end of the study. From time 0 h to time 120 h no statistical significant variation in the population was observed; however, by time 168 h a significant 0.37 log10 cfu/g (P = 0.033) reduction was observed when compared to the initial count indicating that samples should not be held under refrigeration conditions for more than 120 h.

2.4.3. Room Temperature Results At 23°C, there was no observable E. coli O157:H7 growth in the samples. A trend to a decrease in the population was observable; however, it was not statistically significant at any sampling time. At 23°C detection of the pathogen was possible in the inoculated samples from time 0h to 48h and at time 168h. Surprisingly, the use of pre-enrichment and IMS did not allow any recovery of the pathogen in any of the replications that were performed at time 120h. Positive results were also obtained in the control samples, from time 0 h to 120 h. None of the control samples was positive at time 168 h in any of the replications.

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Table 2.1. Least square means values for the population of E. coli O157:H7 (log 10 cfu/g) in inoculated bovine feces held at different t temperature conditions at each of the sampling times.

Treatments

0h

24 h

48 h

120 h

168 h

Control†

NR

NR

NR

NR

NR

Cooler**

4.91

5.00 b

5.06 b

5.05

5.02

4.4°C

5.06

5.03

5.06

4.81

4.69 b

23°C

5.04

4.99

5.04

4.99

4.87

37°C

5.01

4.71

3.44 a

0.00 a

0.00 a

†

Control: Non-inoculated samples subjected to the same temperature

conditions as inoculated feces. ** Cooler: The temperature inside the cooler was 0°C, 4°C, 14°C, 21°C, and 23°C at time 0 h, 24 h, 48 h, 120 h, and 168 h, respectively. NR: No E. coli O157:H7 colonies were recovered by the quantification method at any of the temperature conditions. a

P < 0.001 Difference in LSM compared to time 0 h.

b

P < 0.05 Difference in LSM compared to time 0 h

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2.4.4. 37°C Results At 37°C, the population of E. coli O157:H7 decreased from 5.01 log10 cfu/g at time 0h to 4.71 log10 cfu/g at time 24h (P = 0.0825). After 48h, the population decreased dramatically by 1.57 log10 cfu/g (P <0.0001). After 120 h no colonies were recovered by direct plating (P <0.0001); (Table 2.2.). From time 0h to 48h E. coli O157:H7 colonies were recovered with the use of the pre-enrichment step and IMS, but no pathogen was detectable after 120 h. Control samples at 37°C also were positive with IMS, at 0 h on replications 1 and 2, and at time 24 h on replications 1 and 3. A more complete description of the E. coli O157:H7 population change (log10 cfu/g) together with the probabilities of differences between treatments at any given time can be observed in Appendix A to Appendix D.

2.5. Discussion Results obtained from samples held at 4.4°C, in the cooler with ice packs, and at 23°C indicate that viable E. coli O157:H7 can be cultured for up to 168 h (7 days) both by direct plating and through the use of a pre-enrichment step together with the immunomagnetic separation. Storage of fecal samples at 37°C yielded negative results by both methods after 120 h. After 24 h, samples stored at 37°C resulted in a significant decrease in the population of E. coli O157:H7, with a decrease of approximately 1.57 log10 cfu/g after 48 h at this temperature

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Table 2.2. Final IMS results for E. coli O157:H7 obtained from inoculated bovine fecal samples incubated at 37°C.

Sampling Time

0h

24 h

48 h

120 h

168 h

Replication

Temperature

37°C

37°C

37°C

37°C

37°C

1

Inoculated

+

+

+

-

-

2

Inoculated

+

+

-

-

-

3

Inoculated

+

-

+

-

-

1

Control

+

+

-

-

-

2

Control

+

-

-

-

-

3

Control

-

+

-

-

-

83

conditions. This is likely due to the culture entering the decline phase of the microbial growth curve. At 4.4°C, there was a significant reduction in the populations after 120 h, but the pathogen was still detectable. Because naturally infected samples would not be as high of population load as the inoculated ones in the laboratory, it would be suggested that samples held at refrigeration conditions to be analyzed within 120 h. A few studies report the survival characteristics of E. coli O157:H7 in bovine manure. In one of the earliest studies, it was demonstrated that the pathogen’s survival rates were affected by the temperature at which samples were held, the inoculum level at the beginning of the experiment, and perhaps the moisture content of the fecal sample (Wang et al., 1996). In that study, samples were inoculated at two different amounts of bacteria and stored at three different environmental conditions. At 5°C, no growth was observed either at the low and high inoculum level (103 and 105 cfu/g, respectively); but the pathogen survived for 63 and 70 days under this conditions. Although our experiment was not intended to hold the samples for this long, our results are similar in that the pathogen survived for 7 days at 4.4°C when inoculated at 105 cfu/g. The pathogen was recovered by direct plating and IMS by the end of the study, with a slight decrease in the population after 120 h (P = 0.1419). Wang et al., (1996) reported that at 22°C E. coli O157:H7 survived 49 and 56 days at the low and high inoculum, with an increase between 1.5 to 2.0 log10 cfu/g during the first three days at this temperature condition. Our inoculated samples did not show

84

growth at 23°C, in fact, a small decrease in the population (0.16 log10 cfu/g) was observed by 168 h; however, it was not statistically significant (P = 0.3348). Despite the small decrease in the population, the pathogen was recovered from these samples by both methods for up to seven days. The most noticeable difference between our samples and the results reported by Wang occurred with the samples at 37°C. Wang et al. (1996) reported an increase in about 2 log10 cfu/g by the second day after inoculation, recovering the pathogen with the use of a pre-enrichment step at day 42 and 49 for the low and high inoculum, respectively. Our study revealed a decrease in the population after 24 h, with a decrease of 1.57 log10 cfu/g (P <0.0001) after 48 h. After 120 h (5 days) under this temperature condition the pathogen could not be recovered by neither direct plating nor use of IMS. When comparing both studies, even though the amount of inoculum used in both experiments was similar, two differences could be found: 1) the amount used for microbial analysis and detection in our protocol was 10 times higher than the 1 g used in theirs; and 2) the use of GN-VCC broth in our experiment instead of TSB as pre-enrichment media for the recovery of the pathogen. Other studies have revealed similar long-term survival rates of E. coli O157:H7 in bovine feces when held at similar conditions. Fukushima et al., (1999) reported that the pathogen was detectable in samples collected just after excretion from 4 to 8 weeks at 25°C and for 16 to 18 weeks at 15°C. Kudva et al., (1998) reported a reduction of 2 log cycles in the samples after 48 h at 4°C and a

85

constant trend in the population thereafter. A similar trend was observed in our samples; however, initial reduction was not as big in magnitude as that previously reported, probably due the highest inoculum (6.6 x109 cfu/g) in that study. Kudva et al. (1998) also reported no viable bacteria after approximately 5 days when samples were at 37°C, a similar result to those obtained in our experiment. One of the most surprising results was that obtained from the control fecal samples. Despite being analyzed with IMS for detection of the pathogen prior to addition of BPW, once the experiment started some samples yielded positive IMS results, indicating that the microorganism was naturally occurring in the samples at the time of collection, but maybe in such small quantities that were not detected during the first IMS procedure. These pathogens may be injured when the initial samples were collected and recovered during the holding time. While IMS methods detected the pathogen, the enumeration methods were not sensitive enough to detect it; so it would not have an impact on our final enumeration results. Another possibility is that the pathogen is not evenly distributed in the fecal pat, possibly missing its detection when sampling in the initial step, but being able to be recovered after addition of the BPW and homogenization of the sample through manual mixing for 2 minutes. As expected, at any given treatment none of the control samples analyzed with the quantification method were positive for E. coli O157:H7; however, some of the controls subjected to IMS were as described before. Control samples held at

86

4.4°C yielded positive results from time 0 h to time 120 h on replication 2; at 23°C a positive result was obtained on time 120 h (replication1) and from 0 h to 48 h and from 0 h to 24 h in replication 2 and 3, respectively. When samples were held at 37°C, positive E. coli O157:H7 colonies were isolated from time 0 h to 24 h, at 0 h, and at 24 h time 0 h in replication 1, 2, and 3, respectively; however, none of the control samples at this temperature resulted positive for the microorganism after time 120 h. Finally, control sub-samples subjected to the cooler conditions yielded positive E. coli O157:H7 results at time 120 h in replication 1, from time 0 h to time 168 h in replication 2, and at time 168 h in replication 3, respectively. Because the amount of E. coli O157:H7 in the naturally infected samples was below our detection limits or direct plating, it is unlikely that the natural pathogen loads has an impact on the results of this study. The samples that were enumerated were not subjected to the preenrichment step and this factor together with the use of selective media with antimicrobial agents to suppress the growth of competitive flora may explain why no growth was presented on control samples after subjected to quantification when IMS yielded contrary results. IMS allows injured cells to recover and increase numbers if present in the sample, allowing higher rates of recovery than when other methods are used and accounting for the detection of the bacteria even when no counts were able to be performed with the same sub-samples subjected to quantification (Wright et al., 1994; Foster et al., 2003; Parham et al.,

87

2003; Chapman et al., 1994). Overall, the pathogen was recovered for up to 168 h when all the temperatures were equal or under 23°C but no colonies were recovered after 120 h when samples where incubated at 37°C. The cooler conditions allowed recovering of the pathogen for the course of the study, and from time 24 h to 168 h the population levels even increased as the temperature inside the container raised, indicating that shipping and handling under these conditions before processing is admissible.

88

CHAPTER III NON-UNIFORM DISTRIBUTION OF ESCHERICHIA COLI O157:H7 IN BOVINE FECES AND UNDERESTIMATION OF PREVALENCE

3.1. Abstract Several studies reporting different sampling methods, size, amount of fecal material collected and microbiological analysis protocols have shown a wide range of results in the prevalence of E. coli O157:H7. The objective of this study was to evaluate sampling strategies for fecal pats in order to improve accuracy of E. coli O157:H7 prevalence estimates. A total of 120 bovine fecal pats were used in this study. Five fecal sections for each pat were collected systematically going from west to east (positions 1 to 5, respectively) in north to south lines direction in order to avoid cross contamination and each was cultured for E. coli O157:H7 within two hours after sample collection. Samples were preenriched in GN-VCC broth for six hours and subjected to a sensitive assay using immunomagnetic bead separation to detect E. coli O157:H7. Final positive cultures were confirmed by using an agglutination test kit specific for the pathogen and PCR analysis for shiga toxins. Of the 120 fecal pats, 96 (80%) had no positive sub-samples in any of the 5 positions. Of the 600 total number of subsamples analyzed (5 per fecal pat), 49 were positive with 14, 9, 8, 8, and 10 on position A, B, C, D, and E, respectively. Prevalence estimates may be underestimated as a result of an uneven distribution in fecal material and

89

collection of a small sample size; therefore, sampling procedure plays a critical role in E. coli O157:H7 detection in bovine fecal pats.

3.2. Introduction Escherichia coli O157:H7, one of hundreds of strains of the Escherichia coli group, has become one of the most important emerging foodborne pathogens in recent years, causing an estimate of 74,000 cases of infection and 61 deaths in the United States every year (Mead et al., 1999). In several outbreaks, the pathogen has been isolated from a wide variety of foods (Griffin et al., 1991; Altekruse et al., 1997; Watanabe et al., 1999), especially undercooked ground beef. Cattle are considered the major reservoir of the pathogen in the environment (Cassin et al., 1998; Eisel et al., 1997; Tuttle et al., 1999; Riley et al., 1983; Khaitsa et al., 2003). The most notorious example of infection with this pathogen was a multistate outbreak occurred in early 1993, where 731 persons were affected, 56 cases developed HUS, and 4 children died as a result of the consumption of undercooked hamburgers in a chain restaurant (CDC, 1993; Bell et al., 1994; Dorn et al., 1993; Tuttle et al., 1999; Barrett et al., 1994). Since then, research on distribution and control of this pathogen has exploded. Due the expanding range of foods and pathways associated and involved with infection caused by E. coli O157:H7 (Anonymous, 1996; CDC, 1997; Hancock et al., 1997; Taormina et al., 1999, Besser et al., 1997), a more accurate understanding of the ecology, epidemiology and prevalence of the

90

microorganism in cattle and in the pre-harvest environment is needed in order to appropriately assess the microbial risk posed by this pathogen (Childers et al., 1996; Clough et al., 2003; Duffy, 2003; Callaway et al., 2003). Some studies have shown that E. coli O157:H7 prevalence in beef feedlot cattle is higher than previously reported, with up to 38.6% of individual animals (Chapman et al., 1994), 72% of lots (Elder et al., 2000), and 100% of feedlots (Smith et al., 2001) testing positive for the pathogen. This fact clearly indicates the necessity to reduce the carriage by cattle and other food-producing animals. The huge variation in the reported results could be attributed to several factors including, but not limited to, status of the animal (sampling live animals or immediately after slaughter), sampling season and seasonal variation in the natural rate of shedding (Barkocy-Gallagher et al., 2003; Van Donkersgoed et al., 1999; Khaitsa et al., 2003; Edrington et al., 2004), variation in diet (Hovde et al., 1999; McSweeney et al., 2004; Diez-Gonzalez et al., 1998; Kudva et al., 1997; Russell et al., 2000; Boukhors et al., 2002; Lema et al., 2001; Grauke et al., 2003; Sanderson et al., 1999; Buchko et al., 2000; Younts-Dahl et al., 2004; Van Baale et al., 2004), animals’ age (Paiba et al., 1999; Paiba et al., 2002; Dunn et al., 2004; LeJeune et al., 2004; Garber et al., 1995; Tkalcic et al., 2003; Zhao et al., 1995; Zhao et al., 1998; Zhao et al., 2003; Besser et al., 2001; Laegreid et al., 1999), stress of the animal due changes in the diet and/or transportation conditions (Barham et al., 2002; Clough et al., 2003; Jr. Cray et al., 1998; Miniham et al., 2003; Hallaran et al., 2001; Gansheroff et al., 2000) and variation

91

in sampling methods and microbiological analysis used for the detection of the pathogen. Recently, a new culture method using immunomagnetic bead separation was developed and is much more sensitive in detecting the pathogen in the presence of background flora. In recent studies, the use of more sensitive pre-enrichment methods such as IMS together with different agars have improved the detection, and therefore, the prevalence of the pathogen (Blackburn et al., 2000; Vernozy-Rozand et al., 2000; Wright et al., 1994; Ogden et al., 2001; Safarikova et al., 2001; Drysdale et al., 2004; Cubbon et al., 1996; Chapman et al., 1994; Chapman et al., 1996; Chapman et al., 1997; Restaino et al., 2001; Taormina et al., 1998). In these studies, a wide diversity of sampling methods have been used, including collection of saliva from the oral cavity, collection of fecal material and sponging different locations on the hides and carcasses which are analyzed for the microorganism (Wagner et al., 2002; Byrne et al., 2000; Gill et al., 2000; Kang et al., 2001; Rice et al., 2003b; Dorsa et al., 1997; Chart et al., 2003; Keen et al., 2002; McEvoy et al., 2003; Ransom et al., 2002; Ware et al., 1999; MeyerBroseta et al., 2001, Arthur et al., 2004). Few studies have considered the effect that sample size and/or number of samples per fecal pat could have in the detection and estimation of E. coli O157:H7 prevalence in cattle (Rice et al., 2003b; Ransom et al., 2002; Altekruse et al., 2003). When sampling fecal material, the procedures used to collect the sample and the amount of fecal material has varied greatly, and in some cases

92

the type or source of the samples and/or the quantity used for microbial analysis is not clearly specified (Tutenel et al., 2003; Sanderson et al., 1995; Pradel et al., 2000; McDonough et al., 2000). In some studies, small amounts of feces have been collected through the use of swabs (0.1 – 1.-0 g approximately), either by insertion of a cotton-tipped swab in the rectum of the animal, by digital retrieval (Hancock et al., 1998; Rice et al., 2003; Gill et al., 2000; Hancock et al., 1994; Shere et al. 1998; Stephan et al., 2000; Tkalcic et al., 2003) or by collecting a limited amount directly from the fecal pat (Hancock et al., 1998; Sanderson et al., 1999; Hancock et al., 1994). Fecal grabs collected by rectal palpation have also been used in some studies, after which up to 10 g are measured and analyzed for the microorganism (Magnuson et al., 2000; Smith et al., 2001; Rice et al., 2003b; Lahti et al., 2003; Wang et al., 1996; Kobayashi et al., 2001; Zhao et al., 1995); however, in many other cases, the fecal material was collected directly from intestinal tracts after slaughter of the animals (Omisakin et al., 2003; Shere et al., 2002; Ransom et al., 2002; Naylor et al., 2003; Zhao et al., 2003; Heuvelink et al., 1998b; Ransom et al., 2002) and amounts as high as 20 g of feces have been used for the microbial detection of the pathogen (Heuvelink et al., 1998a). In some studies, the status and prevalence is determined at the farm by collecting samples from fresh fecal pats directly from the ground/floor of the pen after defecation (Renter et al., 2003; Galland et al., 2001; Sargeant et al., 2003; Parham et al., 2003; Zhao et al., 1998). For studies involving large number of

93

animals, such as those in a commercial feedlot, this method may be the most practical and causes the least amount of stress to the animal. The differences that can be found among the type of sample collected, sampling techniques, and point during production in which samples are collected might be influencing prevalence results among these studies. The lack of a standardized sampling protocol does not allow comparison of the results from different studies, and therefore, farms and industry are not provided with the best information available. This material and data is required to direct the resources to the use of the best useful interventions (or combination of interventions in order to create hurdles) that can be implemented to reduce the pathogen. In a recent study, it was found that when animals were experimentally inoculated, the E. coli O157:H7 tend to colonize the mucosa next to the rectoanal-junction (RAJ) (Naylor et al., 2003), having an impact on the numbers of the pathogen being shed and its distribution in the fecal stool. As part of the goal to develop a standard sampling procedure for E. coli O157:H7, fecal sampling efficacy must be evaluated and validated since most reported prevalence estimates in cattle and feedlots are calculated by using this method (APHIS, 1997; APHIS, 2001; Synge et al., 2003; Sargeant et al., 2000; Sargeant et al., 2003). The main objective of this study was to evaluate the potential of variation in the distribution of E. coli O157:H7 within the fecal pat through a new sampling strategy. We investigate the possibility that by increasing the number of samples per pat the estimate of prevalence could also increase. If detection of E. coli

94

O157:H7 increases by using a new approach, an optimum number of samples may be determined in order to have a more precise prevalence of the microorganism.

3.3. Materials and Methods 3.3.1. Study design The purpose of the study was to analyze individual fecal pats to detect the presence and distribution of E. coli O157:H7 within the pat. Four replicates were conducted for the entire experiment, with each consisting of 30 pats for a total of 150 sub-samples analyzed per replication. A replication was defined as the collection of samples in the same sampling day in one of the two feedlots included in the study. No attempt was made to ensure that the fecal samples were exactly from the same animals or pens. Sampling was performed at separate days at the two different feedlots during a three month period.

3.3.2. Sample collection Fresh manure samples were obtained from cattle maintained at two different locations; one being a research feedlot at West Texas A&M University in Canyon, Texas; and the other being a commercial feedlot in the Texas panhandle. Samples were collected between May and July 2003. One hundred twenty fecal pats were analyzed, for a total of 600 sub-samples analyzed. In each replication, five samples (ca. 50-100 g) were obtained systematically from

95

each pat surface going from west to east (positions 1 to 5 respectively) in north to south lines direction and placed in sterile, labeled specimen cups. In order to minimize cross-contamination, each collected sample was taken with spoons that were replaced with new ones when sampling each position. After collection, cups were stored in a plastic cooler with ice packs and transported from the farm to Texas Tech University Food Microbiology Laboratory (TTUFML) within 2 h of collection for processing.

3.3.3. Microbial Analysis for Detection of E. coli O157:H7 In this study, all media were prepared following manufacturers directions. The use of a fast, highly sensitivity method (Chapman et al., 1994; Elder et al., 2000; Meyer-Broseta et al., 2001) was chosen in order to have the best possible results when compared to those obtained through the use of traditional methods such as direct plating. The protocol followed for the isolation and identification of the E. coli O157:H7 in this part of the study has been described in detail (Smith et al., 2001; Brashears et al., 2003; Fegan et al., 2004) and can be found in section 2.3.6. Immediately after arrival to the food microbiology laboratory, 10 g of feces from each sub-sample were transferred for enrichment directly into dilution bottles containing 90 ml of VCC – GN Broth and incubated at 37°C for 6 h (Foster et al., 2003; Barkocy-Gallagher et al., 2002; Taormina et al., 1998). After pre-enrichment, 1 ml aliquots from each sub-sample were analyzed

96

by using IMS (Wright et al., 1994; Chandler et al., 2001; Foster et al., 2003; Safarikova et al., 2001; Chapman et al., 1994; Chapman et al., 1996; Chapman et al., 2001; Tu et al., 2001) following the procedure described earlier and spread onto CT-SMAC plates (Sargeant et al., 2003; Chapman et al., 1994; Chapman et al., 1996). After 18 h – 24 h of incubation time at 37°C, typical colorless sorbitolnegative colonies per plate were picked and streaked for isolation on CT-SMAC and placed overnight at 37°C (McCarthy et al., 1998 March et al., 1986; Fujisawa et al., 2002). A single sorbitol-negative colony (clear or whitish) was selected and inoculated on MAC agar and Fluorocult E. coli O157:H7 agar. After incubation, MUG negative colonies were transferred from the MAC plates into TSB, TSI, and MacConkey broth and incubated overnight in aerobic conditions at 37°C (De Boer, 1998; Manafi et al., 1991; Manafi, 1996; Manafi et al., 2001; Brashears et al., 2001). Presumptive positive E. coli O157:H7 colonies were confirmed with the use a commercial latex agglutination test (Sowers et al., 1996; Chapman et al., 1989; March et al., 1989). Final confirmation was made using the BAX system.

3.3.4. Statistical analysis A sub-sample, or one of the five positions sampled per fecal pat, was categorized as positive for this study after a presumptive E. coli O157:H7 colony obtained, subjected to the microbial protocol described earlier, and confirmed with the agglutination test specific for E. coli O157:H7. Results were obtained,

97

compiled and entered into a spreadsheet. The SAS statistical package was used for this study (SAS, 2001). Prevalence estimates for each position and fecal pat were calculated as the total number of positives obtained with the agglutination test and divided by the total number of sub-samples tested for that position. Sample position within a pat was analyzed as a separate sample from the rest. Hypergeometric probabilities were used to generate the expected number of positive samples and binomial response variable of the expected prevalence given number of samples per pat for each replicate was modeled using GLIMMIX macro for SAS 8.2.

3.4. Results 3.4.1. Overall results A total of 120 fecal pats were sampled for a total of 600 fecal sub-samples that were analyzed. Isolation of E. coli O157:H7 from any given sample position was recorded as a positive for the entire fecal pat. Of the 120 fecal pats examined for E. coli O157:H7, 24 were positive for the pathogen, resulting in an overall 20% positive E. coli O157:H7 prevalence for the fecal pats in this study. A total of 96 pats (80%) returned negative results for the pathogen after microbiological examination.

98

3.4.2. Feedlot Results For each feedlot, 2 replicates were conducted (4 for the whole experiment). In feedlot “A”, a total of 3 pats and 2 pats resulted in positive results for E. coli O157:H7 in replication 1 and 2, respectively; resulting in an overall 8.33% prevalence of the pathogen in feedlot “A”. For feedlot “B”, where the microorganism was isolated from a total of 13 and 6 fecal pats on replication 3 and 4, respectively, the results were higher than the ones obtained in feedlot “A”. The overall pathogen’s prevalence in feedlot “B” was 31.67%. A better description of the prevalence of E. coli O157:H7 in each feedlot involved in the study can be observed in Table 3.1. The distribution of the total number of positive sub-samples within each fecal pat also varied for each replication. In feedlot “A”, during the first replication, a total of 2 and 1 pats had 1 and 2 sub-samples (positions) been positive for the pathogen; during the second attempt the two positive fecal pats had 1 of the subsamples positive for the pathogen. In feedlot “B”, during replication 3, a total of 5, 3, 2, and 3 fecal pats had 1, 2, 3 and 4 out of the five sampled positions with a positive result for the pathogen, respectively. In the 4th replication, feedlot “B” resulted in 4 of the fecal pats yielding positive results for E. coli O157:H7 in one of the positions and a total of 2 of the fecal pats being positive for the pathogen in all 5 of the sub-samples collected within the fecal pat, respectively. Table 3.2 describes the distribution of the total number of positive sub-samples for each of the feedlots involved in the study.

99

Table 3.1. Distribution and prevalence of fecal pats positive for E. coli O157 in each feedlot involved in the study.

Feedlot

Replication

Number of Positive Fecal pats

Prevalence (%) per replication

1

3

10.00

2

2

6.67

3

13

43.33

4

6

20.00

Overall prevalence per Feedlot (%)

8.33 %

A

B

Overall Prevalence for the Study:

31.67 %

20.00 %

.

100

Table 3.2. Distribution and frequency of the total number of sub-samples that returned positive E. coli O157:H7 results for each of the participating feedlots.

Number of pats with E. coli O157:H7 positive results

Total number of positions with positive results within a fecal pat

Feedlot A

Feedlot B Number of Frequency % fecal pats

Rep 1

Rep 2

Rep 3

Rep 4

0 – None

27

28

17

24

96

80.0%

1

2

2

5

4

13

10.8%

2

1

0

3

0

4

3.3%

3

0

0

2

0

2

1.7%

4

0

0

3

0

3

2.5%

5

0

0

0

2

2

1.7%

Σ of positive pats

3

2

13

6

24

20%

% of positive pats

10%

6.67%

43.33%

20%

Total pats sampled

30

30

30

30

120

100%

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3.4.3. Final Results for Fecal Pats The final total distribution of positive fecal pats was in the following manner: Thirteen, 4, 2, 3, and 2 fecal pats had 1, 2, 3, 4, and 5 positive E. coli O157:H7 sub-samples (i.e. positions), respectively. Of the positive 24 fecal pats, E. coli O157:H7 was isolated from a diverse arrangement of positions from the Ato-E sub-samples within the fecal pat; ranging from 1 to all 5 of the sampled positions resulting in a positive outcome for the pathogen. A description of the distribution of positive sub-samples can also be found on Table 3.2.

3.4.3.1. Final Results by sampling position Of the total number of fecal samples analyzed for each of the positions, E. coli O157 was isolated and confirmed 14, 9, 8, 8 and 10 times in positions A (West), B, C, D and E (East), respectively, for a total of 49 sub-samples yielding positive for the pathogen (Figure 3.1). Since each position was analyzed 120 times, the frequency of E. coli O157:H7 was 11.67%, 7.50%, 6.67%, 6.67%, and 8.33% on position A, B, C, D, and E, respectively. The prevalence of positive E. coli O157:H7 by each position within the fecal pats can be observed in Figure 3.2. The 49 confirmed positive sub-samples distributed in the 5 positions account for 8.17% of the total number of samples analyzed (n=600). When analyzing positive positions A, B, C, D and E, they accounted for 28.6%, 18.4%, 16.3%, 16.3% and 20.4% of the total number of positives subsamples, respectively, as seen in Figure 3.2. Although the graph shows that the

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20

16

14

18

14 10

12 y

8

8

8

9

10

6 4 2 0 1 - West

2

3 Position Sampled

4

5 - East

Total Number of Positive Samples Figure 3.1. Total number of positive Escherichia coli O157:H7 sub-samples (By their respective position).

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18.00

14.00

11.67

16.00

6.67

8.00

6.67

7.50

10.00

8.33

12.00

6.00 4.00 2.00 0.00 1 - West

2

3 Position Sampled

4

5 - East

Respective Prevalence For Position (%)

Figure 3.2. Respective Escherichia coli O157:H7 prevalence for each of the positions sampled.

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higher number and percentage of positive isolates were found on position A (n = 14; 28.57%) and E (n = 10, 20.41%), respectively, the chi-square test reflected that for this big sample the size was not significant (P = 0.5995), suggesting that variation in the number of positives between sampling sites is an independent variable and can not be attributable to the samples size (n = 600).

3.4.3.2. Final results for sub-samples within fecal pats The number of positive E. coli O157:H7 positions within the fecal pats contributed to the increase to the pathogen’s prevalence for the study. A total of 13 fecal pats had one position yielding positive results for the pathogen, accounting for 54.17% of the accumulated positives (Figure 3.3.). An increase of approximately 17% in the number of positive fecal pats was achieved when 2 or less than 2 of the positions were positive for the pathogen. When sampling 3 positions, the increase was of approximately 8%. An expected prevalence for any given number of samples collected was also computed for each replication. With this data, an estimated overall value was calculated for the total number of positions sampled within a fecal pat as observed in Table 3.3. The probability of having a positive result for the fecal pat increased as the number of sub-samples/positions increased. However, the increase in the overall prevalence was diminished as the number of samples per fecal pat rose. For example, the overall estimated prevalence increased 4.16% when 2 positions instead of 1 were collected (from an overall 8.17% to 12.33%);

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70.83

54.17

80 60

91.67

79.17

100

100

120

2

3

2

0 1

3

2

4

20

13

40

4

5

Total Number Of Positive E. coli O157:H7 Sub-samples Within the Fecal Pats Total Number of Positive Fecal Pats Cumulative Percentage Of The Total Positive Fecal Pats

Figure 3.3. Cumulative prevalence of E. coli O157 in positive fecal pats according to the number of samples within the pat

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Table 3.3. Estimated E. coli O157:H7 prevalence for any given number of sub-samples (positions) collected.

Estimated prevalence (%)

Number of positions collected / pat

Replicate

Overall

1

2

3

4

Prevalence (%)

Relative Increase

Absolute Increase

1

2.67

1.33

19.33

9.33

8.17

--

--

2

5.00

2.67

29.67

12.00

12.33

4.16

50.92

3

7.00

4.00

35.67

14.67

15.33

3.00

24.33

4

8.67

5.33

40.00

17.33

17.83

2.50

16.31

5

10.00

6.67

43.33

20.00

20.00

2.17

12.17

107

25.0

Prevalence (%)

20.0 20.0 17.8

15.0 15.3 12.3

10.0 5.0

8.2

0.0 1

2

3

4

5

Number of samples collected

Figure 3.4. Variation in E. coli O157:H7 prevalence in bovine feces with number of samples collected.

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but only an increase in the prevalence of 3.00% and 2.50% was achieved when 3 and 4 sub-samples per fecal pat were collected, respectively (Table 3.3.) In a recent article, the distribution of E. coli O157 in bovine feces was analyzed by testing multiple samples from fecal pats (Pearce et al., 2003). In this study, from each of 58 fecal pats in a pen of yearlings (group A), 29 fecal pats in a pen of cows and calves (group B), and 59 fecal pats in a pen of finishing animals (group C) a total of 3, 5 and 10 sub-samples were collected, respectively. The samples, each with approximately 1 g of fecal material, were subjected to pre-enrichment in BPW followed by IMS and plating in SMAC agar plates in order to detect the presence of non-sorbitol fermenting E. coli O157. After microbial analysis, the proportion of positive E. coli O157 pats were 98% (n=57 pats), 17% (n=5 pats) and 75% (n=44 pats) in group A, B and C, respectively. In our study, where 5 samples per fecal pat were collected, yielded a similar result (20% overall prevalence) to those reported by Pearce et al. (2003). In our study, the methodology used to collect the sub-samples is clearly stated, by going in North to South direction from West (position A or 1) to East (position E or 5) and taking care of collecting fecal material from the surface of the pat. However, in the previous study, no clarification on the sampling methodology exists. Other factors that can account for our slightly difference in the prevalence are the amount of fecal material sampled (being ours 10 g instead of 1g), broth used in the pre-enrichment step (VCC-GN broth vs. BPW) and type of animals in the pens where the samples were collected. The limit in sensitivity

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detection of the Dynabeads may also play a role in the differences seen in both studies. Dynabeads can detect E. coli O157 in aliquots containing as low as 100 cells per ml against numbers of background flora as high as 106 organisms per ml (Anonymous, 2003). If the numbers of cells of E. coli O157 in the fecal sample are lower than 100 cfu/g, then the use of a larger amount of sample together with incubation in VCC-GN broth as a pre-enrichment step will reduce the background of non-Gram negative bacteria and the likelihood of the pathogen to recover and grow in numbers enough to be detected after the 6 h incubation period. By obtaining several sub-samples for each fecal pat, an increase in the overall E. coli O157:H7 prevalence was observed. It seems positive E. coli O157 returns in the fecal samples were better achieved by increasing the number of samples/positions per fecal pat collected. We observed that when just one sample was collected per pat the estimated prevalence was 8.17%, reaching a maximum overall prevalence of 20% when 5 positions were sampled. An increase of 2.45 times in the estimated prevalence was achieved when sampling 5 positions in the fecal pat as when just 1 sample was collected. This more accurate prevalence was achieved when using a multiple sampling strategy instead of relying in collecting just one sample per fecal pat, where the pathogen is usually found in low concentrations. If E. coli O157:H7 is not evenly distributed in the fecal material as we observed, following this sampling strategy may lead to more conclusive prevalence results. Results of the pathogen’s prevalence in cattle and feedlots in which just one sample was collected or where small

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amounts of fecal material were tested may have underestimate the real frequency and density of the pathogen. Although the method used is very sensitive in detecting the pathogen, more research is required in order to improve fecal sampling techniques, which proved to be reliable in detecting the pathogen. An increase of 2.45 fold times in the prevalence of E. coli O157:H7 when sampling 5 sites per fecal pat instead of just 1 was achieved in this study. This new method also shows the reliability of the collection technique by the improvement achieved in detection of E. coli O157; however, prolonged sampling time associated to find the proper fecal pat that can be sampled and laboratory costs associated to the volumes of media, reagents and technician time that the samples required remain a concern for this technique.

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CHAPTER IV GENERAL DISCUSSION/SUMMARY OF FINDINGS

4.1. Overview The purpose of this research was to analyze the survival characteristics and distribution of E. coli O157:H7 in bovine fecal material. Cattle are the major reservoir of the pathogen and it is one of the major identified sources of the pathogen at farm; however, other environmental sources such as water troughs and tanks, feed and feed bunks and other non-bovine animals can not be underestimated in the distribution of the pathogen (Heuvelink et al., 2002; Dodd et al., 2003; Cornick et al., 2004; Renter et al., 2001; Renter et al., 2002; Van Donkersgoed et al., 2001; Shere et al., 1998; Garber et al., 1995; Davis et al., 2003; Cobbold et al., 2002; Rasmussen et al., 2001Guan et al., 2003). Transmission of E. coli O157:H7 through the environment and to the food supply has been implicated in several reported cases of infection, many of them leading to severe illnesses and even deaths (Anonymous, 1996; CDC, 1999; CDC, 2000; Ammon et al., 1999; Bell et al., 1994; Davis et al., 1995; Coia et al., 1998; Ackman et al., 1997; Swerdlow et al., 1992; Belongia et al., 1991; Hilborn et al., 1999; Crump et al., 2003; Cowden 1997; Bielaszewska et al., 1997; Chapman et al., 2000; Chapman 2000; Goldwater et al., 1996; Mead et al., 1999; Shukla et al., 1995; Rasmussen et al., 2001; Besser et al., 1993; Barrett et al., 1994). Because of its public health importance, reduction of the amount of the

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pathogen in the farm and control in shedding by the animals is one of the primary goals scientists have established with the use of pre-harvest food safety interventions. Sampling strategies play an important role in the determination of E. coli O157:H7 prevalence among cattle, allowing assessment of the efficacy of the different interventions that are used to control the pathogen (Rice et al., 2003b, De Boer et al., 2000; Ransom et al., 2002; Altekruse et al., 2003). The status of the animals for the pathogen, either positive or negative, needs to be established in order to determine if reduction has occurred. In many cases, this determination is achieved by examining fecal material sent from different locations all over the nation. Survival of the pathogen can be affected by shipping, and therefore, the outcome of the investigation (i.e. status of the animal) and effectiveness of the interventions can also be influenced by these results (Barham et al., 2002; Synge et al., 2003; Fu et al., 2003; Miniham et al., 2003). Factors such as the type and amount of the sample, type of broths, media and pre-enrichment steps used for the recovery of the pathogen may also affect the final outcome (Bird et al., 2001; De Boer et al., 2000; Foster et al., 2003; Johnson et al., 1998; Ransom et al., 2002; Ware et al., 1999; Cubbon et al., 1996; Tortorello et al., 1998; Sanderson et al., 1995; Stephan et al., 2000; Reinders et al., 2002). A variety of conditions such as temperature, time, pH, solid content, aeration, bacterial concentration and moisture can influence the survival of the

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pathogen in fecal material (Wang et al., 1996; Kudva et al., 1998). An estimation of the pathogen’s behavior on feces was achieved by inoculating bovine fecal material with a cocktail of antibiotic resistant E. coli O157:H7 and storing it in different temperature conditions. Simulation of transportation conditions to which samples can be subjected was performed by using a plastic container with frozen ice packs that were not replaced during the course of the study. These conditions were tested in order to replicate temperatures and length of storage during real life shipping conditions. The pathogen’s characteristics were also analyzed in other plausible temperatures that samples can encounter under normal laboratory conditions (4.4°C, 23°C and 37°C). Sub-samples were tested by two different methods on time 0 h, 24 h, 48 h, 120 h, and 168 h. Quantification was performed on the sub-samples to analyze the growth of the pathogen during time at any of the previous mentioned temperature conditions. Direct plating allowed recovering of the pathogen from time 0 h to 168 h in samples held at 4.4°C, 23°C and in the cooler during the course of the study. No colony counts were possible to perform in samples at 37°C after 120 h. The use of a pre-enrichment step together with a sensitive test such as immunomagnetic separation allowed detection of the pathogen for up to 168 h from fecal samples held at 4.4°C, 23°C and in the cooler. The use of IMS also did not permitted recovery of the pathogen at 37°C. Some of the control samples yielded positive results for the pathogen during the length of the study even though they were analyzed with IMS previous

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to the beginning of the experiment. In one occasion on sampling time 168 h, E. coli O157:H7 was isolated from control samples stored at 4.4°C and at 23°C and in two occasions from the sub-samples held in the cooler, respectively; however, none of the control samples held at 37°C were positive with this method after 24 h. The previous results suggest that the fecal samples contained E. coli O157:H7 from naturally infected cattle at the time of collection. The negative results observed at the beginning of the experiment could be explained by 1) a low natural density of the pathogen in the bovine feces, which did not allow to detect the pathogen even with the use of a sensitive method such as IMS and 2), a not evenly distribution of the pathogen in the fecal pat. It is assumed that if the pathogen is naturally present in the animal, the fecal material may content E. coli O157:H7 in an evenly manner and sampling of just a small fraction of the pat could give definite results for the status of the animal. Recovery of the pathogen from control samples after 48 h and 120 h with the use of IMS was probably accomplished due the change in the temperature conditions. These temperatures are probably not the more suitable for the natural background flora within the fecal material, but they may allow E. coli O157:H7 to outcompete and grow to numbers that allow its detection after 48 h. Inside the cooler, average temperatures increased from 0°C on time 0 h to 23°C after 168 h; time at which control samples yielded positive results with IMS in two different occasions. If E. coli O157:H7 is more adapted to survive at the initial cold temperature conditions than the rest of the background flora, allowing

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the pathogen to growth easier, then this would explain its unexpected detection in the control fecal samples after 168 h despite it’s initial absence. The advantages of sampling fecal material by directly collecting samples from the fecal pats has led to its general use in several studies (Wagner et al., 2002; Renter et al., 2003; Rice et al., 2003a; Rice et al., 2003b; Galland et al., 2001; Sargeant et al., 2003; Parham et al., 2003; Zhao et al., 1998). Escherichia coli O157:H7 occurrence and shedding in feedlots and feedlot cattle have been estimated based on the use of this method (USDA, 1995; USDA, 2001); however, these numbers may not reflect adequately the real proportion of animals infected currently with the pathogen. New findings about the pathogen’s colonization site within the bovine host support the idea of an unevenly distribution within the fecal material (Gally et al., 2003; Naylor et al., 2003), which could account for an underestimation of the pathogen in previous studies. The previous data indicates the need to find a new sample strategy that is more accurate. Recent findings examining the patterns of fecal excretion by cattle experimentally challenged with E. coli O157:H7 demonstrated that a region close to the recto-anal junction was the pathogen’s site of colonization (Gally et al., 2003; Naylor et al., 2003), having the greatest impact on the numbers of the pathogen that were shed by the animals. In the same experiments the pathogen was not found in any gastrointestinal tract tissue or digesta that was analyzed, suggesting that most of the fecal material gets contaminated with E. coli O157:H7 on its surface when passing through the terminal rectum. This result

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was also supported by an earlier study where the pathogen was more persistent in the lower gastrointestinal tract (Grauke et al., 2002), and another where recto anal mucosal swabs were more sensitive detecting the pathogen than fecal cultures (Rice et al., 2003b). The advantages of fecal sampling cannot be underestimated. Sampling fecal pats is an easier and faster method compared to collecting mucosal swabs, a time-consuming method in which animals are restrained in a chute and subjected, even for a small amount of time, to stress conditions. We tested 120 fecal pats, each in 5 different surface positions by a new sampling methodology. A total of 24 fecal pats (20%) yielded positive results for the pathogen; with 13, 4, 3, 2 and 3 fecal pats having 1, 2, 3, 4 and 5 positive sub-samples, respectively. 80% of the fecal pats were negative after microbiological examination in all their respective sub-samples. A total of the 49 sub-samples were positive for the pathogen, distributed unevenly within the fecal pat in the following manner: 14, 9, 8, 8 and 10 on positions A (West), B, C, D, and E (East), respectively. An estimated overall prevalence was calculated allowing to determine the prevalence as the number of sub-samples per fecal pat increased. We found that the E. coli O157:H7 estimate prevalence was 8.17% when sampling one position per fecal pat, but that the prevalence increased by 2.45 times when 5 positions were sampled, achieving for the pathogen a maximum expected prevalence of 20%. Studies in which only one sample from fecal pats and/or fecal grabs were collected, such as the overall 11% prevalence in feedlots (USDA, 2001) and the

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0.5% in dairy and 5.0% prevalence in beef in individual animals (USDA, 1997), may have underestimated the microorganism apparent prevalence by 2.45 times. Although this new sampling approach involves more time and economic resources, the increase achieved in detecting the pathogen may be useful and necessary when evaluating the effectiveness of new pre-harvest interventions that are used to reduce the presence of the microorganism in the farm animals and in the pre-harvest environment. Validation of this method and other sampling techniques involving a higher number of sub-samples or more fecal pats to assess differences between sampling locations may be necessary in order to determine the best protocol/techniques to detect the pathogen.

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157

APPENDIX

158

A. 1 Probability of difference between Least Square Means (LSM) (Log 10 CFU/g) for E. coli O157:H7 populations when comparing cooler results to the rest of the treatments during time.

Sampling Time

24 h

0h

48 h

120 h

168 h

159 162 62

Treatment

Compared to

LSM

P>ItI

LSM

P>ItI

LSM

P>ItI

LSM

P>ItI

LSM

P>ItI

Cooler 0 h

4.4°C

-0.15

0.3861

-0.12

0.4778

-0.15

0.3861

0.10

0.5408

0.22

0.1953

Cooler 0 h

37°C

-0.10

0.5539

0.20

0.2463

1.47

<.0001

4.91

<.0001

4.91

<0.0001

Cooler 0 h

23°C

-0.13

0.4538

-0.08

0.6356

-0.13

0.4305

-0.08

0.6497

0.04

0.8279

Cooler 0 h

Cooler

-0.09

0.0349

-0.15

0.0133

-0.14

0.1076

-0.11

0.264

A. 2 Probability of difference between Least Square Means (LSM) (Log 10 CFU/g) for E. coli O157:H7 populations when comparing 4.4°C results to the rest of the treatments during time.

Sampling Time

160 160 162 62

24 h

0h

Treatment

Compared to

4.4°C 0 h

4.4°C

4.4°C 0 h

37°C

0.05

4.4°C 0 h

23°C

0.02

LSM

P>ItI

48 h

120 h

168 h

LSM

P>ItI

LSM

P>ItI

LSM

P>ItI

LSM

P>ItI

0.03

0.8744

0.00

1

0.25

0.1419

0.37

0.033

0.7821

0.34

0.0454

1.61

<.0001

5.06

<.0001

5.06

<.0001

0.9056

0.07

0.6929

0.01

0.937

0.07

0.6784

0.18

0.2795

161 162 0.02

0.8744

23°C

37°C 0 h

-0.03

0.30

37°C

LSM

37°C 0 h

P>ItI

Compared to LSM

0h

Treatment

Sampling Time

0.9056

0.0825

P>ItI

24 h

.0.033

1.57

LSM

0.8434

<.0001

P>ItI

48 h

0.02

5.01

LSM

0.89

<0.0001

P>ItI

120 h

populations when comparing 37°C results to the rest of the treatments during time.

0.14

5.01

LSM

0.4191

<0.0001

P>ItI

168 h

A. 3 Probability of difference between Least Square Means (LSM) (Log 10 CFU/g) for E. coli O157:H7

162 162 62 Compared to

23°C

Treatment

23°C 0 h

Sampling Time

0.05

LSM

0.7821

P>ItI

24 h

populations results at 23°C during time.

-0.01

LSM

0.9685

P>ItI

48 h

0.05

LSM

0.767

P>ItI

120 h

0.16

LSM

0.3348

P>ItI

168 h

A. 4 Probability of difference between Least Square Means (LSM) (Log 10 CFU/g) for E. coli O157:H7

A. 5 Sampling methodology

Fig. 1

Fig. 2

163

Fig. 3

Fig. 4

164

5.10

Population log

10

cfu/g

5.06

5.05

5.05

5.02 5.00

5.00

4.95

4.91 4.90

4.85 0h

24 h

48 h

120 h

168 h

Sampling Time (hours)

A. 6

Change of E. coli O157:H7 population in inoculated bovine manure held under cooler conditions. The temperature inside the cooler was 0°C, 4°C, 4°C, 21°C, and 23°C at time 0 h, 24 h, 48 h, 120 h, and 168 h, respectively.

165

cfu/g

5.00

Population log

5.10

10

5.20

5.06

5.06

5.03

4.90

4.81 4.80

4.69 4.70 4.60 0h

24 h

48 h

120 h

168 h

Sampling Time (hours)

A. 7

Change of E. coli O157:H7 population in inoculated bovine manure held under refrigeration at 4.4°C

166

cfu/g

5.00

Population Log

5.05

10

5.10

5.04

5.04 4.99

4.99

4.95

4.87

4.90 4.85 4.80 0h

24 h

48 h

120 h

168 h

Sampling Time (hours)

A. 8

Trend of E. coli O157:H7 population in inoculated bovine manure held at room temperature conditions (23°C).

167

6.00

Population log 10 cfu/g

5.01 5.00

4.71 3.44

4.00 3.00 2.00 1.00

0.00

0.00

120 h

168 h

0.00 0h

24 h

48 h

Sampling time (hours)

A. 9

Viable E. coli O157:H7 counts in inoculated bovine manure after incubation at 37°C

168

5

E. coli O157:H7 Population (Log

10

cfu/g)

6

4

3

2

1

0 0h

Control*

24 h

48 h 120 h Sampling Time (hours)

Cooler**

4.4°C

23°C

168 h

37°C

Figure A. 10 Escherichia coli O157:H7 behavior in inoculated bovine feces at different temperature conditions (Superposition of Appendix 6 to Appendix 9 results). *Control: Non-inoculated samples subjected to the same temperature conditions as inoculated feces and where no E. coli O1575:H7 was recovered. **Cooler: Temperatures inside the cooler were 0, 4, 14, 21, and 23°C at time 0, 24, 48, 120, and 168 h, respectively.

169

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