On-Farm Strategies to Control Foodborne Pathogens [1 ed.] 9781621004806, 9781621004110

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Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. On-Farm Strategies to Control Foodborne Pathogens, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook Central,

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. On-Farm Strategies to Control Foodborne Pathogens, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook Central,

ADVANCES IN FOOD SAFETY AND FOOD MICROBIOLOGY

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

ON-FARM STRATEGIES TO CONTROL FOODBORNE PATHOGENS

No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

On-Farm Strategies to Control Foodborne Pathogens, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook Central,

ADVANCES IN FOOD SAFETY AND FOOD MICROBIOLOGY Anderson de Souza Sant'Ana and Bernadette D.G.M. Franco - Series Editors Advances in Food Safety and Food Microbiology Anderson de Souza Sant'Ana and Bernadette D.G.M. Franco (Editors) ISBN: 2152-2006 Probiotic and Prebiotic Foods: Technology, Stability and Benefits to Human Health Nagendra P. Shah, Adriano Gomes da Cruz and Jose de Assis Fonseca Faria (Editors) 2011. 978-1-61668-842-4 Stress Response of Foodborne Microorganisms Hin-chung Wong (Editor) 2011. 978-1-61122-810-6

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Advances in Post-Harvest Treatments and Fruit Quality and Safety Manuel Vázquez and José A. Ramírez de Leon (Editors) 2011. 978-1-61122-973-8 Bacteriophages in Dairy Processing Andrea del Luján Quiberoni and Jorge Alberto Reinheimer (Editors) 2012. 978-1-61324-517-0 New Trends in Marine and Freshwater Toxins: Food and Safety Concerns Ana G. Cabado and Juan Manuel Vieites (Editors) 2012. 978-1-61470-324-2 Clostridium Botulinum: A Spore Forming Organism and a Challenge to Food Safety Christine Rasetti-Escargueil and Susanne Surman-Lee (Editors) 2011. 978-1-61470-575-8 Enterococcus and Safety Teresa Semedo-Lemsaddek, Maria Teresa Barreto-Crespo and Rogério Tenreiro (Editors) 2011. 978-1-61470-569-7

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Essential Oils as Natural Food Additives: Composition, Applications, Antioxidant and Antimicrobial Properties Luca Valgimigli (Editor) 2012. 978-1-62100-241-3 On-Farm Strategies to Control Foodborne Pathogens Todd R. Callaway and Tom S. Edrington (Editors) 2012. 978-1-62100-411-0 Molecular Typing Methods for Tracking Foodborne Microorganisms Steven L. Foley, Rajesh Nayak and Timothy J. Johnson (Editors) 2012. 978-1-62100-643-5 Progress on Quantitative Approaches of Thermal Food Processing Vasilis P. Valdramidis and Jan F. M. Van Impe (Editors) 2012. 978-1-62100-842-2 Pathogenic Vibrios and Food Safety Yi-Cheng Su (Editor) 2012. 978-1-62100-866-8

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Foodborne Protozoan Parasites Lucy J. Robertson and Huw V. Smith (Editors) 2012. 978-1-61470-008-1 Predictive Mycology Philippe Dantigny and Efstathios Z. Panagou (Editors) 2012. 978-1-61942-675-7

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Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. On-Farm Strategies to Control Foodborne Pathogens, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook Central,

ADVANCES IN FOOD SAFETY AND FOOD MICROBIOLOGY

ON-FARM STRATEGIES TO CONTROL FOODBORNE PATHOGENS

TODD R. CALLAWAY AND

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved.

TOM S. EDRINGTON EDITORS

Nova Science Publishers, Inc. New York

On-Farm Strategies to Control Foodborne Pathogens, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook Central,

Copyright © 2012 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works.

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Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book.

Library of Congress Cataloging-in-Publication Data On-farm strategies to control foodborne pathogens / editors, Todd R. Callaway and Tom S. Edrington. p. ; cm. Includes bibliographical references and index. ISBN 978-1-62100-480-6 (eBook) I. Callaway, Todd Riley, 1971- II. Edrington, Tom S. [DNLM: 1. Foodborne Diseases--prevention & control. 2. Agriculture--methods. 3. Animal Husbandry--methods. 4. Animals, Domestic--microbiology. 5. Food Microbiology--methods. 6. Food Safety--methods. WC 268] LC classification not assigned 615.9'54--dc23 2011034216

Published by Nova Science Publishers, Inc. † New York

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CONTENTS Preface

xi

Introduction

1

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Todd R. Callaway and Tom S. Edrington Chapter 1

Pre-Harvest Food Safety: The Past and the Future Mary E. Torrence

Chapter 2

Ethics and Preharvest Food Safety B. Rollin

17

Chapter 3

Interventions to Improve the Safety of Milk Production S. P. Oliver and S. E. Murinda

29

Chapter 4

Bacteriophage as a Pre-Harvest Pathogen Reduction Strategy Kim Stanford, Dongyan Niu and Tim McAllister

79

Chapter 5

Probiotics in Swine Production A. V. S. Perumalla, Navam. S. Hettiarachchy, Philip G. Crandall and S. C. Ricke

99

Chapter 6

Vaccination as a Method of E. Coli O157: H7 Reduction in Feedlot Cattle David R. Smith and Amanda R. Vogstad

133

Vaccination to Reduce Foodborne Bacterial Pathogens in Swine, with Emphasis on Salmonella Filip Boyen, Frank Pasmans and Freddy Haesebrouck

143

Chapter 7

Chapter 8

Phytochemicals as a Preharvest Pathogen Reduction Strategy Jeffery A. Carroll, Nicole C. Burdick, Michael A. Ballou and John D. Arthington

Chapter 9

Organic Acids and Their Role in Reduce Foodborne Pathogens in Food Animals Ester Grilli and Andrea Piva

Chapter 10

Stress Effects on Pathogen Colonization of Farm Animals Marcos H. Rostagno

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5

167

183 211

viii Chapter 11

Contents Lesser Mealworms on Poultry Farms: A Potential Arena for the Dissemination of Pathogens and Antimicrobial Resistance Tawni L. Crippen and Toni L. Poole

233 273

Index

275

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About the Authors

On-Farm Strategies to Control Foodborne Pathogens, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook Central,

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. On-Farm Strategies to Control Foodborne Pathogens, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook Central,

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. On-Farm Strategies to Control Foodborne Pathogens, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook Central,

PREFACE

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The economic impact of foodborne illnesses caused by bacteria associated with food animals ranges from $10 to 40 billion (USD) per year, and effects across the EU are similar in scale. Due to the large drain on the GDP, as well as human health and societal impacts, research and regulations over the past 100 years have focused on improving food safety which has resulted in the U.S. and EU currently having the safest food supply in history. Unfortunately, the very safety of the food supply causes increased notice of the sporadic outbreaks of foodborne illness, receiving intensive media coverage and instilling fear and distrust in the public mind in regards to the safety of their food supply. This book encapsulates many of the arenas in which the future improvements in food safety and human health will be explored.

On-Farm Strategies to Control Foodborne Pathogens, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook Central,

Copyright © 2012. Nova Science Publishers, Incorporated. All rights reserved. On-Farm Strategies to Control Foodborne Pathogens, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook Central,

In: On-Farm Strategies to Control Foodborne Pathogens ISBN: 978-1-62100-411-0 Editors: Todd R. Callaway and Tom S. Edrington © 2012 Nova Science Publishers, Inc.

INTRODUCTION Todd R. Callaway and Tom S. Edrington

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USDA, ARS, Food and Feed Safety Research Unit, College Station, TX Foodborne illnesses affect more than 48 million Americans each year (Scallan et al., 2011). The economic impact of these foodborne illnesses caused by bacteria associated with food animals ranges from $10 to 40 billion (USD) per year (Scharff 2010; USDA-ERS 2001), and effects across the EU are similar in scale. Because of the large drain on the GDP, as well as human health and societal impacts, research and regulations over the past 100 years have focused on improving food safety which has resulted in the U.S. and EU currently having the safest food supply in history. Unfortunately, the very safety of the food supply causes increased notice of the sporadic outbreaks of foodborne illness, receiving intensive media coverage and instilling fear and distrust in the public mind in regards to the safety of their food supply (Bryan 1988; Jain et al., 2009; Rangel et al., 2005). Early in the history of food safety research, focus largely remained on spoilage organisms and the result of improper handling. In recent years, the threat to the food supply posed by foodborne pathogenic bacteria associated with food animals has become more apparent. The apparent emergence of Escherichia coli O157:H7 and other EHEC in the 1980‘s and 1990‘s (Rangel et al., 2005) provided the impetus for the development of numerous in-plant strategies (Gyles 2007; Karmali et al., 2010; Strachan et al., 2006). The ―zer o-tolerance‖ /adulterant policy surrounding this organism has caused the accelerated development of HACCP practices in processing plants (McClure 2000) at a cost of more than $1 billion (Kay 2003). In spite of the numerous hurdles that have been erected throughout the food production chain to ensure food safety, including those in processing plants, too many foodborne illnesses still occur. The reason for this is simple, foodborne pathogenic bacteria are relatively common in the environment and in the intestinal tract of food animals. Many of these pathogens do not cause any illness in animals, yet produce illnesses in human consumers. The most common foodborne pathogenic bacteria, Salmonella and Campylobacter, are often also associated with swine and poultry, as well as cattle, where these animals are colonized asymptomatically (Davies 2011; Hanning et al., 2010; Vandeplas et al., 2010). Enterohemorrhagic Escherichia coli (EHEC), are found in the intestinal tract of cattle (Ferens and Hovde 2011; Karmali et al., 2010; LeJeune and Wetzel 2007), but they do not cause illness in cattle (Porter et al., 1997;

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Todd R. Callaway and Tom S. Edrington

Pruimboom-Brees et al., 2000). Thus foodborne pathogenic bacteria enter the processing plant undetected within (and on the hides of) the live animal (Fegan et al., 2009; Kunze et al., 2008) where they can make their way into the food chain. Indirect routes of human infection with foodborne pathogenic bacteria from the live animal have also been discovered. Direct contamination of drinking water supplies in Walkerton, Ontario (Anonymous 2000; Armstrong 2000), contamination of irrigation water used on crops (Manshadi et al., 2001), and indirect contamination via other animal vectors (Kobayashi et al., 1999; Strachan et al., 2001; Talley et al., 2009) have all been reported. Feedlot workers (LeJeune and Kersting 2010), visitors to open farms and petting zoos (Chapman et al., 2000; Keen et al., 2007; Pritchard et al., 2000), and those attending county and state fairs (Durso et al., 2005; Keen et al., 2007; Keen et al., 2006) have all reported foodborne illnesses relating to their animal contact. Thus improving human health cannot be solely reliant upon introducing measures to reduce pathogens once the animal is in the processing stream. It has been suggested that the greatest impact on human health would occur from reducing foodborne pathogenic bacteria in the live animal (Hynes and Wachsmuth 2000). In recent years, the role of the microbial ecosystem in both human and animal health has become more prominent (Finegold 2008; Ley et al., 2006; Murphy 2004; Turnbaugh et al., 2009; Turnbaugh et al., 2006; Xu and Gordon 2003). The ―m icrobial organ‖ is at last getting its due as a playing a part in health as well as production parameters (Lyte 2010). As such, many researchers have approached the issues of preharvest food safety from the perspective of how we can harness the microbial power of competition to reduce foodborne pathogenic bacteria. Thus this book touches upon many of the nutritional, ecological and microbial measures that have been investigated and proposed for use in food animals to reduce foodborne pathogenic bacteria. This book encapsulates many of the arenas in which the future improvements in food safety and human health will be explored.

REFERENCES Anonymous. 2000. Waterborne outbreak of gastroenteritis associated with a contaminated municipal water supply, Walkerton, Ontario, May-June 2000. Can. Commun. Dis. Rep. 26:170-173. Armstrong G.D. 2000. Learning from Walkerton. How do we end the possibility of further deadly E. coli oubreaks? In Time. Bryan F.L. 1988. Risk of practices, procedures and processes that lead to outbreaks of foodborne disease. J. Food Prot. 51:663-673. Chapman P.A., Cornell J. and Green C. 2000. Infection with verocytotoxin-producing Escherichia coli O157 during a visit to an inner city open farm. Epidemiol. Infect. 125:531-536. Davies P.R. 2011. Intensive swine production and pork safety. Foodborne Path. Dis. 8:189201.

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Introduction

3

Durso L.M., Reynolds K., Bauer N. and Keen J.E. 2005. Shiga-toxigenic Escherichia coli O157:H7 infections among livestock exhibitors and visitors at a Texas county fair. Vector-borne Zoo. Dis. 5:193-201. Fegan N., Higgs G., Duffy L.L. and Barlow R.S. 2009. The effects of transport and lairage on counts of Escherichia coli O157 in the feces and on the hides of individual cattle. Foodborne Path. Dis. 6:1113-1120. Ferens W.A. and Hovde C.J. 2011. Escherichia coli O157:H7: Animal reservoir and sources of human infection. Foodborne Path. Dis. 8:465-487. Finegold S.M. 2008. Therapy and epidemiology of autism-clostridial spores as key elements. Med. Hypoth. 70:508-511. Gyles C.L. 2007. Shiga toxin-producing Escherichia coli: An overview. J. Anim Sci. 85:E4562. Hanning I., Biswas D., Herrera P., Roesler M. and Ricke S.C. 2010. Prevalence and characterization of Campylobacter jejuni isolated from pasture flock poultry. J. Food Sci. 75-83. Hynes N.A. and Wachsmuth I.K. 2000. Escherichia coli O157:H7 risk assessment in ground beef: A public health tool. In Proc. 4th Int. Symp. on Shiga Toxin-Producing Escherichia coli Infections. (Kyoto, Japan). pp 46. Jain S., Bidol S.A., Austin J.L. et al., . 2009. Multistate Outbreak of Salmonella Typhimurium and Saintpaul Infections Associated with Unpasteurized Orange Juice-United States, 2005. Clin. Infect. Dis. 48:1065-1071. Karmali M.A., Gannon V. and Sargeant J.M. 2010. Verocytotoxin-producing Escherichia coli (VTEC). Vet. Microbiol. 140:360-370. Kay S. 2003. E. coli O157:H7: The costs during the past 10 years. In Meat and Poultry News. pp 26-34. Keen J.E., Durso L.M. and Meehan T.P. 2007. Isolation of Salmonella enterica and shigatoxigenic Escherichia coli O157 from feces of animals in public contact areas of United States zoological parks. Appl. Environ. Microbiol. 73:362-365. Keen J.E., Wittum T.E., Dunn J.R., Bono J.L. and Durso L.M. 2006. Shiga-toxigenic Escherichia coli O157 in agricultural fair livestock, United States. Emerg. Infect. Dis. 12:780-786. Kobayashi M., Sasaki T., Saito N. et al., . 1999. Houseflies: Not simple mechanical vectors of enterohemorrhagic Escherichia coli O157:H7. Amer. J. Trop. Med. Hyg. 61:625-629. Kunze D.J., Loneragan G.H., Platt T.M. et al., . 2008. Salmonella enterica burden in harvestready cattle populations from the southern high plains of the United States. Appl. Environ. Microbiol. 74:345-351. LeJeune J. and Kersting A. 2010. Zoonoses: An occupational hazard for livestock workers and a public health concern for rural communities. J. Agric. Safe. Health 16:161-179. LeJeune J.T. and Wetzel A.N. 2007. Preharvest control of Escherichia coli O157 in cattle. J. Anim. Sci. 85-97. Ley R.E., Turnbaugh P.J., Klein S. and Gordon J.I. 2006. Human gut microbes associated with obesity. Nature 444:1022-1023. Lyte M. 2010. The microbial organ in the gut as a driver of homeostasis and disease. Med. Hypoth. 74:634-638.

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Todd R. Callaway and Tom S. Edrington

Manshadi F.D., Gortares P., Gerba C.P., Karpiscak M. and Frentas R.J. 2001. Role of irrigation water in contamination of domestic fresh vegetables. In Gen. Mtg. Amer. Soc. Microbiol. pp 561. McClure P. 2000. The impact of E. coli O157 on the food industry. World J. Microbiol. Biotechnol. 16:749-755. Murphy M. 2004. Bacteria could treat symptoms of autism. Chem. Indust. (London):6. Porter J., Mobbs K., Hart C.A., Saunders J.R., Pickup R.W. and Edwards C. 1997. Detection, distribution, and probable fate of Escherichia coli O157 from asymptomatic cattle on a dairy farm. J. Appl. Microbiol. 83:297-306. Pritchard G.C., Willshaw G.A., Bailey J.R., Carson T. and Cheasty T. 2000. Verocytotoxinproducing Escherichia coli O157 on a farm open to the public: outbreak investigation and longitudinal bacteriological study. Vet. Rec. 147:259-264. Pruimboom-Brees I.M., Morgan T.W., Ackermann M.R. et al., . 2000. Cattle lack vascular receptors for Escherichia coli O157:H7 shiga toxins. Proc. Nat. Acad. Sci. (USA) 97:10325-10329. Rangel J.M., Sparling P.H., Crowe C., Griffin P.M. and Swerdlow D.L. 2005. Epidemiology of Escherichia coli O157:H7 outbreaks, United States, 1982-2002. Emerg. Infect. Dis. 11:603-609. Scallan E., Hoekstra R.M., Angulo F.J. et al., . 2011. Foodborne illness acquired in the United States—major pathogens. Emerg. Infect. Dis. 17:7-15. Scharff R.L. 2010. Health-related costs from foodborne illness in the United States. http://www.producesafetyproject.org/admin/assets/files/Health-Related-FoodborneIllness-Costs-Report.pdf-1.pdf. Strachan N.J.C., Dunn G.M., Locking M.E., Reid T.M.S. and Ogden I.D. 2006. Escherichia coli O157: Burger bug or environmental pathogen? Int. J. Food Microbiol. 112:129-137. Strachan N.J.C., Fenlon D.R. and Ogden I.D. 2001. Modelling the vector pathway and infection of humans in an environmental outbreak of Escherichia coli O157:H7. FEMS Microbiol. Lett. 203:69-73. Talley J.L., Wayadande A.C., Wasala L.P. et al., . 2009. Association of Escherichia coli O157:H7 with filth flies (Muscidae and Calliphoridae) captured in leafy greens fields and experimental transmission of E. coli O157:H7 to spinach leaves by house flies (diptera: Muscidae). J. Food Prot. 72:1547-1552. Turnbaugh P.J., Hamady M., Yatsunenko T. et al., . 2009. A core gut microbiome in obese and lean twins. Nature 457:480-484. Turnbaugh P.J., Ley R.E., Mahowald M.A., Magrini V., Mardis E.R. and Gordon J.I. 2006. An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444:1027-131. USDA-ERS. 2001. ERS estimates foodborne disease costs at $6.9 billion per year. http://www.ers.usda.gov/publications/aer741/aer741.pdf. Vandeplas S., Dubois Dauphin R., Beckers Y., Thonart P. and Thewis A. 2010. Salmonella in chicken: Current and developing strategies to reduce contamination at farm level. J. Food Prot. 73:774-785. Xu J. and Gordon J.I. 2003. Honor thy symbionts. Proc. Nat. Acad. Sci. (USA) 100:1045210459.

On-Farm Strategies to Control Foodborne Pathogens, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook Central,

In: On-Farm Strategies to Control Foodborne Pathogens ISBN: 978-1-62100-411-0 Editors: Todd R. Callaway and Tom S. Edrington © 2012 Nova Science Publishers, Inc.

Chapter 1

PRE-HARVEST FOOD SAFETY: THE PAST AND THE FUTURE Mary E. Torrence National Program Leader, Food Safety, USDA, Agricultural Research Service, Beltsville

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ABSTRACT This chapter provides an introduction to the area of food safety with specific focus on preharvest food safety. Preharvest food safety refers to the period of time when a food animal is growing, prior to livestock slaughter. This chapter describes various initiatives and activities that involve preharvest food safety that have occurred over the last 10 years, the progress that has been made, and future directions that are needed. Preharvest food safety is just one phase of a complex food production continuum with the ultimate goal of reduced foodborne disease and risk.

INTRODUCTION TO FOOD SAFETY Food safety remains an important global public health concern even with increased research and targeted initiatives since 1998. These continued efforts highlight the complexity of the food safety issue and the evolution and diversity of food production practices, food distribution, and foodborne pathogens. Foodborne diseases are without barriers as both humans, animals, and food products have become more mobile. There is increased international travel and immigration, globalization of trade, and importation of foods. Even social and economic changes provide impetus for the globalization of food safety. Food systems are diverse among developed and developing countries and there is a broad range of pathogens and contaminants that include bacteria, viruses, parasites, chemical agents and toxins.



Email: [email protected], ph: 301-504-4616.

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6

Mary E. Torrence

The often quoted estimates of 76 million foodborne illness cases and 5,000 deaths from 1999 CDC study were recently updated in 2010 (Mead, 1999; Scallan, et al. 2011; Scallan, et al., 2011). CDC now estimates that 47.8 million illnesses and 3,037 deaths were caused by contaminated food consumed in the U.S (Scallan et al., 2011; Scallan et al., 2011). These recent data indicate lower estimates but cannot be compared with the 1999 studies because of changes in data sources and methods. Although the current estimates show a perceived decrease in numbers, the full report suggests the continued need for research, improved surveillance and detection, and further education and extension efforts in food safety. Estimates are difficult, with only a proportion of illness being captured by actual physician visits, laboratory testing, or reported to public health agencies. Most reported cases are from sporadic outbreaks while large clustered outbreaks attract the most media attention. The first Presidential Food Safety Working Group, Food Safety Initiative, and Produce Food Safety Initiative were started by President Clinton in 1998 (http://www.whitehouse.gov.........). Because of the continued importance of food safety, President Obama re-formed the Food Safety Working Group to advise on upgrading the U.S. Food safety laws, foster coordination of food safety efforts in the government, and to ensure laws were being adequately enforced to keep food safety. The three core guiding principles of the current working group are prevention, effective food safety inspections, and enforcement/surveillance and intervention in foodborne outbreaks (http://www.foodsafety workinggroup.gov).The Food Safety Modernization Act signed into law in January, 2011, is one of the most recent shifts in food safety and provides FDA with new regulatory powers.

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BACKGROUND ON PREHARVEST FOOD SAFETY Previous studies have estimated that the majority of foodborne diseases are zoonotic; that is, diseases that are transmitted from animals to humans (Schlundt et al., 2004). This has created a focus on pre-harvest food safety, the food production environment, and human, animal and environment interactions. As new pathogens emerge or re-emerge, it is also important to evaluate whether they are zoonotic and transmitted by foodborne routes. This epidemiology is important to guide research and potential control or intervention programs. For example, as avian influenza emerged, researchers, policy-makers, and producers, had to determine if there was a foodborne risk. Methicillin-resistant Staphylococcus aureus (MRSA) has traditionally been a human hospital-acquired pathogen, but in recent years, it has also become community-acquired (Boucher and Corey, 2008). Cross sectional studies in animals have shown a low prevalence internationally and in the U.S., but more research is needed on the strain and serotype differences, and route of transmission. At this time, MRSA is not considered a foodborne pathogen, but research and surveillance will continue. It is critical that epidemiologists as well as microbiologists continue to work together in evaluating these pathogens. In 2004, AAM published a colloquium report entitled, ―P reharvest Food Safety and Security‖ (Isaacson and Torrence, 2004). This report provided recommendations that remain relevant in 2011, despite research conducted since then. Most important are the recommendations that preharvest food safety priorities should be set to public health outcomes and that studies are needed to delineate the relationship between preharvest food

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Pre-Harvest Food Safety: The Past and the Future

7

safety and public health risk, specifically , studies to quantify pathogen load and product contaminations. This parallels previous reports by the Institute of Medicine in 1998 and 2003. The 2003 Institute of Medicine (IOM) ―Sci entific Criteria to Ensure Safe Food‖ report provided innovative recommendations on developing science-based food safety criteria that could be linked to public health objectives (Institute of Medicine, 2003). The IOM report outlined specific criteria that could be defined at each production phase. For example, the microbiological criterion matched the microorganisms on product or food lot, and the food safety objective was the concentration of hazard at consumption. The IOM recognized the importance of HACCP principles and Good Agricultural Practices being implemented by industry. This report also recognized that the application of criteria and standards throughout the food production continuum would not be easy because of the many opportunities for the introduction of microorganism and contaminants to enter the food supply. The ease of this application is evident in that industry and government are still struggling with implementation. The IOM report of 1998 ―Ensur ing Food Safety from Production to Consumption‖ reported on the fragmented regulatory statues of food safety. The report also discussed the divergence of surveillance, perception, and research among the different phases of food production (IOM, 1998). In a book entitled, Microbial Food Safety in Animal Agriculture: Current Topics, the entire book concentrated on foodborne pathogens at the preharvest level. Most sections covered the epidemiology and ecology of the pathogen, detection and diagnosis, molecular and genetic aspects, pathogenesis and mechanisms, and prevention and control. (Torrence and Isaacson, 2003). There have been many books and papers since then. Dr. Oliver presented the Development and future outlook of preharvest food safety as the Centennial paper at that Annual American Society of Animal Science meeting in 2008. (Oliver et al., 2008). There are several examples of current initiatives that specifically focus on preharvest food safety and that focus has heightened in the last few years. One program that unbelievably is over 70 years old is the National Poultry Improvement Plan, started by USDA‘s Animal and Plant Health Inspection Service (APHIS) as cooperative industry-stateand federal program that would address both technological and management issues. This includes blood testing and serological laboratory protocols, bacterial procedures, and sanitation programs (http://www.aphis.usda.gov) In addition, the U. S programs against foodborne pathogens, particularly toxoplasmosis and trichinella have been quite successful. USDA‘s APHIS developed the National Trichinae Certification program which is a preharvest program for US Swine producers, and has lowered the prevalence to very low. There are at least 3 examples of primary initiatives that will change preharvest food safety and how we move forward in research and policy. They are : 1) the EU concentration on preharvest interventions for the control of foodborne pathogens, primarily for Salmonella in swine, poultry and eggs, and now for Campylobacter in poultry; 2) FSIS ‗s look at preharvest interventions for E.coli 0157:H7 in cattle; 3) FDA‘s look at antibiotic use in food animals as growth promoters. The EU‘s demonstrated success in interventions at the preharvest level has raised awareness by the policy-makers in the EU and other international countries, as well as the U.S. In 2004, the new EU food hygiene legislation was announced, with a goal of January 2006 to be implemented in the member countries (EUhttp://www.ec....)This legislation looked at applying controls throughout the food chain from primary to finished product, regulations on the hygiene of foodstuffs, for food of animal origin. The EU has set national control plans for Salmonella, and set salmonella reduction

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targets for laying hens, eggs, and broiler flocks (Mulder and Hupkes, 2007). Interestingly, some member states have been able to meet the target, others have not. Baseline prevalence studies that are conducted by the European Food Safety Authority each year demonstrate a trend in lowered Salmonella numbers, yet there are some Salmonella serotypes and other foodborne pathogens that have increased. In fact, the EU had a meeting in the fall of 2009 to discuss potential on-farm interventions for Campylobacter in poultry. A large group of experts could not recommend a single intervention. The success in some member countries has been laudable, although production systems are different in the U.S. However, this has not deterred FSIS from looking at similar measures in the U.S. Before this time, FSIS has set Salmonella standards at the processing level. FSIS has instituted several new activities directed toward preharvest food safety. FSIS has a committee of experts from academia, industry, and government that provide FSIS with guidance on numerous issues, the National Advisory Committee on Meat and Poultry Inspection (FSIShttp//:www.fsis.usda.gov….)This advisory committee formed a Preharvest Food Safety Subcommittee to look at various preharvest controls for several foodborne concerns (FSIShttp://www.fsis..... Preharvest…..). The concerns are antibiotic resistant Salmonella, Salmonella entertitidis, and E.coli 0157:H7. Using the impetus of the President‘s Food Safety Working Group in 2009, FSIS has developed several specific preharvest food safety initiatives. They are still developmental and will require public input. FSIS published preharvest management controls and intervention options for E.coli 0157:H7 in cattle, May, 2010 (FSIS, http://www.fsis......reducing_ecoli.......)To deal with S. enterititidis, FSIS has collaborated with FDA and CDC to develop a set of metrics for SE. SE accounts for about 17% of all Salmonella cases. With these new metrics, FSIS will propose performance standards for Salmonella in broiler and turkey carcasses and FSIS intends to conduct additional and more frequent baselines studies in conjunction with the Salmonella verification sampling (processing). FDA is looking to set a SE rule for egg-laying flocks that will institute management and sanitary measures for on farm practice, including environmental sampling and refrigeration of eggs. FSIS is looking at the EU policies and developing such as criterion for salmonella (absence in 25 gms for meat), monitoring of flocks, SE vaccination, and licensed live vaccines for S. typhimurium.

PREHARVEST FOOD SAFETY INTERVENTIONS, CONTROLS, AND MITIGATIONS The ultimate goal for food safety is to decrease foodborne pathogens and decrease public health risk. This goal focuses our main efforts at interventions and controls throughout the food production chain. This also infers that research on pathogenesis, virulence, detection methods, and the epidemiology and ecology of the disease and the organisms are all focused on the ultimate goal of interventions and control. Pre-harvest food safety is the beginning of the food production cycle, and logically, most feel that reductions of pathogen load at the animal level would translate to lower pathogen load at the end of processing. It also seems logical that improved animal health and welfare would result in animals which are healthier and would be considered ― safer‖ or lower in pathogen load. Although this makes logical sense, research has been slow to prove this scientifically.

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The study of pre-harvest food safety alone is complex. Not only does preharvest food safety consider the food animal and its microbial ecology, but also the interaction of the food animal and its environment. The environment (water, soil, and air, wildlife) and the production environment (management approaches) influences the potential introduction of foodborne pathogens and contaminants into the food animal or from the food animal to potential food products. Control, interventions and mitigations must be considered from numerous perspectives. One weakness in recent years is the consideration or evaluation of one intervention, e.g. vaccination against a single pathogen. While this helpful, it is important to realize that it will take multiple interventions/ controls, either in series or parallel, for the reduction of foodborne pathogens. Future research and evaluations must look at this. And, as emphasized before, effects at the pre-harvest level must be measured further along the production chain. Focus on preharvest food safety interventions/control and mitigations is important for 2 reasons: reduce foodborne pathogens and contaminants (including parasites, toxins, and chemicals) and to reduce pathogens that produce a burden on animal health. Unlike the processing environment that can be contained, food production at the pre-harvest level often involves the movement of people, animals, and equipment which can lead to the movement of pathogens (including the transport/lairage). Biosecurity measures implemented for homeland security and emerging outbreaks like avian influenza have resulted in better control strategies on farm. Preharvest control and intervention strategies can be two-fold. Efforts can be directed within the animal or directed at factors outside the animal, such as the animal‘s interaction with the environment. They are not mutually exclusive. In fact, the ―cont rol points‖ are so diverse at the preharvest level, that there is no single control method or intervention that provides complete reduction. It will take a multiple hurdle approach at the preharvest level in combination with efforts further down the production chain. The ideal goal would be achieve a specific prevalence or pathogen load in the animal at that time that it reaches processing. This measurement would be a level at which processing could assure almost no levels of pathogen at the end product. This ideal puts producers and processing often at different perspectives. Interventions/control programs directed toward the animal depend on the food animal species as well as the microbial pathogen. This area needs research in understanding of the epidemiology of disease, the microbial ecology of the organisms in the gut, as well as pathogenicity, virulence, and transmission. For example, some organisms are species specific, and some organisms may not be pathogenic to the food animal, yet pathogenic to humans. For example, what makes a Salmonella or E.coli serotype become virulent in humans yet remain subclinical in animals? Interventions and controls targeted within the animal may be used to improve the animal‘s resistance, alter the microbial flora of the gut, or the shedding of the foodborne pathogens. Research is being done to enhance the immune status of the animal either through genetics or vaccination, and on understanding the potential stressors on the microbial populations. Vaccines against Salmonella have been used in swine and poultry. There two new vaccines for E. coli 0157:H7 in cattle. Epitopix was given conditional license from USDA and received approval to market it in the U.S. (Thornton et al. 2009). Bioniche is another vaccine that received full licensing approval in Canada in 2009, and recently received approval in the US (2010) (Smith, et al., 2009). Although research is still being conducted on

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the efficacy of the vaccine, early trials have shown some reduction in E. coli 0157:H7 shedding in cattle. It does not completely eliminate the pathogen. Approaches that involve altering food sources continue to be a popular preharvest intervention and control strategy, although with limiting impact. A pivotal study demonstrated that changing diet, from grain to grass, in cattle can decrease shedding of E.coli 0157:H7 (Diez-Gonzalez et al, 1998). This was followed with a flurry of papers and recommendations. The impact on industry has been minimal, since the demand for fast growing beef continues to drive the industry. Some of this first research, however, will be relevant as protein sources change and such byproducts as distiller‘s grains are used. Current research is still ongoing on the use of prebiotics, probiotics, and commensal exclusion products with varied success. Prebiotics can provide nutrients or energy to microbiota or even specific commensals. Probiotics are a broad category of products that contain viable or live microbes in an effort to change the microflora for the better. These products may be live cultures of yeast or bacteria, heat-treated, or fermented. Two useful probiotics for animal health concerns are lactobacillus products and a direct-fed microbial (DFM) of lactobacillus and streptococcus. Several studies showed a specific DFM product helped reduce the shedding of E.coli in the feces (Brashears et al., 2003). Competitive exclusion products, e.g. PREEMPT, have been successful in controlling Salmonella in newly hatched chicks by ―com peting‖ against the foodborne pathogens. However, competitive exclusion products, like pre-and probiotics have had limited use because of the cost-benefit ratio for industry. Approaches that are targeted more toward the specific foodborne pathogen include the use of antibiotics, bacteriocins, or bacteriophages. One weakness of this approach is the inability to specifically target foodborne pathogens without affecting the entire microbial flora. Antibiotic use has declined over the years, and more research is being conducted to look at alternatives to anitibiotics and growth promoters. This is response to growing awareness of antibiotic resistance in animals, humans, and the environment, and the concerns for treatment failures for important animal and human diseases, as well as the public health risk. Antimicrobial proteins, or bacteriocins, can inhibit the growth of foodborne pathogens in the gut microflora. However, the ability to produce bacteriocins in large quantities to be used as feed additives has been a major limiting factor in the research and use of this product. Bacteriophages (which are bacteria thacn be infected by bacterial viruses) with narrow targets, even to a specific strain seem to be the promising products. They are common organisms in the microbial flora yet can be used with a great deal of specificity. The effect of the use of bacteriophages in the gut is variable, but in 2007 a bacteriophage spray produced for use against E.coli 0157:H7 in live cattle before slaughter was approved by FDA has demonstrated effectiveness (Callaway, et al. 2006). The use of a vaccination scheme and this phage spray seems to be the most effective approach to date for E.coli 0157:H7 in cattle. Finally, ARS researchers have developed the addition of chlorate to diets to reduce Salmonella and E.coli in swine, E.coli 0157:H7 in cattle and sheep, and even its use in poultry. This has been going on for over 10 years, and research is stopped because of the lack of response by FDA to provide slaughter approval to do the final analysis. As mentioned before, the ability to control the environment, as opposed to a closed processing environment, is limited. Pathogens may be introduced or transmitted through water, soil, air, manure or fertilizer, wildlife, birds, insects, and rodents. Contamination of carcasses at slauther and food products by animal feces is a principal mode of foodborne disease. Contaminated feed and water by feces is a source for animals, and the use of

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nontreated manure for fertile (or inadequately composted), slurry or recycled wastewater contaminates the environment and cause contamination of animals, plants, and plant crops (such as fruits and vegetables). More focus on these areas of contamination is needed given the increased consumption of raw or minimally processed food products in the U.S. and around the world. FDA is currently looking at animal feeds and possibly setting requirements for testing feeds for Salmonella spp. Biosecurity measures and management practices probably are the most common and have the most impact. The birth of Homeland Security and the evaluation of large production farms and open access focused the attention on biosecurity measures. This has included the limited movement of animals, humans, and equipment across and into farms, limited access to wildlife, rodents, and insects, and promoting less stressful management practices like optimum animal density the reduced contact or commingling between infected and healthy animals, an amenable environment, and bedding. More research is needed in developing and evaluating best management practices or even HACCP like programs at the pre-harvest level. For example the NPIP plan has significantly the poultry industry. Some similar plans have been mandated in the E.U., and it is a matter of time where this model is used in the U.S.

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PREHARVEST: LEARNING FROM THE PAST AND LOOKING TO FUTURE The 2004 AAM report also recommended other broad research activities that are currently being used or still being attempted. Again, this illustrates the complexity of food production and the pre-harvest phase. Recommendations included the need for new and improved tools or methods (e.g. microbial indicators, detection methods for on-farm), longitudinal cohort studies to identify risk factors, risk assessments, and the development and implementation of best management practices. To date, there have been new methods and tools developed. New serotyping methods, microarrays, and now, genomics, have improved our understanding of foodborne pathogens and the relationship between food animals and humans. They also raise new questions about the ability of pathogens to be subclinical or commensal in some food animal species yet become pathogenic in humans. Surveillance is often suggested as an activity, but rarely is it understood about the magnitude of designing a national system, nor the cost. Surveillance has been suggested for baseline prevalence studies in food animals for pathogens of interest and for antibiotic use data and resistance prevalence in food animals. The National Animal Health Monitoring Studies by USDA‘s Animal and Plant Health Inspection Service is an example of cross-sectional surveys done every so many years for specific animal species. The expense has lessened the frequency of the studies. Longitudinal cohort studies suffer the same fate of expense and time. There are examples of research funded through the Epidemiologic Approaches for Food Safety (USDA, now the National Institute of Food and Agriculture) that provided significant impact on some timely issues. These grants, as they were, are no longer funded. Risk assessments continue to be refined and conducted, particularly by policy and regulatory bodies. The greatest need for risk assessments are data. Finally, some best management practice programs instituted by industry have been very successful. For example the National Poultry Improvement Plan uses good

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sanitary practices and biosecurity measures (APHIS). Sadly, one note from the 2004 AAM report is still true. The public and policy makers are still are still ill-informed about food production practices and the fact that zero risk is unattainable. Future challenges for the preharvest food safety are numerous and not clearly solvable. Yet, increased efforts and research are needed to address these issues in a pro-active manner as opposed to reactively. Genetically modified (e.g. salmon) and cloned animals continue to be developed, yet regulatory approval is unclear. As this trend continues, along with the use of nanotechnology in products, researchers as well as policy makers must evaluate the potential foodborne and public health risk. One significant change is the movement toward organically raised, or free range food animals. This is in part a response to consumers‘ desiring ―heal thier‖ products free of pesticides and antibiotics. Recent studies have demonstrated that organically raised food animals when compared to conventionally raised food animals, still have foodborne pathogens and antibiotic resistance present. Swine (as well as other animals) raised outdoors now are more exposed to wildlife, rodents, birds, and insects that carry foodborne, as well as animal, pathogens which are more easily introduced to the animal and herd. Foodborne parasites once contained to a lower level will be more easily introduced from the environment and soil. Niche markets that have emerged with new cultures and tastes, may present foodborne issues that we have not thought about, in food animals like bison, goat, and even ostrich. The use of antibiotics in animals and humans and the public health risk of resistance will continue to grow. Research is needed to develop alternatives to antibiotics, particularly in food animals, as well as potential management strategies that will reduce the need for antibiotics. FDA has attempted to develop surveillance or reporting systems to determine the use of antibiotics in food animals, but to date, it is too onerous from a resource and financial perspectives. Researchers are continually developing new technology and methods, such as microarrays, genomics, and the other ―om ics‖ to help understand the link between animal and human pathogens, and disease mechanisms. This has increased the need for more complicated analysis, more flexible data servers, and scientists trained in bioinformatics. The impact of climate change on food safety and food production is unknown. There are obvious concerns about water and protein sources, but increases in aflatoxins in feeds can have a direct impact on both food animals and humans. Over 10 years of research have been done on preharvest food safety and potential interventions. It is essential that in evaluating intervention and control strategies, researchers focus on understanding and measuring the impact and outcome of these interventions. This will provide direction for future approaches. New methodology and strategies need to evolve as the technology has over time. The analytical and epidemiologic approaches can be used to evaluate the progress and to point out the future, as well as improve the science. For example, Sargeant et al (2009a and b) showed that in 100 preharvest food safety studies, important information was lacking, such as randomization, study criteria, and even sample size. At the same time, there has been a movement in clinical human medicine to improve their clinical trials used in drug development and for research. This movement has led to new standards on how to design clinical trials and to report them. In 2010, Conner et al. developed a similar or a consort statement of 22 specified items that outlined the need for specific standards for use of food animals in randomized clinical trials for food safety and animal research (O‘Connor, 2010, Sargeant, 2010). This paper was published in 6 journals simultaneously and is being used as a standard for journals to evaluate submissions. Evidence-based approaches and

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systematic reviews that have been used in human medicine, are gaining in popularity, and have been used on a limited basis in veterinary medicine. These reviews are not simple and require specialized training and epidemiologists. But, they can provide a scientific approach to past and current research, and develop a plan for future research goals. Preharvest food safety is just one phase in a multi-faceted cycle of food production that requires various disciplines, multiple partners, and a clear goal of reduced foodborne disease and public health risk. The solutions to reduce foodborne disease are not linear or simple, but continued efforts by researchers, producers and processors, and policy-makers in a collaborative approach across the food production continuum will maintain small but impactful progress in reducing public health risk.

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REFERENCES AAM. (2009). American Academy of Microbiology. Global Food Safety: Keeping Food Safe from Farm to Table. American Academy of Microbiology, Washington DC. APHIS- http://www.aphis.usda.gov/animal_health/animal_dis_spec/poultry/. Boucher H.W., Corey G.R. 2008. Epidemiology of methicillin-resistant Staphylococcus aureus. Clin. Infect. Dis. June 1 (46): Suppl. 5: S344-9. Brashears, M.M., Galyean, M.L., Loneragan, G.H., Mann, J.E., Killinger-Mann, K. 2003. Prevalence of Escherichia coli 0157:H7 and performance by beef feedlot cattle given Lactobacillus direct fed microbials. J. Food Prot. 66:748-754. Callaway, TR, Edrington TS, Brabban AD, Keen JE, Anderson RC, Rossman, ML, Engler MJ, Genovese, KJ, Gwartney BL, Reagan JO, Poole TL, Harvey RB, Kutter EM, Nisbet DJ. 2006. Fecal prevalence of Escherichia coli 0157, Salmonella, Listeria, and Bacteriophage infecting E.coli 0157:H7 in feedlot cattle in the Southern Plains region of the United States. Foodborne Pathog. Dis. 3(3); 234-44. Diez-Gonzalex, F, Callaway, T.R., Kizoulis, M.G., and Russell, J.B. 1998. Grain Feeding and the Dissemination of Acid-Resistant Escherichia coli from Cattle. Science. 281:16661668. EU- http://www.ec.europa.eu/food/food/biosafety/hygienelegislation/comm_rules_en.htm. FSIS : http://www.fsis.usda.gov/about/nacmpi/index.asp. FSIS. http://www.fsis.usda.gov/pdf/reducing_ecoli_shedding_in_cattle_0510.pdf. FSIS: www.fsis.usda.gov/oppde.nacmpi/sept....preharvest_subcommittee_final_report.pdf. http://www.foodsafetyworkinggroup.gov. http://www.whitehouse.gov/sites/default/files/microsites/ostp/foodsafetyresearch1999.pdf. IOM. (1998). Institute of Medicine. Ensuring Safe Food from Product to Consumption. National Academy Press, Washington, DC. IOM. (2003). Scientific Criteria to Ensure Safe Food. National Academy Press, Washington DC. Isaacson, RE, Torrence, ME, and Buckley, MR. AAM (2004). American Academy of Microbiology. Preharvest Food Safety and Security. Perthshire, Scotland. Mead, PS, Slutsker, L, Dietz v, McCaig, LF, Bresee JS, Shapiro, C … R. Tauxe. (1999) Food-related illness and death in the United States. Emerging Infectious Diseases 5, 60725.

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Microbial FS in Animal Agriculture. ME torrence and RE Isaacson (eds). Iowa State university Press, Ames, ia. 2003. Torrence, ME. US Federal Activities, initiatives and research in food safety. Pag 3-10. Mulder, R.W.AW, and Hupkes, H. 2007. European Legislation in Relation to Food Safety in Production of Poultry Meat and Eggs. J. Appl. Poult. Research 16:92-98. NACMCF (National Advisory Committee on Microbial Criteria for Foods) 1998. Hazard analsysis and critical control point principoles and application guidelines. J. Food Protection 61, 1246-1259. OConnor, AM, Sargeant, JM, Gardner, IA, Dickson, JS, Torrence, ME et al. 2010. The REFLECT statement: methods and processes of creating reporting guidelines for randomized controlled trials for livestock and food safety by modifying the CONSORT statement. Zoonoses Public Health: Mar. 57(2): 95-104. Oliver, SP, Patel, DA, Callaway, TR, and Torrence ME. 2009. ASAS Centennial Paper: Developments and future outlook for preharvest food safety. J. of Animal Science, 87: 419-437. Sargeant, JM Elgie, R., Valcour, J., Saint-onge, J, Thompson, A., Marcynuk, P., and Snedeker, K. 2009a. Methodological quality and completmeness of reporting in clinical trials conducted in livestock species. Prev Vet. Med. 91, 107-115. Sargeant, JM, O‘Connor, AM, Gardner IA, Dickson JS, Torrence, ME. 2010. The REFLECT statement: reporting guidelines for randomized controlled trials in livestock and food safety: explanation and elaboration. Zoonoses Public Health. Mar 57(2): 105-36. Sargeant, JM, Saint-Oinge, J., Valcour, J., Thompson, A., Elgie, R., Snedeker, K, and Marcynuk. P.., 2009b. Quality of reporting clinical trials of preharvest food safety interventions and associations with treatment effect. Foodborne and Pathogens Disease. 6: 989-999. Scallan, E, Griffin, PM, Angulo, F, Tauxe, R, and Hoekstra, R. ( 2011). Foodborne Illness Acquired in the United States-Unspecified Agents. Emerging Infectious Diseases 17 (1), 16-22. Scallan, E., Hoekstra, R, Angulo, F, Tauxe, R, Widdowson, M, Roy ….. PM Griffin (2011). Foodborne Illness Acquired in the United States- Major Pathogens. Emerging Infectious Diseases 17 (1), 7-15. Schlundt, J., Toyofuku, H., Jansen, J., Herbst SA. 2004. Emerging Food-borne diseases. Rev.Sci. Tech: Aug 23(2) 513-533. Smith, D.R., Moxley, R.A., Klopfenstein, T.J., Erickson, E.G.. 2009. A Randomized Longitudinal Trial to Test the Effect of Regional Vaccination within a Cattle Feedyard on E.coli 0157:H7 Rectal Colonization, Fecal Shedding and Hide Contamination. Foodborne Pathog.Dis. 6(7):885-892. Thornton, A.B., Thomson, D.U., Loneragan, G.H., Fox, J.T., Burkhardt, D.T., Emery, D.A., Nagaraja, T.G. 2009. Effects of a siderophore receptor and porin proteins-based vaccination on fecal shedding of Escherichia coli 0157:H7 in experimentally inoculated cattle. J. Food Prot. 2:866-869. WHO – WHO Global Strategy for Food Safety.2002. Geneva Switzerland. Retrieved from WHO website: http://www.who.int/foodsafety/publications/general/global_strategy/en. WHO- (WHA63.3) Advancing Food Safety Initiatives. 2010. Retrieved from WHO website:http://apps.who.int/gb/ebwha/pdf_files/WHA63/A63_R3-en.pdf.

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Disclaimer: This article presents the opinions of the author and does not represent any official policy or opinion of ARS, or USDA. Proper names or brands may be necessary to report factually on available data: however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product, or exclusion of others that may be suitable.

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In: On-Farm Strategies to Control Foodborne Pathogens ISBN: 978-1-62100-411-0 Editors: Todd R. Callaway and Tom S. Edrington © 2012 Nova Science Publishers, Inc.

Chapter 2

ETHICS AND PREHARVEST FOOD SAFETY B. Rollin

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Dept of Philosophy, Colorado State University, Fort Collins, CO Over the past 50 years, agriculture has responded to a perceived need to ―f eed the world‖. Food production has reached the stage where hunger across the world is determined more by distribution, transportation and economics (i.e. ability to pay) rather than limits on production capacity (except in local situations, such as drought or civil unrest). Our fears of a Malthusian scenario where the food supply becomes a world-wide limitation have been put to rest by the ingenuity and hard work of many people across science and agriculture. Unfortunately, this drive to produce food efficiently and at the lowest external cost has been subject to unforeseen social issues and market forces that have had a major impact on animal agriculture. In recent years (approximately the past 20), market-driven agriculture has rapidly changed, with farm sizes increasing and the numbers of individuals involved in animal agriculture decreasing to less than 1% of the population. Furthermore, the industrialization of agriculture has produced unforeseen results that impact human health and food safety. All segments of the animal production industry strive to produce the safest possible product, and they are keen to do so. Yet animal producers must make an economic profit in order to survive, as do processors and retailers. Thus the ultimate economic expenses occasioned by demands for change and greater food safety must fall directly upon the consumer. In general, 20th Century science has been delinquent in discussing, let alone dealing with, ethical issues following in its wake, resulting in a loss of credibility with the general public. The ethics surrounding food production is an issue that has not been studied in depth, because it has always been patently ―obv ious‖ that more and cheaper food is better for everyone. Yet over the last decade, the public has become increasingly conscious of numerous issues growing out of modern animal agriculture, including sustainability, environmental despoliation, animal welfare, loss of rural communities to agricultural intensification, and food safety . Agriculture must engage these issues in a socially acceptable way, Questions such as ―i s food too cheap?‖ and ―w ho is responsible for the safety of food consumers?‖ are critical to the future direction of animal agriculture.

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The future of animal science and food production must certainly include an understanding of the ethical issues surrounding animal welfare and human food safety. Suggestions from the Pew Commision on Industrial Farm Animal Production address a wide range of ethical issues that impact a large proportion of animal agriculture. These suggestions include the controversial issues of banning non therapeutic antibiotic use, and the phasing out of intensive confinement operations, both of which have direct and indirect impacts on food safety and security. A crucial point that emerged from the Pew report concerns the cost of food. There is no question that food costs at the cash register are extraordinarily low in the U.S. American consumers spend about 9% of their income on food, while the European percentage is double that. What the public has failed to realize is that much of the true cost of food is externalized, i.e. paid for in hidden ways that do not show up at the register but are nonetheless paid for by society. Examples abound, when CAFOs (confined animal feeding operations) pollute (as when lagoons containing hog manure were breached during severe rains), it is the public that pays for the clean-up. Similarly, when people living near CAFOs have impaired health, that cost is not borne by the production facility. The social demand that the ethical issues cited above be addressed by agriculture will often (though not always) increase the cost of production. It is not fair to change the rules in the middle of a game. That is, it is not fair or equitable to demand that producers alone pay for conversion of battery cage systems for hens into open barns when society suddenly decides that battery cages are no longer socially acceptable. In the case in question, consumer demand for a greater degree of food safety than what was hitherto accepted will very likely cost more, a cost rightly borne in part by those making the demands. In short, improving food safety will raise the cost of food paid by consumers. Although we, the authors, have agreed to disagree on some issues addressed herein, we present in this chapter an approach to understanding the ethical dilemmas that face the animal and food production industry in the early part of the 21st century, most specifically as they address food safety. While all of us involved in animal agriculture will not agree on all suggestions from the Pew commission, we must all understand that the commission has provided a social map for the future of agriculture, and that change is coming and that the industry can either embrace and direct these inevitable societal changes, or be swept under a tide of external regulations that may not reflect the realities of animal production.

FOOD SAFETY PROBLEMS ON THE FARM Foodborne pathogenic bacteria are commonly found in food animal species (NAHMS 1997; 2000; USDA/APHIS 2001; 2003). Foodborne pathogens usually only cause illness in human consumers (although Salmonella serotypes can cause illnesses in cattle and swine) so infected animals are typically asymptomatic and cannot be visually identified. Many of these pathogens evolved to inhabit the intestinal tract of these food animals over millennia (Law 2000). Other chapters in this book do a good job of addressing the specific pathogens found in the intestinal milleu of food animals. Suffice it to say that the presence of ―com mensal‖ or transient pathogenic bacteria is widespread in food animals. Nearly all of the most common

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foodborne pathogenic bacteria are found in the intestinal tract of food animals as at least a transient organism. In general, processing plants do a good job at reducing contamination with foodborne pathogenic bacteria (Koohmaraie et al., 2005). However, during the process of transport and lairage, pathogenic bacteria can be spread or amplified in population of animals (Arthur et al., 2007; Arthur et al., 2008; Fegan et al., 2009; Hurd et al., 2001; Minihan et al., 2003; Swanenburg et al., 2001). Some studies indicate social stresses increase shedding of Salmonella in pigs (Callaway et al., 2006b). However other researchers have found inconsistent results in the effects of various stresses that food animals face on foodborne pathogen colonization (Barham et al., 2002; Corrier et al., 1990; Edrington et al., 2004; Marg et al., 2001; Rostagno et al., 2003). Therefore, in recent years researchers in academia and industry have begun to examine ways to reduce foodborne pathogens in food animals before they enter the processing facility. Overall, the economic impact of foodborne illness on the economy is vast, more than $150 billion per year in the U.S. alone (Scharff 2010). The foodborne pathogenic bacteria that are most commonly associated with food animals are presented in Table 1, which estimates the economic cost to individuals and society as a whole of these bacterial species as being in excess of $40 billion per year in the U.S., with a roughly equivalent impact estimated for the EU. Because many of these illnesses can be traced to the pathogens living in the live animal, if the pathogens in the animal can be reduced, then the societal cost of the pathogen can be reduced.

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Table 1. Costs of cases of foodborne illness caused by bacteria commonly associated with food animals. Data excerpted and adapted from Scharff, 2010 Foodborne Bacterial Pathogen

Estimated cases per year

Cost per case (USD)

Enterohemorrhagic E. coli (EHEC) Salmonella (nontyphoidal) Campylobacter Listeria Total

72,273

$14,800

Total cost to U.S. Residents ($ Billions, USD) 1.0

1,597,411

$9,100

14.6

2,112,302 5,200 3,727,186

$8,900 $1,600,000 $11,500 (est)

18.8 8.8 43.2

What Role Do Large-Scale Confinement Facilities Play in Foodborne Pathogen Dissemination? The role of confined animal feeding operations (CAFOs) in the development and dissemination of foodborne pathogens is complicated and in some ways, counter-intuitive. Because CAFOs have such a large footprint and role in animal agriculture, it has become ―com mon knowledge‖ that CAFOs ―cr eated foodborne illnesses‖ or ―h amburger disease‖ (Martens 2000; 2001). However, the science does not support this conclusion at this time.

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While it is intuitively logical that large farms have more pathogens present on them, the prevalence rates are surprisingly similar. The use of large animal facilities has allowed for animals to be produced more efficiently, but it creates opportunities for transient pathogens (especially those that do not cause illness in the host food animal) to be passed freely from animal to animal (Matthews et al., 2006; Nansen and Roepstorff 1999; Van Donkersgoed et al., 2001; Zheng et al., 2007). The contamination of environments and spread of these organisms has been linked at times to animal density and crowding has been shown to affect performance as well as pathogen contamination (Frank et al., 2008; Oetzel et al., 2007; Rachuonyo et al., 2002), though this case is by no means conclusive (Wilhelm et al., 2009). For example, herd size was not associated with shedding of Salmonella in dairy cattle on a variety of conventional and organic farms (Fossler et al., 2005a; 2005b; Fossler et al., 2005c). On a variety of cattle farms in Belgium, farm size did not affect shedding of E. coli O157 on beef or dairy farms (Cobbaut et al., 2009). Conversely, in a study contrasting farm size, it was found that small farms had higher odds of STEC shedding than did larger farms (Cho et al., 2009). Enterohemorrhagic E. coli (EHEC, such as O157:H7) have been around for at least 50,000 years, and have existed in cattle for much of their history (Law 2000; Wick et al., 2005). Furthermore, other foodborne pathogenic bacteria are ancient species, and are not recently emerged pathogens. The natural history and environmental niches of these organisms indicates that they have lived in the gut of animals for millennia, but they are perceived as greater threats to human food safety in recent years. In general, the incidence of these pathogens is quite variable from year to year and farm to farm. One theory surrounding this apparent disconnect involves the ―h ygiene hypothesis‖, that we are too clean as a society resulting in an increased susceptibility to the rare pathogen challenge because our immune systems are not constantly primed to attack transient pathogens (Callaway et al., 2006a). Collectively, the results indicate that the relatively recent introduction of large scale animal production facilities has not had a significant enough impact to alter the ecology of foodborne pathogenic bacteria in the animal intestinal tracts, but the cleanliness of the ―s usceptible pool‖ for these pathogens (i.e., humans, especially children, the elderly and immunocompromised) has apparently had a greater impact in allowing for an increasing incidence of human illnesses. Smaller scale animal production achieved food production goals by raising more animals on more farms, which meant more point sources for potential infection and subsequent spread. This point cuts two ways, in cases of diseases such as avian influenza, the larger farms mean more birds exposed at each farm, yet easier control of the disease from a public health perspective because it is one physical location, not 50. However, it appears that smaller farms would have less impact and less spread of pathogens through smaller population pools. On the other hand, today‘s modern agriculture is so integrated and has such an effective distribution system that food from one processing facility can be disseminated across many states. Furthermore, when meat products from many farms are co-mingled the presence of pathogens from one food source may be spread horizontally, resulting in very low pathogens concentrations but a high and broadly distributed pathogen prevalence. Given the increase in elderly and immunocompromised persons now found in our population, the impacts of these low concentrations can be devastating to consumers and to companies that produce food. Recent large scale food recalls highlight this point, that large scale distribution issues may have broad and far-reaching societal impacts.

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Organic Production is Better than Modern Industrial Systems, Right? Conventional wisdom, at least in the public mind, suggests that organic food production results in better food, safer food, for less money and is better all around; however, scientific data suggests this is simply not the case, especially in regard to foodborne pathogenic bacteria (Pacanoski 2009). In spite of a great deal of research into organic vs. modern production systems, no difference in pathogen levels has been detected. A great deal of research has gone into proving this ―obv ious‖ hypothesis (Martens 2000), but none has been able to demonstrate any true differences between production systems, especially in pre-harvest foodborne pathogen levels in food animal gastrointestinal tracts (Wilhelm et al., 2009). In general, most results comparing organic to conventional food production systems have found little or no differences in most of the food safety quality measures (Hoogenboom et al., 2008; Ludewig et al., 2004; Wilhelm et al., 2009; Young et al., 2009). Research has shown that Salmonella levels in certified organic broilers were lower compared to conventionally produced broilers (Alali et al., 2010). When examining retail cuts of broiler chickens, the incidence of Salmonella was the same between organic and conventional production, however there was a greater degree of antimicrobial resistance from chickens raised in a conventional system (Lestari et al., 2009). In a nice recent study from the Netherlands, researchers compared organic to conventional broiler production methods. They found that the organic system performed better than the conventional system as related to animal welfare issues, and contained lower Salmonella contamination levels, but had a higher level of Campylobacter contamination(Bokkers and de Boer 2009). This same effect of Campylobacter increase and Salmonella decrease in broilers was shown in another comparison from Belgium (Van Overbeke et al., 2006). A meta-analysis indicated that the incidence in broilers of Campylobacter was higher in organic chickens, but not in their carcasses (Young et al., 2009). Other researchers have also found that the differences in Campylobacter populations between organic and conventional broiler carcasses were negligible at the retail level (Hanning et al., 2010). Danish researchers found there were no differences in the proportions of swine seropositive for Salmonella in pigs reared in organic, outdoor or indoor pig farms (Zheng et al., 2007). Studies in the U.S. have found that outdoor swine facilities can transmit pathogens and carry Salmonella in approximately the same proportion as conventional production systems (Callaway et al., 2005). In the UK, it was found that conventional production of pigs resulted in a protective effect from Salmonella infection based on ELISA results (Smith et al., 2010). In other studies from the U.S., it was found that organically-reared cattle farms had an increased incidence of Shiga-toxin producing E. coli (STEC) compared to conventional herds (Cho et al., 2009). Other results found that grass-fed beef did not differ in their carriage of (generic) E. coli or Enterococcus spp (Zhang et al., 2010). However, while most of the antimicrobial resistance did not differ between conventional and organic production, some Enterococcus isolates were more resistant to daptomycin and linezolid. Overall the authors declare ―t aken together, these data indicate that there are no clear food safety advantages to grass-fed beef products over conventional beef products‖ (Zhang et al., 2010). Collectively, these results indicate that there is not a glaring difference in pathogen incidence on organic and conventional farms. Thus, while modern agriculture does have to

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deal with the issue of foodborne pathogenic bacteria in food animals, these pathogens were not created and do not thrive under commercial or modern practices any more than under small farm or organic practices. They are simply adapted to colonize and survive in the gastrointestinal tract of food animals.

WHAT CAN WE DO?

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Several solutions have been posited over the years to improve food safety by reducing foodborne pathogenic bacteria in the live animal while having positive (or at least neutral) impacts on animal welfare and other agricultural parameters. Other changes that impact, or could impact, food safety have been inadvertent experiments in unintended consequences. While many of the changes proposed are political policy decisions (e.g., biofuels, subsidies) that are beyond the scope of this chapter it is clear that all changes that can reduce these issues will come at a direct economic cost to consumers. 1) Local production/marketing: ocally‖ (e.g., New York While many urban areas cannot meet their food needs truly ―l City, London), a return of agriculture to more urban and suburban areas can enhance the quality of life and increase the connection between the true cost and market price of food. As many cities shrink there is an upswing in urban and suburban gardening in cities such as Detroit. The new greenbelts offer a potential for use as crop or pastureland, increasing the urban-agricultural connect and providing direct accountability between food producers and consumers. It is hypothesized that utilizing large centralized processing facilities enhances food safety because these plants can afford the investment in new interventions in plant. However, because these large processors supply meat to large numbers of stores via a highly efficient distribution system, it has a widespread effect. This is best exemplified in the concepts of probability/impact, the larger processors have an extremely low probability but high impact, whereas smaller plants have a low probability and lower impact. In a similar vein, the use of smaller, local processing would appear to assure more care and direct accountability for quality and safety, yet smaller plants are more subject to staffing issues and training, as well as a need for more FSIS inspectors to be in place in a larger number of processing facilities. 2) Reducing impact or dissemination of food outbreaks: Reduction of ground beef chub or lot size would increase the number of batches and tests. Coupled with the currently effective test and hold strategy, this could result in fewer human illnesses by slowing the dissemination of these pathogens into the food supply. This simply builds upon the positive results from FSIS implementation of their ―t est and hold‖ procedure for ground beef lots that should be roundly applauded. 3) Reducing foodborne pathogens in the live animal: While a variety of methods to reduce foodborne pathogens in food animals have been proposed, few of these products are currently in the market (LeJeune and Wetzel 2007; Oliver et al., 2008; Sargeant et al., 2007; Vandeplas et al., 2010). The

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introduction of vaccines (McNeilly 2010) and probiotic products (Stephens et al., 2007) against E. coli O157:H7, and the use of phages (Johnson et al., 2008; Wall et al., 2010) and sodium chlorate in other food animals offer glimpses into potential hurdles that could be erected to reduce entry of pathogens into the food supply.

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WHO WILL BE PAYING FOR THESE IMPROVEMENTS? Food animal producers are morally, ethically and legally obligated to provide as safe of a product as they can, and most producers happily do so because they are also consumers and take pride in their products. Throughout recent history, animal agriculture has focused on improving profitability while still producing as safe and wholesome product as possible, at the lowest possible cost to the consumer. As new interventions come on line to further improve food safety, there will be an added cost to the production of safer food. Because foodborne pathogens often do not cause illnesses in food animals, treatments must be applicable across large numbers of animals simultaneously, increasing the overall costs of implementation. Furthermore, some of these food safety improvements may negatively impact animal productivity, resulting in a loss of potential profit in an industry that survives on the slimmest of margins. Thus the revenue of that loss of production efficiency and implementation must be made up from some other source. Food producers are obliged to provide as safe of a food product as they can, but not at a cheap price. Producers, processing plants and distributors have invested large sums of money in their infrastructure to provide food to the consumer at the cheapest possible price. It is not just or fair to change the rules in middle of the game and expect the food producing industry to lose money by making changes to their infrastructure without recompense. The obvious source of the ultimate compensation is the end consumer. Will the implementation of various food safety interventions and costs create an environment of scales of food safety based on willingness or ability to pay? Would this create a ―payfor safety‖ scenario? Would an increase in the price of animal-based protein sources encourage lower meat consumption as a result? And how would this affect agriculture broadly and animal agriculture in particular? Or is food safety a fundamental ―r ight‖ and as such, should additional costs be borne equally by all consumers? The average foodborne bacterial illness costs around $11,000 per case in direct and indirect financial costs (Scharff 2010), not to mention the unquantifiable personal toll (Table 1). Added costs per pound of finished product to cover the added expenses would be viewed most closely as ―i nsurance‖ to protect consumers and society as a whole. An estimated direct cost of some of the treatments to reduce EHEC in cattle range from $9-$30 per head (broad estimates depending on the number of concurrent treatments applied), which would yield approximately 750 pounds of meat, for a total increase in price from $0.012 to $0.04 per pound. So for an estimated cost of less than a dime per pound in toto (including direct costs and production efficiency effects), an assurance of improved food safety can be purchased by consumers.

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CONCLUSIONS Animal agriculture has done a marvelous job in producing a high quality, wholesome product at the cheapest direct cost in response to consumer demand. However, the safety record is not perfect (nor if the hygiene hypothesis is correct, should it be) because food animals can contain foodborne pathogenic bacteria naturally transient or colonizing their intestinal tracts. Interventions to reduce these foodborne pathogenic bacteria are being brought online or are near-market; however, these interventions will come at a cost to producers, and this cost must eventually be passed on to consumers. Food producers are morally and legally obliged to provide the safest food product as they can, but are not obligated to do so at the cheapest direct price. If progress is improving food safety is to be made, it must come through consumer acceptance of this responsibility, and ultimately bearing the economic burden for the improvements that they seek. Scientific questions still surround the impacts of farm size and organic vs. conventional agriculture on foodborne pathogenic bacteria. To date, no differences in food safety parameters have been consistently proven in animals under any production system. These and more management and dietary impact effects must be elucidated before significant progress to improve live animal food safety can be enhanced.

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REFERENCES Alali W.Q., Thakur S., Berghaus R.D., Martin M.P. and Gebreyes W.A. 2010. Prevalence and distribution of Salmonella in organic and conventional broiler poultry farms. Foodborne Path. Dis. 7:1363-1371. Arthur T.M., Bosilevac J.M., Brichta-Harhay D.M. et al., . 2007. Transportation and lairage environment effects on prevalence, numbers, and diversity of Escherichia coli O157:H7 on hides and carcasses of beef cattle at processing. J. Food Prot. 70:280-286. Arthur T.M., Bosilevac J.M., Brichta-Harhay D.M. et al., . 2008. Source tracking of Escherichia coli O157:H7 and Salmonella contamination in the lairage environment at commercial U.S. beef processing plants and identification of an effective intervention. J. Food Prot. 71:1752-1760. Barham A.R., Barham B.L., Clound C.E. et al., . 2002. Effect of shipping stress in beef cattle on prevalence levels of Escherichia coli O157 and Salmonella spp. from the feedyard to the packing plant. J. Food Prot. 65:280-283. Bokkers E.A.M. and de Boer I.J.M. 2009. Economic, ecological, and social performance of conventional and organic broiler production in the Netherlands. Brit. Poult. Sci. 50:546557. Callaway T.R., Harvey R.B. and Nisbet D.J. 2006a. The hygiene hypothesis and foodborne illnesses: Too much of a good thing, or is our food supply too clean? Foodborne Path. Dis. 3:217-219. Callaway T.R., Morrow J.L., Edrington T.S. et al., . 2006b. Social stress increases fecal shedding of Salmonella Typhimurium by early weaned piglets. Curr. Iss. Intest. Microbiol. 7:65-72.

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Callaway T.R., Morrow J.L., Johnson A.K. et al., . 2005. Environmental prevalence and persistence of Salmonella spp. in outdoor swine wallows. Foodborne Path. Dis. 2:263273. Cho S., Fossler C.P., Diez-Gonzalez F. et al., . 2009. Cattle-level risk factors associated with fecal shedding of Shiga toxin-encoding bacteria on dairy farms, Minnesota, USA. Can. J. Vet. Res. 73:151-156. Cobbaut K., Berkvens D., Houf K., De Deken R. and De Zutter L. 2009. Escherichia coli O157 prevalence in different cattle farm types and identification of potential risk factors. J. Food Prot. 72:1848-1853. Corrier D.E., Purdy C.W. and DeLoach J.L. 1990. Effects of marketing stress on fecal excretion of Salmonella spp. in feeder calves. Am. J. Vet. Res. 51:866-869. Edrington T.S., Schultz C.L., Genovese K.J. et al., . 2004. Examination of heat stress and stage of lactation (early versus late) on fecal shedding of E. coli O157:H7 and Salmonella in dairy cattle. Foodborne Pathog. Dis. 1:114-119. Fegan N., Higgs G., Duffy L.L. and Barlow R.S. 2009. The effects of transport and lairage on counts of Escherichia coli O157 in the feces and on the hides of individual cattle. Foodborne Path. Dis. 6:1113-1120. Fossler C.P., Wells S.J., Kaneene J.B. et al., . 2005a. Herd-level factors associated with isolation of Salmonella in a multi-state study of conventional and organic dairy farms. II. Salmonella shedding in calves. Prev. Vet. Med. 70:279-291. Fossler C.P., Wells S.J., Kaneene J.B. et al., . 2005b. Herd-level factors associated with isolation of Salmonella in a multi-state study of conventional and organic dairy farms: I. Salmonella shedding in cows. Prev. Vet. Med. 70:257-277. Fossler C.P., Wells S.J., Kaneene J.B. et al., . 2005c. Cattle and environmental sample-level factors associated with the presence of Salmonella in a multi-state study of conventional and organic dairy farms Prev. Vet. Med. 67:39-53. Frank C., Kapfhammer S., Werber D., Stark K. and Held L. 2008. Cattle density and shiga toxin-producing Escherichia coli infection in Germany: Increased risk for most but not all serogroups. Vector-Borne Zoo. Dis. 8:635-643. Hanning I., Biswas D., Herrera P., Roesler M. and Ricke S.C. 2010. Prevalence and characterization of Campylobacter jejuni isolated from pasture flock poultry. J. Food Sci. 75-83. Hoogenboom L.A.P., Bokhorst J.G., Northolt M.D. et al., . 2008. Contaminants and microorganisms in Dutch organic food products: A comparison with conventional products. Food Addit. Contam. - Part A 25:1195-1207. Hurd H.S., Wesley I.V. and Karriker L.A. 2001. The effect of lairage on Salmonella isolation from market swine. J. Food Prot. 64:939-944. Johnson R.P., Gyles C.L., Huff W.E. et al., . 2008. Bacteriophages for prophylaxis and therapy in cattle, poultry and pigs. Anim. Health Res. Rev. 9:201-215. Koohmaraie M., Arthur T.M., Bosilevac J.M., Guerini M., Shackelford S.D. and Wheeler T.L. 2005. Post-harvest interventions to reduce/eliminate pathogens in beef. Meat Sci. 71:79-91. Law D. 2000. The history and evolution of Escherichia coli O157 and other shiga toxinproducing E. coli. World J. Microbiol. Biotechnol. 16:701-709. LeJeune J.T. and Wetzel A.N. 2007. Preharvest control of Escherichia coli O157 in cattle. J. Anim. Sci. 85-97.

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Lestari S.I., Han F., Wang F. and Ge B. 2009. Prevalence and antimicrobial Resistance of Salmonella serovars In conventional and organic chickens from Louisiana retail stores. J. Food Prot. 72:1165-1172. Ludewig M., Palinsky N. and Fehlhaber K. 2004. Quality of organic and directly marketed conventionally produced meat products. Öko- und direkt vermarktete konventionelle fleischerzeugnisse 84:105-108. Marg H., Scholz H.C., Arnold T., Rösler U. and Hensel A. 2001. Influence of long-time transportation stress on re-activation of Salmonella Typhimurium DT104 in experimentally infected pigs. Berlin. Munch. Tier. Woch. 114:385-394. Martens M.H. 2000. Debunking the industrial agriculture myth that organic foods are more likely to be carriers of dangerous bacteria such as E. coli O157:H7 or plant fungus such as fuminosin. http://www.purefood.org/Organic/ecolimyths.cfm. Martens M.H. 2001. Scientists expose myths that organic farming produces dangerous E. coli and plant diseases. www.purefood.org/Organic/ecolimyths.cfm. Matthews L., McKendrick I.J., Ternent H., Gunn G.J., Synge B. and Woolhouse M.E.J. 2006. Super-shedding cattle and the transmission dynamics of Escherichia coli O157. Epidemiol. Infect. 134:131-142. McNeilly T.N., Mitchell, M. C., Rosser, T., McAteer, S., Low, J. C., Smith, D. G., Huntlye, J. F., Mahajan, A., Gally, D. L. 2010. Immunization of cattle with a combination of purified intimin-531, EspA and Tir significantly reduces shedding of Escherichia coli O157:H7 following oral challenge. Vaccine 28:1422-1428. Minihan D., O'Mahony M., Whyte P. and Collins J.D. 2003. An investigation on the effect of transport and lairage on the faecal shedding prevalence of Escherichia coli O157 in cattle. J. Vet. Med. 50:378-382. NAHMS. 1997. Shedding of Salmonella by finisher hogs in the U.S. http://www.aphis.usda. gov/vs/ceah/cnahs/nahms/swine/swine95/sw95salm.pdf. NAHMS. 2000. Swine 2000. http://www.aphis.usda.gov/vs/ceah/cnahs/nahms/swine/ swine.htm. Nansen P. and Roepstorff A. 1999. Parasitic helminths of the pig: factors influencing transmission and infection levels. Int. J. Parasitol. 29:877-891. Oetzel G.R., Emery K.M., Kautz W.P. and Nocek J.E. 2007. Direct-fed microbial supplementation and health and performance of pre- and postpartum dairy cattle: A field trial. Journal of dairy science 90:2058-2068. Oliver S.P., Patel D.A., Callaway T.R. and Torrence M.E. 2008. ASAS Centennial Paper: Developments and future outlook for preharvest food safety. J. Anim Sci. 87:419-437. Pacanoski Z. 2009. The myth of organic agriculture. Plant Prot. Sci. 45:39-48. Rachuonyo H.A., Pond W.G. and McGlone J.J. 2002. Effects of stocking rate and crude protein intake during gestation on ground cover, soil-nitrate concentration, and sow and litter performance in an outdoor swine production system. J. Anim. Sci. 80:1451-1461. Rostagno M.H., Hurd H.S., McKean J.D., Ziemer C.J., Gailey J.K. and Leite R.C. 2003. Preslaughter holding environment in pork plants is highly contaminated with Salmonella enterica. Appl. Environ. Microbiol. 69:4489-4494. Sargeant J.M., Amezcua M.R., Rajic A. and Waddell L. 2007. Pre-harvest interventions to reduce the shedding of E. coli O157 in the faeces of weaned domestic ruminants: a systematic review. Zoonos. Pub. Health 54:260-277.

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Scharff R.L. 2010. Health-related costs from foodborne illness in the United States. http://www.producesafetyproject.org/admin/assets/files/Health-Related-FoodborneIllness-Costs-Report.pdf-1.pdf. Smith R.P., Clough H.E. and Cook A.J.C. 2010. Analysis of meat juice ELISA results and questionnaire data to investigate farm-level risk factors for Salmonella infection in UK pigs. Zoonoses Pub. Heal. 57:39-48. Stephens T.P., Loneragan G.H., Karunasena E. and Brashears M.M. 2007. Reduction of Escherichia coli O157 and Salmonella in feces and on hides of feedlot cattle using various doses of a direct-fed microbial. J. Food Prot. 70:2386-2391. Swanenburg M., Urlings H.A.P., Keuzenkamp D.A. and Snijders J.M.A. 2001. Salmonella in the lairage of pig slaughterhouses. J. Food Prot. 64:12-16. USDA/APHIS. 2001. Salmonella in United States feedlots. http://www.aphis.usda.gov/vs/ ceah/ncahs/nahms/feedlot/Feedlot99/FD99salmonella.pdf. USDA/APHIS. 2003. Salmonella and Campylobacter on U. S. Dairy Operations. http://www.aphis.usda.gov/vs/ceah/ncahs/nahms/dairy/Dairy02/Dairy02SalCampy.pdf. Van Donkersgoed J., Berg J.L., Potter A. et al., . 2001. Environmental sources and transmission of Escherichia coli O157 in feedlot cattle. Can. Vet. J. 42:714-720. Van Overbeke I., Duchateau L., De Zutter L., Albers G. and Ducatelle R. 2006. A comparison survey of organic and conventional broiler chickens for infectious agents affecting health and food safety. Avian Dis. 50:196-200. Vandeplas S., Dubois Dauphin R., Beckers Y., Thonart P. and Thewis A. 2010. Salmonella in chicken: Current and developing strategies to reduce contamination at farm level. J. Food Prot. 73:774-785. Wall S.K., Zhang J., Rostagno M.H. and Ebner P.D. 2010. Phage therapy to reduce preprocessing Salmonella infections in market-weight swine. Appl. Environ. Microbiol. 76:48-53. Wick L.M., Qi W., Lacher D.W. and Whittam T.S. 2005. Evolution of genomic content in the stepwise emergence of Escherichia coli O157:H7. J. Bacteriol. 187:1783-1791. Wilhelm B., Rajić A., Waddell L. et al., . 2009. Prevalence of zoonotic or potentially zoonotic bacteria, antimicrobial resistance, and somatic cell counts in organic dairy production: Current knowledge and research gaps. Foodborne Path. Dis. 6:525-539. Young I., Rajić A., Wilhelm B.J., Waddell L., Parker S. and McEwen S.A. 2009. Comparison of the prevalence of bacterial enteropathogens, potentially zoonotic bacteria and bacterial resistance to antimicrobials in organic and conventional poultry, swine and beef production: A systematic review and meta-analysis. Epidemiol. Infect. 137:1217-1232. Zhang J., Wall S.K., Xu L. and Ebner P.D. 2010. Contamination rates and antimicrobial resistance in bacteria isolated from "grass-fed" labeled beef products. Foodborne Path. Dis. 7:1331-1336. Zheng D.M., Bonde M. and Sørensen J.T. 2007. Associations between the proportion of Salmonella seropositive slaughter pigs and the presence of herd level risk factors for introduction and transmission of Salmonella in 34 Danish organic, outdoor (non-organic) and indoor finishing-pig farms. Livestock Sci. 106:189-199.

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In: On-Farm Strategies to Control Foodborne Pathogens ISBN: 978-1-62100-411-0 Editors: Todd R. Callaway and Tom S. Edrington © 2012 Nova Science Publishers, Inc.

Chapter 3

INTERVENTIONS TO IMPROVE THE SAFETY OF MILK PRODUCTION 1

S. P. Oliver1 and S. E. Murinda2 UT AgResearch, Tennessee Agricultural Experiment Station, Knoxville, Tennessee Animal and Veterinary Sciences and Center for Antimicrobial Research and Food Safety, California State Polytechnic University, Pomona, California

2

ABSTRACT

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Milk is regarded by many as one of Mother Nature‘s most perfect foods. Consequently, several regulations are in place to ensure access to a safe, wholesome, nutritious, and readily available milk supply for human consumption. Production of maximum quantities of high quality milk is an important goal of every dairy operation. Poor milk quality affects all segments of the dairy industry ultimately resulting in milk with decreased manufacturing properties and dairy products with reduced shelf-life. Production of high quality milk places a much greater emphasis on strategies for the prevention and control of mastitis. In this chapter, we focus on the following topics: 1) antibiotic use for treatment and prevention of mastitis 2) issues associated with antibiotic use including residues in milk and antimicrobial resistance 3) assessing milk quality and monitoring for pathogens in bulk tank milk 4) use of sanitizers and disinfectants, teat dips; farm hygiene, good husbandry and production practices 5) vaccines to prevent mastitis 6) vaccines to reduce shedding of foodborne pathogens 7) hazards associated with consumption of raw milk, and 8) what is on the horizon, such as potential new vaccines, creation of transgenic cattle, identification of disease resistant cattle, etc.



Author for correspondence: Stephen P. Oliver, Ph.D. Assistant Dean and Assistant Director, UT AgResearch, 103 Morgan Hall, 2640 Morgan Circle, The University of Tennessee, Knoxville, TN 37996, USA Tel. (865) 9747340; Fax: (865) 974-3394; E-mail: [email protected].

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S. P. Oliver and S. E. Murinda Advances in biotechnology have resulted in some exciting new technologies that can/will be used to solve complex problems confronting animal agriculture. Improved disease surveillance; enhanced disease resistance; and advances in mastitis treatment and prevention will dramatically improve dairy herd health programs and result in production of maximum quantities of high quality milk at lower costs. Future scientific breakthroughs will no doubt have a profound impact on production of high quality milk. A safe, wholesome, abundant and nutritious milk supply should be the goal of every dairy producer in the world. Use of interventions to improve the safety of milk production will better enable dairy producers to achieve these important goals and continue to ensure the production of high quality, wholesome and nutritious milk for human consumption.

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INTRODUCTION The quality of milk has been and continues to be a topic of intense debate. An important issue in this ongoing debate is establishing export markets of United States produced milk. One important measure of milk quality is the number of somatic cells in milk, referred to as the somatic cell count (SCC). Milk with a high SCC is produced by cows with mastitis and is of inferior quality. In the United States, the current regulatory limit for somatic cells defined in the Grade A Pasteurized Milk Ordinance (US Food and Drug Administration, Center for Food Safety and Applied Nutrition (2007) Grade ― A‖ pasteurized milk ordinance: 2007 revision) is 750,000/ml of milk. There is continuing pressure from a variety of advocacy groups to reduce the regulatory limit for somatic cells in milk from the current 750,000/ml to 400,000 or less to be competitive in the global dairy marketplace. Global standards are considerably lower (400,000 somatic cells/ml), and as low as 200,000 somatic cells/ml in some of the Scandinavian countries. This disparity in SCC makes it difficult, if not impossible, to export United States produced milk/milk products to other developed countries. Thus, SCC limits for raw milk to be acceptable at dairy processing plants may decrease to levels much lower than they are now, making it increasingly problematic for some dairy producers to meet these higher standards. Another important issue relates to human health. Opponents of reducing the regulatory limit of somatic cells in milk claim there is no human health risk associated with high bulk tank SCC milk, therefore the SCC limit in the PMO should not be lowered. However, milk with a high SCC is associated with a higher incidence of antibiotic residues in milk (Ruegg, 2005), and the presence of pathogenic organisms and toxins in milk (Oliver et al., 2005b). Last, but certainly not least, is the fact that poor quality milk is an inferior product with reduced processing properties resulting in dairy products with a reduced shelf-life. Thus, milk with a high SCC is associated with indirect health risks to the consumer and is an inferior quality product. Good quality milk has a longer shelf-life, tastes better, and is more nutritious and wholesome. These issues are the basis for animal health advocacy groups to lower the SCC regulatory limit. Production of better quality milk will place a much greater emphasis on strategies for the prevention and control of mastitis to reduce the number of somatic cells in milk. Mastitis is the most important factor associated with reduced milk quality. Mastitis is an inflammation of the udder that affects a high proportion of dairy cows throughout the world. Mastitis differs from most other animal diseases in that several diverse bacteria are capable of infecting the udder. These pathogens invade the udder, multiply there and produce harmful

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substances that result in inflammation, reduced milk production and altered milk quality. Because mastitis can be caused by many different pathogens, control is extremely difficult and economic losses due to mastitis can be immense. These losses include reduced milk production, discarded milk, replacement costs, extra labor, treatment, and veterinary services. Alterations in milk composition associated with mastitis are due to several factors including impaired milk synthesis and secretion, mammary epithelial cell death and degeneration, and transport of substances from blood to milk and from milk to blood (Oliver and Calvinho, 1995). The most notable changes in milk composition associated with mastitis are decreased concentrations of fat, lactose, casein and calcium; and increased concentrations of albumin, sodium and chloride. Concentrations of lipases, proteases, oxidases, plasmin and plasminogen increase, which may adversely influence milk stability, milk flavor, and overall quality of processed dairy products. In addition, factors not normally found in milk such as inflammatory mediators and bacterial enterotoxins and endotoxins have been detected in milk from cows with mastitis (Oliver and Calvinho, 1995). From a dairy manufacturing perspective, mastitis decreases concentrations of desirable components and increases concentrations of undesirable components all of which influence milk shelf-life and organoleptic qualities, such as taste. The National Mastitis Council (2006) estimates that mastitis costs dairy producers in the United States over two billion U.S. dollars annually. Thus, mastitis continues to be one of, if not, the most significant limiting factor to profitable dairy production in the United States and worldwide. Objectives of this chapter are to discuss the importance of high quality milk, and how dairy producers can produce high quality milk by controlling mastitis using proven methods of mastitis prevention and control. The following topics will be covered: 1) antibiotic use for treatment and prevention of mastitis, 2) issues associated with antibiotic use, 3) assessing milk quality and monitoring for pathogens in bulk tank milk, 4) milking time hygiene and good husbandry/production practices, 5) vaccines used to prevent mastitis, 6) vaccines to reduce shedding of foodborne pathogens, 7) hazards associated with consumption of raw milk, and 8) intervention strategies on the horizon to improve the safety of milk production.

ANTIBIOTIC USE FOR TREATMENT AND PREVENTION OF MASTITIS Many factors can influence development of mastitis; however, inflammation of the mammary gland is usually a consequence of invasion and colonization of mammary secretory tissue by one or more mastitis pathogens such as Staphylococcus aureus, Streptococcus agalactiae, Streptococcus uberis, or Escherichia coli. Microorganisms that most frequently cause mastitis can be divided into two broad categories: contagious pathogens, which are spread from cow to cow primarily during the milking process; and environmental pathogens, which are found throughout the habitat of dairy cows. Contagious mastitis is caused primarily by Staph. aureus and Strep. agalactiae. Mycoplasma bovis and other Mycoplasma species have been increasingly reported as another genus of importance to contagious mastitis. The primary source of these organisms is the udder of infected cows. Contagious mastitis pathogens spread from infected cows to uninfected cows primarily at milking. Some characteristics of herds with a contagious

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mastitis problem include: (1) a high prevalence of intramammary infection (IMI) during lactation, (2) a high bulk tank SCC (BTSCC), (3) infections of long duration, (4) a low proportion of infections that result in clinical mastitis (infections mostly subclinical), and (5) a low prevalence of infection during the dry period. Environmental mastitis is caused primarily by environmental streptococci including Strep. uberis, Streptococcus dysgalactiae subsp dysgalactiae, and coliforms including E. coli and Klebsiella species. The primary source of environmental mastitis pathogens is the environment of the cow. Infections generally occur between milkings and during the milking process. Some characteristics of herds with an environmental mastitis problem include: (1) a low prevalence of IMI during lactation, (2) a low BTSCC, (3) infections of short duration, (4) many IMI result in clinical mastitis, and (5) a high prevalence of infection during the dry period. Current mastitis control programs are based on hygiene and include teat disinfection, antibiotic therapy and culling of chronically infected cows. Acceptance and application of these measures throughout the world has led to considerable progress in controlling mastitis caused by contagious mastitis pathogens such as Strep. agalactiae and Staph. aureus. However, as the prevalence of contagious mastitis pathogens was reduced, the proportion of IMI caused by environmental pathogens such as E. coli and Strep. uberis has increased markedly (Oliver and Mitchell, 1984). Therefore, it is not surprising that mastitis caused by coliforms and environmental Streptococcus species has become a major problem in many well-managed dairy farms that have successfully controlled contagious pathogens. Contagious mastitis pathogens are controlled effectively by procedures that prevent spread of bacteria at milking time, which include good udder hygiene, and premilking and postmilking teat disinfection with effective teat disinfectants. In the U. S., the general recommendation is that all mammary quarters of all cows be infused with antibiotics approved for use in nonlactating cows after the last milking of lactation to eliminate existing infections and to prevent new infections during the early dry period which is a time that the udder is highly susceptible to new infection. It may be necessary to cull chronically infected cows. Control of environmental mastitis pathogens is best achieved by maintaining a clean, dry environment for lactating and nonlactating cows and heifers. Premilking and postmilking teat disinfection are recommended. Antibiotic dry cow therapy is recommended also. Dry cow therapy helps control new infections during the early dry period caused by environmental streptococci. However, dry cow therapy has little effectiveness in controlling coliforms and is not effective in preventing new infections that occur near calving. Vaccines to reduce the severity and duration of coliform mastitis are available and are useful in herds with environmental mastitis. Mastitis often requires antibiotic treatment. Antibiotic therapy of clinical mastitis involves: (1) detection of the infected quarter, (2) prompt initiation of treatment, (3) administration of the full series of recommended treatments, (4) maintaining a set of treatment records, (5) identification of treated cows, and (6) making sure the milk is free of antibiotic residues before adding to the bulk tank. There has been and continues to be concern over the low efficacy of antibiotic mastitis therapy against certain mastitis pathogens. This is due to bacterial factors, pharmacologic and pharmacokinetic limitations, and pathobiologic circumstances of the infected mammary gland. Efficacy of mastitis therapy is extremely low for chronic Staph. aureus infections; ß-

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lactamase production may be partly responsible for the low cure rate. However, even with antibiotics to which the bacteria were sensitive in vitro, the cure rate was still low (Owens et al., 1997). This suggests the presence of some other mechanisms that interfere with therapy such as formation of microabscesses in mammary tissues and internalization into phagocytic and epithelial cells (Almeida et al., 1996). Most antibiotics used in mastitis therapy do not penetrate into the infected area and have poor intracellular penetration. Pirlimycin has been studied extensively to treat cows with chronic Staph. aureus IMI because of its lower minimum inhibitory concentration and its tissue-penetrating ability. Extended therapy with pirlimycin greatly improved the cure rate against chronic Staph. aureus IMI during lactation (Belschner et al., 1996; Deluyker et al., 2001). Results from our laboratory have shown that extended therapy with pirlimycin is an effective procedure for treatment of chronic environmental Streptococcus species (Strep. uberis and Strep. dysgalactiae) IMI in lactating dairy cows (Gillespie et al., 2000). We have also had much success with extended therapy using ceftiofur hyrochloride for treatment of cows with naturally-occurring subclinical mastitis and experimentally induced clinical Strep. uberis mastitis (Oliver et al., 2004a, 2004b). Results of research on extended therapy (Owens et al., 1997; Deluyker et al., 2000; Gillespie et al., 2000; Oliver et al., 2004a; 2004b) support the concept that extended intramammary antimicrobial therapy is significantly more effective at eliminating natural and experimentally induced mastitis than standard intramammary treatment regimens. It would appear that lengthening the duration of antibiotic therapy increases treatment efficacy. This has been demonstrated for ceftiofur and pirlimycin against a variety of mastitis pathogens including Strep. uberis, other environmental Streptococcus species, Staph. aureus, Corynebacterium bovis and coagulase negative Staphylococcus species (CNS). Effectiveness of extended antimicrobial therapy must be weighed against several factors including the price of the antibiotic, loss of milk due to withholding time, marketability of the milk, potential of infecting the cow through repeated infusions via the teat canal, increased milk production following elimination of the chronic infection, reduced spread of contagious mastitis pathogens, and reduced culling because of a greater emphasis on milk quality. Caution should be taken to avoid extended antibiotic therapy during stressful situations for the animals such as heat stress. Studies to evaluate economic benefits of extended antimicrobial therapy need to be conducted to fully evaluate costs and benefits associated with this type of therapy. It is still controversial whether to treat or not treat cows with coliform mastitis. Clinical signs of coliform mastitis are mainly due to the effects from endotoxin. There are few antibiotics suitable for treating cows with coliform mastitis, however, ceftiofur hydrochloride has good in vitro activity against a wide variety of Gram-negative mastitis pathogens, and could prove useful for intramammary treatment of cows with clinical mastitis caused by Gram-negative bacteria. When treating cows with clinical or subclinical mastitis, dairy producers must recognize that administration of antibiotics in a manner inconsistent with the label instruction is considered extra-label use, and MUST be carried out under the supervision of the herd veterinarian. Furthermore, milk and meat for human consumption from antibiotic-treated cows must be free of drug residues. The importance of the nonlactating (dry) period in the control of mastitis in dairy cows has been recognized for 60 yr. A classic study by Neave et al. (1950) demonstrated that udders were markedly susceptible to new IMI during the early dry period. The rate of new

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infection during the first 21 days of the dry period was over 6 times higher than the rate observed during the previous lactation. Studies have also shown that udders are highly susceptible to new IMI during the periparturient period (Smith et al., 1985a; 1985b; Oliver 1988a; 1988b; Oliver and Mitchell, 1983; Oliver and Mitchell, 1984; Oliver and Sordillo, 1988). Increased susceptibility to new IMI is likely associated with physiological transitions of the mammary gland either from or to a state of active milk production. Many IMI that occur at this time persist throughout the dry period and are often associated with clinical mastitis after calving. Thus, the early dry period was identified as an extremely important time for the control of mastitis in dairy cows. Since the early work by Neave et al. (1950), effective procedures were developed to control infections during the dry period. Most dairy advisors recommend that all mammary quarters of all cows be infused with antibiotics approved for use in dry cows following the last milking of lactation. Objectives of dry cow therapy are twofold: (1) to eliminate infections present during late lactation, and (2) to prevent new infections during the early dry period when mammary glands are highly susceptible to new IMI. Antibiotic therapy at drying off plays an important role in the control of mastitis during the dry period. Dry cow therapy is particularly effective against streptococci and to a lesser extent against Staph. aureus. Smith et al. (1985a; 1985b) demonstrated that antibiotic therapy at drying off reduced the rate of new environmental streptococcal infection during the early dry period only and that the rate of new coliform IMI was not affected at all. Thus, two significant limitations of present antibiotic formulations used for dry cow therapy are: 1) ineffectiveness against coliform bacteria, which can cause a high proportion of IMI during the early dry period and near calving, and 2) ineffectiveness in preventing new IMI by a broad spectrum of mastitis pathogens during the period near calving when mammary glands are highly susceptible to new infection (Oliver, 1988a; 1988b; Oliver and Sordillo, 1988; 1989). Dry cow antibiotic preparations are formulated primarily to maintain persistent activity during the early dry period and most likely provide little protection during the late dry period. Oliver et al. (1990), using the Bacillus stearothermophilus disc assay to detect antibiotic residues, demonstrated that dry cow antibiotics persisted for only 14 to 28 days after infusion, and some persisted for shorter periods. Thus, based upon present methods of formulation, it would appear that antibiotic preparations currently available for use in dry cows will not control IMI that occur during the late dry period based on a dry period length of 6 to 8 weeks. Experimental evidence suggests that dry cow therapy is effective in controlling IMI due to Strep. agalactiae and somewhat effective against Staph. aureus. However, dry cow therapy appears to be less effective against streptococci other than Strep. agalactiae and ineffective against coliform bacteria (Smith et al., 1985a; 1985b). Differences in effectiveness of dry cow antibiotic therapy to prevent new IMI are most likely related to several factors. Strep. agalactiae and Staph. aureus are thought to be transmitted primarily during the milking process, and transmission can be controlled by hygiene and antibiotic therapy. The sources of these two organisms are infected mammary glands, colonized teat ducts, and teat lesions. Extramammary sources of contagious mastitis pathogens have been identified but appear to be relatively unimportant in the pathogenesis of infection. Thus, exposure of mammary glands to contagious pathogens during the dry period is reduced in the absence of regular milking and therapy at drying off tends to control these pathogens effectively. Research has also shown that mastitis in breeding age and pregnant heifers is much higher than previously thought. A review on this topic was published recently (Oliver et al.,

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2005a). Many IMI in heifers can persist for long periods of time, are associated with elevated SCC, and may impair mammary development during gestation and affect milk production after calving. Presence of mastitis before calving increased the risk of infection during lactation, increased the risk of clinical mastitis in the first week after calving, and increased the risk of further cases of mastitis and culling during the first 45d of lactation. In some studies, prepartum intramammary antibiotic infusion of heifer mammary glands was shown to be an effective procedure for eliminating many IMI in heifers during late gestation and for reducing the prevalence of mastitis in heifers both during early lactation and throughout lactation. Prepartum antibiotic-treated heifers also produced significantly more milk than control heifers and had significantly lower SCC scores than untreated control heifers. These observations are likely due to the lower prevalence of mastitis pathogen isolation in prepartum antibiotic-treated heifers throughout lactation (Oliver et al., 2005a). While much has been learned about mastitis in heifers, many issues remain unanswered such as: (1) identification of herds where this strategy would be most advantageous and cost effective, (2) should all heifers in the herd be treated or only certain heifers? (3) are there certain bacteria that are more problematic than others? and (4) identification of key risk factors that could have a significant impact on prevention of heifer mastitis so that antibiotic treatment could be minimized. Additional studies are needed to address these fundamentally important questions. Use of internal teat sealants is a relatively new concept and much of the early data came from studies conducted in New Zealand (Woolford et al., 1998). Results of those studies showed that internal teat sealants were effective in preventing new IMI during the dry period. A total of 528 cows in late lactation with SCC 200,000/ml suggests that an inflammatory response has been elicited, that a mammary quarter is infected or is recovering from an infection, and is a clear indication that milk has reduced manufacturing properties. It is not uncommon for milk from cows with subclinical and/or clinical mastitis to contain several hundred thousand and even millions of somatic cells/ml of milk. Thus, an increase in the SCC of milk is a good indicator of mastitis or inflammation in the udder. Infection of the udder by mastitis pathogens alters milk composition and reduces milk yield. Most studies that evaluated the influence of mastitis on the composition of milk used SCC as the basis for determining the infection status of udders and for determining the degree of inflammation. The bulk tank SCC (BTSCC) can be used to gauge the udder infection status of a dairy herd, and also gives a good indication of the loss in milk production in a herd due to mastitis. As the BTSCC increases, the percent of mammary quarters infected increases and the percent production loss increases. Small increases in SCC can impact production. Most herd milk contains between 200,000 to 500,000 somatic cells/ml of milk (Norman et al., 2011). These herds are losing at least 8% in potential milk production. Thus, methods of mastitis control that reduce SCC will not only improve milk yield and composition but will also decrease economic losses due to mastitis. A recent report published by the USDA Animal Improvement Program Laboratory (Norman et al., 2011) summarized SCC data from all herds in the United States enrolled in

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the Dairy Herd Improvement (DHI) testing program for 2010. The national SCC average for 2010 was 228,000 cells/ml of milk, which is 5,000 cells/ml lower than in 2009. However, the percentage of herd test days that exceeded 750,000, 600,000, 500,000, and 400,000 cells/ml during 2010 was 2.7, 5.7, 10.0, and 18.0, respectively, which was lower for 3 of the 4 levels than during 2009. The 2.7% of 2010 DHI herd test days that were higher than the present legal limit for bulk tank SCC may overestimate the percentage of herds that shipped milk exceeding the legal limit because milk from cows treated for mastitis is excluded from the bulk tank even though this information is included in DHI test data. The SCC of milk produced by dairy farms in the Southern Region of the U. S. over the last 10 years was about 35% higher than the U.S. average with a yearly range of ~30% higher in 2000 to almost 41% higher than the U. S. average in 2003. These data demonstrate quite clearly that there is much room for improving milk quality in the U. S., and this is particularly the case for milk produced on dairy farms in the Southeast. The standard plate count (SPC) is an estimate of the total number of viable aerobic bacteria in raw milk. This test is done by plating milk on a solid agar, incubating plates for 48 hours at 32°C followed by counting bacteria that grow on plates. The SPC is used to monitor progress since consistent application of proper milking system cleaning practices, proper milking practices, udder hygiene and good mastitis prevention and control practices should allow dairy producers to produce milk with a low SPC (< 5,000 colony forming units (cfu) of bacteria/ml). Federal regulations defined in the Pasteurized Milk Ordinance mandate that the milk SPC should not exceed 100,000 cfu/ml. However, most segments of the dairy industry feel that more stringent standards (SPC ≤ 10,000 cfu/ml) will result in higher quality milk. Though it is impossible to eliminate all sources of bacterial contamination of milk; milk from clean, healthy cows that has been properly collected generally has a SPC < 1,000 cfu/ml. Consistent application of proper milking practices, udder hygiene and good mastitis prevention and control practices should allow dairy producers to produce milk with a SPC of ≤ 5,000 cfu/ml, while most farms can produce milk with counts of < 10,000 cfu/ml. High bacterial counts (> 10,000 cfu/ml) suggest that bacteria are entering milk from a variety of possible sources (Gillespie et al., 2007; Gillespie et al., 2008; Jayarao et al., 2004). The most frequent cause of high SPC‘s is poor cleaning of milking systems. Milk residues on equipment surfaces provide nutrients for growth and multiplication of bacteria that contaminate milk of subsequent milkings. Cows with mastitis (streptococcal and coliforms), soiled cows, unclean milking practices, failure to cool milk rapidly to < 4.4C (40F), failure of the water heater, and extremely wet and humid weather can also contribute to high SPC‘s in raw milk. Some limitations of the SPC method include: 1) no indication of the bacterial types present, 2) no indication of the specific source of high counts, and 3) the SPC does not give a complete count of all bacteria as some bacteria only grow at lower temperatures. The preliminary incubation count (PI) count is an estimate of the number of pyschrotrophic (cold-loving) bacteria in milk. The PI count is not a regulatory test and results of this test are interpreted as a general reflection of milk production practices on the farm and are used as a tool to identify inadequate on-farm sanitation practices and holding temperatures of milk in the bulk tank. The PI count is conducted by holding milk at 13C (55F) for 18 hours. Bacteria that grow under refrigerated conditions are enumerated using the SPC method described above. PI counts are generally higher than SPC‘s. Selection of a PI count cut-off and interpretation of PI count results are difficult because variability in PI counts negatively influences repeatability (Boor et al., 1998; Murphy and Boor, 2000; Jayarao et al., 2004).

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Some milk plants use a specific cut-off number while others use PI counts in relation to SPC‘s. PI counts < 10,000 cfu/ml are considered low, while PI counts > 20,000 cfu/ml are considered high (Jayarao et al., 2004). A PI count 3 - 4 fold higher than the SPC is suggestive of potential problems related to cleaning and sanitation of the milking system or poor udder preparation before milking. Failure to cool milk rapidly, marginal cooling, prolonged storage times, milking cows with wet teats, and/or extremely wet and humid weather conditions may also result in high PI counts (Gillespie et al., 2007; Gillespie et al., 2008). A PI count equal or slightly higher than a high SPC (> 50,000 cfu /ml) may suggest that the high SPC is possibly due to mastitis. The PI count has been used by some as an indicator of the shelf-life of processed dairy products. However, research conducted at Cornell University (Boor et al., 1998; Murphy and Boor, 2000) and Penn State University (Jayarao et al., 2004) has shown that the PI count alone cannot be directly correlated with the flavor quality of raw milk OR quality OR shelf-life of processed dairy products. PI counts are most useful with data from other tests and additional information such as farm observations and inspections. The laboratory pasteurization count (LPC), also known as the thermoduric count, is an estimate of the number of bacteria that can survive laboratory pasteurization at 62.8C (143F) for 30 minutes. This process destroys most of the mastitis causing pathogens, selecting for those bacteria that can survive pasteurization temperatures (thermoduric bacteria). This is not a regulatory test required by state or federal agencies; however, some milk processors perform this test to ensure quality of the final product. Bacteria not killed by pasteurization are enumerated using the SPC method. LPC‘s are generally much lower than SPC‘s (Gillespie et al., 2007; Gillespie et al., 2008; Jayarao et al., 2004). An LPC of > 200 cfu/ml is considered high. A high LPC is most often seen with persistent cleaning problems; faulty milking machines or worn out parts such as leaky pumps, old pipe line gaskets, inflations and other rubber parts; and milkstone deposits. Significant contamination from soiled cows can also contribute to high LPC‘s. The coliform count (CC) is a test that estimates the number of bacteria that originate from manure or a contaminated environment. Milk samples are plated on Violet Red Bile Agar or MacConkey‘s agar and incubated for 48 hours at 32C (90F), after which typical coliform colonies are counted. Coliform counts reflect hygiene and sanitation practices followed on the farm. Coliforms enter the milk supply as a consequence of milking dirty cows or dropping the milking claw into manure during milking. Coliform counts > 100 cfu/ml suggest poor milking practices, dirty equipment, contaminated water, dirty milking facilities, and/or cows with subclinical or clinical coliform mastitis (Jayarao et al., 2004).

MILKING TIME HYGIENE AND GOOD HUSBANDRY/PRODUCTION PRACTICES Because of the large number of pathogens capable of causing mastitis and the fact that these pathogens behave quite differently, a one size fits all approach to mastitis management is not feasible. Since pathogenic bacteria gain entrance into the mammary gland through the teat canal, the greater the bacterial load at the teat end, the greater the probability of an infection occurring thus emphasizing the importance of maintaining a clean dry environment and udder hygiene at milking time. Any procedure that reduces the number of bacteria to

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which the teat end is exposed will likely be beneficial. Proper milking hygiene and good milking practices consist of the following elements: (1) milk in a clean stress-free environment, (2) check foremilk and udder for signs of clinical mastitis, (3) minimize use of water, (4) wash teats with a warm sanitizing solution, if necessary, (5) apply premilking teat disinfection, (6) dry teats thoroughly 30 to 45 seconds after premilking teat disinfectant application, (7) attach teat cups within one minute after cleaning, (8) provide stable vacuum at claw during peak milk flow, (9) avoid squawking or slipping of teat cup liners during milking, (10) adjust milking units as necessary, (11) shut off vacuum before removing machine, and (12) apply postmilking teat disinfectant shortly after milking machine removal (National Mastitis Council, 2006). Premilking teat disinfection has been adopted by several dairy producers and is intended to combat environmental pathogens that may have been transmitted to the teat at some point after the last milking. Studies have shown that premilking teat disinfection in combination with postmilking teat disinfection was more effective in preventing new infections than postmilking teat disinfection only. Premilking teat disinfection appears to be effective against environmental pathogens and may also influence contagious pathogens (Oliver et al., 1993; 1994). Dairy producers using this mastitis control procedure must make sure that the premilking teat disinfectant is removed from teats before milking to prevent contamination of milk. There are several good teat disinfectants on the market. However, when choosing a teat disinfectant, require the sales representative to provide evidence that the product is safe, effective and registered. Furthermore, make sure that manufacturer's recommendations are followed. Finally, do not assume that all postmilking teat disinfectants would be effective as a premilking teat disinfectant. The NMC publishes a summary of peer-reviewed publications on efficacy of premilking and postmilking teat disinfectants that is updated annually that provides information that may be useful to dairy advisors and producers when making decisions on teat disinfectants (National Mastitis Council, 2011). This information is available online at www.nmconline.org. Postmilking teat disinfection has been shown repeatedly to be an effective technique for preventing new IMI during lactation. This procedure destroys mastitis pathogens on teats after milking. In general, effective postmilking teat disinfectants reduce the rate of new infection by 50% or more when used in conjunction with other components of mastitis control. Postmilking teat disinfection has been adopted widely in major milk-producing countries worldwide as an essential component of mastitis control programs. However, postmilking teat disinfection is generally not as effective in preventing new IMI by environmental pathogens such as coliforms and Strep. uberis. This may be due to decreased germicidal activity in the period between milkings. For this reason, efforts have been made to examine premilking teat disinfection and to develop barrier-type teat dips to prevent new IMI by environmental pathogens during the intermilking interval. Barrier-type teat dips were developed with the goal of reducing exposure of teat ends to environmental pathogens during the intermilking period. Barrier dips are generally more viscous. However, their efficacy for prevention of environmental mastitis pathogens is equivocal. The incidence of new IMI actually increased with some barrier-type teat disinfectants when evaluated under conditions of experimental challenge with Strep. agalactiae and Staph. aureus (Nickerson and Boddie, 1995). Persistent barrier-type dips have also been used to prevent mastitis during the early dry period and near calving when cows are at high risk for new IMI (Timms, 2000). One problem has been persistence of the barrier on teat ends.

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VACCINES TO PREVENT MASTITIS Given today‘s public health and food safety concerns regarding antimicrobial resistance and antibiotic residues in dairy products associated with treatment of diseases like mastitis, approaches to enhance the cow‘s immunity to prevent disease and thus minimize use of antibiotics has gained considerable attention. Yet, for a variety of reasons, vaccines developed for the prevention and control of mastitis have achieved only limited success. The multiplicity of pathogens capable of causing mastitis, and knowledge of mammary gland immunology, bacterial virulence factors, and mechanisms of pathogenesis are factors that have hindered development of effective mastitis vaccines. However, some progress has been made in these areas in the last decade or so. Commercial mastitis vaccines are currently available in the United States for immunization against mastitis caused by Staph. aureus and E. coli. There are two Staph. aureus bacterins, essentially the same product, marketed as Somato-Staph® and Lysigin® which are labeled as somatic antigen containing phage types I, II, III, IV and miscellaneous groups of Staph. aureus (Ruegg, 2001). There are three common coliform mastitis vaccines and two of them are identical, i.e., J-5 Bacterin and Mastiguard™, and contain coliform bacterins; a third, J Vac®, is a bacterin-toxoid. Another gram-negative mastitis vaccine, Endovac-Bovi®, contains re-17 mutant Salmonella Typhimurium bacterin toxoid. All coliform mastitis vaccine formulations use gram-negative bacterial core antigens to produce non-specific immunity directed against endotoxic disease (Ruegg, 2001). According to a recently published survey, J VAC is the most preferred E. coli/coliform mastitis vaccine among U.S. dairy producers (Holsteinworld, Available online). A recent review of the literature to assess the efficacy of vaccines for bovine mastitis caused by Staph. aureus suggested that vaccines that employ new technologies (DNA and/or recombinant protein vaccines) and some long-standing bacterins have achieved good results, and this supports their continued use in the prevention and control of Staph. aureus bovine mastitis (Pereira et al., 2010). In another review on vaccines against bovine mastitis in New Zealand, Denis et al. (2009) argued that novel approaches must be considered to search for vaccine candidates, and vaccines need to be designed and constructed within the special framework of their uses, in the mammary gland, which offers a unique immunological environment. In addition, effective vaccines against mastitis due to Streptococcus uberis may be more likely to emerge from strategies that target the cell-mediated arm of the immune response rather than strategies that target specific antibody responses.

Mastitis Caused by Gram-Positive Pathogens Most of the early vaccine research focused on Staph. aureus vaccines based on bacterins derived from in vitro grown bacteria. With increased knowledge of bacterial virulence factors, different approaches to vaccine formulation have been attempted. Watson et al. (1996) developed a Staph. aureus mastitis vaccine consisting of killed bacteria bearing pseudocapsule and toxoid exotoxins. The vaccine was efficacious in reducing the incidence of clinical mastitis and prevalence of subclinical staphylococcal mastitis. Nordhaug et al. (1994) tested a vaccine containing whole-inactivated Staph. aureus with pseudocapsule, and α- and ß-toxoids in heifers. There was a potential protective effect on general udder health during the

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entire first lactation period. Nickerson et al. (2000) suggested a positive effect of vaccination with a polyvalent Staph. aureus vaccine by increasing antistaphylococcal antibody titers in preventing new Staph. aureus infections when the program was initiated at an early age in heifers from a herd with a high exposure to Staph. aureus. More recently, Nickerson et al. (2008) reported that the percentage of heifers with Staph. aureus IMI at calving was significantly lower in heifers vaccinated with a commercially available vaccine containing a lysed culture of polyvalent Staph. aureus somatic antigens containing 5 phage types than in unvaccinated heifers. Somatic cell counts were also lower in vaccinated heifers during the first week of lactation. Guidry et al. (1998) randomly sampled the national herd and found three Staph. aureus capsule serotypes were responsible for 100% of bovine Staph. aureus mastitis in the U.S. They formulated a vaccine, using the 3 serotypes, and tested its ability to cure chronic Staph. aureus infections. In preliminary field trials, the trivalent Staph. aureus vaccine with antibiotics was as effective as the autogenous vaccine with antibiotics for curing chronic Staph. aureus infections (Sears et al., 2000). This would allow for treatment of cows chronically infected with Staph. aureus without the necessity of preparing a herd-specific vaccine. Further testing was suggested to determine the effect of duration of infection on cure rate. Middleton et al. (2006) compared the efficacy of two experimental Staph. aureus mastitis bacterins and Lysigin with unvaccinated controls. Cattle vaccinated with Lysigin had a lower mean duration of clinical mastitis and lower total mastitis score post-challenge than controls. None of the vaccinated groups had lower mean somatic cell counts (SCC) than controls. There were no differences in milk yield between vaccinates and controls post-challenge. Although there was no evidence the vaccines reliably prevented Staph. aureus IMI, Lysigin reduced clinical severity and duration of disease post-challenge and was superior to the other bacterins. Denis et al. (2009) concluded there are currently no effective vaccines that have been designed to prevent or mitigate IMI. Additionally, novel approaches must be considered to search for vaccine candidates, and vaccines need to be designed and constructed within the special framework of their uses in the mammary gland which offers a unique immunological environment. Furthermore, effective vaccines against mastitis due to Streptococcus uberis may be more likely to emerge from strategies that target the cell-mediated arm of the immune response than strategies that target specific antibody responses. Since few studies had evaluated vaccination of heifers against staphylococcal mastitis, Middleton et al. (2009) evaluated the efficacy of a commercially available Staph. aureus bacterin in protecting against staphylococcal IMI Staph. aureus and CNS. The effect of vaccination on milk SCC, and antibody isotype response to vaccination was investigated. No animals in vaccinated and control groups developed new Staph. aureus IMI after vaccination. The numbers of mammary quarters that developed a new CNS IMI, time to new CNS IMI, milk somatic cell count, and milk antibody isotype sample-to-positive ratio did not significantly differ between groups (P>0.05). The vaccine did not reduce the new staphylococcal IMI rate. There may have been insufficient vaccine-induced opsonizing antibody in milk to facilitate phagocytosis and clearance of staphylococci from the mammary gland. A vaccine composed of three field isolates of Staph. aureus derived from cases of mastitis in cows was developed (Leitner et al., 2003a). All cows were challenged with a

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S. P. Oliver and S. E. Murinda

highly virulent Staph. aureus strain administered into two quarters. No systemic effects were observed in any of the vaccinated or control cows. Vaccinated cows had 70% protection from infection compared with fewer than 10% in the controls. Moreover, all quarters challenged in the vaccinated cows, regardless of whether they were successfully infected or not with Staph. aureus, exhibited very mild inflammatory reactions, identified by their low SCC (104 per g feces, as has been documented for Salmonella, E. coli O157:H7 and Campylobacter (Atterbury et al. 2005; Atterbury et al. 2007; Hagens and Offerhaus 2008). A final advantage of phage biocontrol is that development of phage treatments is inexpensive relative to production of a new antibiotic (Hanlon 2007; Owens et al. 2007), although studies are required to document efficacy and ensure the safety of phage therapy (Sulakvelidze and Pasternack 2010).

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3. REGULATORY ENVIRONMENT FOR PHAGE BIOCONTROL - CONCERNS AND SOLUTIONS Even though phages are virtually ubiquitous in the environment, each new application of phages for biocontrol of pathogenic bacteria must meet a few key criteria before use in the pre-harvest control of bacteria in livestock. As the Shiga toxins found in Escherichia coli O157 and the cholera toxin in Vibrio cholerae are thought to have been acquired though viral transduction (Tobe et al. 2006, Hanlon 2007), temperate/lysogenic phages are not recommended for use in phage biocontrol. Use of lysogenic phages may facilitate transfer of genes encoding virulence factors and lead to development of new strains of pathogenic bacteria (Saunders et al. 2001), an outcome not in the interest of food safety. In contrast, virulent/lytic phages are unlikely to transfer genes as the host undergoes lysis and fails to multiply (Waddell et al. 2009). Consequently, it is necessary to screen phage candidates for biocontrol of pathogenic bacteria to ensure selection of strictly lytic phages. Plaque morphology may provide preliminary information to aid in distinguishing lytic from lysogenic phages. Plaques from lysogenic phages may have turbid centres compared to clear plaques from lytic phages (Ceyssens and Lavinge 2010). However, alterations to phage/host environment may also affect plaque morphology (Hanlon 2007) and a number of generally lytic phages have demonstrated transduction upon exposure to high temperature within a stationary-phase host (Waddell et al. 2009). Consequently, the most effective means of avoiding lysogenic phages and phages with other undesirable characteristics such as bacterial toxins or antibiotic resistance genes would be through DNA sequencing and subsequent bioinformatics analyses of candidate phages (Deresinski 2009; Sulakvelidze and Pasternack 2010). Use of non-pathogenic alternate host bacteria for phage propagation is also advised (Bielke et al. 2007) along with effective purification of phages to remove endotoxins (Boratynski et al. 2004). As additional phage products become registered for use, the steps for approval of their use in pathogen control will likely become more streamlined, as evidenced by the generally regarded as safe (GRAS) status awarded to a phage propagated in nonpathogenic Listeria targeted at Listeria monocytogenes (Center for Food Safety and Applied Nutrition 2007). Very few adverse reactions to phage therapy in human subjects have been reported (Matsuzaki et al. 2005, Hanlon 2007, Abedon 2009), although documentation is limited mainly to 20th century studies undertaken in Eastern Europe (Barrow 2001, Hanlon 2007). Precautions to ensure safety of phages selected for pathogen biocontrol are clearly warranted, but due to the expanding number of phage preparations currently commercially available (Sulakvelidze and Pasternack 2010) the procedures to obtain approval are by no means insurmountable.

4. FACTORS AFFECTING THE SUCCESS OF PHAGE BIOCONTROL Although complete eradication of pathogenic zoonotic bacteria would be ideal, the predator-prey dynamics of phages and their hosts makes such an achievement unlikely (Payne and Jansen 2001; Abedon 2009). In pre-harvest food safety, a reduction in bacterial populations to a point that ensures the integrity of carcass hygiene procedures (Elder et al.

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2000) is a more realistic goal for phage biocontrol. Considering this as the optimum outcome, a number of factors may potentially affect the success of phage biocontrol.

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A. Replication Threshold and Physical or Biological Barriers Given the biology of phages, one would assume that the biocontrol of pathogenic bacteria should be easily assured, but the ease of biocontrol in vitro is often not directly transferable to in vivo condition owing to a number of challenges (Loc Carrillo et al. 2005). One factor which may limit the success of phage biocontrol in vivo is a replication threshold, defined as the minimal population of phages and/or host required for effective biocontrol (Owens et al. 2007). Wiggins and Alexander (1985) demonstrated a replication threshold of 104 host cells per mL, but existence of a replication threshold has been disputed (Kasman et al. 2002; Cairns and Payne 2008), likely due to the diversity of phage biocontrol applications or differences among in vitro and in vivo studies. Wilkinson (2001) proposed a decoy theory, whereby phage biocontrol loses efficacy if there are an overwhelming number of non-target bacteria or numerous particles which may impair adsorption of phage to the targeted host. As the ruminant digestive tract contains billions of bacteria per mL of rumen fluid (Fonty et al. 1989) and rafts of undigested fiber (Holden et al. 1994), the structural complexity and rich microflora in that environment may increase the difficulty of successful oral phage biocontrol in ruminants. Abedon (2009) suggested that a phage multiplicity of infection of 10, which equals 1 bacterium out of 20,000 left untargeted by phage, would result in effective biocontrol. However, phage survival in vivo is difficult to predict (Hurley et al. 2008). The acidic environment of the gastrointestinal tract (GIT) may substantially lower phage infectivity (Johnson et al. 2008; Atterbury 2009). When phages are orally administered to monogastrics (Loc Carrillo et al. 2005; El-Shibiny et al. 2009) or young pre-ruminants (Waddell et al. 2000), buffers are often included as a drench to reduce the potentially detrimental impact of acid on phage activity. Encapsulation has also been used to protect phages from acidic intestinal contents in an attempt to deliver active phages to intestinal sites colonized by pathogenic bacteria (Ma et al. 2008; Stanford et al. 2010). Encapsulation requires a delicate balance between over-protection, where encapsulated phages are unable to access target bacteria, and under-protection where phages are exposed to inactivating environmental conditions before contacting the target bacteria. Development of genetic resistance by bacteria to phages is another important factor in influencing the success of phage biocontrol. Atterbury et al. (2007) obtained a > 4 log reduction in Salmonella Enteriditis and S. Typhimurium, but after 72 hours the chicken GIT became re-colonized with phage-resistant mutants. Similarly, Rozema et al. (2009) detected increased resistance to phages over time in E. coli O157:H7 as phage-resistant isolates showed altered pulse field gel electrophoresis profiles as compared to isolates that were originally inoculated into cattle (Figure 2). Consequently, development of bacterial resistance to phages may restrict the window for optimum phage biocontrol and should always be considered in biocontrol strategies. Another factor which would influence phage survival in vivo would be development of host antibodies against phages. Huff et al. (2010) recently demonstrated that prior exposure of chickens to phages targeted at E. coli limited phage efficacy due to a host immune response.

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Single-time dosing with phages may help to limit the development of antibodies in the host. Huff et al. (2010) used repeated intravenous injections of phages, which may trigger a more rapid antibody response than repeated oral dosing (Atterbury 2009). Additional studies are required to determine to what extent immune responses to phages may influence their efficacy at biocontrol.

Figure 2. Changes in pulse field gel electrophoresis profiles in E. coli O157:H7 after resistance developed to phages. Lanes 1 through 5 are E. coli O157:H7 strains inoculated in cattle. Lanes 6 and 7 are phage resistant strains of E. coli O157:H7. Lanes M are molecular weight markers ranging from 48.5 to 485 kb.(Rozema et al. 2009).

Establishment of bacterial biofilms may impact efficacy of phage biocontrol as biofilms often include extracellular polymeric substances which shield binding sites on the bacterial cell surface from phages (Hanlon 2007). Environmental biofilms generally contain multiple bacterial species which may also limit the effectiveness of phages for biocontrol (Hughes et al. 1998), although Hanlon et al. (2001) has shown promising results in using phage enzymes to disrupt mature biofilms.

B. Method of Phage Administration Each method of phage administration has its own specific challenges. Intra-muscular or intravenous injection avoids exposing phages to the acidic environment of the GIT, but results in their rapid elimination by phagocytosis in the liver even without eliciting an increase in antibody production (Hanlon 2007; Atterbury 2009). Rapid phage evolution provides a solution to this problem as Merrill et al. (1996) were able to successfully select for mutant phages which were at least 13,000 times more resistant to elimination in the liver than parental strains.

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Administration of phages by topical sprays requires balancing water content with expected environmental desiccation as phage efficacy is highest in liquid media where phage particles can freely diffuse within the environment (Gunether et al. 2009). As well, a large and uneven surface area may limit contact between phages and target bacteria. Topical sprays have been successfully used to control pathogens in fruits and vegetables (Leverentz et al. 2003; Sharma et al. 2009), but to date, few studies have demonstrated their efficacy in preharvest livestock (Borie et al. 2009). Topical control of pathogens in complex environments such as manure may prove challenging if contact of the phage with the targeted host can not be assured. Oral administration of phages makes them susceptible to inactivation by the acidity of the GIT and the abundance of non-target bacteria within this environment may further limit their effectiveness (Wilkinson 2001). Compared to nasal spray or anal bolus, oral use of phages would better control dosage as loss of phages due to sneezing or defecation would be avoided. Administration of phages within feed (Stanford et al. 2010) remains an attractive option as requirements for animal handling immediately prior to slaughter may limit adoption of more invasive phage biocontrol strategies.

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C. Competitive Interference and Endogenous Phage Studies from our laboratory where sheep or cattle were inoculated with E. coli O157:H7, have resulted in an increase in the isolation of endogenous phages (Rozema et al. 2009; Stanford et al. 2010) which upon examination with electron microscopy were morphologically different from orally-administered experimental phages. These endogenous phage were presumptively identified as members of the Siphoviridae family (Figure 3). After populations of E. coli O157 declined below 103 CFU/g of feces, these endogenous phages were no longer isolated from the feces of inoculated cattle (Stanford et al. 2010). As these phage were not detected prior to inoculation with E. coli O157:H7, it seems likely that an abundance of the host resulted in the proliferation of these endogenous phages. Others have also isolated a variety of phages with activity against E. coli O157:H7 from beef feedlots (Oot et al. 2007; Niu et al. 2009) and against Salmonella in poultry house environments (Connerton et al. 2004; Higgins et al. 2008). Recently, we have characterized a T5-like phage with activity against E. coli O157:H7 (Niu et al., 2010; Figure 4). Host range and microplate phage virulence assays showed this phage exhibited high virulence against 30 common phage types of E. coli O157:H7 reference strains and 27 E. coli O157:H7 bovine isolates endemic to the feedlot pen from which the phage originated. This T5-like phage is also highly specific for E. coli O157:H7 with no lytic activity against the 72 strain E. coli reference collection established by Ochman and Selander (1984). Studies examining the interactions among different types of phages which share a common host have not been conducted, although it has been proposed that endogenous phages negatively influence the efficacy of experimentally inoculated phages (Rozema et al. 2009; Stanford et al. 2010). Callaway et al. (2008) described the phenomenon of competitive interference, where adsorption of one phage can inhibit adsorption of other phages. Similarly, presence of prophages in bacterial cells can direct the synthesis of repressor proteins that block the transcription of its own genes and those of closely related phages, conferring a form of immunity to the bacterial cell to infection with other phages (Hanlon 2007).

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Figure 3. Duplicate bacteriophage plates of the same dilution showing A. small plaques of experimental phages and B. large plaques of endogenous phage. C. transmission electron micrographs of experimental phage wV7 and D. endogenous (large-plaque) phage. (Rozema et al. 2009).

Figure 4. Negatively stained phage vB_EcosS_AKFV_33. Phosphostungstate; the scale bar represents 200nm. By Hans-W Ackermann, Department of Microbiology, Faculty of Medicine, Laval University, Quebec. On-Farm Strategies to Control Foodborne Pathogens, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook Central,

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D. Dosage Rates for Effective Phage Biocontrol Determination of appropriate dose rates for phage biocontrol is not a simple task as competitive interference, development of phage-resistant mutants, antibodies against phage or other environmental factors can lead to phage inactivation thereby reducing the effective dose. Phage proliferation thresholds and the time allotted for phage therapy as a means of pathogen control prior to slaughter of the livestock are also factors that must be simultaneously considered. Due to the complexity of these factors, there is no substitute for carefully planned and executed in vivo efficacy studies to determine appropriate dosage rates for pre-harvest phage biocontrol.

5. PAST IN VIVO PHAGE BIOCONTROL STUDIES AND BACTERIAL PATHOGENS TARGETED

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A. Chickens Compared to other livestock, use of phages for biocontrol of pathogenic bacteria is most advanced in poultry (Table 1). The two enteric pathogens of greatest concern for pre-harvest control in poultry are Salmonella spp. and Campylobacter spp. (Cox and Pavic 2010), although the utility of phages for controlling Salmonella in vivo has been inconsistent. Hurley et al. (2008) orally dosed newly hatched chicks with 108 CFU Salmonella enterica serotype Typhimurium followed by oral inoculation with 106 plaque-forming units (PFU) of phage SP6 on days 2, 3 and 28, or a single dose of phage SP6 on day 28. Phage treatment did not reduce fecal shedding of Salmonella. Accordingly, Fiorentin et al. (2005) did not significantly reduce caecal S. enterica counts in 7-day-old broilers treated with a 1011 PFU mixture of 3 phages. Higgins et al. (2008) isolated seven Salmonella-lytic phages from a commercial broiler house, but concluded that the phages had little or no effect on the presence of Salmonella within that environment. Some of these studies showed reduced phage efficacy in vivo after promising in vitro results. In contrast, Borie et al. (2009) treated 6-day-old chicks with an aerosol body spray containing a total of 103 PFU of a mixture of 3 phages 1 day prior to oral inoculation with 105 CFU of S. enterica. These researchers were able to significantly reduce fecal S. enterica by approximately 2-log CFU after 8 days of phage treatment. Similarly, Atterbury et al. (2007) were able to significantly reduce S. Enteritidis and S. Typhimurium in cecal contents by oral treatment with a high concentration (1011 PFU) of one phage per serotype, although S. Hadar was not affected by phage treatment. Of all Salmonella biocontrol studies, that of Atterbury et al. (2007) is the closest to mirroring commercial poultry production conditions as phage efficacy was evaluated using broilers at slaughter age (40 to 42 days) in the UK. The successful biocontrol of several Salmonella enterica serotypes by Atterbury et al. (2007) is encouraging, but in order to ensure effective phage biocontrol of Salmonella in poultry, additional studies will be required to further refine application methods and optimize time of dosing. Commercial phage products targeted at Salmonella in poultry are limited, but would include Salmo-Pro™ released in 2008 by Biophage Pharma of Montreal for control of

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Salmonella DT108 (Sulakvelidze and Pasternack 2010). To date, studies on the efficacy of Salmo-Pro™ have yet to be reported in the literature. Table 1. Summary of in vivo poultry pre-harvest phage biocontrol studies Target Pathogen

Phage dosage (PFU) 1011

# phage doses 1

Method applied

Phages used

Outcome – reduction in target organism

Reference

oral

No impact

Fiorentin et al. 2005

S. Enteritidis S. Typhimurium S. Hadar S. Typhimurium S. Enteritidis

1011

1

oral

Mix of CNPSA1, CNPSA3, CNPSA4 Φ 151 Φ 10 Φ 25

4.2 log in 24 h 2.2 log in 24 h No impact

Atterbury et al. 2007

106

1, 2

oral

SP6

No impact

108

2

aerosol

2 log in 8 d

C. jejuni

105-107

1

oral

C. jejuni

10101011

10, 6

oral

Mix of BP1, BP2, BP3 Mix of CP8, CP34 71

C. jejuni C. coli

107 109

4 1

oral

Mix of 69, 71 CP220

1.5 log in 24 h 2 log in 48 h

Hurley et al. 2008 Borie et al. 2009 Loc Carillo et al. 2005 Wagenaar et al. 2005

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S. enterica

2 log in 24-48 h 3 log in 24-28 h

El-Shibiny et al. 2009

In contrast to results with Salmonella, phage biocontrol for Campylobacter in poultry has been more consistent. Phages targeted at Campylobacter jejuni were first isolated from broiler chickens (Connerton et al. 2004). Loc Carrillo et al. (2005) then determined that a dose of 109 PFU was less effective than one of 5 or 7 –log PFU and achieved a 2 log reduction in C. jejuni 24 h after treatment. As a 2 log-reduction in Campylobacter on retail chicken carcasses could potentially reduce the incidence of Campylobacter-mediated enteric infection by up to 30 times (Rosenquist et al. 2003), such an outcome may make this a viable approach for reducing food-borne disease. More recently, El-Shibiny et al. (2009) further refined phage therapy and achieved a 2-log reduction in cecal C. jejuni, after a single 7-log PFU dose of phage CP220, with a similar reduction in Campylobacter coli after a 9-log PFU dose of CP220. Timing of phage application for biocontrol of Campylobacter in poultry may be critical as decline in the target organism after treatment with phages is rapid and possibly transitory. Accordingly, Wagenaar et al. (2005) produced an initial 3-log decline in C. jejuni counts which stabilized at a 1-log decline after 5 days of phage biocontrol. Although biocontrol of Campylobacter in poultry appears promising, commercial phage products that target this pathogen have yet to be released.

B. Pigs For pigs, a wide variety of zoonotic organisms have been investigated for pre-harvest biocontrol, although in vivo studies are limited. O‘Flynn et al. (2006) isolated a promising phage for oral biocontrol of S. enterica in pigs, but in vitro results still require confirmation in

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vivo. Lee and Harris (2001) reduced S. Typhimurium in the tonsils and ceca of pigs 6 h after treatment with 109 PFU of phage Felix-O1, although S. Typhimurium remained at high levels in the rectal contents. Calvo et al. (1981) isolated phages for control of Yersinia enterocolitica from feces of swine, but this work was not pursued for biocontrol, possibly as some of the isolated phages were of a temperate nature. Kaszubkiewicz et al. (1982) reported increased survival of piglets treated with oral doses of phages as compared to antibiotics (95.6 vs. 90.4%) during an enterohemorrhagic E. coli outbreak, although E. coli strains with resistance to phage were isolated. Similarly, Smith and Huggins (1983) found increased survival of piglets treated with a two-phage cocktail at the onset of diarrhoea after challenge with E. coli strain O9:K30.99. Multiple phages have been isolated that target E. coli O149 in pigs (Jamalludeen et al. 2007), although treatment of pigs with these phages did not cause a significant reduction in shedding of this organism (Johnson et al. 2008). Most recently, Mazarheri et al. (2010) isolated phages targeting Enterococcus faecalis, Enterococcus faecium and Enterococcus gallinarum from piggery waste, but further in vivo evaluations of efficacy for these phages are required. In summary, despite the abundance of organisms considered, additional studies will be required to ensure efficacy of phage biocontrol in pigs. Likely some of these studies have been performed, but may remain proprietary information. Biophage Pharma of Montreal is marketing Coli-Pro™ for control of E. coli diarrhoea in piglets (Sulakvelidze and Pasternack 2010), but the in vivo efficacy of this product has yet to be reported.

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C. Cattle and Sheep In contrast to studies with pigs where multiple zoonotic pathogens have been considered, phage biocontrol in ruminants has focused on E. coli. The earliest phage studies concerned control of E. coli diarrhoea in pre-ruminant calves (Smith and Huggins et al. 1983; Smith et al. 1987), but subsequent ruminant phage biocontrol studies have predominantly attempted to control E. coli O157 (Table 2). As with phage biocontrol for other pathogenic organisms in other farm animal species, in vitro studies have been largely positive, while in vivo results have been more mixed. Sheng et al. (2006) were able to significantly reduce fecal shedding of E. coli O157:H7 in steers treated with 106 PFU/mL of a 2-phage cocktail in drinking water and 102 PFU at the rectal-anal junction. An interesting feature of this study was that the E. coli O157:H7 (1010 CFU) was also administered rectally. These authors also tested the same phages in sheep with oral dosing of both phages (1011 PFU) and E. coli O157:H7 (1010 PFU) and in contrast to the steer study carriage of E. coli O157:H7 was unaffected in sheep. Oral inoculation of a single phage into sheep challenged with E. coli O157:H7 also failed to reduce the shedding of this pathogen (Bach et al. 2003). Oral dosing of sheep with E. coli O157:H7 likely increased its distribution along the GIT, possibly reducing its contact with phages, whereas rectal administration of both phages and E. coli O157:H7 increased the likelihood of phage-host contact. The rectal-anal junction had been proposed as the principal site of E. coli O157:H7 colonization in ruminants (Naylor et al. 2003), but Keen et al. (2010) recently determined that E. coli O157 was distributed along the entire GIT in cattle with variable inter-animal

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distribution. Consequently, phage biocontrol in the ruminant GIT will likely not succeed if solely targeted at E. coli O157:H7 at the rectal-anal junction. Table 2. Summary of in vivo ruminant E. coli O157 phage biocontrol studies Livestock species

# phage doses

Method applied

Phages used

1 1 Multiple 1

oral oral oral + rectal oral

Sheep

Phage dosage (PFU) 105 1011 106 + 102 1011 109

Reference

DC22 CEV1 SH1, KH1 KH1

Outcome – reduction in target organism No impact 2 log in 2 d 1.5 log in 24 h No impact

2

oral

EDL 933

1 log in 24 h

5

oral

1011

5

oral rectal oral + rectal

No impact on population, fewer positive fecal samples No impact

Cattle

1011

3

oral

Cattle

109

5

oral

Mixture rV5 wV8 wV11 Mixture rV5 wv7 wV8 wV11 Mixture e11/2 e4/1c Mixture encapsulated rV5 wv7 wV8 wV11

Callaway et al. 2008 Bach et al. 2009

Sheep

1010

Cattle

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Sheep Sheep Cattle Sheep

Bach et al. 2003 Raya et al. 2006 Sheng et al. 2006

Rozema et al. 2009

No impact

Rivas et al. 2010

No impact

Stanford et al. 2010

Multiple applications of 1010 PFU of a 3-phage cocktail significantly reduced proportion of fecal samples positive for E. coli O157:H7 in sheep inoculated with 109 CFU (Bach et al. 2009). However, multiple oral, rectal or oral + rectal applications (1011 PFU) of the same 3 phages along with 1 additional phage did not reduce fecal shedding in cattle orally inoculated with 1010 CFU E. coli O157:H7 (Rozema et al. 2009). Possibly, the lack of efficacy in both the study of Rozema et al. (2009) and a recent study by Rivas et al. (2010) may be attributed to transference of experimental phages to control cattle. Experimental designs of Rozema et al. (2009) and Rivas et al. (2010) were similar as both used multiple oral doses of a phage cocktail (1011 PFU) and shedding of E. coli O157:H7 was not affected in steers inoculated with 1010 CFU of this bacterium. Stanford et al. (2010) encapsulated the phages used by Rozema et al. (2009) to protect them from acid inactivation (Smith et al. 1987), but similar to Rozema et al. (2009) found no reduction in the shedding of E. coli O157:H7 (oral inoculation 1011 CFU) after multiple oral treatments of phage (1010 to 1011 PFU). As Raya et al. (2006) were able to significantly reduce E. coli O157:H7 in sheep inoculated with 1010 CFU after a single oral dose of phages, repeated phage treatments used in other E. coli O157:H7 biocontrol studies might have led to an immune response that limited phage efficacy (Atterbury 2009). However, Callaway et al. (2008) produced a 1-log reduction in E. coli O157:H7 in sheep after two oral inoculations

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with phages. Due to only intermittently positive results for phage biocontrol of E. coli O157:H7 in the ruminant GIT, the call by Rivas et al. (2010) for additional in vivo studies would seem well-advised. Currently the only commercial product for phage biocontrol of E. coli O157:H7 is Finalyse®, a hide-wash applied to cattle a minimum of 1 h prior to slaughter which was developed by OnmiLytics Inc. of Salt Lake City, Utah (Sulakvelidze and Pasternack 2010). A surface spray would likely function through ―l yses from without‖, possibly avoiding some of the complications associated with oral administration of phage. As with other pre-harvest phage biocontrol products efficacy data remain proprietary and third-party validation will be necessary. A key test of this technology would be the ability of phages applied in a surface spray to contact target organisms which are potentially embedded in solid feces on the hide.

6. FUTURE DIRECTIONS A. Multivalent Phages

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The ideal phage would be able to effectively control multiple strains or possibly multiple species of pathogenic bacteria perhaps by recognizing a commonality in binding sites or by genetic manipulation of the host-binding profile (Hanlon 2007). With such a broad host range, extreme care would have to be taken to ensure that the phages did not impact nontarget bacteria as well as give consideration to the precautions outlined in Section 3. Recently, Santos et al. (2010) have isolated a phage which is able to lyse almost all Salmonella subtypes except IIIa. As well, a phage capable of controlling multiple enterohemorrhagic serotypes of E. coli would be a welcome advance. Studies to determine if a broad host-specifity is retained after pathogenic bacteria are repeatedly exposed to phage have not been conducted.

B. Phages in Aquaculture Although discussion of pre-harvest use of phages has been limited to poultry, pigs and ruminants, future expansion of phage applications in aquaculture seems likely. Most of the antibiotics formerly used for aquaculture have been restricted resulting in the use of elaborate and expensive water treatment systems including sand filters, cartridge filters and UV light (Karunasagar et al. 2007). Work with phage biocontrol in aquaculture is as yet limited, although dispersion and efficacy of phages is heightened in liquids (Gunether et al. 2009). Such an approach could have the added attraction of reducing environmental loads of the pathogen concurrently with loads in the fish (Morrison and Rainnie 2004). Karunasagar et al. (2007) isolated phages which reduced Vibrio harveyi in both larval shrimp and in biofilms formed on high-density polyethylene surfaces. In contrast, VernerJeffreys et al. (2007) were not able to control Aeromonas salmonicida by intra-peritoneal injection of a 3-phage mixture into salmon and phage-resistant isolates of this organism were recovered. Phage therapy will likely have the greatest application in commercial aquaculture facilities as dispersion of phages from the targeted host would likely limit their usefulness in aquaculture cages within the ocean.

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C. Phages for the Environment After isolation of phages with demonstrated efficacy in controlling livestock pathogens, the logical next step is to investigate the ability of this approach to reduce the transmission of pathogens through the environment. One of the earliest animal studies (Smith and Huggins 1983) demonstrated biocontrol of E. coli by moving naïve calves onto bedding used by a phage-treated cohort. Similarly, Niu et al. (2009) demonstrated significant reductions of E. coli O157 in pens of feedlot cattle where phages were also isolated. In contrast, Connerton et al. (2004) found C. jejuni to be insensitive to resident phages in three poultry houses, emphasizing that selection of suitable phages is key to the success of biocontrol strategies. Ideally, treating a barn or pen with phages targeted to key pathogenic bacteria could limit colonization of livestock by reducing environmental transmission of the pathogen. Stephens et al. (2008) found pens of naïve cattle were not colonized with E. coli O157 after repeated introduction of 102 CFU of the organism to the pen floor. In contrast, Stanford et al. (2011) found between 50 and 75% of naïve steers were colonized when 106 CFU of E. coli O157 were introduced onto pen floors. As phages have a higher likelihood of successful adsorption to host when host levels are high (Payne and Jansen 2001), environmental application of phages may have greatest impact when environmental contamination with pathogenic organisms is also high. As well, contamination of the environment with E. coli O157 and Salmonella spp. originating from livestock has also led to human disease via the adulteration of fresh fruit and vegetables (Berger et al. 2010). Phage biocontrol may offer a solution to fruit and vegetable contamination either by application of phages to irrigation water or direct treatment of fruit or vegetable products.

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D. Toward the Goal of Consistently Effective Phage Biocontrol The inconsistency noted in some studies of phages for pre-harvest biocontrol of zoonotic pathogens is not surprising given the numerous factors which concurrently impact efficacy. To date, more consistent efficacy has been noted for control of Campylobacter in poultry than for either Salmonella or E. coli O157 in other livestock species. Biocontrol of E. coli O157:H7 in the ruminant GIT is particularly difficult due to the resident rich microflora, structural complexities and presence of feed particles. Likely many phage biocontrol efficacy issues will be resolved by selection of improved candidate phages. Other concerns such as antibodies against phages, competitive interference among phages and timing, frequency and concentration of phage doses will require future careful in vivo studies for optimization of phage biocontrol.

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Atterbury, R.J., Van Bergen, M.A., Ortiz, F., Lovell, M.A., Harris, J.A., De Boer A, Wagenaar, J.A., Allen V.M. and Barrow, P.A., 2007. Bacteriophage therapy to reduced Salmonella colonization of broiler chickens. Appl. Environ. Microbiol. 73: 4543-4549. Atterbury, R.J., Dillon, E., Swift, C., Connerton, P.L., Frost, J.A., Dodd, C.E.R., Rees, C.E.D. and Connerton, I.F. 2005. Correlation of Campylobacter bacteriophage with reduced presence of hosts in broiler chicken ceca. Appl. Environ. Microbiol. 71: 4885-4887. Bach, S., Johnson, R.P., Stanford, K. and McAllister, T.A. 2009. Bacteriophage reduce Escherichia coli O157:H7 levels in experimentally inoculated sheep. Can J. Anim. Sci. 89: 285-293. Bach, S.J., McAllister, T.A., Veira, D.M., Gannon, V.P.J. and Holley, R.A. 2003. Effect of bacteriophage DC22 on Escherichia coli O157:H7 in an artificial rumen system (Rustitec) and inoculated sheep. Anim. Res. 52: 89-101. Barrow, P.A. 2001. The use of bacteriophages for treatment and prevention of bacterial disease in animals and animal models of human infection. J. Chem. Tech. Biotech. 76: 577-682. Berger, C.N., Sodha, S.V., Shaw, R.K., Griffin, P.M., Pink, D., Hand, P. and Frankel, G. 2010. Fresh fruit and vegetables as vehicles for the transmission of human pathogens. Environ. Micro. 12: 2385-2397. Bielke, L.R., Higgins, S.E., Donoghue, A.M., Kral. T., Donoghue, D.J., Hargis, B.M. and Tellez, G. 2007. Evaluation of alternative host bacteria as vehicles for oral administration of bacteriophages. Int. J. Poultry Sci. 6: 758-761. Biswas, B., Adhya, S., Washart, P., Paul, B., Trostel, A.N., Powell, B., Carlton, R. and Merril, C.R. 2002. Bacteriophage therapy rescues mice bacteremic from a clinical isolate of vancomycin resistant Enterococcus faecium. Infect Immun. 70: 204-210. Boratynski, J., Syper, D., Weber-Dabrowska, B. Lusiak-Szelachowska, M., Pozniak, G. and Gorski, A., 2004. Preparation of endotoxin-free bacteriophages. Cell. Molecular Biol. Letts. 9: 253-259. Borie, C., Sanchez, M.L., Navarro, C., Ramirez, S., Morales, M.A., Retamales, J. and Robeson, J. 2009. Aerosol spray treatment with bacteriophages and competitive exclusion reduces Salmonella enteritidis infection in chickens. Avian Dis. 53: 250-254. Cairns, B.J. and Payne, R.J.H. 2008. Bacteriophage therapy and the mutant selection window. Antimicrob. Agents Chemo. 52: 4344-4350. Callaway, T.R., Edrington, T.S., Brabban, A.D., Anderson. R.C., Rossman, M.L., Engler, M.J., Carr, M.A., Genovese, K.J., Keen, J.W., Looper, M.L. Kutter, E.M. and Nisbet, D.J. 2008. Bacteriophage isolated from feedlot cattle can reduce Escherichia coli O157:H7 populations in ruminant gastrointestinal tracts. Foodborne Path. Dis. 5: 183191. Calvo, C., Brault, J., Alonso, J.M. and Mollaret, H.M. 1981. New waterborne bacteriophages active on Yersinia enterocolitica. Appl. Environ. Microbiol. 42: 35-38. Center for Food Safety and Applied Nutrition. 2007. GRAS notice no. GRN 000218, US Food and Drug Administration, Rockville, MD. Ceyssens, P.-J. and Lavigne, R. 2010. Introduction to bacteriophage biology and diversity, In Bacteriophages in the Control of Food- and Waterborne Pathogens. P.M. Sabour and M.W. Griffiths, Eds., ASM Press, Washington DC., pp 11-29. Connerton, P.L., Loc Carrillo, C.M., Swift, C., Dillon, E., Scott, A., Rees, C.E.D., Dodd, C.E.R., Frost, J. and Connerton, I.F. 2004. Longitudinal study of Campylobacter jejuni

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bacteriophages and their hosts from broiler chickens. Appl. Environ. Microbiol. 70: 38773883. Cox, J.M. and Pavic, A. 2010. Advances in enteropathogen control in poultry production. J. Appl. Micro. 108: 745-755. d‘Hérelle, F. 1926. The Bacteriophage and Its Behaviour. Williams and Wilkins, Baltimore, MD. Deresinski, S. 2009. Bacteriophage therapy: exploiting smaller fleas. Clin. Infect. Dis. 48: 1096-1101. Deveau, H., Barrangou, R., Garneau, J.E., Labonte, J., Fremaus, C., Boyaval, P., Romero, D.A. Horvath, P. and Moineau, S. 2008. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J. Bacteriol. 190: 1390-1400. Elder, R.O., Keen, J.E., Siragusa, G.R., Barkocy-Gallagher, G.A., Koohmaraie, M. and Lagreid, W.W. 2000. Correlation of enterohemorrhagic Escherichia coli O157 prevalence in fees, hides and carcasses of beef cattle during processing. Proc. Nat. Acad. Sci USA. 97: 2999-3003. El-Shibiny, A., Scott, A., Timms, A., Metawea, Y., Connerton, P. and Connerton, I., 2009. Application of a group II Campylobacter bacteriophage to reduce strains of Campylobacter jejuni and Campylobacter coli colonizing broiler chickens. J. Food Prot, 72: 733-740. Fiorentin, L., Vieira, N.D. and Barioni, W. 2005. Oral treatment with bacteriophages reduces the concentration of Salmonella Enteritidis PT4 in the caecal contents of broilers. Avian Path. 34(3) 258-263. Fonty, G., Goulet, P. and Nebout, J.M. 1989. Development of the cellulolytic microflora in the rumen of lambs transferred into sterile isolators a few days after birth. Can. J. Microbiol. 35: 416-422. Forde, A. and Girxgerald, G.F. 1999. Bacteriophage defence systems in lactic acid bacteria. Antonie van Leeuwenhoek 76: 89-113. Gunether, S., Huwyler, D., Richard, S. and Loessner, M.J. 2009. Virulent bacteriophage for efficient biocontrol of Listeria monocytogenes in ready-to-eat foods. Appl. Environ. Microbiol. 75: 93-100. Hagens, S. and Offerhaus, M.L. 2008. Bacteriophages – new weapons for food safety. Food Tech. 4: 46-54. Hagens, S. and Loessner, M.J. 2007. Application of bacteriophages for detection and control of foodborne pathogens. Appl. Microbiol. Biotechnol. 76: 513-519. Hagens, S., Habel, A., von Ahsen, U., von Gabain, A., and Blasi, U. 2004. Therapy of experimental Pseudomonas infections with a nonreplicating genetically modified phage. Antimicrob. Agents and Chemo. 48: 3817-3822. Hanlon, G.W. 2007. Bacteriophages: an appraisal of their role in the treatment of bacterial infections. Int. J. Antimicrobial Agents 30: 118-124. Hanlon, G.W., Denyer, S.P., Olliff, C.J., and Ibrahim, L.J. 2001. Reduction in exopolysaccharide viscosity as an aid to bacteriophage penetration through Pseudomonas aeruginosa biofilms. Appl. Environ. Microbiol. 67: 2746-2753. Higgins, J.P., Filho, R.L.A., Higgins, S.E., Wolfenden, A.D., Tellez, G. and Hargis, B.M. 2008. Evaluation of Salmonella-lytic properties of bacteriophages isolated from commercial broiler houses. Avian Dis. 52: 139-142.

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Holden, L.A., Glenn, B.P. Erdman, R.A. and Potts, W.E. 1994. Effects of alfalfa and orchardgrass on digestion by dairy cows. J. Dairy Sci. 77: 2580-2594. Huff, W.E., Huff, G.R., Rath, N.C. and Donoghue, A.M. 2010. Immune interference of bacteriophage efficacy when treating colibacillosis in poultry. Poultry Sci. 89: 895-900. Hughes, K.A., Sutherland, I.W. and Jones, M.V. 1998. Biofilm susceptibility to bacteriophage attack: the role of phage-borne polysaccharide depolymerise. Microbiol. 144: 3039-3047. Hurley, A., Maurer, J.J. and Lee, M.D. 2008. Using bacteriophages to modulate Salmonella colonization of the chicken‘s gastrointestinal tract: lessons learned from in silico and in vivo modelling. Avian Dis. 52: 599-607. Jamalludeen, N., Johnson, R.P., Friendship, R., Kropinski, AM, Lingorh, E.J. and Gyles, C.L. 2007. Isolation and characterization of nine bacteriophages that lyse O149 enterotoxigenic Escherichia coli. Vet. Micro. 124: 47-57. Johnson, R.P., Gyles, C.L., Huff, W.E., Ojha, S., Huff, G.R., Rath, N.C. and Donoghue, A.M. 2008. Bacteriophages for prophylaxis and therapy in cattle, poultry and pigs. Anim Health Res. Rev. 9: 201-215. Karunasagar, I., Shivu, M.M., Girisha, S.K., Krohne, G. and Karunasagar, I. 2007. Biocontrol of pathogens in shrimp hatcheries using bacteriophages. Aquaculture 268: 288-292. Kasman, L.M., Kasman, A., Westwater, C., Dolan, J., Schmidt, M.G. and Norris, J.S. 2002. Overcoming the phage replication threshold: a mathematical model with implications for phage therapy. J. Virol. 76: 5557-5564. Kaszubkiewicz, C., Kucharewicz-Krukowska, A., Michaliski, Z., Bochianowski, M., Soltysiak, Z. and Durlak, I. 1982. Use of bacteriophage in the treatment of Escherichia infection in piglets. Medycyna Weterynaryjna 38: 281-282. Keen, J.E., Laegreid, W.W., Chitko-McKown, C.G., Durso, L.M. and Bono, J.L. 2010. Distribution of shiga-toxigenic Escherichia coli O157 in the gastrointestinal tract of naturally-O157-shedding cattle at necropsy. Appl. Environ. Microbiol. 76: 5278-5281. Labrie, S. and Moineau, S. 2010. Bacteriophages in industrial food processing: incidence and control in industrial fermentations. In Bacteriophages in the Control of Food- and Waterborne Pathogens. P.M. Sabour and M.W. Griffiths, Eds., ASM Press, Washington DC., pp 199-216. Lee, N. and Harris, D.L. 2001. The effect of bacteriophage treatment as a pre-harvest intervention strategy to reduce the rapid dissemination of Salmonella typhimurium in pigs. Am. Assoc. Swine Vet. (AASV) Perry, IA, AASV pp 555. Leverentz, B., Conway, W.S., Camp, M.J., Janisiewicz, W.J., Abuladeze, T., Yang, M., Saftner, R. and Sulakvelidze, A. 2003. Biocontrol of Listeria monocytogenes on fresh-cut produce by treatment with lytic bacteriophages and a bacteriocin. Appl. Environ. Microbiol. 69: 4519-4526. Loc Carrillo, C., Atterbury, R.J., El-Shibiny, A., Connerton, P.L., Dillon, E., Scott, A. and Connerton, I.F. 2005. Bacteriophage therapy to reduce Campylobacter jejuni colonization of broiler chickens. Appl. Environ. Microbiol. 71: 6554-6563. Ma, Y., Pacan, J.C., Wang, Q., Xu, Y., Huang, X., Korenevsky, A. and Sabour P.M. 2008. Microencapsulation of bacteriophage Felix O1 into chitosan-alginate microspheres for oral delivery. Appl. Environ. Microbiol. 74: 4799-4805. Matsuzaki, S., Rashel, M., Uchiyama, J., Sakurai, S., Ujihara, T., Kuroda, M., Ikeuchi, M., Tani, T., Fujieda, M., Wakiguchi, H. and Imai, S. 2005. Bacteriophage therapy: a

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revitalized therapy against bacterial infectious diseases. J. Infect. Chemother. 11: 211219. Mazaheri, R., Fard, N., Barton, M.D. and Heuzenroeder, M.W. 2010. Novel bacteriophages in Enterococcus spp. Curr. Micobiol. 60: 400-406. Merill, C.R., Biswas, B., Carlton, R., Jensen N.C., Creed, G.J., Zullo S. and Adhya, S. 1996. Long-circulating bacteriophage as antibacterial agents. Proc. Natl. Acad. Sci. USA 93: 3188-3192. Morrision, S., and Rainnie, D.J. 2004. Bacteriophage therapy: an alternative to antibiotic therapy in aquaculture. Can. Tech. Rep. Fish. Aquat. Sci. 2532, 23 pp. Naylor, S.W., Low, J.C. Besser,T.E., Mahajan, A., Gunn, G.J., Pearce, M.C., McKendrick, L.J.,Smith , D.G. and Gally, D.L. 2003. Lymphoid follicle dense mucosa at the terminal rectum is the principal site of colonization of enterohemorrhagic Escherichia coli O157:H7. Infect. Immun. 71: 1505-1512. Niu, Y.D., McAllister T.A., Johnson R.P., Kropinski, A. M., Xu Y. and Stanford K. A newly isolated lytic bacteriophage AKFV33 is highly virulent against Shiga toxin-producing Escherichia coli O157:H7. Canadian Society of Microbiologists 60th Annual Conference, Hamilton, Canada, 2010: http://www.csm-scm.org/english/abstracts/public/ view_abs.asp?id=3129. Niu, Y.D., Xu, Y., McAllister, T.A., Stephens, T.P., Johnson, R.P. and Stanford, K. 2009. Prevalence and impact of bacteriophage on the presence of Escherichia coli O157:H7 in feedlot cattle in Alberta. App. Environ. Microbiol. 75: 1271-1278. Ochman, H. and Selander, R.K. 1984. Standard reference strains of Escherichia coli from natural populations. J. Bacteriol. 157:690-693. O‘Flynn, G., Coffey, A., Fitzgerald, G.F. and Ross, R.P. 2006. The newly isolated lytic bacteriophages st104a and st104b are highly virulent against Salmonella enterica. J. Appl. Micro. 101: 251-259. O‘Flynn, G., Ross, R.P., Fitzgerald, G.F. and Coffey, A. 2004. Evaluation of a cocktail of three bacteriophages for biocontrol of Escherichia coli O157:H7. Appl. Environ. Microbiol. 70: 3417-3424. Oot, R.A., Raya, R.R., Callaway, T.R., Edrington, T.S., Kutter, E.M. and Brabban, A.D. 2007. Prevalence of Escherichia coli O157 and O157:H7-infecting bacteriophages in feedlot cattle feces. Letts. Appl. Micro. 45: 445-453. Owens, J., Heuzenroeder, M. and Barton, M.D. 2007. Therapeutic use of bacteriophages in animals and foods to reduce contamination. CAB reviews 2: 1-8. Payne R.J.H. and Jansen, V.A. 2001. Understanding bacteriophage therapy as a densitydependent process. J. Theor. Biol. 208: 37-48. Raya, R.R., Varey, P., Oot, R.A., Dyen, M.R., Callaway, T.R., Edrington, T.S., Kutter, E.M. and Brahban, A.D. 2006. Isolation and characterization of a new T-even bacteriophage CEV1 and determination of its potential to reduce Escherichia coli O157:H7 levels in sheep. Appl. Environ. Microbiol. 72: 6403-6410. Rivas, L., Coffey, B., McAuliffe, O., McDonnell, M.J., Burgess, C.M., Coffey, A., Ross, R.P. and Duffy, G. 2010. In vivo and ex vivo evaluations of bacteriophages e11/2 and e4/1c for use in the control of Escherichia coli O157:H7. Appl. Environ. Microbiol. 76: 72107216.

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Rosenquist, H. Nielsen, N.L., Sommer, H.M., Norrung, B. and Christensen, B.B. 2003. Quantitative risk assessment of human campylobacteriosis associated with thermophilic Campylobacter species in chickens. Int. J. Food Micro. 83: 87-103. Rozema, E.A., Stephens, T.P., Bach, S.J., Okine, E.K., Johnson, R.P., Stanford, K. and McAllister, T.A. 2009. Oral and rectal administration of bacteriophages for control of Escherichia coli O157:H7 in feedlot cattle. J. Food. Protect.72: 241-250. Santos, S.B., Fernades, E., Carvalho, C.M., Sillankorva, S., Krylov, V.N., Pleteneva, E.A., Shaburova, O.V., Nicolau, A., Ferreira, E.C. and Azeredo, J. 2010. Selection and characterization of a multivalent Salmonella phage and its production in a nonpathogenic Escherichia coli strain. Appl. Environ. Microbiol. 76: 738-7342. Saunders, R, Allison, H, James, C.E., McCarthy, A.J. and Sharp, R. 2001. Phage-mediated transfer of virulence genes. J. Chem. Technol. Biotech. 76: 662-666. Sharma, M., Patel, J.R., Conway, W.S., Ferguson, S. and Sulakvelidze, A. 2009. Effectiveness of bacteriophages in reducing Escherichia coli O157:H7 on fresh-cut cantaloupes and lettuce. J. Food Protect. 72: 1481-1485. Sheng, H., Knecht, H.J., Kudva, I.T. and Hovde, C.J. 2006. Application of bacteriophages to control intestinal Escherichia coli O157:H7 levels in ruminants. Appl. Environ. Microbiol. 72: 5359-5366. Smith, H.W. and Huggins, M.B. 1983. Effectiveness of phage in treating experimental Escherichia coli diarrhoea in calves, piglets and lambs. J. Gen. Microbiol. 129: 26592675. Smith, H.W., Huggins, M.B. and Shaw, K.M. 1987. The control of experimental Escherichia coli diarrhoea in calves by means of bacteriophages. J. Gen. Micro. 133: 1111-1126. Stanford, K., McAllister, T.A., Niu, Y.D., Stephens, T.P., Mazzocco, A., Waddell, T.E. and Johnson, R.P. 2010. Oral delivery systems for encapsulated bacteriophage targeted at E. coli O157:H7 in feedlot cattle. J. Food Protect. 73: 1304-1312. Stanford, K., Stephens, T.P. and McAllister, T.A. 2011. Use of model super shedders to define the role of pen floor and hide contamination in the transmission of Escherichia coli O157:H7. J. Anim. Sci. 89: 237-244. Stephens, T.P., McAllister, T.A. and Stanford, K. 2008. Development of an experimental model to assess the impact of super-shedders on the transmission of Escherichia coli O157:H7 within the feedlot environment. J. Food Prot. 71: 648-652. Sulakvelidze, A. and Pasternack, G.R. 2010. Industrial and regulatory issues in bacteriophage application in food production and processing. In Bacteriophages in the Control of Foodand Waterborne Pathogens. P.M. Sabour and M.W. Griffiths, Eds., ASM Press, Washington DC., pp 297-326. Suttle, C.A. 2007. Marine viruses – major players in the global ecosystem. Nat. Rev. Microbiol. 5: 801-812. Tobe, T., Beatson, S.A., Taniguchi, H., Abe, H., Bailey, C.M., Fivian, A, Younis, R., Matthews, S., Marches, O, Frankel, G., Hayashi, T. and Pallen, M.J. 2006. An extensive repertoire of type III secretion effectors in Escherichia coli O157 and the role of lambdoid phages in their dissemination. Proc. Natl. Acad. Sci. USA 103: 14941-14946. Ventura, M., Sozzi, T., Turroni, F., Matteuzzi, D and van Sinderen, D. 2010. The impact of bacteriophages on probiotic bacteria and gut microbiota diversity. Genes Nutr. In press: DOI 10.1007/s12263-010-0188-4.

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Verner-Jeffreys, P.W., Algoet, M., Pond, M.J., Hardeep, K. V., Bagwell, N.J. and Roberts, E.G. 2007. Furniculosis in Atlantic salmon (Solna sola L.) is not readily controllable by bacteriophage therapy. Aquaculture 270: 475-484. Waddell, T.E., Franklin, K. Massocco, A., Kropinski, A.M. and Johnson, R.P. 2009. Generalized transduction by lytic bacteriophages. In: Bacteriophages, Methods and Protocols, Volume 1: Isolation, Characterizations and Interactions. M.R.J. Clockie and A.M. Kropinski, Eds, Humana Press, pp 293-303. Waddell, T., Mazzocco, A., Johnson., R.P., Pacan, J. Campbell, S., Perets, A., MacKinnon, J., Holtstander, B. and Poppe, C. 2000. Control of Escherichia coli O157:H7 infection of calves by bacteriophage. In: Proceedings of the 4th International Symposium and Workshop on Shiga Toxin (verocyotoxin)-producing Escherichia coli (VTEC 2000) Kyoto, Japan October 29-November 2. Wagenaar, J.A., Van Bergan, M.A.P., Mueller, M.A., Wassenaar, T.M. and Carlton, R.M. 2005. Phage therapy reduces Campylobacter jejuni colonization in broilers. Vet. Micro. 109: 275-283. Wiggins, B.A. and Alexander M. 1985. Minimum bacterial density for bacteriophage replication: implications for significance of bacteriophages in natural ecosystems. Appl. Environ. Microbiol. 49: 19-23. Wilkinson, M.H.F. 2001 Predation in the presence of decoys: an inhibitory factor on pathogen control by bacteriophages or Bdellovibrios in dense and diverse ecosystems. J. Theor. Bio. 208: 27-36.

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In: On-Farm Strategies to Control Foodborne Pathogens ISBN: 978-1-62100-411-0 Editors: Todd R. Callaway and Tom S. Edrington © 2012 Nova Science Publishers, Inc.

Chapter 5

PROBIOTICS IN SWINE PRODUCTION

1

A. V. S. Perumalla1, Navam. S. Hettiarachchy1, 2, Philip G. Crandall1, 2 and S. C. Ricke1, 2 Department of Food Science, University of Arkansas, Fayetteville 2 Center for Food Safety, University of Arkansas, Fayetteville

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ABSTRACT Foodborne pathogens such as Salmonella, Campylobacter and Escherichia transmitted through swine production including pork products pose an ongoing threat to the consumers as well as the processors. Swine production has changed from small herds to confinement of large numbers in feedlots. Increased demand in production, use of antibiotics, and prevalence of antimicrobial-resistant pathogens and the subsequent impact of foodborne outbreaks has necessitated the demand for natural and safe alternatives. Under the present market conditions, using alternative interventions or strategies that would improve the overall food safety status of the meat products represents a potential opportunity. There is increasing evidence that using probiotic supplemented diets can reduce the load of enteric pathogens such as Salmonella, Escherichia coli, and Campylobacter. Consequently, with the need to ultimately limit the use of direct fed antibiotics, administration of alternatives such as probiotics in swine nutrition is a promising strategy to improve the preharvest food safety of swine. This review focuses on the gastrointestinal microflora of swine, their beneficial effects, and their influence on the probiotic microflora on the gastrointestinal microbial ecosystem as well as their implications for swine nutrition and health.

Keywords: Probiotics, Swine, Preharvest, Salmonella, Escherichia coli, Campylobacter.



Corresponding author: Dr. Steven C. Ricke. Center for Food Safety, Department of Food Science, University of Arkansas, 2650 N. Young Ave, Fayetteville, AR 72704. E-mail: [email protected]. Phone (479)575-4678. Fax (479)575-6936.

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A. V. S. Perumalla, Navam. S. Hettiarachchy, Philip G. Crandall et al.

INTRODUCTION Swine production like other meat and poultry farming systems follows a continuous multi-step approach from farm to a meat processor and further processing and retail to distribution channels prior to public consumption. At each step, there is considerable potential for introduction of foodborne pathogens during production or processing. Antibiotics have been incorporated into swine diets for over 50 years to improve the growth performance and prevent diseases in swine rearing environments (Humphrey et al., 2007). This has led to the potential increase in antibiotic-resistance of the foodborne pathogens associated with swine and residual contamination in the food chain (Corpet, 1996; Williams and Heymann, 1998; Roselli et al., 2005). Furthermore, the use of antibiotics as growth promoters has been forbidden since 2006 by the European Union (EU) and this in turn has directly impacted U.S. exports (Chen et al., 2005). These factors have triggered the adoption of natural alternative sources to serve as substitutes to potentially replace antibiotics in swine farming (Castillo et al., 2008). Various strategies such as probiotics, prebiotics, organic acids, plant extracts, and enzymes alone or in combination have been explored as alternatives to antibiotic growth promoters. One such alternative is the use of probiotics in swine nutrition that can exert health benefits via introduction of beneficial microorganisms thereby improving animal health and growth performance. This review will focus on various foodborne pathogens associated with swine, the use of probiotics and their applications as alternatives for improving the preharvest food safety of swine products.

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FOODBORNE PATHOGENS ASSOCIATED WITH SWINE Over the years, swine production has considerably changed from backyard to small operations and eventually to large animal production to meet the protein requirements of a growing human population (Graham et al., 2008). This has led to the rearing of larger swine herds and ultimately predisposed potential concerns regarding the spread of foodborne pathogens associated with swine production and its processing (Graham et al., 2008). The primary foodborne pathogens that are associated with swine and/or pork products include Salmonella, Campylobacter, and Escherichia (Table 1). Several stress factors such as nutrition, housing environment (Madec et al., 2000; Laine et al., 2008), age of piglets at during weaning (Svensmark et al., 1989), change in feed (Bailey et al., 1992), and insufficient space in pens (Amezcua et al., 2002) contribute to the susceptibility for successful infection in the host.

Salmonella Swine is one of the major reservoirs of Salmonella contaminating food and the environment as they can act as both carriers (pork) as well as shedders (feces) (Callaway et al., 2005; Roesler et al., 2004). Pigs become colonized and infected with Salmonella by ingesting contaminated feed and feces, and the respiratory system (tonsils can also assist in colonization) (Miller et al., 2005). Once ingested, Salmonella is challenged by gastric

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Probiotics in Swine Production

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antimicrobial barriers such as porcine epithelial beta-defensin 1 in the dorsal section of the tongue (Shi et al., 1999), increase in acid-stress by a lowering of pH (Smith, 2003), and bile salts repression of the invasion of Salmonella by decreasing virulence gene expression (Prouty and Gunn, 2000) prior to colonization in the small intestine. In intestinal colonization, Salmonella use both conserved and host-specific colonization factors such as Salmonella pathogenicity island (SPI)-1-encoded type III secretion system (T3SS-1), fimbriae, and adhesins (Boyen et al., 2006). These colonization factors vary depending on the serotype; Type-I fimbriae have been implicated in S. Typhimurium intestinal colonization (Althouse et al., 2003) as well as specific pathogenicity island (SPI-1) transcriptional regulator hilA and a number of SPI-1 and SPI-2 genes essesntial for S.Cholerasuis (Ku et al., 2005; Lichtensteiger and Vimr, 2003). The hilA gene is a major transcriptional gene regulator of genes (orgA, prgH, invF, and sspC) in SPI-1 that promotes host invasion and is controlled by environmental factors such as pH, osmolarity, oxygen saturation, and cell density (Darwin and Miller, 1999; Rakeman et al., 1999). Table 1. Foodborne pathogens isolated from swine herds and associated products

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Food borne pathogen Salmonella

Campylobacter

Escherichia coli

Species/ Serotype Derby

Stage of production cycle Finishing swine and pork-products

Identification method Pulse Field Gel Electrophoresis (PFGE) Enzyme linked Immunosorbent Assay (ELISA)

Typhimurium

Weaned piglet, Nursery pigs

Infantis

Finishing swine

PFGE

Typhimurium definitive type (DT) 104

Finishing swine

PFGE

Mbandanka

Finshing swine

PFGE

Typhimurium DT12 jejuni

Swine herds

PFGE

New-born, gilts, pregnant sows, Finsmarket-age

PFGE

Siemer et al., 2005 Young et al., 2000

coli

New-born, gilts, pregnant sows, market-age

Multilocus sequence typing

Harvey et al., 1999; Young et al., 2000; Cliver, 2002; Thakur et al., 2006

Neonatal and recently weaned pig

Multiplex polymerase chain reaction (PCR)

Francis, 2002

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Reference Jayarao, 1992; Valdezate et al., 2005 Fedorka-Cray et al., 1995; Rajic et al, 2005, Kich et al., 2007. Rajic et al, 2005, Lettellier et al., 1999. Anderson et al, 2003; Lee and Lee, 2007; Langvad et al., 2006 Callaway et al., 2005; Filiousis et al., 2008 Spencer, 2004

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Following adhesion, Salmonella invades the porcine absorptive enterocytes, M-cells and goblet cells, and mesenteric lymph nodes (Pospischill et al., 1990; Schauser et al., 2005). In response to the enteric invasion, the porcine gut produce several cytokines; IL-8 in particular (at least 24 hours after inoculation of weaned piglets) (Skjolaas et al., 2006) triggers the release of neutrophils to the lamina propria and eventually to the intestinal lumen forming the first line of defense (Foster et al., 2005). However, an inefficient defense system provides an opportunity for colonization and /or replication that can ultimately facilitate a carrier or infectious state in the infected swine (Stabel et al., 2002). Factors contributing to the shedding of Salmonella in swine production include contaminated feed along with the use of pelleted feeds, antibiotic use, number of live animals that carry Salmonella spp. in feces, transportation to the slaughter house and holding in pens, vaccination, and biosecurity measures (Berends, et al., 1997; Funk et al., 2001; Funk and Gebreyes, 2004; Mikkelsen et al., 2004). Among these factors, a physico-chemcal characteristic of the feed such as ready-mixed pelleted dry feed and coarse grinding appears to increases the risk of Salmonella susceptibility in pigs (Wingstrand et al., 1997; Jorgensen et al., 1999; Stege et al., 2001; Leontides et al., 2003). Pigs fed coarse nonpelleted diets (C-NP) exhibited more solid gastric content with high dry matter content than pigs fed with fine nonpelleted, coarse pelleted, or fine pelleted diets (Mikkelsen et al., 2004). Furthermore, feeding such diets (C-NP) has demonstrated several advantages when compared to aforementioned feeds: a) significant increase in anaerobic bacterial load along with increases in organic acids and undissociated lactic acid which in particular exhibits inhibitory activities against S. enterica serovar Typhimurium DT12, b) reduced pH in the stomach, c) lower coliform bacterial populations in the distal small intestine, ceceum, and mid colon, d) and increased butyric acid concentration in the cecum and colon (Mikkelsen et al., 2004). Salmonella serotypes commonly involved in contaminating swine herds and pork products include, Typhimurium, Infantis, Derby, and Mbandanka (Ojha and Kostrzynska, 2007). Although several antibiotics or antimicrobials have been used to reduce the carriage of intestinal pathogens, dissemination of drug-resistant zoonotic pathogens can still occur (Chapin, 2005). Serotypes such as Typhimurium definitive type (DT) 104 and Typhimurium DT12 are rapidly emerging and dissemination as multi-drug resistant Salmonella serotypes can occur (Anderson, 2003). In the U.S. approximately 48 % of the swine herds possess Salmonella as asymptomatic carriers (Callaway et al., 2005). Furthermore, salmonellosis causes nearly 1.4 million foodborne illnesses every year costing more than $ 2.9 billion (including medical costs due to illness, value of time lost from work, and the economic value of premature deaths) to the U.S. economy (Montagne, 2004). The economic impact of Salmonella associated with swine production and its products have been estimated at $81.5 million which highlights the importance of effective control measures in the pork supply chain (Miller, 2005).

Campylobacter Campylobacter is also a major foodborne pathogen transmitted through swine that can cause acute gastroenteritis (Friedman et al., 2001). Campylobacter is unique from other foodborne pathogens as they are essentially microaerophilic (require a 10% CO2 and

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approximately 5% O2 atmosphere) (Nachamkin, 1995; Humphrey et al., 2007). Two species have been recognized as more prevalent in swine, namely Campylobacter jejuni and Campylobacter coli (Saenzo et al., 2000; Allos, 2001). Swine is an asymptomatic carrier of C. coli and C. jejuni; however, C. coli are predominantly found in pigs (Cliver, 2002) whereas C. jejuni mostly occurs in poultry (Siemer et al., 2005; Horrocks et al., 2009). C. jejuni can be differentiated from C. coli on the basis of hippurate hydrolysis as the former species are positive for this test (International Organization for Standardization, 2006) (Table 2). One possible reason for the occurrence of C. jejuni in swine herds or flocks may be due to its transmission from primary reservoir hosts such as poultry and cattle where herds are maintained in mixed production systems (Boes et al., 2005). Campylobacter infection in swine can be transmitted from the sow to the piglets as early as the first 24 hours after birth (Harvey, 2010). Foodborne transmission of Campylobacter occurs through fecal contamination of food, water and carcasses at slaughter (USDA, 2008). Table 2. Differences between Campylobacter jejuni and Campylobacter coli Attribute Foodborne illness Primary host Hippurate hydrolysis Erythromycin

C. jejuni Major (90 %) Poultry Positive

C. coli Minor (5- 10 %) Swine Negative

Reference Tam et al., 2003

Susceptible

Resistant

Aartstrup et al., 1997

Adzitey and Corry, 2011

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C. coli is only the second most isolated species of Campylobacter in foodborne outbreaks (5 to 10 %) (Gillespie et al., 2002) and are extremely rare (Miller and Mandrell, 2005b).

Escherichia Coli One of the most frequent causes of economic impact losses in swine farming is due to post weaning diarrhoea (PWD) caused by Escherichia coli and specific members of Clostridium, Lawsonia and Brachyspira genera (Amezcua et al., 2002; Vondruskova et al., 2010). Enterotoxigenic Escherichia coli (ETEC) is the predominant cause of enteritis, sometimes leading to death in neonatal and recently weaned pigs (6 to 14-week-old) especially after dietary change (Francis, 2002; Fairbrother and Gyles, 2006). Two types of virulent factors namely adhesins (K88, K99, 987P, F41, and F18) (Wilson et al., 1986; Casey et al., 1992) and enterotoxins [heat labile (LT); heat stable enterotoxin type A (STa); heat stable enterotoxin type B (STb); Shiga toxin type 2e, Stx2e, and enteroaggregative E coli heat-stable enterotoxin 1 (EAST1) (Wilson et al., 1986; Imberechts et al., 1997; Choi et al., 2011) are responsible for the morbidity and mortality incidence in neonatal and post-weaned pigs. These enterotoxins act locally in the gut and thereby stimulating hypersecretion of water and electrolytes by enterocytes (Post et al., 2000). Fimbrial antigens such as K88, in particular mediate the adhesion of ETEC strains to the intestinal epithelial mucosa lining of the intestine, and thereby lead to the formation of the enterotoxins, which induces massive fluid and electrolyte secretion into the gut lumen (Alexander, 1994; Hampson, 1994). In older swine such as the grower stage, E. coli occasionally causes enteritis when it is associated with

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other diseases such as porcine reproductive and respiratory syndrome (PRRS), and porcine circovirus associated disease (PCVAD) (Pittman, 2010). Pathogenic strains of E. coli attach to the intestinal epithelia and interfere with the colonization of indigenous microflora of small intestine to induce the pathogenicity (McAllister et al., 1979). Indigenous microflora changes in post-weaning include the decline (about one-tenth of pre-weaning levels) in populations of aerobes, lactobacilli and Bacteriodes-Clostridia (McAllister et al., 1979). In the same study, the author reported the following changes in indigenous microflora population of post-weaning pigs: a) post-weaning (24 and 48 hr) pigs had lower numbers of lactobacilli (104 CFU/g) and streptococci (103 CFU/g) in the small intestine when compared to nursing pigs (106 to107 CFU/g and 105 to106 CFU/g). However, there were a) no changes in trends of the bacterial population following 3rd to 8th day postweaning, b) an increase in the E. coli populations throughout the small intestine specifically duodenum and jejunum (averaged 106 CFU/g) when compared to pre-weaning levels (104.5 CFU/g), c) an increase in other microflora include Bacteroides-Clostridia, oxygen-tolerant anaerobes in small intestine, d) an increase in hemolytic E. coli in the pig feces after weaning. All of these dynamic changes in the microflora population following post-weaning initiated disturbances ultimately resulted in the increase of hemolytic E. coli by displacing non hemolytic E. coli, streptococci, and possibly some oxygen intolerant anaerobes (Kenworthy and Allen, 1966; Freter and Abrams, 1972; Savage and McAllister, 1970). In addition to the dynamic changes in the microflora, factors such as strong intestinal peristalisis, villi pumping, flow of ingesta and mucus secretion that serve to propel the bacteria through the intestine regulate the establishment of hemolytic E. coli (Moon et al., 1979). Once established and attached, the pathogenic E. coli multiply to a significant number of organisms, successfully evade the host‘s natural defense system, and colonize intestinal mucosa to produce enterotoxins. Consequently, this increases secretion of water and electrolytes with minimal change in epithelial morphology while competing for nutrients and space with normal gut flora of the host (Moon, 1997; Nataro, 1996). ETEC strains utilize specific fimbrial or nonfimbrial adhesins that can facilitate the adherence to the colonization sites in the small intestine (Levine, 1987; Levine et al., 1984; Nataro, 1987; Vial, 1988). The specifity of the virulence and adherence factors is influenced by age (neonatal or adult), site (small or large instesine), and the host (Moon, 1997). The economic impact caused by the enterotoxigenic strains of E. coli in suckling and weaned pigs, morbidity, loss of productivity, and mortality together cost millions of dollars every year (Harvey et al, 2005).

SWINE GASTROINTESTINAL MICROFLORA Introduction Various intervention strategies have been used to reduce the impact of these foodborne pathogens and their subsequent contamination of the pork products. Stringent control regulations have been implemented by the U.S. Department of Agriculture (USDA) to improve the preharvest food safety of swine products. Using probiotics is one approach to improve the preharvest food safety of the swine so that the intestinal problems encountered

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during rearing would be minimized (Vanbelle et al., 1990). For a better understanding of the effect of probiotics in swine, a comprehensive knowledge of the swine digestive tract and its indigenous microflora is needed to understand which bacteria might represent the best candidates as potential probiotic strains.

Porcine Gastrointestinal Tract (GIT) A typical gastrointestinal tract (GIT) in the newborn pig is only 2 meters whereas in a mature animal it measures approximately 20 meters (Slade, 2004). The gastrointestinal tract of swine can be divided into 5 parts, namely, the mouth, the esophagus, the stomach, the small intestine and the large intestine. The GIT of swine supports dense and complex microbial communities such as bacteria (aerobic, facultative anaerobic, and strictly anaerobic) protozoa, and fungi (Chaucheyras-Durnad and Durnad, 2010) (Table 3). The role of these intestinal commensal microbiota has been widely recognized with a focus on beneficial effects on the physiological functions of the host (Cummings and Macfarlane, 1997; Sevin, 2004).

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Table 3. Gut microflora of swine with predominant microflora observed in primary gastrointestinal sections Density (CFU log10/g)

Representative Gut microflora

Indigenous microflora with potential probiotic properties

1.5 to 2.5

4.0 to 9.5

Lactobacilli, Bifidobacteria, and Streptococci

Acid tolerant1 Lactobacilli and streptococci

Proximal

5.1 to 6.5

4.0 to 8.5

Caudal

6.6 to 8.4

5.0 to 9.5

Ceca

5.6 to 6.7

5.0 to 9.5

Colon

6.4 to 7.8

5.5 to 9.5

GI tract section

pH2

Stomach Small intesine

Large intestine

1 2

Facultative anaerobes (Lactobacilli, Streptococci, and Enterobacteria), anaerobes (Bifidobacterium spp.,

Lactobacilli

Bacteroides spp., and Clostridia spp), Eubacterium, and Coliforms

Lactobacilli

Facultative anaerobes (Streptococci, Coliforms, and Proteus), Obligate anaerobes (Clostridium), Eubacterium, Bacteroides, and Lactobacillus

Bacteroides, Enterobacter Bifidobacteria, Lactobacilli

Dibner and Richards, 2005. Smith and Jones, 1963.

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In swine, gut microflora density and composition vary significantly by GI site due to differences in the physico-chemical characteristics such as pH, oxidation-reduction potential, viscosity, and the biological characteristics (nutrient supply and the concentration of biological active compounds) of the luminal contents (Jensen and Jorgensen, 1994). The stomach is lined by keratinized squamous non-secreting epithelium as is the esophagus and colonized with intestinal bacterial cells including Lactobacilli (Lipkin, 1987; Noakes, 1971). In the stomach, the pH may be as low as 2.0 in an adult pig, but as high as 5.0 in milk-fed piglets due to gastric juices and HCl (Slade, 2004). The subsequent components of the small intestine such as the duodenum and ileum, factors including pH and movement of digesta play an important role in bacterial attachment and colonization (Friend et al., 1963; Vodovar et al., 1964). In the large intestine (cecum, spiral colon, and distal colon), the mean retention time of the digesta is slower (2 – 4 days) when compared to the small intestine (2.5 hours) and therefore can be a conducive environment for the establishment of dense and complex anaerobic microflora (Kidder and Manners, 1978). In swine, immediately after birth, the neonate acquires bacteria during parturition and nursing and from the environment, colonizing the sterile mucus lining of the GIT (Kelly, 2001). By 10 to 12 hours after birth the number of microorganisms in piglets ranges from 108 to 109 colony forming units (CFU/g) of feces and this population becomes stabilized within 24 to 48 hours (Ducluzeau, 1983). Nevertheless, the microflora composition varies during the weaning period (Mikkelsen et al., 2003; Roselli et al., 2005). The pig stomach and the proximal small intestine contain relatively low numbers of bacteria (approximately 105/g CFU) than the ileum and the large intestine (approximately 109 to 1012 CFU /g); the principal regions of bacterial colonization (Jensen and Jorgensen, 1994). Beneficial intestinal microflora constitutes a wide range of anaerobic or facultative anaerobic species (approximately 1011 to 1014 CFU/g of digesta) (Ghnassia, 1979; Makala et al., 2000). Potentially beneficial microflora have been isolated from various sites in the intestinal tract of swine (Table 3). Over 500 different species of microorganisms have been identified to date in swine, with a 10:1 ratio of the bacterial cells to eukaryotic cells of the host (Rojas and Conway, 1996; Budino et al., 2005; Dibner and Richards, 2005). Of these, common genera include Bacteroides, Bifidobacterium, Clostridium, Enterobacter, Enterococcus, Eubacterium, Fusobacterium, Peptostreptococcus, Porphyromona, Lactobacillus, and Streptococcus. In initial stages of colonization, the GIT of the neonatal piglets are predominantly occupied by aerobic microorganisms and facultative anaerobes such as Escherichia coli, Lactobacilli, and Streptococci (Smith and Jones, 1963). In later stages, obligate anaerobes such as Bifidobacteria, Bacteroides, Clostridium, Fusobacterium, Butyrivibrio, and Selenomonas spp. become established as a part of stable indigenous microflora (Dibner, 2005). In the stomach, Lactobacilli are the predominant group located in the non-secretory part of the stomach due to the relatively high pH value (1.5 to 2.5) and they further disseminate into the small intestine (Rojas and Conway, 1996). In the cecum, Gram negative bacteria such as Bacteroides and Enterobacter are predominant due to favorable environmental conditions such as an anaerobic atmosphere, temperature, and slow passage of the digesta while Gram-positive bacteria such as Lactobacillus and Bifidobacterium are predominant in colon (Robinson et al., 1981; Rojas and Conway, 1996; Mikkelsen et al., 2003). In addition, methanogenic bacteria of the genus Methanobrevibacter are also found in

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cecum and colon (30 times more dense than that of the cecum) (Miller et al., 1986; Mao et al., 2011).

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Identification of Specific Swine Gut Microflora and Their Development in the GIT Composition of the microbial community in the swine GIT can be obtained directly from the phylogenetic analysis of 16S ribosomal DNA sequences through PCR amplification, cloning and sequencing (Farelly et al., 1995; Resenback et al., 1992; Wang et al., 1996). In a study conducted by Leser et al. (2002), a total of 375 phylotypes were identified using a 97% similarity criterion. Of the total phylotypes, 304 (81%) belonged to the low Guanine (G) + Cytosine (C) gram-positive (Clostridium spp, Eubacterium spp), and 42 phylotypes (11.2%) were associated with the Bacteriodes and Prevotella group. In the stomach, the epithelium is predominantly colonized by lactobacilli with other bacteria including Bifidobacteria, Streptococci, Clostridia and Enterobacteria being present (Henriksson et al., 1995). Similarly, the small intestine is also colonized by Lactobacilli, Streptococci, Clostridia and Enterobacteria (Jensen, 2001). More advanced molecular techniques such as Chaperonin-60 gene sequence analysis and quantitative analysis, and quantitative Polymerase Chain Reaction (PCR) have revealed that low G+C gram-positive organisms such as Lactobacillus spp., Pediococcus spp., Clostridium spp., Bacillus spp., and small numbers of γ-proteobacteria are part of the swine ileal micoflora community (Hill et al., 2005). In the subsequent parts of swine GIT such as the ceca and the colon, the predominant microbiota are more diverse and contain high numbers of Gram-negative bacteria such as Bacterioides (Leser et al., 2002; Konstantinov et al., 2004; Hill et al., 2005). Molecular techniques such as temperature gradient gel electrophoresis (TGGE) and denaturing gradient gel electrophoresis (DGGE) have been used for elucidating the diversity of the microbial ecology in the swine GIT especially from birth to post-weaning period (Muyzer, 1993; Inoue et al., 2005; Hanning and Ricke, 2011). In general, the acquisition and the succession of the intestinal microbiota in swine from birth to post weaning can be observed in three stages (Inoue et al., 2005). The first change is the rapid decline of Clostridium perfringens load in piglets between the 4th and 7th day after birth (Uchida et al., 1965; Melin et al., 1997). This may be due to the mucosal protective effect of the piglet‘s immune system through production of IgA that replaces IgG within 7 days postpartum (Brandtzaeg, 2002; Butler and Brown, 1994). In addition, displacing aerobes and facultative anaerobes by obligate anaerobes may be a possible reason for the initial transition. However, the latter are predominant until the 5th day postpartum and they begin to gradually decrease afterwards (Swords et al., 1993). The second change is the succession phase where anaerobic bacteria such as Eubacterium, Fusobacterium, and Propionibacterium start appearing in the colon from 7th day to 22nd day after birth (Swords et al., 1993). This has been demonstrated by increased TGGE bands (from day 16) as well as increases in the major short chain fatty acids (SCFAs) such as acetate, propionate and butyrate in pig colon (from day 13 to 16 after birth) (Murray et al., 1987). The third change is the predominance of anaerobic bacteria such as Bacteroides spp. with simultaneous reduction of aerobic bacteria such as E. coli and coliforms (22nd to 25th day) (Swords et al., 1993). These changes could be due to the maturation of the swine immune system that includes presence of CD4+ cells in the core of

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villi as seen in the mature intestine (Bailey et al., 2001) along with the predominance of IgA and IgM secretions in the lamina propria of the adult swine until 28 days after birth (Butler and Brown, 1994), and may be due to the nature of the weaning diet (Favier et al., 2002; Inoue and Ushida, 2003). The number and the composition of the GIT microflora may vary considerably (Leser et al., 2002). Development of the intestinal microbiota in the piglet is a gradual and sequential process and is influenced by dietary (suckling pigs, weaning and post-weaning diets) and nondietary (morphological and immunological) factors (Inoue and Ushida, 2003). Furthermore, microbial composition in the swine intestine is determined by two factors, namely autogenic and allogenic. Autogenic factors include mutual interactions between the host and the microorganisms and among different microorganisms (Fuller, 1978; Jensen et al., 1998; Budino et al., 2005). Allogenic factors include pH in the stomach, digestive enzymes, peristaltic movements in the intestine, nutrient availability, and host immunity (Jensen et al., 1998; Budino et al., 2005; Roselli et al., 2005). The density and the composition of the microflora may vary considerably in different microhabitats of the large intestine, including lumen, mucus layer and mucosal surface (Salanitro et al., 1977). In addition to bacteria, yeasts are also the common inhabitants of swine GIT especially in the cecum and the colon (approximately 5.2 log CFU/g of digesta; Canibe et al., 2005). Other factors including feeding practices, diet composition such as incorporation of dietary fiber and fermentable substrates, animal husbandry or farm management practices can also influence the gut microbial balance and ecology of the host.

PROBIOTICS IN SWINE

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General Concepts Colonized gut microflora in swine develop a relatively stable complex microbial ecosystem that have beneficial effects on the host health (Cummings and Macfarlane, 1997; Mackie et al., 1999; Isolauri et al., 2004). These probiotic microorganisms exhibit structural, metabolic, immunologic, and protective functions on the intestinal health of the host (Guarner and Malagelada, 2003). Probiotic microorganisms that have been used frequently in swine diets include bacteria (Lactobacillus spp., Pediococcus spp., Bacillus spp., and Streptococcus spp.), yeast (Saccharomyces spp. and Aspergillus spp.) and their combinations that in some cases can colonize the hindgut (cecum and colon) (Turner et al., 2001). The common routes of probiotic supplementation are through feed and water. Increasing the frequency of the dose of probiotics for a given time period appears to achieve a stable and high level of probiotic bacteria in the GIT as high frequency doses increase the mean number of probiotic bacteria in the cecum (Ohashi et al., 2004). Previous studies have shown that a probiotic feed supplementation in pregnant sows before parturition, during lactation and in neonatal piglets reduced the intestinal microbial load and thereby improved the digestive functions and decreased mortalities (Martin et al., 2009; Schierack et al., 2009). The resident microflora contributes to several health benefits such as overall health improvement, growth and performance of the animal and protective barrier against enteric pathogens.

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Mechanism of Action Several mechanisms have been proposed attributing diverse health benefits to probiotics. Ding et al. (2005) proposed that probiotic bacterial interference activity against pathogens is due to the inhibition of pathogens as evidenced by the fact that lactobacilli strain extracts caused down-regulation of flaA sigma 28 promoter involved in flagella assembly in Campylobacter jejuni. Similar inhibitory results of bacterial adhesion by butyric and lactic acids were observed when probiotic strain Clostridium butyricum was coincubated with ETEC O157:H7 (Takahashi et al., 2004). Commercial probiotics claim beneficial effects such as improving growth performance, immunomodulation, and protective effects based on several hypotheses. This includes alteration of intestinal microflora, modification of intestinal morphology and transport properties, and by influencing the immune system (Simon et al., 2001). More detailed and specific action of probiotics is discussed in the following section.

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Structural and Functional Properties of Intestinal Mucosa Gut microbiota colonizing the swine intestine modifies the intestinal microstructure and induces functional changes in the intestinal mucosa (Vitini et al., 2000). There are different opinions on modes of actions for the probiotic effect exhibited by microorganisms in the intestinal epithelium. One possible mode of action is focused on the intestinal villi in the form of increased villi height and mucosal surface in the jejunum of probiotic supplemented swine which in turn would possibly influence nutrient absorption. Probiotic cultures such as Saccharomyces boulardii and Bacillus cereus var. toyoi have been shown to elicit a beneficial influence on the epithelial structure, crypt morphology, number of Ki-67 positive cells and total mucosal cell counts, including acid and neutral mucopolysaccharide secreting cells (Baum et al., 2002; Budino et al., 2005). Furthermore, probiotic supplementation in piglets has been shown to stimulate Na-coupled glucose and peptide transport in the small intestine thus increasing glucose and nutrient absorption (Breves et al., 2000). However, a few studies have reported that probiotic supplementation had no significant effects on gut morphology and histochemistry as they had no significant effect on villi height, crypt depth, enlargement factor, and number of goblet cells (Scharek et al., 2005; Reiter et al., 2006). These inconsistent results might be due to variation in the breed and age of the pig. Furthermore, scanning electron microscopic studies on intestinal villi have shown a high variability and therefore could not be associated with nutritional treatment (Wiese et al., 2003).

Immunologic Functions Indigenous bacterial colonization in the GIT of swine is important for the development and homeostasis of the host immune system (Ohashi et al., 2006). Following probiotic supplementation, bacteria interact with gut epithelial cells and stimulate the production of pro-and anti-inflammatory cytokines possibly in a strain-dependent manner (Lu and Walker, 2001; Hase and Ohno, 2006; Delcenserie et al., 2008). However, some parts of ingested probiotic bacteria induce mucosal immunity by stimulating T cells and B cells to produce IgG or IgA (Rescigno et al., 2001; Vaarala, 2003). Immunologic effects of indigenous probiotic

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microflora have been well studied in germ-free models. Beneficial effects include increased intraepithelial cells (IELs) and Peyer‘s patches (Umesaki and Setoyama, 2000) and an altered phenotype percentage of TCRαβ IELs (Imaoka et a., 1996; Umesaki and Setoyama, 2000). Furthermore, the number of immunoglobulin-producing cells in the lamina propria and their concentration in serum has been shown to increase (Cebra et al., 1998; Butler et al., 2000). The mode of action of probiotic cultures appears to be species specific or even strain-specific. Incorporation of E. faecium NCIMB 10415 in the feed led to significant changes on the immune system such as reduction in cytotoxic cells (CD8+) in jejunal epithelium and reduced IgG levels after weaning (Scharek et al., 2005, 2007a,b; Schierack et al., 2007, 2009). However, feeding Bacillus cereus var. toyoi had significantly improved systemic immune response of piglets such as enhanced ratio of CD4+ to CD8+ T cells. Factors such as hemolyisn production and existence as non-indigenous intestinal microflora of pigs are responsible for the immunologic functions (Schirack et al., 2007).

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Metabolic Functions Gastrointestinal bacteria ferment the indigestible carbohydrates and endogenous substances such as mucus, to produce SCFAs and vitamin K which in turn have beneficial effects for the swine host (Cherbut et al., 1997; Edwards et al., 1997; Sakata et al., 1997). Increase in the probiotic microflora in the large intestine promotes indigenous Lactobacilli, thus in turn stimulating lactate production. Lactate produced would normally be metabolized to acetate or propionate by lactate-utilizing bacteria (Desulfovibrio spp., Clostridium propionicum, Selenomonas spp., Veillonella spp., Propionibacterium spp., and Anaerovibrio spp.) and to butyrate by Megasphaera elsdenii, some Clostridium spp., Anaerostipes caccae, and Eubacterium hallii (Holdeman et al. 1974; Mackie and Gilchrist 1979; Kuchta and Abeles 1985; Gibson 1990; Ricke et al., 1996; Seeliger et al. 2002; Duncan et al. 2004; Bourriaud et al. 2005; Belenguer et al., 2006). Inclusion of Lactobacilli strains in swine diets has been reported to have several beneficial effects in the host including lowering of pH by lactic acid production, production of bacteriocins (Fayol-Messaoudi et al., 2005), and stimulation of macrophage activity (Bengmark, 2000). In swine, the cecum and colon are the predominant fermentation sites; generating energy in the form of SCFAs which can meet 30 % of the daily energy requirement (Stevens and Hume, 1995). Production of SCFAs especially butyrate promotes epithelial cell growth which in turn leads to an increase in cecal and colonic mucosal cell thickness (Tsukahara et al., 2003). However, production of SCFAs also acts as a chemical stimulus that controls intestinal motility i.e lower concentration promotes colonic motility while higher concentrations exhibit inhibitory activity (Cherbut et al., 1997; Edwards et al., 1997). Thus, in the large intestine (high SCFAs levels) the intestinal motility can be controlled by inhibitory activity of SCFAs and therefore facilitate the adherence of indigenous microflora against a rapid moving digesta (Ohashi and Ushida, 2009). The mode of action of organic acids and their salts can be attributed to (Mroz, 2005): (1) Diffusion of undissociated form across cell membrane of pathogens and thereby inhibiting the growth by inactivating decarboxylases and catalases, (2) pH barrier against the colonization on the brush border, (3) inhibitory action on the pathogens due to low pH, (4) SCFAs acting as as substrate or modulator for mucosal development, epithelial cell growth and thereby increasing the absorptive capacity, (6) providing precursors

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for synthesis of non-essential amino acids, DNA and higher lipids for intestinal growth, and (7) promoting blood flow and hypocholesterolemic effect. However, exposure of SCFAs to Salmonella Typhimurium along with other environmental conditions such as low pH and anaerobic conditions can also further enhance acid-resistance, and thereby increasing the persistence in food supply and ultimately affect the pre-harvest quality (Ricke, 2003; Kwon and Ricke, 1998; Kwon et al., 2000).

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Protective Functions Gastrointestinal microflora of the swine promote various beneficial activities to the host such as host-protective effects against pathogenic microorganisms by adhering to intestinal mucosa and thereby competing for nutrients, nutrient binding sites, and bacteriocin production (Teitelbaum and Walker, 2002). Probiotics enhance the mucosal barrier by stimulating innate immune activities such as phagocytosis, secretion of β-defencin and natural killer activity, secretion of IgA, and by stimulating the anti-inflammatory effect (Isolauri et al., 2001). Protective functions can also be attributed by the competitive exclusion (for space and nutrients) of exogenous pathogens from the intestinal lumen (Gueimonde et al., 2007). The GI microflora of swine have the ability to resist the colonization of certain intestinal pathogens such as coliforms (Hilman et al., 1994), and it has been found that addition of lactic acid bacteria (LAB) in vitro, demonstrated inhibitory activity against the viability of pathogens (Hillman et al., 1995). Several studies had demonstrated that LAB such as Lactobacill, Enterococcus or Bifidobacteria inhibited the attachment of pathogenic bacteria in both in vitro and in vivo (Chan et al., 1985; Blomberg et al., 1993; Bernet et al., 1994). In confirmation with aforesaid finding, Conway (1989) had investigated that pretreating the ileal cells with whole Lactobacillus cells can inhibit the adhesion of pathogenic E. coli. These inhibitory activities have been attributed to altering pH (Lehto et al., 1997), steric hindrance of binding sites (Ouwehand and Conway, 1996), or certain components of lysed cell wall (Ouwehand and Conway, 1996; Lehto et al., 1997). The protein nature of the mucus receptor in the small intestine of swine is responsible for the steric hindrance of the binding receptors of the E.coli and Enterococcus (Jin et al., 2000a,b). The other important factor that can inhibit the adhesion to the mucosa is the pH of the culture (Lehto and Salminen, 1997).

APPLICATIONS OF PROBIOTICS N SWINE PRODUCTION Probiotic cultures such as bacteria and yeasts singly or in combination play a vital role in swine health and nutrition that has the potential to improve the preharvest quality.

Bacterial Based Probiotics Probiotics cultures based on indigenous bacteria and their mechanism of action and beneficial effects are listed in Table 4. Jasek et al (1992a) suggested that adding Lactobacilli to weanling pig diets would enhance the growth performance and decrease the incidence of E.

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coli in feces. Furthermore, it was shown that Lactobacilli would decrease the finishing costs by 17 % when added to grower finished diets (Jasek et al., 1992b). In contrast to these findings, Harper et al., (1983) reported that there were no significant differences in growth performance when weanling pigs were fed with Lactobacillus spp. Feeding growing pigs with a combination of Lactobacilli and yeast improved average daily gain and feed efficiency (Gombo et al., 1995). This may be due to the positive influence of this combination on gut health and nutrient digestibility in growing pigs (Turner et al., 2001). Inclusion of L. casei cultures had significantly reduced the risk of post weaning diarrhea in piglets caused by ETEC (Kyriakis et al., 2001). In addition, other beneficial effects include production of lactate with simultaneous reduction in intestinal pH (Herrick, 1972), colonization to the intestinal tract (Muralidhara et al., 1977), and prevention of toxic amine synthesis (Hill et al., 1970a, b) Table 4. Summary of beneficial effects of probiotic supplementation and its mechanism of action in swine pre-harvest food safety Probiotic culture

Mechanism of action

Beneficial effects

Reference

Enterococcus faecium and E. faecalis

Reduction of intestinal bacterial colonization (E. coli) in suckling piglets. Decline of virulence gene expression and inflammatory response in the host Modulation of intestinal microflora, antibody defences of the gut, and regulation of systemic inflammatory cytokines production

Daily oral supplementation to piglets from birth to weaning reduced post-weaning diarrhea and improved growth performance.

Scharek et al., 2005; Zeyner and Boldt, 2006; Vahjen et al., 2007.

Reduced levels of Enterotoxigenic Escherichia coli (ETEC) in ileum. Improved daily weight gain and reducing diarrhea (E. coli K88) in postweaned piglets. Decreased incidence of necrotizing enterocolitis casued by Clostridium perfringens in neonatal piglets

Konstantinov et al., 2008; Zhang et al., 2010

Reduced the adherence of Salmonella, E. coli, and Clostridium spp.

Collado et al., 2007

Reduction in scours (K88 positive ETEC) within 24 hours after challenge Reduced morbidity and mortality in recently weaned piglets. Improved carcass quality in fattening pigs. Modulated the development of swine mucosal immunity and reduced intestinal bacterial translocation

Bhandari et al., 2008

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Lactobacillus plantarum L. sorbius L. rhamnosus

Bifidobacterium and Lactobacillus

Bifidobacterium lactis and L. rhamnosus

Promoting of beneficial commensal microbiota colonization that are capable of limiting mucosal atrophy, dysfunction and pathogen load. Decreased the adherence of pathogenic bacteria Lactic acid production. Competitive binding sites.

Bacillus subtilis

B. licheniformis and B. subtilis

Competition for binding sites, nutrients, and lactic acid production

Saccharomyces cerevisiae and/or Pediococcus acidilacti

Modulated the establishment of lymphocyte populations and IgA secretion in the gut and reduced bacterial translocation to mesenteric lymph nodes following ETEC infection

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Siggers et al., 2008.

Alexopolous et al., 2004. Lessard et al., 2009

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Bacillus spp. promote indigenous GI microflora by competing with pathogenic microorganisms such as Bacteroides, Enterococci and coliforms (Link et al., 2005; Link and Kovac, 2006; Simon, 2010). Feeding Bacillus spp. to growing pigs has yielded variable results. There have been reported improvements in growth rate, feed efficiency, better nitrogen retention and utilization, and decreases in mortality rates, incidence of diarrhea, and shedding of E. coli in the feces (Bonomi, 1992; Kyriakis et al., 1999; Scheuermann, 1993; Succi et al., 1995; and Tardani et al., 1996). Incorporation of Bacillus cereus var. toyoi in gestating sows had demonstrated the transfer of probiotic cultures to suckling piglets before pre-starter feed supplementation; possibly through contact with maternal feces (Simon, 2010). Incorporating Streptococcus sp. in weanling, growing, and finishing diets achieved enhanced growth rates, feed conversion, and survival (Koriewicz et al., 1992; Kumprecht et al., 1998; Roth et al., 1986; Underahl, 1983). However, some research findings demonstrated that there was no effect on feed conversion and average daily gain in weight; and such differences could be due to the discrepancy in breed, growing stage, complexity of the diet and environmental conditions (Cline et al., 1976; Pollman et al., 1980). Oral administration of Streptococcus faecium suppressed E. coli induced diarrhea and improved growth rate of gnotobiotic (germ-free) pigs (Underahl, 1983). The most common species of Enterococcus found in the normal intestinal microflora of the swine includes E. faecium, E. faecalis, E. hirae, and E. cecorum (Devriese et al., 1994). Administration of competitive exclusion cultures such as Enterococcus faecium in swine had demonstrated reduced mortality associated with enterotoxigenic E. coli diarrhea, Salmonella Cholerasuis in piglets (Anderson et al., 1999; Genovese et al., 2000; Harvey et al., 2003). Furthermore, probiotic preparations based on Enterococcus faecium (18C23) efficiently inhibited the adhesion of ETEC strains (K88, K99, or 987P-expressing strains) to the small intestine mucus; that frequently cause piglet diarrhea during the preweaning and immediate postweaning periods (Jin et al., 2000b). Pollmann et al. (2005) reported that dietary supplementation (50 mg/kg) of microencapsulated E. faecium SF68 (NCIMB 10415) (9 x 109 CFU/g) reduced both the rate of natural infection and the shedding of Chlamydia in swine. In addition, Enterococcus faecium (18C23) also demonstrated anti-adhesion properties to the pathogenic E. coli strains (K88) in porcine small intestine mucus (Jin et al., 2000b). These inhibitory properties can be attributed to the steric hindrance in binding sites of pathogenic E.coli and Enterococcus, (Jin et al., 2000b) and pH of the culture used in the in vitro studies (Lehto et al., 1997). A similar mechanism of action was also observed with Lactobacillus spp. inhibiting E. coli K88 (Chan et al., 1985; Chauviere et al., 1992; Bernet et al., 1994).

Application of Yeasts as Probiotics in Swine Yeasts are eukaryotic and unicellular fungi that have been used for both preventive and therapeutic effects against diarrhea and other gastrointestinal disturbances (Auclair, 2000). Yeast have unique growth characteristics and host beneficial effects (Table 5) in the digestive tract when compared to other probiotic bacteria due to factors such as a barrier effect by the resident gut flora, presence of antibiotics, and failure to induce morphological alteration of the intestinal mucosa (Buts et al., 1986) and to hydrolysis of bile acids (El Hennawy et al., 1994).

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In a probiotic context, Saccharomyces cerevisae is more popular because of its various health benefits followed by administration alone or in combination with other probiotic bacteria (Bertin et al., 1997a; Jurgens et al., 1997; Maloney et al., 1998; Bradley et al., 1994). Furthermore, S. cerevisae has GRAS (Generally Recognized As Safe) status from the US Food and Drug Administration. S. cerevisiae can adapt to the intestinal conditions but cannot colonize unlike conventional probiotic bacteria (Auclair, 2000). Yeast supplementation in monogastric animal diets established several beneficial effects in the host. These effects are mainly due to the mechanisms of action that includes brush border disaccharides, antiadhesive effect against pathogens, stimulation of non-specific immunity, toxin action inhibition, and antagonistic effects against pathogenic microorganisms (Table 5).

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Table 5. Summary of mechanism of action involved in yeast supplemented diets and their beneficial effects Activity

Mechanism of action

Beneficial effects in the host

References

Brush border disacharridases stimulation

Live yeast supplementation may mediate increased disacharridases (sucrase, maltase and lactase) activity through endoluminal realease of polyamines (spermine and spermidine)

Increase disacharridases activity and thereby decrease the incidence of diarrhea in the host

Buts et al., 1994

Mannans and Antiadhesive properties

Solutions of D-mannose can inhibit the agglutination property of bacteria that would interact with mannan in yeast cell walls.

Ofek et al., 1977; Oyofo et al., 1989

Immune system stimulation

Glucans (beta-(1-3)-linked) present in yeast cell wall stimulate the glucan receptors on peripheral blood leukocytes and extravascular macrophages, and thereby amplifies the host defense system that involves a cascade of interactions primarily mediated by cytokines.

Reduce the colonization of S. Typhimurium in broilers due to competition between yeast and the pathogen to bind the intestinal cells

Inhibiting toxic activities

Competitive binding to intestinal cell wall and receptors between yeasts and toxins produced by pathogens

Antagonsim against pathogenic microorganisms in vitro

Dose dependent inhibition activity of yeast in both solid and culture media.

Czop, 1986; Cuaron, 1999.

Enhanced growth performances of growing pigs followed by yeast supplementation

Proteases produced by S. cerevisiae can hydrolyze toxin A from Clostridium difficile and thereby inhibits binding of this toxin to its brush border glycoprotein receptor Antagonistic activities against Candida albicans, S. Typhimurium K88, E. coli

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Rodrigues et al., 1996; Castagliulo et al., 1996

Brugier and Patte, 1975; Auclair, 2001

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Metabolites produced from yeast fermentation includes enzymes, vitamins, saccharides may aid in growth, metabolism, and health of pigs (Shen et al., 2009). Incorporating S. cerevisae in the diet of piglets has demonstrated increased feed intake and body weight gain (Mathews et al., 1998). However, these beneficial effects were not reportedly apparent in studies conducted by Burnett and Neil (1997), Dusel et al., 1994, and Jurgens et al. (1997). These differences might be due to the differences in yeast, strain, mode of application, concentration of active culture, and growing stage of the swine. Carlson et al. (2005) demonstrated that incorporating yeast extract protein improved growth performance in grower and finisher pigs swine when compared to spray-dried animal plasma protein feeding.

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Limitation of Swine Probiotics It is well known that commercial probiotic cultures often have variable results from herd to herd. This led to skepticism on the functionality of the probiotics in the farming and frontline veterinary communities (Turner, 2001; Damgaard and McLaren, 2006). Two possible reasons for discrepancies are the probiotic strain losing its potency over time and commercial probiotics that do not meet standards (composition and viability of the strains) stated on the labels (Weese, 2002; Fasioli et al., 2003). Major issues interfere with the identification of specific health benefits of the probiotic cultures (Klaenhammer and Kullen, 1999): First of all, host-pathogen interactions and the viability of the probiotic culture in the host to produce beneficial effect are extremely complex. Secondly, identification of specific health benefits by specific probiotic strains, viability, and specific properties of the probiotic strain are difficult to elucidate. Finally, identification of effects caused by mono-strain and multi-strain probiotics can be multifaceted (Timmerman et al., 2004). Physico-chemical factors of probiotics that influence beneficial effects in the host depend on their ability to tolerate heat, osmotic stress, and oxygen stressors during processing and storage (Ross et al., 2005). In general, comparing different studies based on the efficacy and beneficial effects of probiotics is always a challenging task; often giving contradictory, inconsistent and irreproducible results. This depends on several factors such as probiotic culture composition, viability, administration level, mode of application (spray, feed, or water), and frequency of application (Cole, 1994; Chesson, 1994). In addition, age, environmental conditions, health status, farm and animal hygienic conditions may influence the efficacy of probiotics (Lessard et al., 2009). Several factors such as production environment (cleanliness, history of diseases in the farm, health status), source of probiotic, number of viable cells in the probiotic and their consistency, survivability and metabolic capabilities in the host gut, probiotic‘s host specificity, influence of feed processing (e.g., steam conditioning and pelleting) on survivability of the probiotic in the final prepared diet, and differences in the experimental conditions appear to play an important role in the effective responses observed following administration of probiotics (Hathaway, 1996; Williams, 1997). In addition, low doses of probiotic bacteria, undesirable interactions with supplemented antibiotics or therapeutics, genetic predisposition, health and age of the host can contribute to the inconsistent results (Kyriakis, 1999; Reiter et al., 2006).

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116

A. V. S. Perumalla, Navam. S. Hettiarachchy, Philip G. Crandall et al. Table 6. List of commercial probiotic in the market with the probiotic cultures and their beneficial effects

Commercial product

Active components

Company

CenBiot

Bacillus cereus

Centro de Biotecnologia (CenBiot), Brazil

BioPlus 2B

B. licheniformis and B. subtilis at the concentration of 3.2 x 109 CFU/g of preparation 5 x 106 Lactobacillus and Streptococcus CFU/mL

(Chr. Hansen A/S, Horsholm, Denmark

Levucell® SB

Saccharomyces cerevisiae boulardii CNCM I1079

Lallemand Animal Nutrition North America, WI

LSP 122

B. lichenifornis

Alpharma

Toyocerin®

B. toyoi (106viable spores)

Toyo Jozo Co. Japan

Ferlac-2

L. acidophilus, L.rhamnosus, E.faecium, S.thermophilus, and L.bulgaricus Sterptococcus faecium type Cernelle 68 Enterococcus faecium SF 68 NCIMB 10415

Rosell Institute, Montreal, Qubec

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Probios

Feed-Mate 68 Cylactin®

Chr. Hansen Biosystems, Milwaukee, WI

Anchor Labs, Inc., St. Joseph, MO. DSM Nutritional products, Switzerland

Beneficial effects in the host

References

Reduced the prevalence of diarrhea Significant increase in feed conversion, daily weight gain, and total weight gain in the nursery phase Improved feed efficiency and average daily gains in weaned pigs.

Zani et al., 1998

Enhanced average daily gain and feed consumption in pigs that were weaned into pens with non-littermates Improved average daily gain in weight in young growing pigs. Enhanced growth performance when combined with Bioplus 2B

Estienne et al., 2005

Improved weight gain, feed conversion ratio. Reduced and controlled the incidence of post weaning diarrhea (ETEC) in piglets

Kyriakis et al., 1999 Kyriakis et al., 1999 Rekiel et al., 2010 Letllier et al., 2000

Improved feed conversion

Pollman et al., 1980

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Link and Govac, 2006

Barker et al., 2003

Reiter et al., 2005

Probiotics in Swine Production

117

FUTURE DIRECTIONS Probiotics are alternative feed additives that can provide various health benefits for a sustainable and profitable swine production. Consistent results and their efficacy can be demonstrated by conducting several controlled field trials under various production conditions. Reporting the experimental data regarding the effects of probiotic supplementation should be standardized, and research involving multiple herds at farm-level is required to address and validate the safety, quality, and regulatory requirements of the commercial probiotics. a Furthermore, a better understanding the mode of action using advanced molecular biological techniques such as gene sequencing tools (to study the microbial populations before and after probiotic supplementation) and genetic engineering (to improve the probiotic abilities such as acid stability, adhesion factors and metabolite profile) will be crucial to develop better products. In depth research should be conducted or focused to determine the dose-response relationship in the host, dose required to maintain balance between the resident microflora and probiotic culture to achieve beneficial effects, and lag time required to observe the effect of probiotic following administration. Factors including environmental, seasonal variations, health status, and stage of host life-cycle must also be considered and be well communicated to the farming community for consistent results following probiotic supplementation.

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CONCLUSIONS Swine farming has evolved from backyard to commercial industry; thereby a huge impact on the quality and safety of the produce. Increase in antiobiotic resistance and regulatory compliance for trade (imports and exports) has led to the use of probiotics in swine farming. Probiotic supplementation in swine diets has potential to improve the preharvest quality and therefore has a promising future in swine nutrition. Several research studies had demonstrated the positive balance and impact on the GI microbiota, and thereby promoting animal health and production. Nevertheless, proper care must be taken while selecting the appropriate probiotic strain and considering the interrelationship between host and microflora. It is possible that selected probiotics along with other natural alternatives such as plant extracts, prebiotics, and bacteriocins would further strengthen the protective effects against the foodborne pathogens.

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amplified fragment length polymorphism analysis and Penner serotyping. Applied and Environmental Microbiology, 71, 1953–1958. Simon, O. (2010). An interdisciplinary study on the mode of action of probiotics in pigs. Journal of Animal and Feed Sciences, 19, 230–243. Slade, R. (2004). Food tube. Pig Intestine, 34, 22-24. Skjolaas, K.A., Burkey, T.E., Dritz, S.S., and Minton, J.E. (2006). Effects of Salmonella enterica serovars Typhimurium (ST) and Choleraesuis (SC) on chemokine and cytokine expression in swine ileum and jejunal epithelial cells. Veterinary Immunology and Immunopathology, 111, 199-209. Smith, J.L. (2003). The role of gastric acid in preventing foodborne disease and how bacteria overcome acid conditions. Journal of Food Protection, 66, 1292-1303. Smith, H.W. and Jones, J.E.T. (1963). Observation on the alimentary tract and its bacterial flora in healthy and diseased pigs. Journal of .Pathology and Bacteriology, 86, 387-412. Spencer, J.L., and Guan, J. (2004). Public health implications related to spread of pathogens in manure from livestock and poultry operations. Methods in Molecualr Biology, 268, 503–515. Stabel, T.J., Fedorka-Cray, P.J., and Gray, J.T. (2002). Neutrophil phagocytosis following inoculation of Salmonella Choleraesuis into swine. Veterinary Research Communications, 26, 103-109. Stege, H., Christensen, J., Nielsen, J.P. and Willeberg, P. (2001). Data-quality issues and alternative variable-screening methods in a questionnaire-based study on subclinical Salmonella enterica infection in Danish pig herds. Preventive Veterinary Medicine, 48, 35–54. Stevens, C.E., and Hume, I.D. (1995). Comparative Physiology of the Vertebrate Digestive System, 2nd ed, Cambridge University Press, Cambridge, UK. Svensmark, B., Nielsen, K., Willeberg, P., and Jorsal, S.E. (1989). Epidemiological studies of piglet diarrhoea in intensively managed Danish sow herds. II. Post-weaning diarrhoea. Acta Veterinaria Scandinavica, 30, 55–62. Swords, W.E., Wu, C.C., Champlin, F.R., and Buddington, R.K. (1993). Postnatal changes in selected bacterial groups of the pig colonic microflora. Biology of the Neonate, 63, 191– 200. Takahashi, M., Taguchi, H. Yamaguchi, H., Osaki, T., Komatsu, A. and Kamiya, S. (2004). The effect of probiotic treatment with Clostridium butyricum on enterohemorrhagic Escherichia coli O157:H7 infection in mice. FEMS Immunology and Medical Microbiology, 41, 219–226. Tam, C.C., O‘Brien, S.J., Adak, G.K., Meakins, S.M., and Frost, J.A. (2003). Campylobacter coli—an important foodborne pathogen. Journal of Infection, 47, 28–32. Teitelbaum, J.E., and Walker, W.A. (2002): Nutritional impact of pre- and probiotics as protective gastrointestinal organisms. Annual Review of Nutrition, 22, 107–138. Timmerman, H.M., Koning, C.J.M., Mulder, L., Rombouts, F.M., and Beynen, A.C. (2004). Monostrain, multistrain and multispecies probiotics – A comparison of functionality and efficacy. International Journal of Food Microbiology, 96, 219-233. Tsukahara T., Iwasaki, Y., Nakayama, K., and Ushida, K. (2003). Stimulation of butyrate production in the large intestine of weaning piglets by dietary fructooligosaccharides and its influence on the histological vartiables of the large intestinal mucosa. Journal Nutritional Science and Vitaminology, 49, 311–314.

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Umesaki, Y, and Setoyama, H. (2000). Structure of the intestinal flora responsible for development of the gut immune system in a rodent model. Microbes and Infection, 2, 1343–1351. Vaarala O. (2003). Immunological effects of probiotics with special reference to lactobacilli. Clinical and Experimental Allergy, 33, 11634–11640. Vial, P.A., Robins-Browne, R. , Lior, H., Prado, V., Kaper, J.B., Nataro, J.P., Maneval, D., Elsayed, A., and Levine, M.M. (1988). Characterization of enteroadherent-aggregative Escherichia coli, a putative agent of diarrheal disease. Journal of Infectious Diseases, 158, 70-79. Vitini, E., and Alvarez, S., Medina, M., Medici, M., de Budeguer, M.V., and Perdigon, G. (2000). Gut mucosal immunostimulation by lactic acid bacteria. Biocell, 24, 223-232. Vodovar, N., Flanzy, J., and François, A.C. (1964). Intestin grele du porc. I. – Dimensions en fonction de l‘age et du poids, etude de la junction du canal cholédoque et du canal pancreatic a celui-ci. Ann. Biol. Anim. Biochem. Biophys. 4, 27-34. Vondruskova, H., Slamova, R., Trckova, M., Zraly, Z., and Pavlik, I. (2010). Alternatives to antibiotic growth promoters in prevention of diarrhoea in weaned piglets: a review. Veterinarni Medicina, 55, 199–224 Wang, G.C.Y., and Wang, Y. (1996). The frequency of chimeric molecules as a consequence of PCR co-amplification of 16S rRNA genes from different bacterial species. Microbiology, 142, 1107–1114. Weese, J.S. (2002). Microbiological evaluation of commercial probiotics. Journal of American Veterinary Medicine Association, 220, 794-797. Wiese F., Simon O., Weyrauch, K.D. (2003). Morphology of the small intestine of weaned piglets and a noval method for morphometric evaluation. Anatomia Histologia Embryologia, 32, 102-109. Williams, R. J., and Heymann, D.L. (1998). Containment of antibiotic resistance. Science, 279, 1153-1154 Wilson, R.A. and Francis, D.H. (1986). Fimbriae and enterotoxins associated with E. coli serogroups isolated from clinical cases of porcine colibacillosis. American Journal of Veterinary Research, 47, 213–217. Wingstrand, A., Dahl, J., Thomsen, L.K., Jørgensen, L. and Jensen, B.B. (1997). Influence of dietary administration of organic acids and increased feed structure on Salmonella typhimurium infection in pigs, p. 170–172. In S. Bech-Nielsen and J. P. Nielsen (ed.), Proceedings of the 2nd International Symposium on Epidemiology and Control of Salmonella in Pork. Federation of Danish Pig Producers and Slaughterhouses, Copenhagen, Denmark. Young, C. R., Harvey, R., Anderson, R., Nisbet, D., and Stanker, L.H. (2000). Enteric colonization following natural exposure to Campylobacter in pigs. Research in Veterinary Science, 68, 75–78. Zani, J.L., da Cruz, F.W., dos Santos, A.F. and Gil-Turnes, C. (1998). Effect of probiotic CenBiot on the control of diarrhoea and feed efficiency in pigs. Journal of Applied Microbiology,84, 68–71.

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In: On-Farm Strategies to Control Foodborne Pathogens ISBN: 978-1-62100-411-0 Editors: Todd R. Callaway and Tom S. Edrington © 2012 Nova Science Publishers, Inc.

Chapter 6

VACCINATION AS A METHOD OF E. COLI O157:H7 REDUCTION IN FEEDLOT CATTLE David R. Smith and Amanda R. Vogstad Schoolof Veterinary Medicine and Biomedical Sciences, University of Nebraska - Lincoln, Lincoln, Nebraska, US

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ABSTRACT Escherichia coli O157:H7 (STEC O157) is ubiquitous to cattle populations, making cattle an important reservoir for human exposure to STEC O157 via direct contact or through contaminated food or water. In addition to post-harvest strategies for control, there may be benefits to the beef industry and public health from reducing STEC O157 carriage by live cattle. One strategy for reducing the prevalence of carriage by cattle isto use vaccines to create an unfavorable gut environment for STEC O157. Vaccines directed against antigens involved with STEC O157 enteric colonization or iron uptake have shown efficacy in animal challenge studies and under feedlot conditions of natural exposure on various measures of fecal and hide contamination. The level of effectiveness afforded by vaccination depends on how the products are used to control environmental transmission within groups of cattle, and by methods to maintain pre-harvest treatment effect throughout the food system; for example, by preventing cross-contamination of cattle or beef product.Risk models suggest the level of efficacy of current vaccines may be sufficient to reduce the load of STEC O157 carried to the abattoir with live cattle and ultimately reduce public health risk.It is not yet clear if vaccines or other pre-harvest food safety interventions will be widely adopted by the beef industry because there is not yet an economic signal to indicate that cattle receiving these interventions are valued over cattle without.

INTRODUCTION Infection with the Shiga toxin producing Escherichia coli (STEC) and subsequent human illnessoriginates from direct or indirect exposure of people to contaminated human or animal feces [1].It is widely held that cattle populations serve as an important reservoir of STEC for

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human exposure.This notion is supported by evidence that: 1) STEC O157, the most studied STEC, is ubiquitous to fed cattle populations [2]; 2) that non-intact beef products are the source of approximately one-third of human STEC O157 infections [3]; and 3) that other foods, water, and environments may become contaminated with STEC O157 that likely originated from cattle feces[4].The highly correlated seasonal pattern of human infection, ground beef contamination, and cattle fecal shedding prevalence, all being greater in summer months, provides additional circumstantial evidence of the important role of live cattle in human STEC O157 exposure[5].Various risk models suggest a potential for public health benefit by efforts to reduce STECO157 carriage by live cattle[3,5,6,7]. There is evidence that the prevalence of carriage of STEC O157 by live cattle is a correlate of subsequent rates of carcass contamination [8,9].Some have speculated that efforts to control STEC O157 prevalence in cattle production systems would improve food safety and reduce opportunities for human environmental exposure to the pathogen[8]. Prevalence of STEC O157 fecal shedding by cattle is highly variable and is a function of the incidence at which cattle are exposed to the organism and the duration of colonization and shedding[2,10]. The prevalence of STEC O157 carriage by live cattle in various production systems differs by season [11,12,13,14,15].There is also meaningful variation in prevalence of STEC O157 carriage within seasons [2,10]and this has been explained by environmental factors that favor bacterial survival and subsequent fecal-oral transmission [12].However, to date, efforts to reduce exposureof cattle to STEC O157 in the environmenthas been unrewarding; for example, by chlorinating water sources, or scraping feedlot pen surfaces, [16,17,18].This is likely because of the difficulty in simultaneously controlling all environmental sources of the agent, including primarily the cattle themselves [17].Successful efforts to reduce the prevalence of STEC O157 carriage by live cattle have done so by creating a gut environment which is unfavorable to STEC O157 survival, colonization, and subsequent fecal shedding[19,20,21]. Vaccinating cattle against STEC O157 creates an unfavorable gut environment for the pathogen, thereby reducing duration of carriage.The desired outcome from vaccination is to reducethe prevalence of STEC O157 carriage in discrete cattle populations(e.g. pens or herds of cattle).If vaccination successfully reduces the duration of carriage by making the gut unfavorable to STEC O157, this in theory should result in reduced shedding of the pathogen into the cattle environment, reduced rates of fecal-oral exposure, reduced contamination of cattle hides, and reduced carriage of the pathogen at harvest.To be useful as a pre-harvest intervention the benefits of vaccination must not be undone during subsequent management practices such as transportation to the abattoir[22] or holding in lairage[9].Finally, to be adopted, preharvest interventions must be sufficiently valued to offset the cost of implementation. Some potential cattle vaccines against STEC O157 have been tested in animal challenge studies or under field conditions of natural exposure. These vaccines have either undefined antigen targets in the form of bacterial extracts, or they are directed against specific antigens that function to enable bacterial colonization or iron uptake. The enterohemorrhagic E.coli(EHEC), including STEC O157, colonize intestinal epithelial cells by a type III secreted protein (TTSP) system. Components of the TTSP system include intimin, an outer membrane bacterial receptor; translocatedintimin receptor (Tir),a receptor injectedinto the host epithelial cell membrane; Esp A, which acts as an injection filament for delivering Tir to the host cell membrane; and Esp B/EspD, which form a pore in

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the host cell membrane [23,24,25].The H7 flagellin is also believed to function in STEC O157:H7 colonization [26,27,28].

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STEC O157 VACCINE CHALLENGE EXPERIMENTS Vaccines which target antigens associated with the TTSP have been tested in experimental challenge studies. Vaccination of sows with an intimin vaccine protected suckling pigs from colonization and microscopic intestinal damage when challenged with 106 CFU of a Shiga toxin-negative strain of EHEC O157:H7 [29].A bacterial supernatant with TTSP reduced the probability, magnitude, and duration of shedding by calves challenged with STEC O157[19].In a follow-up study calves receiving the same vaccine product were less likely to shed STEC O157 in the feces and shed at a lower fecal concentration [30]. Calves vaccinated with Esp A responded with high antigen specific antibody titers but were not protected against colonization with STEC O157 following challenge [31].Following an oral challenge with STEC O157, two month old calves vaccinated intramuscularly with H7 flagellin had reduced rates of colonization and delayed peak bacterial shedding, but did not show a reduction in total bacterial shedding[32].A vaccine prepared with intimin,EspA, and Tir significantly reduced STEC O157 colonization and bacterial counts of orally inoculated calves [33].Also, following an oral challenge with STEC O157, lambs which had been vaccinated with intimin, EspA, and Esp B shed fewer bacteria in feces than placebo treated controls [34]. When iron is in low supply Gram-negative bacteria utilize an iron transport system to acquire the nutrient.Siderophore receptor and porin (SRP) vaccines create an immune response against the bacterial cell membrane proteins responsible for iron transport[35].Preventing iron intake puts STEC O157 at a competitive disadvantage to other gut microbiota[36].In a study of beef calves orally inoculated with STEC O157 the SRP vaccine reduced fecal prevalence and bacterial concentration to a level approaching statistical significance [35].

FIELD EFFICACY OF STEC O157 VACCINES The results of experimental challenge studies may not predict vaccine performance in field conditions because in cattle production settings the dynamics of transmission and sources of exposure are more complex.Only a few vaccine products have been evaluated for efficacy in conditions of natural exposure.An undefined bacterial extract was not effective at reducing STEC O157 carriage in feedlot cattle[37]. However, two vaccine products, targeting TTSP or SRP, have demonstrated efficacyin multiple field trials conducted in dry-lot beef feedlots with environmental conditions typical of the Great Plains region of the United States and Canada.A systematic review and meta-analysis of STEC O157 cattle vaccines indicated sufficient data to conclude that both vaccines effectively reduced the probability for cattle to shed STEC O157 in feces [38]. From among a population of feedlot cattle initially screened for STEC O157 carriage, those steers vaccinated twice with eithertwo or three millilitersof the SRP vaccine were, 14

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percent and 47 percent less likely than placebo treated steers to have STEC O157 detected in either feces or recto-anal mucosa swab samples, respectively.Cattle receiving the threemilliliter dose regimen were significantly less likely than controls to shed the agent,did so for fewer days, and were less likely to shed the organism at high levels [39].In another study, feedlotcattle receiving a two-milliliter, two-dose SRP vaccine regimen did not differ from controls in STEC O157 carriage over the post-vaccination period;however, STEC O157 carriage was less likely among vaccinates on the last day of the study [36]. In a trial testing a two-milliliter,three-dose SRP vaccine regimen against placebo-treated cattle, the vaccine was 85 percenteffective at reducing the probability to detect STEC O157 in fecesand reduced STEC O157 concentration 1.7 logs compared to controls56 days after the last dose of vaccine [36].The vaccine has not affected cattle performance [36]. An initial field study of TTSP vaccine failed to find a significant effect [40].However, efficacy improved after the vaccine product was reformulated.A three dose regimen of TTSP vaccinehas significantly reduced the probabilityfor feedlot cattle to shed STEC O157 by 43 percent to 73 percentin several randomized controlled trials [19,41,42,43].In addition, the vaccine was 92 percent and 98 percent effectiveat reducing the probability for colonization of the terminalrectumusing two [44] or three [45] dose regimens, respectively.Two doses of the same vaccine product has significantly reduced carriage of STEC O157 by feedlot cattle[44,46,47]; however,three doses of vaccine were more effective than two doses in two separate trials with direct comparisons [41,42].The vaccine does not appear to affect growth performance[42,45]or carcass quality [42,45,47].

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POPULATION EFFECTS OF VACCINATION Beyond the effectiveness of the vaccine to induce immunity in individual cattle, grouplevel factors are important considerations for understanding and optimizingSTEC O157 vaccine effect.For example, there is evidence that fecal-oral transmission of STEC O157 is reduced within pens of vaccinated cattle.In a longitudinalstudy, non-vaccinated cattle housed in pens with vaccinated cattle were less likely to shed STEC O157 compared to contemporaneously sampled cattle in the same feedyard housed in pens where none of the cattle received vaccine.These results suggested evidence of herd-immunity [42].Vaccinated cattle in large commercial pens were less likely to have oral exposure to STEC O157 compared to cattle in non-vaccinated pens, based on culture of ropes hung on feedbunk rails for cattle to chew [46].Culture of STEC O157 from ropes is correlated to fecal prevalence[48], but more directly measures oral exposure [49].Further evidence of the importance of considering group-level vaccination effects is thatsignificantly greater efficacy in reducing hide contamination was observed when all of the cattle in a region of the feedyard were vaccinated compared to the efficacy observed when vaccinated and unvaccinated cattle were commingled within pens [47]. The efficacy of pre-harvest interventions may be lost due to events occurringduring later stages of the food system. For example, the reduction in prevalence that occurs in live cattle post-vaccination might be undone by cross contamination of cattle hides with STEC O157 during transportation and lairage [9,22].Therefore, to preserve pre-harvest treatment effects it may be necessary to devise systems for cattle handling so that pre-harvest benefits are

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retained post-harvest.There is evidence that the efficacy of pre-harvest interventions can persist into the abattoir.In a randomized clinical trial of a STEC O157 cattle vaccine, there was a significant increase in the prevalence of hide contamination between the time periodimmediately prior to loading at the feedyardversus just prior to hide removal in the abattoir.However, vaccination treatments were equally effective at reducing hide contamination in the feedyard and at the abattoir.The preservation of vaccine efficacy into the abattoir may have been because treatment groups were maintained as cattle were loaded into clean trucks for transportation [47].

MODELING THE VALUE OF VACCINATION

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The goal of vaccinating cattle against STEC O157 is to improve public health and reduce costs to the beef industry due to recalls, lost product value, and liability.Therefore, although there is value in preventing human illness due to direct contact with cattle environments, the primaryfood-safety value from vaccinating live cattle must continue downstream to the postharvest sectors of the food system.

Figure 1. Box and whisker plot of observed STEC O157 fecal shedding prevalence from 44 Nebraska feedlot pens (n= 4,952 cattle) tested May through October (Summer) and 30 feedlot pens (n= 2,941) tested January through March (Winter) compared to the predicted prevalence of summer-fed pens after using an intervention with 65 percent efficacy (pert distribution with mean 65%, low 50%, high 80%, 5,000 pen replications).Plus signs indicate mean pen prevalence, whiskers indicate range of 90 percent of prevalence observations (5th to 95th percentiles) and boxes indicate 75th, 50th, and 25th percentiles.The model predicts that the mean intervention prevalence would approximate the winter mean, but variability of prevalence among vaccinated summer-fed cattle would be less than that observed for winter or summer-fed cattle.

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If the costs to use an intervention exceed the benefits to industry or to public health then the intervention is not likely to be used.For example, public health policy analysts might compare the cost of human illness to the cost to implement a pre-harvest intervention. If the marginal costs of vaccinating cattle was held equivalent to the marginal benefit to public health, then as the cost of a vaccine intervention increases, fewer cattle would bevaccinated, and as a resultfewer human illnesses would beprevented. Similarly, the marginal number of cattle that must receive an intervention in order to prevent a human illness, and therefore the pre-harvest cost, changes with the effectiveness of the product[3]. The beef industry might view the value of pre-harvest intervention on the basis of how the prevalence of cattle carrying STEC O157 into the abattoir affects subsequent food safety costs. For example, an important source of STEC O157 carcass contamination is the hide[9] and fecal shedding prevalence above 20 percent has been associated with higher levels of hide contamination prevalence[50].Therefore, post harvest sectors of the beef industry might benefit from preharvest interventions that supplied cattle with lower and less variable fecal shedding prevalence while in the feedlot because hide contamination at harvest would be less likely.A simulation model(Figure 1) suggests that a pre-harvest intervention with 65 percent efficacy for reducing fecal shedding might reduce the distribution of pen-level fecal shedding prevalence to levels comparable to the differences observed between winter and summer [51].Because ofthe correlated seasonal variation in STEC O157 prevalence in live cattle, prevalence of STEC O157 in ground beef, and human incidence of STEC O157 illness, this level of efficacy might be valuable to public health as well [5].

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CONCLUSION For pre-harvestinterventions against STEC O157 to meaningfully improve food safety they must be: 1) efficacious –demonstrate that cattle are less likely to carry the organism; 2) effective –work as a food safety intervention within the beef production system; and 3) economical –add sufficient value to pay for the cost of the intervention.Some vaccine products have demonstrated efficacy to reduce the prevalence of cattle carrying STEC O157 by making the gut environment unfavorable to the organism.The level of efficacy afforded by vaccination depends on how the products are used to control environmental transmission within groups of cattle and by methods to maintain pre-harvest treatment effect throughout the food system, for example, by preventing cross-contamination of cattle or beef product.It is not yet clear if vaccines or other pre-harvest food safety interventions will be widely adopted by the beef industry because there is not yet an economic signal to indicate that cattle receiving these interventions are valued over cattle without.

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Smith, D.R., Blackford, M.P., Younts, S.M., Moxley, R.A., Gray, J.T., Hungerford, L.L., Milton, C.T., and Klopfenstein, T.J. (2001). Ecological relationships between the prevalence of cattle shedding Escherichia coli O157:H7 and characteristics of the cattle or conditions of the feedlot pen. J.Food Prot., 64, 1899-1903. Withee, J., Williams, M., Schlosser, W., Bauer, N., and Ebel, E. (2009). Streamlined analysis for evaluating the use of preharvest interventions intended to prevent Escherichia coli O157:H7 illness in humans. Foodborne Path Dis, 6, 817-825. Renter, D.G. and Sargeant, J.M. (2002). Enterohemorrhagic Escherichia coli O157: epidemiology and ecology in bovine production environments. Anim Health Res.Rev., 3, 83-94. Williams, M.S., Withee, J.L., Ebel, E.D., Bauer, N.E., Scholosser, W.D., Disney, W.T., Smith, D.R., and Moxley, R.A. (2010). Determining relationships between the seasonal occurrence of Escherichia coli O157:H7 in live cattle, ground beef, and humans. Foodborne Path Dis, 7, 1-8. Jordan, D., McEwen, S.A., Lammerding, A.M., McNab, W.B., and Wilson, J.B. (1999). Pre-slaughter control of Escherichia coli O157 in beef cattle: a simulation study. Preventive Veterinary Medicine, 41, 55-74. Ebel, E., Schlosser, W., Kause, J., Orloski, K., Roberts, T., Narrod, C., Malcolm, S., Coleman, M., and Powell, M. (2004). Draft risk assessment of the public health impact of Escherichia coli O157:H7 in ground beef. Journal of Food Protection, 67, 19911999. Elder, R.O., Keen, J.E., Siragusa, G.R., Barkocy-Gallagher, G.A., Koohmaraie, M., and Laegreid, W.W. (2000). Correlation of enterohemorrhagic Escherichia coli O157 prevalence in feces, hides, and carcasses of beef cattle during processing. Proc.Natl.Acad.Sci.U.S.A, 97, 2999-3003. Arthur, T.M., Bosilevac, J.M., Nou, X., Shackelford, S.D., Wheeler, T.L., Kent, M.P., Jaroni, D., Pauling, B., Allen, D.M., and Koohmaraie, M. (2004). Escherichia coli O157 prevalence and enumeration of aerobic bacteria, Enterobacteriaceae, and Escherichia coli O157 at various steps in commercial beef processing plants. J.Food Prot., 67, 658-665. Khaitsa, M.L., Smith, D.R., Stoner, J.A., Parkhurst, A.M., Hinkley, S., Klopfenstein, T.J., and Moxley, R.A. (2003). Incidence, duration, and prevalence of Escherichia coli O157:H7 fecal shedding by feedlot cattle during the finishing period. J Food Prot., 66, 1972-1977. Hancock, D., Besser, T.E., Rice, D.H., Herriot, D.E., and Tarr, P.I. (1997). A longitudinal study of Escherichia coli O157:H7 in fourteen cattle herds. Epidemiol Infect., 118, 193-195. Smith, D.R., Moxley, R.A., Clowser, S.L., Folmer, J.D., Hinkley, S., Erickson, G.E., and Klopfenstein, T.J. (2005). Use of rope devices to describe and explain the feedlot ecology of Escherichia coli O157:H7 by time and place. Foodborne.Pathog.Dis., 2, 5060. Renter, D.G., Smith, D.R., King, R., Stilborn, R., Berg, J., Berezowski, J., and McFall, M. (2008). Detection and determinants of Escherichia coli O157:H7 in Alberta feedlot pens immediately prior to slaughter. Can J Vet Res., 72, 217-227.

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[14] Chapman, P.A., Siddons, C.A., Cerdan Malo, A.T., and Harkin, M.A. (1997). A 1-year study of Escherichia coli O157:H7 in cattle, sheep, pigs and poultry. Epidemiology and Infection, 119, 245-250. [15] Van Donkersgoed, J., Graham, T., and Gannon, V. (1999). The prevalence of verotoxins, Escherichia coli O157:H7, and Salmonella in the feces and rumen of cattle at processing. Can.Vet J, 40, 332-338. [16] LeJeune, J.T., Besser, T.E., Rice, D.H., Berg, J.L., Stilborn, R.P., and Hancock, D.D. (2004). Longitudinal study of fecal shedding of Escherichia coli O157:H7 in feedlot cattle: predominance and persistence of specific clonal types despite massive cattle population turnover. Appl.Environ.Microbiol., 70, 377-384. [17] Smith, D.R., Klopfenstein, T., Moxley, R.A., Milton, C.T., Hungerford, L.L., and Gray, J.T. (2002). An evaluation of three methods to clean feedlot water tanks. The Bovine Practitioner, 36, 1-4. [18] Folmer, J., Macken, C., Moxley, R., Smith, D., Brashears, M., Hinkley, S., Erickson, G., and Klopfenstein, T. (2003). Intervention strategies for reduction of E. coli O157:H7 in feedlot steers. Nebraska Beef Cattle Report, MP 80-A, 22-23. [19] Potter, A.A., Klashinsky, S., Li, Y., Frey, E., Townsend, H., Rogan, D., Erickson, G., Hinkley, S., Klopfenstein, T., Moxley, R.A., Smith, D.R., and Finlay, B.B. (2004). Decreased shedding of Escherichia coli O157:H7 by cattle following vaccination with type III secreted proteins. Vaccine, 22, 362-369. [20] Peterson, R.E., Klopfenstein, T.J., Erickson, G.E., Folmer, J., Hinkley, S., Moxley, R.A., and Smith, D.R. (2007). Effect of Lactobacillus acidophilus strain NP51 on Escherichia coli O157:H7 fecal shedding and finishing performance in beef feedlot cattle. J.Food Prot, 70, 287-291. [21] Younts-Dahl, S.M., Galyean, M.L., Loneragan, G.H., Elam, N.A., and Brashears, M.M. (2004). Dietary supplementation with Lactobacillus- and Propionibacterium-based direct-fed microbials and prevalence of Escherichia coli O157 in beef feedlot cattle and on hides at harvest. J.Food Prot., 67, 889-893. [22] Miller, M.F., Loneragan, G.H., Harris, D.D., Adams, K.D., Brooks, J.C., and Brashears, M.M. (2008). Environmental dust exposure as a factor contributing to an increase in Escherichia coli O157:H7 and Salmonella Populations on cattle hides in feedyards. Journal of Food Protection, 71, 2078-2081. [23] Garmendia, J., Frankel, G., and Creprin, V.F. (2005). Enteropathogenic and enterohemorrhagic Escherichia coli infections: translocation, translocation, translocation. Infection and Immunity, 73, 2573-2585. [24] Goosney, D.L., Knoechel, D.G., and Finlay, B.B. (1999). Enteropathogenic E.coli, Salmonella, and Shigella: masters of host cell cytoskeletal exploitation. Emerg Infect Dis, 5, 216-223. [25] Moxley, R.A. (2004). Escherichia coli O157:H7: an update on intestinal colonization and virulence mechanisms. Anim Health Res.Rev., 5, 15-33. [26] Mahajan, A., Currie, C.G., Mackie, S., Tree, J., McAteer, S., McKendrick, I., McNeilly, T.N., Roe, A., La Ragione, R.M., Woodward, M.J., Gally, D.L., and Smith, D.G.E. (2009). An investigation of the expression and adhesin function of H7 flagella in the interaction of Escherichia coli O157:H7 with bovine intestinal epithelium. Cell Microbiol, 11, 121-137.

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[27] Erdem, A.L., Avelino, F., Xicohtencatl-Cortes, J., and Giron, J.A. (2007). Host protein binding and adhesive properties of H6 and H7 flagella of attaching and effacing Escherichia coli. Journal of Bacteriology, 189, 7426-7435. [28] Bretschneider, G., Berberov, E.M., and Moxley, R.A. (2007). Reduced intestinal colonization of adult beef cattle by Escherichia coli O157:H7 tir deletion and nalidixixacid-resistant mutants lacking flagellar expression. Veterinary Microbiology, 125, 381386. [29] Dean-Nystrom, E.A., Gansheroff, L.J., Mills, M., Moon, H.W., and O'Brien, A.D. (2002). Vaccination of pregnant dams with intimin(O157) protects suckling piglets from Escherichia coli O157:H7 infection. Infect.Immun., 70, 2414-2418. [30] Allen, K.J., Rogan, D., Finlay, B.B., Potter, A.A., and Asper, D.J. (2011). Vaccination with type III secreted proteins leads to decreased shedding in calves after experimental infection with Escherichia coli O157. The Canadian Journal of Veterinary Research [31] Dziva, F., Vlisidou, I., Creprin, V.F., Wallis, T.S., Frankel, G., and Stevens, M.P. (2007). Vaccination of calves with EspA, a key colonisation factor of Escherichia coli O157:H7, induces antigen-specific humoral responses but does not confer protection against intestinal colonisation. Veterinary Microbiology, 123, 254-261. [32] McNeilly, T.N., Naylor, S.W., Mahajan, A., Mitchell, M.C., McAteer, S., Deane, D., Smith, D.G.E., Low, J.C., Gally, D.L., and Huntley, J.F. (2008). Escherichia coli O157:H7 colonization in cattle following systemic and mucosal immunization with purified H7 flagellin. Infection and Immunity, 76, 2594-2602. [33] McNeilly, T.N., Mitchell, M.C., Rosser, T., McAteer, S., Low, J.C., Smith, D.G.E., Huntley, J.F., Mahajan, A., and Gally, D.L. (2010). Immunization of cattle with a combination of purified intimin-531, EspA and Tir significantly reduces shedding of Escherichia coli O157:H7 following oral challenge. Vaccine, 28, 1422-1428. [34] Yekta, M.A., Goddeeris, B.M., Vanrompay, D., and Cox, E. (2011). Immunization of sheep with a combination of intimin, EspA and EspB decreases Escherichia coli O157:H7 shedding. Veterinary Immunology and Immunopathology, 140, 42-46. [35] Thornton, A.B., Thomson, D.U., Loneragan, G.H., Fox, J.T., Burkhardt, D.T., Emery, D.A., and Nagaraja, T.G. (2009). Effects of a siderophore receptor and porin proteinsbased vaccination on fecal shedding of Escherichia coli O157:H7 in experimentally inoculated cattle. Journal of Food Protection, 72, 866-869. [36] Thomson, D.U., Loneragan, G.H., Thornton, A.B., Lechtenberg, K.F., Emery, D.A., Burkhardt, D.T., and Nagaraja, T.G. (2009). Use of a siderophore receptor and porin proteins-based vaccine to control the burden of Escherichia coli O157:H7 in feedlot cattle. Foodborne.Pathog.Dis., 6, 871-877. [37] Woerner, D.R., Ransom, J.R., Sofos, J.N., Scanga, J.A., Smith, G.C., and Belk, K.E. (2006). Preharvest processes for microbial control in cattle. Food Protection Trends, 26, 393-400. [38] Snedeker, K.G., Campbell, M., and Sargeant, J.M. (2011). A systematic review of vaccinations to reduce the shedding of Escherichia coli O157 in the faeces of domestic ruminants. Zoonoses and Public Health, 10.1111/j.1863-2378.2011.01426.x [39] Fox, J.T., Thomson, D.U., Drouillard, J.S., Thornton, A.B., Burkhardt, D.T., Emery, D.A., and Nagaraja, T.G. (2009). Efficacy of Escherichia coli O157:H7 siderophore receptor/porin proteins-based vaccine in feedlot cattle naturally shedding E. coli O157. Foodborne.Pathog.Dis., 6, 893-899.

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[40] Van Donkersgoed, J., Hancock, D., Rogan, D., and Potter, A.A. (2005). Escherichia coli O157:H7 vaccine field trial in 9 feedlots in Alberta and Saskatchewan. Can.Vet.J., 46, 724-728. [41] Moxley, R.A., Smith, D.R., Luebbe, M., Erickson, G.E., Klopfenstein, T.J., and Rogan, D. (2009). Escherichia coli O157:H7 vaccine dose-effect in feedlot cattle. Foodborne Path Dis, 6, 879-884. [42] Peterson, R.E., Klopfenstein, T.J., Moxley, R.A., Erickson, G.E., Hinkley, S., Rogan, D., and Smith, D.R. (2007). Efficacy of dose regimen and observation of herd immunity from a vaccine against Escherichia coli O157:H7 for feedlot cattle. J.Food Prot, 70, 2561-2567. [43] Rich, A.R., Jepson, A.N., Luebbe, M., Klopfenstein, T.J., Smith, D.R., and Moxley, R.A. (2010). Vaccination to reduce the prevalence of Escherichia coli O157:H7 in feedlot cattle fed wet distillers grains plus solubles. Nebraska Beef Cattle Report, 9495. [44] Smith, D.R., Moxley, R.A., Peterson, R.E., Klopfenstein, T.J., Erickson, G.E., Bretschneider, G., Berberov, E.M., and Clowser, S. (2009). A two-dose regimen of a vaccine against Type III secreted proteins reduced Escherichia coli O157:H7 colonization of the terminal rectum in beef cattle in commercial feedlots. Foodborne Pathog.Dis, 6, 155-161. [45] Peterson, R.E., Klopfenstein, T.J., Moxley, R.A., Erickson, G.E., Hinkley, S., Bretschneider G., Berberov, E.M., Rogan, D., and Smith, D.R. (2007). Effect of a vaccine product containing type III secreted proteins on the probability of Escherichia coli O157:H7 fecal shedding and mucosal colonization in feedlot cattle. J.Food Prot, 70, 2568-2577. [46] Smith, D.R., Moxley, R.A., Peterson, R.E., Klopfenstein, T., Erickson, G.E., and Clowser, S.L. (2008). A two-dose regimen of a vaccine against Escherichia coli O157:H7 type III secreted proteins reduced environmental transmission of the agent in a large-scale commercial beef feedlot clinical trial. Foodborne.Pathog.Dis., 5, 589-598. [47] Smith, D.R., Moxley, R.A., Klopfenstein, T.J., and Erickson, G.E. (2009). A randomized longitudinal trial to test the effect of regional vaccination within a cattle feedyard on Escherichia coli O157:H7 rectal colonization, fecal shedding, and hide contamination. Foodborne.Pathog.Dis., 6, 885-892. [48] Smith, D.R., Gray, J.T., Moxley, R.A., Younts-Dahl, S.M., Blackford, M.P., Hinkley, S., Hungerford, L.L., Milton, C.T., and Klopfenstein, T.J. (2004). A diagnostic strategy to determine the Shiga toxin-producing Escherichia coli O157 status of pens of feedlot cattle. Epidemiol.Infect., 132, 297-302. [49] Irwin, K.E., Smith, D.R., Gray, J.T., and Klopfenstein, T.J. (2002). Behavior of cattle towards devices to detect food-safety pathogens in feedlot pens. Bovine Pract., 36, 5-9. [50] Arthur, T. M., Keen,J., Bosworth,B.T., Brichta-Harhay,D.M., Kalchayanand,N., Shackelford,S.D., Wheeler,T.L., Nou,X., and Koohmaraie,M. (2009). Longitudinal study of Escherichia coli O157-H7 in a beef cattle feedlot and role of high-level shedders in hide contamination. 7, 6515-6523. [51] Smith, D.R. (2010). Is vaccination of cattle against E. coli O157 effective? International Conference on Emerging Infectious Diseases, Atlanta, GA.

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Chapter 7

VACCINATION TO REDUCE FOODBORNE BACTERIAL PATHOGENS IN SWINE, WITH EMPHASIS ON SALMONELLA Filip Boyen, Frank Pasmans and Freddy Haesebrouck Laboratory of Veterinary Bacteriology and Mycology, Faculty of Veterinary Medicine, Ghent University, Salisburylaan 133 B9820 Merelbeke – Belgium

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ABSTRACT The main microbiological risk hazards related to the consumption of pork are of bacterial origin. Bacterial vaccines for use in pigs aiming to reduce infections in humans are only available for Salmonella. Scientific literature suggests that Salmonella vaccination is associated with reduced Salmonella prevalence in swine and that it can have an economic incentive for pig producers. Best results are expected from live attenuated vaccines. Vaccination of both fattening pigs and sows could play a role in lowering the number of Salmonella positive slaughter pigs. Current vaccines, however, do not allow the differentiation of infected from vaccinated animals. Information on the pathogenesis of non-typhoidal Salmonella infections in pigs and on the protective role of the different immunological responses should form the basis to create new or improve existing vaccines.

INTRODUCTION The main microbiological risk hazards related to the consumption of pork are of bacterial origin: Salmonella, Yersinia, Listeria and Campylobacter are considered to be the 4 most important pork-related foodborne pathogens (Fosse et al., 2009). An overview of bacterial zoonotic pathogens that can be transmitted to humans through consumption of pork is listed in Table 1. This list includes both bacteria that are commonly accepted as important foodborne zoonotic agents in pork, and bacterial species that are less frequently described as such. In pigs, very little epidemiological research and randomized controlled trials on

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important bacterial food borne pathogens such as Campylobacter, Yersinia or Listeria have been performed to assess the influence of control measures, such as vaccination, on human infection risk (Davies, 2011). In addition, vaccines aimed to reduce the transmission of zoonotic bacterial pathogens from pigs towards humans are missing, except for Salmonella. The current chapter will therefore focus predominantly on Salmonella. Table 1. pork-related Zoonotic agent Salmonella enterica Yersinia spp. Campylobacter spp. Listeria spp. Streptococcus suis Mycobacterium spp. STEC Bacillus cereus

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Bacillus anthracis Clostridium perfringens Clostridium botulinum Staphylococcus aureus Brucella suis Burkholderia pseudomallei Helicobacter suis Brachyspira pilosicoli Erysipelothrix rhusiopathiae Arcobacter spp.

References on the agent as a pork-related zoonosis Mataragas et al., 2008 ; Fosse et al., 2011; Mataragas et al., 2008 ; Fosse et al., 2008; Mataragas et al., 2008 ; Fosse et al., 2011; Fosse et al., 2008; Mataragas et al., 2008 Wertheim et al., 2009; Gottschalk et al., 2010 Fosse et al., 2008; Fosse et al., 2008; Mataragas et al., 2008 Fosse et al., 2008; Mataragas et al., 2008 Redmond et al., 1997 ; Fosse et al., 2008 Mataragas et al., 2008 ; Juneja et al., 2010 ; Fosse et al., 2011 Roblot et al., 1994 ; Fosse et al., 2008; Mataragas et al., 2008 ; Fosse et al., 2011; Pu et al., 2011 Fosse et al., 2008 ; Meng et al., 2009 Choy et al., 2000 ; Fosse et al., 2008 Haesebrouck et al., 2009 ; Flahou et al., 2010 Smith, 2005 ; Hampson et al., 2006 Wang et al., 2002 ; Wang et al., 2010 Ohlendorf and Murano, 2002; Van Driessche and Houf, 2007

References on vaccination against the agent See Table 2 Autenrieth and Autenrieth, 2008 Lin, 2009 / Fittipaldi et al., 2007 ; Baums and Valentin-Weigland, 2009 Hines et al., 1998 ; Ballesteros, 2009 Dean-Nystrom et al., 2002 / Chitlaru et al., 2011 Springer and Selbitz , 1999 ; Titball, 2009 / Ohlsen and Lorenz, 2010 Lord et al., 1998; Stoffregen et al., 2006 Sarkar-Tyson and Titball, 2010 Flahou et al., 2009 Hampson et al., 2000 ; Movahedi et al., 2010 Neumann et al., 2009; Eamens et al., 2006 /

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SALMONELLA AS A ZOONOTIC PATHOGEN IN PIGS AND PORK Salmonella enterica subspecies enterica serotype Choleraesuis (Salmonella Choleraesuis) was the first Salmonella serotype to be isolated from pigs, only 2 years after the first isolation ever of Salmonella, performed by Gaffky in 1884 (Le Minor, 1994). In the course of time, more than 2400 different serotypes were isolated from different animal species, including pigs. During 1950s and 1960s, Salmonella Choleraesuis, including variant Kunzendorf, was the predominant serotype isolated from pigs worldwide (Fedorka-Cray et al., 2000). Porcine disease associated with this host-adapted serotype is characterised by septicemia, enterocolitis or bacteremic localisation such as pneumonia, hepatitis, meningitis, encephalitis or abortion (Schwartz, K.J., 1999. Salmonellosis. In: Straw, B.E., D‘Allaire, S., Mengeling, W.L., Taylor, D. (Eds.), Diseases of Swine. Iowa State University Press, Ames, IA, pp. 535–551.Schwartz, 1999). At the present time, Salmonella Choleraesuis is still highly prevalent in North America and Asia, but is found rarely in Australia and Western European countries (Fedorka-Cray et al., 2000, Chiu et al., 2004, Davies et al., 2004, Chang et al., 2005). Pigs can be infected by several broad-host range (non-typhoidal) Salmonella serotypes and the occurrence of these serotypes is partly geographically determined (Fedorka-Cray et al., 2000 and Loynachan et al., 2004). Worldwide, the most commonly isolated non-typhoidal serotypes in pigs and pork are Salmonella Typhimurium, including the monophasic 4,[5],12:i:– and Copenhagen variants, and Salmonella Derby (Davies et al., 2004, Gebreyes et al., 2004, Valdezate et al., 2005, EFSA, 2006 and Rostagno et al., 2007; Hauser et al., 2010). All serotypes isolated from pigs are considered a hazard for public health by the European food safety authority (EFSA) (EFSA, 2006), even though infections in humans with Salmonella Choleraesuis tend to be more severe than infections with non-typhoidal serotypes. Since its spectacular rise in the nineties until recently, Salmonella Enteritidis remained the most important serotype causing salmonellosis in humans in many countries. However, due to a remarkable drop in egg-related Salmonella Enteritidis infections since 2005/2006 in different European countries (Gillespie and Elson, 2005, Mossong et al., 2006 and Collard et al., 2008), Salmonella Typhimurium has now become the predominant serotype isolated from humans in Europe and pigs are probably the most important source of infection with this serotype in these countries. It is estimated that more than 90 million cases of gastroenteritis due to Salmonella occur globally each year of which 80 million cases are estimated to be foodborne (Majowicz et al., 2010). It has been estimated in various European countries that 15–23% of all cases of salmonellosis in humans, are related to the consumption of pork (Borch et al., 1996, Berends et al., 1998, Steinbach and Hartung, 1999 and Van Pelt et al., 2000) and pork-related outbreaks of non-typhoidal salmonellosis in humans with fatal outcome have been described (Jansen et al., 2007). The relative importance of Salmonella contaminated pork as a cause of salmonellosis in humans in the European countries is likely to be even higher today, due to the decline in cases of disease caused by Salmonella Enteritidis. In USA, statistical models have predicted that every year approximately 100,000 human cases of salmonellosis are related to the consumption of pork, with a corresponding annual social cost of approximately 80 million dollar (Miller et al., 2005).

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PATHOGENESIS OF SALMONELLA INFECTIONS IN PIGS In order to be able to come up with efficient control measures to combat Salmonella infections in pigs, for example by means of vaccination, detailed knowledge of the pathogenesis should be the starting point. Transmission of Salmonella between pigs is thought to occur mainly via the faecal–oral route. Depending on the inoculation dose, experimental oral infection of pigs with Salmonella Typhimurium may result in clinical signs and faecal excretion of high numbers of bacteria (Loynachan and Harris, 2005 A.T. Loynachan and D.L. Harris, Dose determination for acute Salmonella infection in pigs, Appl. Environ. Microbiol. 71 (2005), pp. 2753–2755. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (5)Loynachan and Harris, 2005 and Boyen et al., 2009). During ingestion, Salmonella enters the tonsils in the soft palate and persists in the tonsillar crypts (Fedorka-Cray et al., 1995 and Horter et al., 2003). The palatine tonsils are often heavily infected in pigs and should, therefore, not be underestimated as a source of Salmonella contamination during slaughter (Wood et al., 1989 and Kühnel and Blaha, 2004). Surprisingly, little information has been gathered on how Salmonella interacts with and persists in the porcine tonsillar tissue. Persistence of Salmonella on the superficial epithelium of the tonsillar crypts has been reported (Horter et al., 2003, Boyen et al., 2006a; Van Parys et al., 2010). The mode of colonization of the tonsils is probably very different from the mechanism of colonization of the intestines (Boyen et al., 2006a). Very recently, various genes that are expressed in the porcine tonsils during persistent infection have been identified using the in vivo expression technology (Van Parys et al., 2011). Bacteria that are swallowed and survive passage through the stomach, reach the gut. In the distal parts of the intestine, adherence to the intestinal mucosa is generally accepted as the first step in the pathogenesis of Salmonella infections in pigs. It has recently been shown that in epithelials cells, reversible adhesion of Salmonella Typhimurium is Fim mediated and irreversible adhesion (docking) is Salmonella Pathogenicity Island 1 (SPI-1) mediated (Misselwitz et al., 2011). Both type 1 fimbriae and SPI-1 have been shown to contribute to the colonization of the porcine intestinal tract (Althouse et al., 2003; Boyen et al., 2006a). In a signature-tagged mutagenesis assay, Salmonella atypical fimbriae (saf), located on Salmonella Pathogenicity Island 6 (SPI-6), were shown to play a role in porcine gut colonization, even though no statistically significant reduction was seen in the magnitude or duration of faecal excretion of a safA mutant (Carnell et al., 2007). Following adhesion, Salmonella invades the intestinal epithelium. Salmonella Typhimurium can be found within the porcine enterocytes and mesenteric lymph nodes at 2 h after oral inoculation (Reed et al., 1986). Recently, it has been shown that the virulence genes encoded in the SPI-1 mediate this invasion step and that these genes are crucial for the colonization of the porcine gut and GALT (Boyen et al., 2006a and Brumme et al., 2007). Using signature tagged mutagenesis, also several other virulence genes, including SPI-2 associated genes, have been identified as being important for the short-term colonization of the epithelium of the porcine gut (Carnell et al., 2007). The rapid growth of Salmonella Typhimurium in the porcine gut and subsequent induction of pro-inflammatory responses may explain why pigs in most cases confine Salmonella Typhimurium infection to the intestines, whereas slow replication of Salmonella Choleraesuis may enable it to evade host immunity and subsequently spread beyond the intestinal boundaries (Paulin et al., 2007 S.M. Paulin, A. Jagannathan, J. Campbell, T.S. Wallis and M.P. Stevens, Net replication of

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Salmonella enterica serovars Typhimurium and Choleraesuis in porcine intestinal mucosa and nodes is associated with their differential virulence, Infect. Immun. 75 (2007), pp. 3950– 3960. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (15)Paulin et al., 2007). The systemic part of a Salmonella Typhimurium infection in pigs is not welldocumented. It is generally accepted that Salmonella can spread throughout an organism using the blood stream or the lymphatic fluids and infect internal organs, although this has not yet been studied in detail in swine. The colonization of the gut associated lymphoid tissue (GALT), spleen and liver can result in prominent systemic and local immune responses (Dlabac et al., 1997; Skjolaas et al., 2006; Meurens et al., 2009; Collado-Romero et al., 2010). Macrophages are the cells of interest for host-restricted or -adapted Salmonella serotypes to disseminate to internal organs of different host species. The bacteria replicate rapidly intracellularly and cause the systemic phase of the infection, while interfering with the antibacterial mechanisms of the macrophages and inducing cell death (Waterman and Holden, 2003 S.R. Waterman and D.W. Holden, Functions and effectors of the Salmonella pathogenicity island 2 type III secretion system, Cell. Microbiol. 5 (2003), pp. 501–511. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (157)Waterman and Holden, 2003 and Hueffer and Galan, 2004). In pigs, non-typhoidal serotypes such as Salmonella Typhimurium, can reach liver and spleen shortly after experimental inoculation, but are cleared from these organs a few days after inoculation (Boyen et al., 2009). At this time, the bacteria are still found in the gut, gut-associated lymph nodes and tonsils. These infections may result in long-term asymptomatic carriage of the bacterium (Wood et al., 1991). Since this carrier state in pigs is difficult to detect in live animals, either by bacteriological or serological methods (Baggesen and Wegener, 1993; Nollet et al., 2005), these pigs can bias monitoring programmes. Very few researchers have made an attempt to unravel the mechanism of the concealed, but prolonged infection in carrier pigs (Boyen et al., 2006b; Wang et al., 2007; Van Parys et al., 2011). There are indications that Salmonella Typhimurium interferes with seroconversion in pigs, and that this phenomenon might be related to strain-dependent persistency capacities (Van Parys et al., unpublished results). Stress-induced excretion of Salmonella Typhimurium by carrier pigs transported to the slaughterhouse may cause contamination of shipping equipment and holding areas, resulting in preslaughter transmission of Salmonella to non-infected pigs (Isaacson et al., 1999, Larsen et al., 2003 and Boughton et al., 2007). Although the mechanism of this stress-induced excretion is not known, there are some indications that catecholamines and/or cortisol may play a role. It has been shown that Salmonella Typhimurium can ―s ense‖ catecholamines and as a result increase its growth rate (Williams et al., 2006; Methner et al., 2008; Bearson et al., 2010; Pullinger et al., 2010). It has very recently been shown that the presence of cortisol has marked effects on the intracellular fate of Salmonella Typhimurium in porcine macrophages (Verbrugghe et al., unpublished results). The mechanism and the bacterial factors playing a role in this phenomenon are currently under investigation (Verbrugghe et al., unpublished results).

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VACCINATING AGAINST SALMONELLA IN PIGS Even though a few risk factors for Salmonella carriage in pigs have been determined in several epidemiological studies, there is very limited direct proof obtained from randomized controlled trials that Salmonella prevalence can be controlled in pigs using conventional preharvest interventions such as hygiene and biosafety measures, feeding practices and the use of feed/water additives (Boyen et al., 2008; Davies et al., 2011). Considering the positive effects of vaccination of laying hens on prevalence of Salmonella Enteritidis in eggs (Gantois et al., 2006) and Salmonella Enteritidis infections in humans (Collard et al., 2008; Anonymous, 2010), vaccination could also be a major tool to control Salmonella in pigs. The evidence available in scientific literature suggests that Salmonella vaccination is in fact associated with reduced Salmonella prevalence in swine at or near harvest (Denagamage et al., 2007). In addition, there are indications that even subclinical Salmonella infections can lead to weight gain losses in pigs indicating that vaccination to control salmonellosis in pigs may have an economic incentive for pig producers as well (Boyen et al., 2009; Farzan and Friendship, 2010). Table 2 gives an overview of the experimental and commercial Salmonella vaccines that have been tested in pigs. Table 2.

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Salmonella serotype

Mode of attenuation/inactivation Rough LPS

References

Salmonella Choleraesuis

Attenuated / inactivated Live attenuated

Salmonella Choleraesuis

Live attenuated

Schöll et al., 1980

Salmonella Choleraesuis

Live attenuated

Δthiamine Δadenine rough LPS rifampicine resistance

Salmonella Choleraesuis

Live attenuated

Plasmid cured

Salmonella Choleraesuis

Live attenuated

Δcya Δcrp derivatives

Kramer et al., 1992; Roof and Doitchinoff, 1995; Letellier et al., 2000; 2001; Kolb et al., 2003 Kennedy et al., 1999

Salmonella Choleraesuis

Live attenuated

Δcya Δ(crp-cdt)

Salmonella Choleraesuis

Live attenuated

Unknown

Charles et al., 2000; Maes et al., 2001 Groninga et al., 2000

Salmonella Choleraesuis

Live attenuated

ΔssaV; Δgifsy-1

Ku et al., 2005

Salmonella Choleraesuis

Live attenuated

Plasmid cured, Δcrp

Chu et al., 2007

Salmonella Choleraesuis

Live attenuated

ΔphoP, ΔrpoS

Dominguez-Bernal et al., 2008

Salmonella Typhimurium

Live attenuated

ΔaroA

Salmonella Typhimurium

Live attenuated

Δcya Δcrp (χ4233)

Lumsden et al., 1991; Lumsden and Wilkie, 1992 Coe and Wood, 1992

Salmonella Typhimurium

Live attenuated

Δhistidine Δadenine

Springer et al., 2001; Eddicks et al., 2009; De Ridder et al., 2011

Salmonella Typhimurium

Live attenuated

ΔgyrAΔcpxAΔrpoB

Roesler et al., 2004

Salmonella Typhimurium

inactivated

Roesler et al., 2006

Salmonella Typhimurium

Live attenuated

Inactivation by heat + aluminium hydroxide ΔcpxR Δlon + E. coli LT enterotoxin

Hanna et al., 1979

Kramer et al., 1987

Hur and Lee, 2010 ; Hur et al., 2011

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Activating Innate, Mucosal, Humoral and/or Cellular Immune Response? Vaccination might be able to reduce porcine carcass contamination and subsequently infections in humans in different ways, by interfering at different stages of the pathogenesis: inhibit early colonization, reduce excretion thereby lowering infection pressure at farm level, decrease spread between pigs by increasing the infective dose threshold, interfere with the development of the carrier state in the various target organs (gut, GALT and tonsils) and prevent the stress-related re-excretion at slaughter. To inhibit early colonization, adhesion to and/or invasion in epithelial intestinal cells should be blocked. This could probably be best achieved by attaining high levels of mucosal IgA. There are no indications which porcine immune response(s) are most important for reducing excretion shortly after infection, during the carrier state or in stress-related re-excretion periods. To interfere with the development of the carrier state, it can be expected that both the humoral and cellular immune response will be important. The onset of clinical symptoms, but also colonization efficiency and persistence, are dose-dependent (Gray et al., 1996; Boyen et al., 2009). Probably innate, mucosal, humoral and cellular immunity all play a role in increasing the infective dose threshold. There are indications that competitive exclusion effects and the influx and activation of heterophils induced by Salmonella vaccine strains may provide a very early protection (within 24 hours) in poultry (Van Immerseel et al., 2005). Similar observations were made in pigs (Foster et al., 2005; Splichal et al., 2005; Trebichavsky et al., 2006), but it is not clear what the impact of this phenomenon might be on colonization and/or excretion at subclinical infection levels in pigs. In addition, it can be expected that these effects will not be able to protect pigs throughout the fattening period, which is much longer than for broilers. The importance of mucosal IgA in the protection against Salmonella in pigs is not well studied. It has been shown that IgA is able to interfere with Salmonella Typhimurium motility and invasion in intestinal epithelial cells (Forbes et al., 2008). In pigs vaccinated with a live attenuated Salmonella Choleraesuis strain, the production of intestinal IgA against Salmonella Typhimurium was significantly higher in the vaccinated group in comparison to uninfected control pigs (Letellier et al., 2001). Very recently, it was shown that a live attenuated vaccine strain secreting the Escherichia coli heat-labile enterotoxin as a live form of mucosal adjuvant was able to induce intestinal mucosal IgA production in sows and that it could be transferred towards the offspring (Hur and Lee, 2010). For protection against pathogenic bacteria, both antibodies as well as cell-mediated immunity are usually involved. Generally speaking, facultatively intracellular bacteria such as Salmonella, can replicate in an intracellular environment free of antibodies and therefore cellular immunity is often considered most important for protection (Curtiss et al., 1993). This, however, does not mean that antibodies do not at all play a role in protection against these bacteria. At some point in the infectious cycle, or in certain organs such as the tonsils (Horter et al., 2003; Van Parys et al., 2010), most intracellular pathogens reside in the extracellular space, where they are vulnerable to antibody action. Cell-mediated immune responses involve production of lymphokines by Th1-lymphocytes that cause activation of macrophages and cytotoxic T-lymphocyte responses. While macrophages from unimmunised animals are normally incapable to destroy facultatively intracellular bacteria, this capacity is increased after activation by lymphokines. Cytotoxic T-lymphocytes destroy bacteriacontaining host cells resulting in release of the bacteria in the extracellular space where they

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can be killed. T-cells and antibodies are not essential for the control of the early stages of a systemic Salmonella infection, but the concerted action of cellular and humoral immune response is probably needed for the clearance of the bacterium in the later phase of the infection and for acquired immunity in vaccinated animals (Mastroeni and Ménager, 2003; Ménager et al., 2007). It has been shown, using human B-cells, that a B-cell receptor mediated internalization of Salmonella leads to a fast and specific humoral immune response (Souwer et al., 2009). In addition, it has been shown that the impaired immune response against non-typhoidal Salmonella in HIV patients is caused by an excessive production of anti-LPS antibodies, which are not protective. Serum induced killing of Salmonella, however, is mediated by antibodies which are directed against outer membrane proteins (MacLennan et al., 2010). A Salmonella strain lacking the O-antigen of the LPS showed an increased rate of uptake by murine dendritic cells, altered intracellular processing, increased degradation and also boosted the activation of the immune functions of dendritic cells (Duerr et al, 2009; Zenk et al., 2009). Until now, there are no indications whether specific subsets of antibodies might provide better protection than other subsets against Salmonella infections in pigs. This can be an important issue to improve the efficacy of Salmonella vaccines. As Salmonella is able to interfere with both endocytotic and exocytotic cellular transport, it was not surprising to find that Salmonella is also able to interfere with the antigen presentation in dendritic cells (Cheminay et al., 2005). Although the exact mechanism of this interference is not entirely understood, it has been proposed that a Salmonella transporter system might prevent peptide loading of phagosomal MHC class I molecules by flooding the vacuole with competing short peptides (Qimron et al., 2004). Interestingly, Salmonella escape from antigen presentation can be overcome by targeting bacteria to Fcγ receptors on dendritic cells (Tobar et al., 2004). In addition, work in mice indicates that targeting Salmonella to FcγR is needed for the expression of vaccine-induced acquired immunity, but is not essential for the production of T- and B-cell immunity to the bacterium in vivo (Ménager et al., 2007). A mechanism has recently been proposed in which antibodies and FcR engagement subverts the strategies by which intracellular bacterial pathogens evade lysosomal degradation (Joller et al., 2010).

Using Live Attenuated or Inactivated Vaccines for Optimal Effects? At present, live vaccine strains are considered to offer a better protection against Salmonella infections compared to inactivated vaccines, probably due to the more pronounced cellular immune response and the induction of mucosal IgA production (Haesebrouck et al., 2004 ; Meeusen et al., 2007 E.N. Meeusen, J. Walker, A. Peters, P.P. Pastoret and G. Jungersen, Current status of veterinary vaccines, Clin. Microbiol. Rev. 20 (2007), pp. 489–510. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (24)Meeusen et al., 2007). However, it has been shown that inactivated vaccines can also induce cell-mediated immunity, depending on the adjuvant used in the vaccine (Haesebrouck et al., 2004). Nevertheless, most promising results for Salmonella vaccines in pigs are reported in scientific literature using live attenuated strains (Table 2). Additional advantages of live vaccine strains are the possibility to administer these vaccines at a very young age, despite the presence of maternal antibodies (Eddicks et al., 2009), the flexibility of a live

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vaccine strain to switch to different colonization strategies at different target organs (guttonsils) and the possibility to administer it by mixing it in feed and/or water supply. The most difficult aspect of creating a good live attenuated vaccine is finding the perfect balance between attenuation to assure safety and residual potency to assure the induction of a protective immune response. Bacterial attenuated vaccine strains can be divided in three types: (1) strains, which are attenuated without the attenuation being localised or characterised, (2) strains with mutations in genes that are important for the bacterial metabolism, for example auxotrophic mutant strains, (3) strains in which specific (virulence) genes were removed. The advantage of the latter group is that the vaccine strains are very well characterised and that reversion to the wild-type phenotype is extremely unlikely. Strains that lack one or more virulence genes important for clinical salmonellosis or for the induction of persistent infections in pigs might represent promising candidates for future vaccine development. It has been suggested that virulence-gene deleted vaccines may be less immunogenic than metabolic-gene deleted vaccines (Meeusen et al., 2007). Nevertheless, it has been shown in pigs that protection against Salmonella Typhimurium is not dependent on secreted proteins of SPI-1 (Carnell et al., 2007). Recent research has identified various virulence genes, playing a role in different stages of the pathogenesis of Salmonella Typhimurium infections in pigs (Carnell et al., 2007; Boyen et al., 2008; Stevens et al., 2009; Bearson et al., 2010; Van Parys et al., unpublished results). These findings may contribute to the development of more efficient and safer live attenuated vaccines.

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Vaccinating Sows or Piglets? Various researchers have reported that Salmonella prevalence at slaughter age is predominantly a result of infection during the finishing period, especially in multi-site herds (Davies et al., 1998; Nielsen et al., 1995; Schwartz, 1999). Therefore, currently, most control measures, including vaccination, are mainly focussed on older piglets and slaughter pigs (Schwartz, 1999; Hill et al., 2008). These strategies may indeed influence Salmonella prevalence and shedding at the end of the production cycle, but are not able to interfere with infection pressure and spread of the bacterium at younger stages of life. These early stages of the production cycle, can nevertheless be a crucial factor for the initial contamination of pig batches, especially in pig farms where biosecurity and cleaning-disinfection protocols are good to excellent. Based on sensitivity analysis, the studies of Lurette et al. (2008, 2009) showed that early infection, occurring between birth and weaning, seemed to be a critical point for the Salmonella spread within a pig batch, and possibly within a herd. A change in Salmonella shedding pattern of sows during gestation, farrowing and lactation could not be observed in a study of Nollet et al. (2005). However, shortly after weaning, a significant increase in Salmonella shedding was noticed and it was concluded that sows may play an important role in the maintenance of Salmonella infections in farrow-to-finish herds (Nollet et al., 2005).

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Interference of Vaccination with Salmonella Monitoring Programmes The purpose of monitoring and control programs is to reduce the risk of public health problems arising from the consumption of contaminated pork, reducing human disease and maintaining consumer confidence. Salmonella control measures can be implemented at three levels: the pre-harvest level (on farm), the harvest level (transport to and procedures in the slaughterhouse) and the post-harvest level (cutting, processing, retail and food preparation at home). Implementation of monitoring programs and coordination of control measures at harvest and post-harvest, are being used worldwide to prevent non-typhoidal Salmonella infections in humans from contaminated pork (Mossel et al., 2003, Chen et al., 2006, Padungtod and Kaneene, 2006, EFSA, 2006b EFSA, Opinion of the Scientific Panel on Biological Hazards on the request from the Commission related to ― Risk assessment and mitigation options of Salmonella in pig production‖, EFSA J. 341 (2006), pp. 1–131.EFSA, 2006, Hamilton et al., 2007, Larsen et al., 2007 and Rajic et al., 2007). Extensive national monitoring and control programs at the farm level (preharvest) are mostly conducted in the European countries (regulation [EC] 2160/2003, Asai et al., 2006, EFSA, 2006, Hamilton et al., 2007, Larsen et al., 2007 and Rajic et al., 2007). Only the Scandinavian countries have been given low prevalence status by EFSA. In Sweden, preharvest and harvest monitoring programmes are being implemented on both a compulsory and a voluntary basis, using mainly bacteriological isolation to assess Salmonella contamination (Wahlström et al., 2000 and EFSA, 2006). The Danish, British, Irish and German programmes are based on serological testing of meat juice samples taken at the slaughterhouse, thus categorising the pig herds according to their assessed risk of carrying Salmonella into the slaughter plant (Nielsen et al., 2001, Davies et al., 2004, EFSA, 2006 and Merle et al., 2007). Belgian and Dutch monitoring programmes are similar, but the serological testing is currently performed on blood or serum samples collected on the farm (EFSA, 2006, Bollaerts et al., 2007 and Hanssen et al., 2007). Farmers with herds belonging to the category with the highest risk of introducing Salmonella into the slaughterhouse are assisted by the national governments to reduce the Salmonella load of their herd (EFSA, 2006; Anonymous, 2008). There is currently one Salmonella vaccine registered for use in pigs in Europe which is a live attenuated vaccine (Springer et al., 2001; Eddicks et al., 2009). Even though this vaccine has promising features to decrease the Salmonella load on farm, the induced immune response interferes with the national control programmes of most of the European member states. A variant of this vaccine that enables differentiation of infected from vaccinated animals (DIVA) has been described by Selke et al. (2007). This DIVA strain was created by deleting the gene encoding a major outer membrane protein (OmpD). Infected and vaccinated piglets can be differentiated by measuring the antibodies in blood or meat juice that are directed against the OmpD proteins, using a newly designed Enzyme Linked ImmunoSorbent Assay (ELISA). However, to differentiate vaccination with this DIVA strain from infection in the European monitoring systems, this new detection system should be implemented and validated in all European member states, which is very time consuming and expensive. A marker vaccine that would not interfere with the European (and/or other) control programmes would mean a huge step forward. Recent research by Leyman et al. (2011) has shown that the

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deletion of an LPS encoding gene might create a DIVA marker strain that will not interfere with the current European control programmes.

CONCLUSION In conclusion, there are serious indications that vaccination can play an important role in the control of Salmonella infections in pigs. Effective vaccines are currently already available or are under construction. The absence of a DIVA marker in these vaccine strains, however, might limit their use in countries that are running serologic monitoring programmes. Information on the pathogenesis of non-typhoidal Salmonella infections in pigs (Bearson and Bearson, 2011) and on the protective role of the different immunological responses in the target species (Scharek and Tedin, 2007) should form the basis to create new or improve existing vaccines.

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REFERENCES Althouse, C., Patterson, S., Fedorka-Cray P.and Isaacson, R. E. (2003). Type 1 fimbriae of Salmonella enterica serovar Typhimurium bind to enterocytes and contribute to colonization of swine in vivo. Infection and Immunity, 71, 6446-6452. Anonymous. Trends and Sources of Zoonoses and Zoonotic Agents and Food-borne Outbreaks in the European Union in 2008. Last visited 21/02/2011. Available from: http://www.efsa.europa.eu/en/efsajournal/doc/1496.pdf. Asai, T., Esaki, H., Kojima, A., Ishihara, K., Tamura Y. and Takahashi, T. (2006). Antimicrobial resistance in Salmonella isolates from apparently healthy food-producing animal from 2000 to 2003: the first stage of Japanese veterinary antimicrobial resistance monitoring (JVARM). The Journal of Veterinary Medical Science, 68, 881–884. Autenrieth, S. E. and Autenrieth, I. B. (2008). Yersinia enterocolitica: subversion of adaptive immunity and implications for vaccine development. International Journal of Medical Microbiology, 298, 69-77. Baggesen, D. L. and Wegener, H. C. (1993). Detection of Salmonella in Swine. Dansk veterinaer tidsskrift, 76, 1017–1022. Ballesteros, C., Garrido, J. M., Vicente, J., Romero, B., Galindo, R. C., Minguijón, E., Villar, M., Martín-Hernando, M.P., Sevilla, I., Juste, R., Aranaz, A., de la Fuente, J. and Gortázar, C. (2009). First data on Eurasian wild boar response to oral immunization with BCG and challenge with a Mycobacterium bovis field strain. Vaccine, 27, 6662-6668. Baums, C. G. and Valentin-Weigand, P. (2009). Surface-associated and secreted factors of Streptococcus suis in epidemiology, pathogenesis and vaccine development. Animal Health Research Reviews, 10, 65-83. Bearson, B. L. and Bearson, S. M. (2011). Host specific differences alter the requirement for certain Salmonella genes during swine colonization. Veterinary Microbiology, In Press. Bearson, B. L., Bearson, S. M., Lee, I. S. and Brunelle, B. W. (2010). The Salmonella Microbial Pathogenesis, 48, 214-219.

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Tobar, J. A., González, P. A. and Kalergis, A. M. (2004). Salmonella escape from antigen presentation can be overcome by targeting bacteria to Fc gamma receptors on dendritic cells. Journal of Immunology, 173, 4058-4065. Trebichavsky, I., Splichalova, A., Rychlik, I., Hojna, H., Muneta, Y., Mori, Y., Splichal, I. (2006). Attenuated aroA Salmonella enterica serovar Typhimurium does not induce inflammatory response and early protection of gnotobiotic pigs against parental virulent LT2 strain. Vaccine, 24, 4285-4289. Valdezate, S., Vidal, A., Herrera-Leon, S., Pozo, J., Rubio, P., Usera, M. A., Carvajal, A. and Echeita, M. A. (2005). Salmonella Derby clonal spread from pork. Emerging Infectious Diseases, 11, 694–698. Van Driessche, E. and Houf, K. (2007). Characterization of the Arcobacter contamination on Belgian pork carcasses and raw retail pork. International Journal of Food Microbiology, 118, 20-26. Van Immerseel, F., Methner, U., Rychlik, I., Nagy, B., Velge, P., Martin, G., Foster, N., Ducatelle, R. and Barrow, P. A. (2005). Vaccination and early protection against nonhost-specific Salmonella serotypes in poultry: exploitation of innate immunity and microbial activity. Epidemiology and Infection, 133, 959-978. Van Parys, A., Boyen, F., Leyman, B., Verbrugghe, E., Haesebrouck, F. and Pasmans, F. (2011). Salmonella Typhimurium genes expressed during persistence in pigs. PLoS one, Accepted for publication. Van Parys, A., Boyen, F., Volf, J., Verbrugghe, E., Leyman, B., Rychlik, I., Haesebrouck, F., Pasmans, F. (2010). Salmonella Typhimurium resides largely as an extracellular pathogen in porcine tonsils, independently of biofilm-associated genes csgA, csgD and adrA. Veterinary Microbiology, 144, 93-99. Van Parys, A., Boyen, F., Leyman, B., Verbrugghe, E.,Maes, D., Haesebrouck, F. and Pasmans F. Salmonella Typhimurium interferes with the porcine adaptive immune response. Unpublished results. Van Pelt, W., Van Giessen, A., Van Leeuwen, W., Wannet, W., Henken, A., Evers, E., De Wit M. and Van Duynhoven, Y. (2000). Oorsprong, omvang en kosten van humane salmonellose, Infectieziekten Bulletin, 11, 4–8. Wahlström, H., Eriksson, E., Noll, B., Plym Forsell, L., Wierup, M. and Wollin, R. (2000). The Swedish control of pig and pork production during 1999. Proceedings of the 16th IPVS Congres, Melbourne, Australia, 215. Wang, Q., Fidalgo, S., Chang, B. J., Mee, B. J. and Riley, T. V. (2002). The detection and recovery of Erysipelothrix spp. in meat and abattoir samples in Western Australia. Journal of Applied Microbiology, 92, 844-850. Wang, Q., Chang, B. J. and Riley, T. V. (2010). Erysipelothrix rhusiopathiae. Veterinary Microbiology, 140, 405-417. Waterman, S. R. and Holden, D. W. (2003). Functions and effectors of the Salmonella pathogenicity island 2 type III secretion system. Cellular Microbiology, 5, 501–511. Wertheim, H. F., Nguyen, H. N., Taylor, W., Lien, T. T., Ngo, H. T., Nguyen, T. Q., Nguyen, B. N., Nguyen, H. H., Nguyen, H. M., Nguyen, C. T., Dao, T. T., Nguyen, T. V., Fox, A., Farrar, J., Schultsz, C., Nguyen, H. D., Nguyen, K. V. and Horby, P. (2009). Streptococcus suis, an important cause of adult bacterial meningitis in northern Vietnam. PLoS One, 4, e5973.

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Williams, P. H., Rabsch, W., Methner, U., Voigt, W., Tschape, H. and Reissbrodt, R. (2006). Catecholate receptor proteins in Salmonella enterica: role in virulence and implications for vaccine development. Vaccine, 24, 3840–3844. Wood, R. L., Pospischil, A. and Rose, R. (1989). Distribution of persistent Salmonella Typhimurium infection in internal organs of swine. American Journal of Veterinary Research, 50, 1015–1021. Wood, R. L., Rose, R., Coe, N. E. and Ferris, K. E. (1991). Experimental establishment of persistent infection in swine with a zoonotic strain of Salmonella Newport. American Journal of Veterinary Research, 52, 813–819. Zenk, S. F., Jantsch, J. and Hensel, M. (2009). Role of Salmonella enterica lipopolysaccharide in activation of dendritic cell functions and bacterial containment. Journal of Immunology, 183, 2697-2707.

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In: On-Farm Strategies to Control Foodborne Pathogens ISBN: 978-1-62100-411-0 Editors: Todd R. Callaway and Tom S. Edrington © 2012 Nova Science Publishers, Inc.

Chapter 8

PHYTOCHEMICALS AS A PREHARVEST PATHOGEN REDUCTION STRATEGY

1

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Jeffery A. Carroll1, Nicole C. Burdick1, Michael A. Ballou2 and John D. Arthington3

Livestock Issues Research Unit, USDA-ARS, Lubbock, TX Department of Animal and Food Sciences, Texas Tech University, Lubbock, TX 3 Range Cattle Research and Education Center, University of Florida, Ona, FL

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ABSTRACT Phytochemicals are naturally occurring non-nutritive chemicals produced by plants to limit pathogenic bacteria growth. Thousands of phytochemicals have been isolated from a variety of plants, and some have been documented to possess antimicrobial, antibacterial, and immunostimulatory properties. As there is an increasing demand from consumers to remove sub-therapeutic levels of antibiotics from livestock feeds in order to reduce the potential development of antibiotic-resistant pathogens, as discussed in an earlier chapter, there has been an increased scientific effort to identify non-antibiotic alternatives to enhance animal health and to reduce the potential risk of increasing antibiotic-resistant bacteria. Additional, there have been efforts focused on the potential benefit of using phytochemicals as a means to reduce livestock pathogen loads prior to harvest, thus decreasing the potential for bacterial contamination during harvest and associated foodborne illnesses in humans. Although post-harvest methodologies have been employed to reduce pathogen transfer in the harvest facility, there is increasing interest in reducing pathogen loads in the live animals before harvest in order to further reduce potential pathogen contamination. The benefits of phytochemical addition to livestock feedstuffs have been demonstrated in several livestock species including swine, cattle, and poultry. While many phytochemicals have already been discovered and evaluated, it is expected that there will be a continued effort to identify even more phytochemicals as efforts continue to find feasible alternatives for sub-therapeutic levels of antibiotics in livestock production systems. This chapter will focus on some of the most characterized phytochemicals: citrus pulp and citrus peel, saponins, beta-glucan, flavonoids, and carotenoids, as well as other less-studied phytochemicals. The chapter concludes with a brief discussion on the potential toxic effects of phytochemicals in livestock.

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INTRODUCTION Plants produce a variety of chemical compounds known as phytochemicals that provide a level of protection from invading microbes including bacteria, fungus, parasites, and viruses. In essence, these phytochemicals serve as somewhat of an immune system for plants, thus allowing the plant to grow and thrive. While certain phytochemicals produced by plants serve as insect repellants, or as chemoattractants for pollinating insects, others have been scientifically documented to possess antimicrobial, antibacterial, and immunostimulatory properties in humans and livestock. Phytochemicals are plant secondary metabolites (Booker and Acamovic, 2005), many of which have no nutritional value, and may even be toxic. However, some phytochemicals, such as those described in this chapter, have the potential to benefit animal and human health (Dorman and Deans, 2000; Callaway et al., 2011). Phytochemicals, and more specifically volatile oils, extracted from plants have demonstrated antimicrobial and antibacterial properties against bacteria, fungi, yeasts, and viruses (Dorman and Deans, 2000). Certain phytochemicals, such as beta-glucan, can also exhibit immunostimulatory properties. There is an increasing demand from consumers to remove sub-therapeutic levels of antibiotics from livestock feeds in order to reduce the potential development of antibiotic-resistant pathogens, as discussed previously by Callaway and colleagues (Callaway et al., 2004). Therefore, the demand for practical alternatives to the use of sub-therapeutic antibiotics in livestock production is increasing, and scientific efforts to identify economically feasible alternatives for livestock production have increased in recent years. In addition to the potential use as an alternative to sub-therapeutic antibiotics, there has also been an increased interest in the potential benefit of using phytochemicals as a means to reduce pathogen loads prior to harvest, thus decreasing the potential for carcass contamination during the harvest process. As discussed in an earlier chapter, pathogenic bacteria, transferred into the harvest facility in animal feces or on the hide, remain a main cause of foodborne illnesses in humans. Although post-harvest methodologies have been employed to reduce pathogen transfer in the harvest facility, there is increasing interest in reducing pathogen loads in the live animal before arrival to the harvest facility (Callaway et al., 2004). Reducing the overall pathogen loads in livestock could greatly reduce the incidence of foodborne pathogens transferred from the animal to the food supply during and immediately after harvest, thus reducing the overall incidences of foodborne illnesses. While numerous phytochemicals have already been isolated and evaluated as a potential alternatives to the use of sub-therapeutic levels of antibiotics in several livestock species including swine, cattle, and poultry, it is expected that there will be a continued scientific effort to identify even more phytochemicals in the future (Booker and Acamovic, 2005). The intent of this chapter is to provide a brief overview of some of the most characterized phytochemicals that have been evaluated in various livestock species including citrus pulp and citrus peel, saponins, beta-glucan, flavonoids, and carotenoids, as well as a few other less-studied phytochemicals.

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CITRUS PULP AND CITRUS PEEL Citrus pulp and citrus peel products are readily available as low cost by-products of the citrus industry in several regions. The most commonly used citrus by-product is citrus pulp (Arthington et al., 2002). Due to its low cost, high nutritive value, and inherent antimicrobial effects, citrus by-products could potentially be used to modulate, in a beneficial manner, bacterial populations in livestock (Tripodo et al., 2004; López et al., 2010). Orange byproducts such as orange peel have been used as a nutritive source for cattle, specifically as a nutrient source for rumen microbes (López et al., 2010). Citrus products contain essential oils, including citrullene, linalool, and limonene, that are toxic to bacteria (Lota et al., 2002; Fisher and Phillips, 2006). Given that the U.S. Food and Drug Administration has classified citrus oils as ―G enerally Recognized as Safe‖, these naturally occurring by-products may serve as a feasible alternative to synthetic antibiotics due to the antioxidant and antimicrobial activities. Exposure to citrus essential oils causes an increase in bacterial cell wall permeability leading to cell death (Cvetnić and VladimirKnežević, 2004; Di Pasqua et al., 2007). Both in vitro and in vivo studies have demonstrated that citrus essential oils exhibit antimicrobial activity against a broad range of bacteria including Escherichia coli (E. coli; Callaway et al., 2008; Nannapaneni et al., 2008), Enterococcus (Fisher and Phillips, 2009), Listeria (Fisher and Phillips, 2006), and Salmonella (Parish et al., 2003; Nams et al., 2006; Nannapaneni et al., 2008). Therefore, given the availability and antimicrobial properties, there is a great potential to use citrus by-products as feed additives in order to reduce pathogen loads and enhance the health status of livestock. Nannapaneni et al. (2008) demonstrated that the citrus essential oils limonene and terpeneless inhibited E. coli O157:H7 using an in vitro disk-diffusion assay. Additionally, incubation of grapefruit seed extract, derived from grapefruit seeds and pulp, inhibited the growth of a large variety of gram negative and gram positive bacteria, yeast, and fungi (Cvetnić and Vladimir-Knežević, 2004). The in vitro addition of citrus pulp and citrus peel to both cattle rumen fluid fermentations and pure bacterial cultures decreased the populations of E. coli 0157:H7 and Salmonella typhimurium (Callaway et al., 2008). In addition, in vivo data from weaned pigs indicated that the inclusion of citrus pulp at 10% as fed decreased ileal and cecal recovery of E. coli F18, and completely eliminated fecal recovery of E. coli F18 (Carroll et al., 2010). Collectively, these data suggest the addition of citrus by-products can reduce the shedding of pathogenic bacteria from livestock species, and may be a viable tool for reducing bacterial contamination of livestock carcasses and associated foodborne illnesses.

SAPONINS Saponins are chemicals found in many plants and exhibit natural detergent properties (Cheeke, 2000). Desert plants, such as Yucca schidigera and Quillaja saponaria, are rich in saponin content. Yucca schidigera is an herbaceous plant native to the deserts of the southwestern United States and northern Mexico which has been traditionally used by Native Americans to treat arthritis and other common ailments. The plant is a rich source of vitamins A, B-complex, and C, as well as copper, calcium, manganese, and potassium. Yucca

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schidigera contains several physiologically active phytochemicals including steroidal saponins and polyphenoics such as resveratrol and stilbenes. Steroidal saponins, which are precursors to cortisone, are found in high concentrations in the Yucca schidigera plant (10% dry matter in the stems), which may contribute to its anti-arthritic and anti-inflammatory actions. The anti-inflammatory and anti-arthritic properties associated with Yucca schidigera were the basis of a recent review by Cheeke and colleagues (Cheeke et al., 2006). Quillaja saponins are natural surfactants derived from the bark of the tree Quillaja saponaria, which is indigenous to Chile. Quillaja saponins have been reported to possess antiinflammatory, antimicrobial, cytotoxic, anti-hypercholesterolemic, expectorant, hemolytic, and immunostimulantory properties. For more than 3 decades, Quillaja saponaria has been extensively used as a vaccine adjuvant in large animals (Kensil, 1996). The effectiveness as a vaccine adjuvant may stem from the ability of Quillaja saponaria to induce interleukin-6 (Behboudi et al., 1997) and interleukin-12 production (Villacres-Eriksson et al., 1997). Interleukin-6 is known to promote differentiation of B lymphocytes into antibody-secreting cells and to increase production of Immunoglobulin G (IgG), IgA and IgM (Wong and Clark, 1988). Interleukin-12 plays a primary role by controlling the differentiation of T helper cells and favoring the expansion of Type 1 T helper cells which in turn promotes cell-mediated immunity. Additional research in mice has demonstrated not only an increase in antibody production following antigen exposure, but an increased survivability to a lethal dose of rattlesnake venom when mice were given Quillaja saponaria (Gebara et al., 1995). These authors also noted an increase in the pro-inflammatory cytokines tumor necrosis factor-alpha and interferon-gamma as a result of saponin exposure. In general, saponins have been characterized to exhibit anti-inflammatory, antifungal, antiparasitic, antitumor, and antimicrobial properties (for review see Sparg et al., 2004). Saponins can be grouped into either steroidal saponins or triterpenoid saponins, based on their aglycone skeleton (Sparg et al., 2004). They exhibit their antiprotozoal actions through binding irreversibly to cholesterol in the cell membrane of protozoans, thus causing breakdown and lysis of the cell membrane resulting in cell death (Cheeke, 2000). The resulting decrease in protozoans in the rumen is believed to reduce rumen ammonia concentrations. To have their antiprotozoal effects, saponins must be intact (i.e., nucleus and side chain(s) present). While saponins can have effects on protozoa, they can also affect the growth of bacteria in the gut. In contrast to the effect of saponins on protozoa, the effect on bacteria is concentration-dependent (Cheeke, 2000). Rumen microbes quickly break down saponins within 6 hours, suggesting that the antiprotozoal action occurs quickly upon feeding. However, rumen bacteria may also adapt to saponins, and therefore, they should be fed intermittently (Cheek, 2000). The use of quillaja may be more beneficial than Yucca as an antiprotozoan feed additive, as higher concentrations of yucca are necessary to elicit its antiprotozoal effects. Yucca extract has also been demonstrated to modulate the growth of rumen bacteria. Specifically, Wallace et al. (1994) found that yucca extract enhanced Prevotella ruminicola growth while suppressing the growth of Streptococcus bovis. In contrast, Eryavuz and Dehority (2004) reported that yucca extract had no effect on ruminal microbial concentrations when fed to sheep for 3 weeks at 5, 10, 20, and 30 g/day. Therefore, the effects of saponins on ruminal bacteria growth may be dependent on the saponin source. Regarding pathogenic bacteria, Hassan et al. (2010) reported that yucca and quillaja extracts expressed antibacterial activities in vitro against Staphylococcus aureus and Salmonella Typhimurium, while only quillaja

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extracts demonstrated antibacterial activities against E. coli. Similar antimicrobial activities were observed during in vitro studies using ruminal fluid medium supplemented with steroidal saponins from yucca extracts on the growth of Streptococcus bovis, Prevotella bryantii, and Ruminobacter amylophilus (Wang et al., 2000). While there is a large amount of literature describing the effects of saponins in vitro, there are limited data on the effects of saponins on bacterial populations in vivo. Regardless, the in vitro data suggest that feeding of saponins, in particular those extracted from yucca or quillaja, may have the ability to decrease rumen concentrations of protozoans and some bacteria. In pigs, we have previously demonstrated that yucca and quillaja can have significant effects on the innate immune system following a provocative challenge with an endotoxin derived from gram-negative bacteria, and the weaned pig‘s response to a Mycoplasma hyponeumoniae vaccine (Carroll and Haydon, 2007). In weaned pigs supplemented with 0.4% yucca, average daily gain was significantly increased during the 12 days following endotoxin exposure as compared to control-fed pigs. While research in this arena is still relatively new, our data demonstrates that supplementing the weaned pig‘s diet with Yucca schidigera and Quillaja saponaria can increase the acute phase cytokines in response to a provocative immune challenge, which may indeed enhance both cell-mediated and humoral immunity. Additional research is needed to evaluate the optimal inclusion rate of these supplements and the potential immunological protective effect of these supplements when animals are exposed to live bacterial challenges. These preliminary data do suggest, however, that inclusion of Yucca schidigera and Quillaja saponaria in the weaned pig‘s diet may prove to be viable alternatives to antimicrobials.

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BETA-GLUCAN Beta-glucans are one of the most common polysaccharides found in the cell wall of bacteria and fungi, including yeast. In addition, β-glucans are found in high concentrations in the bran of many cereal grains such as oat and barley. Beta-glucans have been extensively researched for their immunomodulatory potential (Lowry et al., 2005; Eicher et al., 2006; Chan et al., 2009) as well as their ability to reduce pathogen shedding among livestock (Lowry et al., 2005; Eicher et al., 2010). Beta-glucans have previously been reported to enhance innate immunity, thereby providing immunological protection against bacterial (Onderdonk et al., 1992), protozoan (Goldman and Jaffe, 1991), viral (Rouchier et al., 1995), and fungal diseases (Browder et al., 1984). Both in vitro (Hoffman et al., 1993) and in vivo -glucans can activate macrophages, neutrophils, and natural killer (NK) cells, and increase phagocytosis and cytokine production. Beta-glucans are a diverse class of compounds. All β-glucans are glucose polymers linked together with β-1,3 glycosidic bonds and the diversity among the various β-glucan sources originates from their length as well as the degree and type of branching. The β-1,4 and β-1,6 glycosidic bonds are the 2 major types of branching that occur among the various beta-glucans. Generally, the β-1,6 is more commonly found among fungi; whereas β-1,4 branching are observed among bacteria and cereal grains (Yun et al., 2003). The solubility of β-glucans in water is influenced by the type and degree of branching such that β-glucans with more β-1,6 bonds and more branching are associated with reduced solubility in water.

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Furthermore, it has been suggested that a higher degree of structural complexity is associated with more potent immunomodulatory effects (Chan et al., 2009). Most of the research on the influence of β-glucans on immune responses has been conducted in monogastrics (Lowry et al., 2005; Eicher et al., 2006) or milk-fed calves (Eicher et al., 2010) because it was assumed that β-glucans were completely digested in the rumen (Engstrom et al., 1992). However, data reported by Grove et al. (2006) indicated that fecal recovery of β-glucans when expressed per kg of body weight in finishing cattle fed a diet with 80% barley were close to concentrations reported to have immunomodulatory effects in monogastrics. In that study the authors reported a large variation in the in situ ruminal digestion kinetics of β-glucans from various barely sources; therefore, future research should determine ruminal digestion kinetics as well as the influence of dose on recovery of β-glucans from the lower gastrointestinal tract of ruminants supplemented with various β-glucan sources. In monogastrics, and conceivably in ruminants, ingested β-glucans are recognized by the gut-associated mucosal immune system as foreign. Tissue resident macrophages, neutrophils, dendritic cells, and (NK) cells recognize the β-glucans through a variety of cell-associated receptors, and their diverse innate and adaptive immune responses are subsequently stimulated (Taylor et al., 2002). Beta-glucans have been reported to induce macrophage nitric oxide and cytokine production (Eicher et al., 2006), increase neutrophil/heterophil phagocytosis and oxidative burst (Lowry et al., 2005; Eicher et al., 2006), increase blood mononuclear cell proliferation (Chan et al., 2007), enhance maturation of dendritic cell populations (Lin et al., 2007), and alter salivary immunoglobulin A concentrations (Lehne et al., 2005). While the proof of principle for the influence of β-glucans on various immune cell populations has been validated, the specific role of the various β-glucans in promoting innate and adaptive immune responses among various livestock species warrants further investigation. Stimulation of both innate and adaptive immune responses at the level of the gastrointestinal tract via -glucan supplementation could potentially reduce the colonization and shedding of pathogens that could enter the food-chain upon slaughter. Roosters fed purified β-glucan beginning at one day of age had reduced liver and spleen Salmonella enterica invasion after they were orally challenged at 3 days of age (7 vs 76% invasion for the β-glucan supplemented vs non-supplemented, respectively; Lowry et al., 2005). In a more recent study, supplementing β-glucan to pre-weaned Holstein calves increased the percentage of calves that were positive for spontaneous fecal E. coli 0157:H7 (50 vs 0% for the β-glucan supplemented vs non-supplemented, respectively; Eicher et al., 2010). In the latter study by Eicher et al. (2010), the authors proposed that the higher fecal recovery of the E. coli 0157:H7 in calves supplemented with the β-glucan potentially could be attributed to the clearance from intestinal colonization. Continued research on the ability of various β-glucans to reduce the colonization and shedding of potential pathogens among livestock in needed before more definitive conclusions can be made. In addition to the -glucans that are found in many cereal grains fed to livestock, there -glucans that are are many yeast commercially available as a feed supplement for livestock. However, more research is needed on the immunomodulatory potential of these various β-glucan sources as well as their ability to reduce gastrointestinal colonization and fecal shedding of pathogens that can be vertically transmitted to the human population during slaughtering.

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FLAVONOIDS

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Flavanoids are a group of polyphenols, isolated from herbs, spices, fruits, nuts, seeds, flowers, and vegetables, that possess antioxidant, antiviral, antimicrobial, antiinflammatory, anticancer, and antiallergic properties (Cushnie and Lamb, 2005; Choi et al., 2007; Mandalari et al., 2007). Flavanoids encompass flavanones, flavones, flavan-3-ols, flaonols, and anthocyanins (Mandalari et al., 2007). Citrus peel, discussed earlier, is also a major source of flavanoids (Choi et al., 2007). Flavonoids act upon cells through various mechanisms, including prohibiting cell proliferation as well as acting as direct antioxidants and free radical scavengers (Pietta, 2000). Plants containing flavonoids are becoming of greater interest due to the consumer shift towards consuming foods with natural antimicrobial activity instead of chemical preservatives. Flavonoids extracted from bergamot peel have been reported to posses antimicrobial activity against E. coli and Salmonella enterica in vitro (Mandalari et al., 2007). Sohn et al. (2004) reported that a variety of prenylated flavonoids that were isolated from medicinal plants exhibited varying degrees of antimicrobial activities against Candidate albicans, Saccaromyces cerevisiea, E. coli, Salmonella typhimurium, Staphylococcus epidermis and Staphylococcus aureus. However, it should be noted that even though a particular flavonoid many demonstrate significant antimicrobial activities in vitro, results often are conflicting when examining available in vivo data. For example, larch extract is a by-product obtained from processing the bark of the larch tree and has been reported to contain high concentrations of the flavonoid taxifolin that possesses antimicrobial activity (Hara-Kudo et al., 2004). However, an in vivo study by Wells et al. (2009) reported that feeding larch extract at concentration of 2000 ppm to weaned pigs had no effect on fecal shedding of E. coli or Campylobacter.

CAROTENOIDS Carotenoids are a diverse group of compounds that are commercially available and are widely distributed in many feedstuffs fed to livestock. A large number of the carotenoids have provitamin A activity, and much of the initial research attributed the primary immunomodulatory effects of dietary carotenoids to the provitamin A activity (Green and Mallanby, 1930). However, it was later reported that many carotenoids do not possess provitamin A activity, but do retain their immunomodulatory capabilities (Bendich and Shapiro, 1986). The immunomodulatory potential of the various carotenoids is likely to be influenced by the species, physiological state, and the specific carotenoid that is under investigation (Koutsos et al., 2006). The immunomodulatory effects of the various carotenoids have lead to the hypothesis that supplemental carotenoids may reduce the colonization and/or shedding of potential pathogens among livestock (Van Oort and Dawson, 2005; Cernicchiaro et al., 2010). The immunomodulatory effects of carotenoids among many species are diverse and they have been reported to influence both innate and adaptive immune responses. Vitamin A is known to be involved in cellular differentiation, and a recent study in children in India indicated that supplemental vitamin A improved the gastrointestinal epithelial integrity and

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hematology (McCullough et al., 1999). Therefore, it is feasible to speculate that direct effects of provitamin A carotenoids on epithelial integrity may reduce the prevalence of disease. In addition to influencing the physical barrier, carotenoids also affect innate immune responses. Blood neutrophils from cattle supplemented with β-carotene had higher phagocytic and bacterial killing capacity than cattle that were not supplemented with β-carotene (Tjoelker et al., 1988; Michal et al., 1994). Leukocytes from the lymphoid lineage are also influenced by carotenoids including NK cells, and T and B lymphocytes. Increased number of NK cells (Watson et al., 1991) as well as enhanced NK cell cytotoxicity has been observed when humans were supplemented with β-carotene (Prabhala et al., 1991). Additionally, supplemental β-carotene to pigs (Hoskinson et al., 1992) and cattle (Daniel et al., 1990) has been shown to stimulate lymphocyte blastogenesis and increase the weight of the thymus gland. As noted before, much of the initial research focused on β-carotene and the effect was attributed to the provitamin A activity. However, more recent research using carotenoids that do not have provitamin A activity has demonstrated that the immunomodulatory effects previously described for β-carotene are independent of provitamin A activity (Chew and Park, 2004). Therefore, the carotenoids are a group of compounds that have been reported to have beneficial effects on immune responses and have the potential to reduce microbial colonization and shedding. To date, the primary research focus with the carotenoids has been the immunomodulatory roles, which has served as the foundation for the hypotheses that supplemental carotenoids will reduce the incidence of disease (Van Oort and Dawson, 2005) and shedding of pathogenic microorganisms (Cernicchiaro et al., 2010). Van Oort and Dawson (2005) reported that among adult finches with an improved carotenoid status there was a reduced risk of mortality due to Salmonellosis. Although carotenoids were not fed in the study by Cernicchiaro et al. (2010), they observed among feedlot cattle that 2200 IU of supplemental retinol acetate was not associated with a reduction in the prevalence of E. coli 0157:H7 (Cernicchiaro et al., 2010). Future research should address the role of the various carotenoids in reducing both the colonization and shedding of pathogens among livestock.

OTHER PHYTOCHEMICALS In addition to the phytochemicals reviewed above, there is also literature on the use of seaweed and seaweed extract, hops and distiller‘s grains, and capsaicin to reduce pathogen loads in livestock. Seaweed contains indigestible polysaccharides, such as laminarin, fucoidan, and alginic acid, which possess antimicrobial properties (Zvyagintseva et al., 2003; Lynch et al., 2010). These antimicrobial effects have been demonstrated in cattle, lambs, and pigs. Specifically, Bach et al. (2008) reported that feeding sun-dried seaweed at 10 g/kg or 20 g/kg for 7 days following oral inoculation with E. coli O157:H7 reduced fecal shedding in yearling Continental x British steers. Additionally, top-dressing feed with sun-dried seaweed for 28 days decreased the fecal shedding of generic E. coli populations in newly weaned Canadian Arcott lambs (Bach et al., 2008). Seaweed extract from Laminaria digitata fed to newly weaned pigs also decreased E. coli fecal counts (O‘Doherty et al., 2010; Dillon et al., 2010). While the exact mechanism(s) by which seaweed reduces fecal shedding of pathogenic

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bacteria remains elusive, it does appear to exert a direct antimicrobial effect on at least E. coli O157:H7 in cattle (Braden et al., 2004). The push for alternative fuel sources, particularly ethanol, has resulted in an increase in the availability of distillers grains that can be used as a feedstuff for livestock production. The two most common types of distillers grains are wet distillers grains with solubles (WDGS) and dried distillers grains with solubles (DDGS), both of which are by-products of the production of ethanol from cereal grains such as wheat, corn, and barley. In crossbred heifers fed WDGS, fecal shedding of E. coli O157 was greater compared to control-fed heifers on one sample day, with other sample days showing no difference between WDGS and control heifers (Jacob et al., 2008). Additionally, when cattle were fed a diet consisting of various concentrations of triticale dried distillers grain with solubles (TDDGS), no difference was found between control and treatment diets on the amount of E. coli shedding (Sharma et al., 2010). A study in which DDGS was supplemented to crossbred pigs found a decrease in gastrointestinal lesions in the ileum and colon, yet greater fecal shedding when challenged with Lawsonia intracellularis, a bacteria known to cause ileitis in pigs (Whitney et al., 2006). Whitney et al. (2006) reported that there was a trend for pigs supplemented with DDGS to have greater small intestine weights, suggesting a greater turnover in intestinal cells and thus reducing the potential of microorganisms to bind to the intestinal wall. It is clear that the conflicting results between types of distillers grains warrants further research into potential beneficial effects of distillers grains on reducing fecal shedding of pathogens. There is some evidence of an effect of mixed plant extracts on the gastrointestinal ecology. Specifically in pigs, Castillo et al. (2006) demonstrated that feeding a mixture of plant extracts (5% carvacrol, 3% cinnamaldehyde, and 2% capsicum oleoresin) was able to increase the ratio of desirable to undesirable bacteria (lactobacilli:enterobacteria ratio) in the cecum of early weaned crossbred pigs. The increase in the lactobacilli:enterobacteria ratio was mainly due to an increase in the number of lactobacilli. Therefore, plant extracts can alter the microbiota ecology; however, the effect of mixed plant extracts on shedding of bacteria needs to be elucidated. Mitsch et al. (2004) found a decrease in the concentration of Clostridium perfringens in the intestine and in the feces of Ross broiler chickens treated with 2 different essential oil blends (100 ppm) compared to control chickens. Additionally, a study in young Hubbard HI-Ye broiler hybrids found that feeding the same plant extract mixture resulted in an increase in mucus production in the gastrointestinal tract (Jamroz et al., 2006). As the mucosal epithelium of the intestine is the attachment point for pathogenic microorganisms (Manzanilla et al., 2004), these results would suggest that the feeding of these plant extracts could provide a barrier against the attachment of pathogenic microorganisms to the mucosa of the gastrointestinal tract (Jamroz et al., 2005; Jamroz et al., 2006).

TOXIC IMPACT OF PHYTOCHEMICALS The use of phytochemicals at relatively low concentrations can be beneficial with regard to increasing animal health and potentially reducing foodborne illnesses associated with pathogen shedding, as described above. However, adding these supplements at greater concentrations has been demonstrated to have toxic effects in certain situations. For example,

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feeding excessive amounts of saponins could potentially have detrimental consequences given the effects on the permeability of tissues, in particular the intestine when saponins are fed directly to the animal, and therefore should be fed at limited concentrations. Citrus pulp has also been reported to exhibit toxic effects, similar to symptoms of hairy vetch toxicosis, suggesting that citrus pulp stimulated a type IV hypersensitivity reaction (Saunders et al., 2000). In a report by Saunders et al. (2000), 13 out of a group of 650 dairy cows died following consumption of citrus pulp-supplemented feed, with the first death occurring after the cows had been supplemented for 6 weeks. Only cows that were lactating and fed citrus pulp at 6% as fed were affected, thus suggesting a potential relationship between the metabolic state of the animal and the ability to utilize citrus pulp. Additionally, it has been reported that ruminal parakeratosis with associated enlargement of ruminal papillae can occur if too much orange peel is added to cattle diets (López et al., 2010). Therefore, greater attention should also be given to processing and storage procedures associated with phytochemicals that are being utilized as nutritional supplements in livestock production. Shelf life, for example, of phytochemicals that are composed of volatile oils should be thoroughly evaluated as mycotoxins may develop over time resulting in toxic affects if consumed (López et al., 2010).

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CONCLUSION While the medicinal use of phytochemicals has been a common practice in many cultures for centuries, research associated with the use of phytochemicals as a potential alternative to synthetic antimicrobials and as a natural means to reduce/eliminate fecal shedding of pathogens in livestock production systems is a relatively young area of research (Windisch et al., 2008). An increasing amount of research into phytochemicals has resulted in a broader understanding of the antimicrobial activities and potential for reducing livestock pathogen loads. Simultaneously, these efforts have also demonstrated that the use of phytochemicals could potentially cause an imbalance in the gut microbial mileau, thereby jeopardizing the health of animals and potentially causing immunesuppression which would ultimately increase the incidence of disease (Dorman and Deans, 2000). Therefore, further research is needed regarding the use of phytochemicals, with an increased emphasis on appropriate inclusion rates, duration of feeding these supplements, and overall health and performance evaluations to prevent any potential negative effects in livestock production systems.

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Bendich, A, Shapiro, SS. (1986). Effect of β-carotene and canthaxanthin on the immune responses of the rat. J. Nutr. 116, 2254-2262. Braden KW, Blanton JR Jr., Allen VG, Pond KR, and Miller MF. (2004). Ascophyllum nodosum supplementation: a preharvest intervention for reducing Escherichia coli O157:H7 and Salmonella spp. in feedlot steers. J. Food Prot. 67:1824–1828. Brooker, JD, Acamovic, T. (2005). Phytochemicals in livestock production systems. Anim. Feed Sci. Tech. 121, 1-4. Browder, IW, Williams, DL, Kitahama, A, Di Luzio, NR. (1984). Modification of postoperative C. albicans sepsis by glucan immunostimulation. Int. J. Immunopharmacol. 6, 19-26. Callaway, TR, Anderson, RC, Edrington, TS, Genovese, KJ, Harvey, RB, Poole, TL, Nisbet, DJ. (2004). Recent pre-harvest supplementation strategies to reduce carriage and shedding of zoonotic enteric bacterial pathogens in food animals. Anim. Health Res. Rev. 5, 35-47. Callaway, TR, Carroll, JA, Arthington, JD, Pratt, C, Edrington, TS, Anderson, RC, Galyean, ML, Ricke, SC, Crandall, P, Nisbet, DJ. (2008). Citrus products decrease growth of E. coli O157:H7 and Salmonella Typhimurium in pure culture and in fermentation with mixed ruminal microorganisms in vitro. Foodborne Path. Dis. 5, 621-627. Callaway, TR., Carroll, JA, Arthington, JD, Edrington, TS, Anderson, RC, Ricke, SC, Crandall, P, Collier, C, Nisbet, DJ. (2011). Citrus products and their use against bacteria: Potential health and cost benefits. In: Nutrients, Dietary Supplements, and Nutriceuticals: Cost Analysis versus Clinical Benefits. Watson, R, Preedy, G, Gerald, L. (Ed‘s). Springer and Humana Press. New York. (In Press). Carroll, JA, Collier, C, Callaway, TR, Arthington, JD. (2010). Oral administration of citrus pulp reduces gastrointestinal recovery of orally dosed Escherichia coli F18 in weaned pigs. J. Anim. Sci. 88 (E-Suppl 3), 19 (Abstract # 60). Carroll, JA, Haydon, K. (2007). Non-nutrient additives alter aspects of the innate and adaptive immune responses in nursery pigs. 38th American Association of Swine Veterinarians Annual Meeting, Orlando, FL, March 4, 2007. Castillo, M, Martín-Orúe, SM, Roca, M, Manzanilla, EG, Badiola, I, Perez, JF, Gasa, J. (2006). The response of gastrointestinal microbiota to avilamycin, butyrate, and plant extracts in early-weaned pigs. J. Anim. Sci. 84, 2725-2734. Cernicchiaro, N, Pearl, DL, McEwen, SA, Zerby, HN, Fluharty, FL, Loerch, SC, Kauffman, MD, Bard, JL, LeJeune, JT. (2010). A randomized controlled trial to assess the impact of dietary energy sources, feed supplements, and the presence of super-shedders on the detection of Escherichia coli O157:H7 in feedlot cattle using different diagnostic procedures. Foodborne Path. Disease. 7, 1071-1081. Chan, WK, Law, HK, Lin ZB, Lau, YL, Chan GC. (2007). Response of human dendritic cells to different immunomodulatory polysaccharides derived from mushroom and barley. Int. Immunol. 19, 891-899. Chan, GC, Chan, WK, Sze, DM. (2009). The effects of β-glucan on human immune and cancer cells. J. Hematol. Oncol. 25, 1-11. Cheeke, PR. (2000). Actual and potential applications of Yucca schidigera and Quillaja saponaria saponins in human and animal nutrition. J. Anim. Sci. 77, 1-10. Cheeke, PR, Piacente, S, Oleszek, W. (2006). Anti-inflammatory and anti-arthritic effects of yucca schidigera: A review. J. Inflamm. 3, 6.

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Hara-Kudo Y, Kobayashi A; Sugita-Konishi Y; Kondo K. (2004). Antibacterial activity of plants used in cooking for aroma and taste. J. Food Prot. 67(12), 2820-2824. Hassan, SF, Byrd, JA, Cartwright, AL, Bailey, CA. (2010). Hemolytic and antimicrobial activities differ among saponin-rich extracts from guar, quillaja, yucca, and soybean. Appl. Biochem. Biotechnol. 162, 1008-1017. Hoffman, OA, Olson, EJ, Limper, AH. (1993). Fungal -glucan modulate macrophage release of tumor necrosis factor alpha in response to bacterial lipopolysaccharide. Immunol. Letters 37, 19-25. Hoskinson, CD, Chew, BP, Wong, TS. (1992). Effects of injectable β-carotene and vitamin A on lymphocyte proliferation and polymorphonuclear neutrophil function in piglets. Biol. Neonate. 62, 325-336. Jacob, ME, Fox, JT, Narayanan, SK, Drouillard, JS, Renter, DG, Nagaraja, TG. (2008). Effets of feeding wet corn distillers grains with solubles with or without monensin and tylosin on the prevelance and antimicrobial susceptibilities of fecal foodborne pathogenic and commensal bacteria in feedlot cattle. J. Anim. Sci. 86, 1182-1190. Jamroz, D, Wiliczkiewixz, A, Wertelecki, T, Orda, J, Skorupińska, J. (2005). Use of active substances of plant origin in chicken diets based on maize and domestic grains. Br. Poult. Sci. 46, 485-493. Jamroz, D, Wertelecki, T, Houszka, M, Kamel, C. (2006). Influence of diet type on the inclusion of plant origin active substances on morphological and histochemical characteristics of the stomach and jeunum walls in chicken. J. Anim. Physiol. Nutr. 90, 255-268. Kensil, CR. (1996). Saponins as vaccine adjuvants. Crit. Rev. Therap. Drug Carrier Systems 13, 1-56. Koutsos, EA, García López, JC, Klasing, KC. (2006). Carotenoids from in ovo of dietary sources blunt systemic indices of the inflammatory response in growing chicks. J. Nutr. 136, 1027-1031. Lehne, G, Haneberg, B, Gaustad, P, Johansen, PW, Preus, H, Abrahamsen, TG. (2005). Oral administration of a new soluble branched β-1,3-D-glucan is well tolerated and can lead to increased salivary concentrations of immunoglobulin A in healthy volunteers. Clin. Exp. Immunol. 143:65-69. Lin, YL, Lee, SS, Hou, SM, Chiang, BL. (2006). Polysaccharide purified from Ganoderma lucidum induces gene expression changes in human dendritic cells and promotes T helper I immune response in BALB/c mice. Mol. Pharmacol. 70, 637-644. López, JÁS, Li, Q, Thompson, IP. (2010). Biorefinery of waste orange peel. Crit. Rev. Biotech. 30, 63-69. Lota, ML, de Rocca Serra, D, Tomi, F, Jacquemond, C, Casanova, J. (2002). Volative components of peel and leaf oils of lemon and lime species. J. Agric. Food Chem. 50, 796-805. Lowry, VK, Farnell, MB, Ferro, PJ, Swaggerty, CL, Bahl, A, Kogut, MH. (2005). Purified βglucan as an abiotic feed additive up-reglates the innate immune system response in immature chickens against Salmonella enterica serovar Enteritidis. Int. J. Food Microbiol. 98, 309-318. Lynch, MB, Sweeny, T, Callan, JJ, O‘Sullivan, JT, O‘Doherty, JV. (2010). The effect of dietary laminaria derived laminarin and flucoidan on nutrient digestibility, nitrogen

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Wong, G. G. and S. C. Clark. 1988. Multiple actions of interleukin 6 within a cytokine network. Immunol. Today. 9:1379. Yun, CH, Estrada, A, Van Kessel, A, Park, BC, Laarveld, B. (2003). β-glucan, extracted from oat, enhances disease resistance against bacterial and parasitic infections. Immunol. Med. Microbiol. 35, 67-75. Zvyagintseva, TN, Shevshenko, NM, Chizhov, AO, Krupnova, TN, Sundukova, EV, Isakov, VV. (2003). Water-soluble polysaccharides of some far-eastern brown seaweeds. Distribution, structure, and their dependence on the developmental conditions. J. Exp. Mar. Biol. Ecol. 294, 1-13.

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In: On-Farm Strategies to Control Foodborne Pathogens ISBN: 978-1-62100-411-0 Editors: Todd R. Callaway and Tom S. Edrington © 2012 Nova Science Publishers, Inc.

Chapter 9

ORGANIC ACIDS AND THEIR ROLE IN REDUCE FOODBORNE PATHOGENS IN FOOD ANIMALS Ester Grilli and Andrea Piva University of Bologna, Italy

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ABSTRACT Organic acids (OA) are used since long as food preservative agents, and, in more recent times, as feed additives. The compelling need to use OA in feed date back to the last two decades since the extensive use of antibiotics in farming, both growth promoters (AGP) and therapeuticals, caused concerns on the incidence of antibiotic-resistant bugs and posed the urge to find alternative solutions. Feeding dietary OA to animals produced positive effects over a 25-years period, allowing the achievement of good results, even though not as good as AGP. Nevertheless, OA demonstrated to own all the characteristics necessary to be the most promising alternative to AGP and to have the highest cost-benefit ratio compared to other alternative compounds available on the market (Mroz et al., 2003). The efficacy of OA as growth promoters has been linked to impairment of growth of intestinal microflora and therefore to their antimicrobial properties. Nowadays there is an increasing interest in looking forward to the use of OA also from a food-safety perspective, ie to take advantage of their ―n atural‖ antimicrobial power and target it toward the prevention or treatment of intestinal colonization by specific foodborne pathogens, such as Salmonella, Campylobacter, E. coli, etc. even though this application of OA is still under investigation and far to be conclusive. The effectiveness of OA as antimicrobials is strictly dependent on the type of acid and its chemical structure, and relies on molecular weight, pKa, and partition coefficient. From a generic standpoint, OA are defined as organic compounds with acidic properties that do not completely dissociate in water. Not all OA have antimicrobial properties, but many of interest from this perspective easily fall into 3 categories: short chain fatty acids (SCFA), medium chain fatty acids (MCFA), and TCA cycle acids. SCFA and MCFA are carboxylic acids with a short or medium unbranched aliphatic chain, respectively. In particular, SCFA have aliphatic chains with less than 6 atoms of carbon, whereas MCFA have tails with a number of carbons between 6 and 12. SCFA, such as acetic, propionic, butyric and iso-butyric, valeric and iso-valeric, are produced in millimolar concentrations in the lower gut by the microbiota, which ferments the substrates (sugars and

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Ester Grilli and Andrea Piva amminoacids) escaping digestion in the small intestine. Those acids are produced in variable concentrations, differing upon animal species and intestinal tract, and in a ratio which is, under physiological circumstances, almost fixed at 60:25:15 for acetic, propionic, and butyric (Cummings, 1981). Molar proportions of valeric acid and iso-acids are usually minor. Each acid has a special function in the host: acetic is the energetic source for skeletal muscles, including the cardiac one; propionic is the precursor for gluconeogenesis, and butyric is essential for the development and maintenance of colonic mucosa (Shepacch et al., 1994). MCFA of particular interest are caproic, caprylic, and capric acid (hesanoic, octanoic, and decanoic acids). They are present in coconut and palm kernel oil, and they are named after goats because of their characteristic and pungent odour; they can also be found in significant concentrations in the milk of goats and other mammals. They can be incorporated in the membrane as phospholipids, and it seems they have an higher bactericidal activity than SCFA (Nakai and Siebert, 2002; Van Immersel et al.,2004a; Van Immersel et al., 2006). Citric acid, α-ketoglutaric acid, succinic acid, fumaric acid, and malic acid are all intermediates of the Krebs cycle, and, as a such, can be used to produce energy by aerobic cells. Depending on concentrations, they can also have antimicrobial properties. In addition to the above mentioned categories, there are a few OA that are commonly used both in food preservation and in animal feeding with great results and high efficacy: benzoic and sorbic acid. Benzoic acid is an aromatic carboxylic acid, and precursor of many other organic substances. Benzoic acid and its salts are used as a food preservative, represented by the E-numbers E210, E211, E212, and E213; they inhibits the growth of molds, yeasts, and some bacteria (Krebs et al., 1983). Sorbic acid is a straight chain unsaturated fatty acid which was first isolated in 1859 from the oil of the berries of the tree Sorbus aucuparia and since the late forties it was used as anti-fungal agent. Afterwards its antibacterial properties were also discovered and since then it is used for a variety of industrial applications in food preservation because, compared to other acids such as benzoic or propionic, is much more effective at less acidic pH, due to its relatively high pKa value ( = 4.76). Inhibition of bacterial growth by sorbic acid or its salts could be exerted through different mechanisms such as the alteration of cell membranes, inhibition of transport systems and key enzymes, creation of a proton flux into the cell or a combination of those (Sofos et al., 1986), even though the most accounted one is the inhibition of enzymes involved in the metabolism of carbohydrates (enolase and LADH), in the TCA cycle, or enzymes containing SH-groups, and catalases or peroxidases (Sofos et al., 1986).

ORGANIC ACIDS MECHANISM OF ACTION The Anion Model The inhibitory effect of OA has been reported to be linked to the ion model, thus being highly related to their undissociated form (Russell and Diez-Gonzalez, 1998). In particular, The antibacterial effect of OA might be explained by the protons (H+ ions) and anions (RCOO- ions) into which OA are divided after passing the bacterial cell wall and which have a disruptive effect on bacterial protein synthesis. Weak acids, including those produced as a result of intestinal fermentation, can diffuse across the bacterial cell membrane in the uncharged, protonated form and dissociate inside the cell, lowering internal pH (pHi). Since OA are able to pass the bacterial cell wall in their undissociated more lipophilic form,

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their rate of dissociation is very important. The effect of pH on organic acid dissociation is given by the Henderson-Hasselbach equation, where A- and HA are the dissociated and undissociated species, respectively (pHe = pKa + log(A-)/(HA), where pHe is the external pH). Figure 1 shows how the rate of dissociation of the acid depends on the pKa. The lower the external pH, the more undissociated weak acid will be available (based upon pKa values; Figure 1) to cross the membrane and affect pHi. To overcome the lowering of pHi, several systems ATP-dependent to elevate the pHi by consuming protons are induced, finally impairing bacterial protein syntesis; furthermore, as the pHi remains high, the pH (pHi-pHe) becomes larger allowing a logarithmic intracellular accumulation of anions, which is believed to be the real toxicity mechanism (Russell and Diez-Gonzalez, 1998).

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Figure 1. Acids rate of dissociation depending on their pKa (In Piva et al., 2006; Elsevier permission awaiting).

Russell and Diez-Gonzalez (1998) observed also that cells with higher concentrations of intracellular potassium as a potential counteraction (Gram-positive bacteria) would be able to accumulate higher concentrations of fermentation anions, and the decrease in intracellular pH would not be so great. Virtually, all bacteria have a constitutive, low-affinity potassium transport system that operates as a potassium/proton symport mechanism to dissipate pH (Kashket and Barker, 1977). If a bacterium has a very high concentration of intracellular potassium, membrane potential remains high and pH is low, and vice versa (Kajikawa and Russell 1992). Fermentation acid-resistant bacteria have low ΔpH and are able to generate ATP and grow with a low intracellular pH; resistant bacteria would eventually allow intracellular pH to decrease rather than pumping out protons that would cause anions accumulation as per acidic-sensitive ones It seems therefore that acidic (Russell and Diez-Gonzalez, 1998). This model explains why bacteria differ in sensitivity to OA but does not explain the antibacterial effect of one acid versus another one (Van Immersel et al., 2006). It has been shown that also the dissociated form can also contribute to the antimicrobial effect of sorbic acid at pH levels above 6 therefore indicating that there must be other mechanisms involved in these reactions (Elkund, 1983). As suggested by Hsiao and Siebert (1999), in fact, OA that are typically used in food and feed preservation systems are members of different classes or subfamilies that can differ in number of carboxylic groups, hydroxylic

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groups, carbon-carbon double bonds, aromatic or aliphatic structures, numbers and type of side chains and therefore it is impossible to predict an antimicrobial effect taking into account the ―ani on model‖ only. For this purpose Hsiao and Siebert (1999) applied principal components analysis (PCA) to 11 different properties of 17 OA and developed a system to predict the effect of OA taking into account the combination of different characteristics as emerged from the principal component analysis. The results demonstrated that four fundamental properties can represent the information contained in the 11 acids that were analyzed, ie presence of polar groups, number of double bonds, molecular size, and solubility in non polar solvents (Hsiao and Siebert, 1999).

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Acid Shock Tolerance Response (ATR) An important feature associated to the pathogenic role of foodborne pathogens is their ability to resist to acidic conditions and acidic stress. Acidic stress is defined as the combination of low pH and high concentrations of weak OA in the environment (Bearson et al., 1997). Once the pathogen is ingested, in fact, it must face the acidity of gastric secretions and its ability to survive them determines its infection dose (Lin et al., 1996). At intestinal level, pH is less acidic than in the stomach, but the high concentration of SCFA increases the stress to high level for acid-sensitive bacteria such as Salmonella and E. coli. But as infection occurs, it must be evident that they can develop system to survive the acidic stress and confer protection to the bacterium itself. Acid resistance, acid tolerance, and acid habituation are terms used to describe the survival of enteric pathogens to acidic conditions (Bearson et al., 1997). Acid tolerance response in S. typhimurium is a process that relies upon induction of a protection system at different pH levels. The first stage of protection is activated when Salmonella encounters environmental pH slightly acidic (pH = 6, pre-acidic shock;) which causes the synthesis of emergency pH homeostasis systems that restore pH to neutrality when periods of extreme acidic stress are encountered (pH = 3). The second stage of protection (post acidic shock) is activated when pH drop off below 4.5, when more than 50 acid shock proteins responsible of macromolecular repair are synthesized (Bearson et al., 1997). Inducible homeostasis is exemplified by the lysine decarboxylase (CadA) coupled with the lysine-cadaverine antiporter (CadB). To overcome the lowering of pHi the CadA decarboxylates intracellular lysine to cadaverine and consumes a proton in the process. Cadaverine is then exchanged for fresh lysine from the surrounding environment via the CadB antiporter (Park et al., 1996). Similar inducible systems, with arginine and glutamate decarboxylases, have been described for Escherichia coli (Lin et al., 1995). Emergency pH homeostasis alone is not effective in protecting Salmonella by low acidic conditions, when acid shock proteins (such as RpoS, Fur, and PhoP) synthesis during the second stage is essential for survival. Audia et al. (2001) reviewed how induced ATR response at pH 4.5-5.8 allowed S. typhimurium to survive at pH 3 for hours, whereas E. coli, adapted to grow with concentrations of 11.3 or 13.5 mM of butyrate and propionate at pH 6.5, survived to a challenge at pH 3.5. Many other enteric pathogens, along with Salmonella and E. coli, have been shown to possess ATR systems.

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ORGANIC ACIDS IN VITRO

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Effect of Organic Acids on Virulence Genes of Pathogenic Bacteria Beside the antimicrobial effect, explained by the anion model, some organic acid may also exert their action through inducing or depressing the expression of specific virulence genes. The best studied case is SCFA, such as acetic, propionic, and butyric regulation of invasion and virulence genes of Salmonella. Salmonella is a facultative intracellular enteric pathogen that requires invasion to exploit its pathogenesis. This invasion is a complex process involving 28 specific genes located in a 40 kilobases region named specific pathogenicity island (SPI-1) (Durant et al ., 1999). SPI-1 genes encode regulatory proteins, structural components of the needle complex, essential for invasion, and additional effector proteins (Van Immersel et al., 2006). In particular, hilA and invF, two genes encoded in the SPI-1 island, are responsible for the regulation of the activation and expression of invasion genes (Durant et al., 1999). BarA/SirA are positive regulators of hilA transcription located outside SPI-1, and are both required for invasion gene expression and enteropathogenesis; loss of either barA or sirA alters the expression of SPI-1 invasion genes, and while sirA seems to work independently, the presence of sirA is required for barA to function (Lawhon et al., 2002). In vivo, the induction of invasion genes is regulated by environmental signals such as neutral pH, low oxigen tension, and increased osmolarity (Durant et al., 2000a; Lawhon et al., 2002). These environmental conditions are tipically found in the small intestine where colonization occurs; also SCFA play a crucial role in regulating invasion genes: low SCFA concentrations are found in the small intestine (20-40 mmol/L), whereas high concentrations of SCFA are present at cecal and colonic level, ranging from 130 mmol/L to 300 mmol/L , depending on the animal specie and diet (Cummings, 1981). Durant et al., (2000b, 2000c) investigated the role of pH in determining the expression of hilA and invF genes in S. typhimurium both in vitro and in Hep-2 cell cultures in presence of SCFA and the results substantiated the hypothesis that changes in pH conditions and SCFA concentrations can modulate the expression of invasion genes: an increased gene expression at pH around 6 indicates in fact an anion accumulation as the cause of this activation. These results led Lawhon and collegues (2002) to conduct a study to investigate the role of SCFA at ileal or cecal conditions in the expression of Salmonella invasion genes. Acetate in concentrations and at pH found in the distal ileum of mammals (10-30 mM; pH = 6.7) induced the expression of invasion genes in Salmonella barA mutants, devoid of barA expression, but not SirA ones. This acetate-related effect was observed in barA mutants only at pH 6.7 but not pH 8, implying that the restoring of expression capacity was effective only at pH-dependent conditions that allowed acetate to accumulate intracellularly. It seems therefore that acetate is directly involved in the regulation of invasion genes since acetate in the bacterial cell is converted in acetylphosphate and, as a such, it may phosphorylate barA or sirA or both. The same test was conducted with 30 mM of propionate and butyrate at pH 6.7 that, conversely, were not able to restore hilA and invF expression in the barA mutant. Moreover, propionate and butyrate significantly reduced invasion genes expression also in the wild type strain. Finally, the last experiment was conducted with a mixture of SCFA that mimics ileal or colonic conditions, both in molar proportions and absolute concentrations, ie

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30 mM composed of 85% acetate, 7.5% propionate and 7.5% butyrate for ileum, and 200 mM of which 55% acetate, 35% propionate and 10% butyrate for colon, both at pH 6.7. Results confirmed that in ileal conditions invasion genes of wild type strains were significantly promoted, similarly to what happened with the only acetate, whereas in colonic conditions they were reduced from 3 to 26-fold, similarly to the effect found for both propionate and butyrate. The authors therefore concluded that ileal conditions, with a predominant proportion of acetate, may induce expression of invasion genes, whereas colonic concentrations of propionate and butyrate have an inhibitory effect on Salmonella (Lawhon et al., 2002). In addition to that, butyrate was found to specifically down-regulate the expression of 19 genes common to both Salmonella enteritidis and typhimurium, of which 17 were localized on the SPI-1 (Gantois et al., 2006); although the mechanism of action of butyrate remains unknown, it has been hypothized that it could interfere with hilA-dependent regulation of SPI-1 by altering the regulation of hilD transcription (Gantois et al., 2006). Formic acid, which is the smallest and the simplest carboxylic acid, is produced by microflora during anaerobic fermentation in significant amounts in the small intestine, whereas it is almost absent in the large intestine (Jensen, 1998). Beside that, formic acid is also one of the most important organic acid used as a feed additive to control intestinal microflora and to ameliorate growth performance of animals. Studies on pigs demonstrated that beside acetate, formic acid is predominant in the distal small intestine (40 mmol/day; Jensen, 1998; Argenzio and Southworth, 1975), whereas propionate and butyrate are higher in cecum and colon. As reported above, it as been demonstrated that while acetate promotes Salmonella invasion genes, propionate and butyrate downregulate them. But what about formate? To understand the role of formic acid in the regulation of invasion genes of Salmonella, Huang et al (2008) tested the effect of formate on the expression of sipC, one of SPI-1 effector proteins secreted from Salmonella, in ackA-pta mutant, a strain with deleted acetate kinase and phosphotransacetylase genes, deficient in invasion and SPI-1 expression. This mutant also excreted 68-fold less formate into the culture medium and the addition of 10 mM of formate or formic acid restored sipC expression to a level greater that the wild type strain, suggesting that formate, which is normally produced by anaerobic fermation of Salmonella wild type, was required for invasion (Huang et al., 2008). The same addition of formate to barA or sirA mutants, however, did not restored sipC expression. To determine the concentration of formate required to stimulate the invasion, growing doses of formate were tested in the ackA-pta mutant and results demonstrated that as little as 1 mM of formate was enough to restore the induction of sipC to values greater than the wild type strain. The next step was the evaluation of the level of invasion of Hep-2 cells by the ackA-pta, sirA, and barA mutants in presence of sodium formate at 10 mM, and, similarly to the first experiment, the addition of formate restored the invasion capacity of the ackA-pta mutant to levels comparable to the wild type strain, but not of the barA or sirA mutants. Additionally, as previously reported for acetate, formate required low pH levels (6.7) to induce sipC expression both in the ackA-pta mutant and in the wild type, whereas in alkaline conditions sipC expression was not modified in either strain, thus demonstrating that formate is required to enter the cell to exploit its action. Formate produced by intestinal microflora in the small intestine acts as a signal that induces Salmonella invasion, further substantiating the theory that OA present in the distal small intestine environment may contribute to those conditions necessary to the infection to occur, and that distal ileum is the election site of infection of

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Salmonella. Still unknown remains formate mechanism of action, which acts through the regulators hilA and hilD, but not through barA/sirA regulator systems found outside SPI-1 (Huang et al., 2008). Furthermore, formate, along with succinate, has been demonstrated to protect stationary phase E. coli and Salmonella cells form the action of the bactericidal/permeability-increasing protein (BPI), which is a potent protein with high affinity for gram-negative LPS and is produced as a defense system by the Paneth cells of the host (Barker et al., 2000). BPI-derived peptide P2 blocked the consumption of oxygen by stationary phase cells pre-incubated with glucose, pyruvate or malate, causing a 9 log10 reduction within 90 minutes of co-incubation, whereas cells viability and oxygen consumption were not significantly affected by formate or succinate addition (Barker et al., 2000). Medium-chain fatty acids were also investigated in vitro in an invasion assay, an the results showed a significant reduction of cell invasion percentage in T84 cells by Salmonella enteritidis, as well as a depression of hilA due to the addition to the medium of a 2 mM concentration of capric, caproic, or caprylic acid (Van Immersel et al., 2004).

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Organic Acid on Microflora and Foodborne Pathogens In vitro antimicrobial properties of OA are measured through the minimal inhibitory concentration (MIC) for target species, which is the ability of OA to prevent their growth, either on an agar plate or in liquid culture, after an incubation period. The MIC of different acids can vary upon type of acid (molecular weight, number of –COOH residues, Ka), media pH, target strain and growth conditions, and inoculum concentration. Numerous data are available about in vitro results, even though they are not always consistent in literature due to the high number of variables that the technique imposes. In a study conducted by our research group, the antibacterial activity against S. typhimurium was studied for citric, sorbic, malic, fumaric, benzoic, lactic, heptanoic, and octanoic acids. A MIC broth dilution assay was performed with OA solutions at concentrations ranging from 0.49 mM to 500 mM at pH 6.5. The MIC for benzoic acid was identified in 125 mM, which completely inhibited the growth of Salmonella. Other acids were effective in reducing optical density, and therefore growth, without determining a complete inhibition of the strain though. In particular, heptanoic and octanoic acids strongly inhibited the growth of the Salmonella by 84% at 1.82 mM, whereas sorbic acid by -92% at 50 mM; citric and malic acids reduced OD by -51% and -85% at 250 mM and 500 mM, respectively. Fumaric and lactic acids did not inhibit Salmonella growth. Linear regression analysis showed an interesting dose-effect for citric, malic, heptanoic and octanoic acid (R2=0.99; R2=0.98; R2=0.94; R2=0.91, respectively, P1000 >1000 Benzoic 31.25 31.25 Caprylic 7.81 62.50 Citric 62.50 >1000 Formic >1000 >1000 Fumaric 500 >1000 D-Gluconic 500 >1000 Heptanoic 7.81 31.25 Iso-butyric 500 >1000 Lactic 1000 >1000 DL-Malic 125 250 Propionic >1000 62.50 Sorbic 31.25 500 Succinic 500 >1000 Tartaric 1000 >1000 n-Valeric 125 >1000 MIC are expressed as mmol/L of the substance that did not increase optical density after 24 h incubation.

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Taken together, those results are a clear indication that gram positive bacteria seem to be much more sensitive than gram negative to the action of OA, which is not a surprise. In gram positive bacteria, in fact, the fewer barriers and the different structural and chemical composition of the cell wall make them more permeable to large and polar molecules, whereas the inner and outer membrane of gram negative modulate their entrance (Nikaido and Vaara 1985; Brul and Coote, 1999). These results were in accordance with Skrivanova and colleagues (2006) who conducted MIC tests with OA on different strains of C. perfringens, Salmonella, and E. coli. They also found MCFA to more effective than lactic and Krebs intermediates acids, with the exception of citric acid which exhibited a weak antibacterial activity. In our study we did observe an higher efficacy of benzoic, MCFA, and sorbic acids in comparison to the others. The reason could be associated to the fact that while malic, lactic and fumaric acid have pKa values between 3 and 4, MCFA and sorbic acid have pKa values around 4.8, and as both experiments were conducted at pH slightly below neutrality (6-6.5), this factor could have affected the results. Following this explanation, the antibacterial power of benzoic acid must be therefore not exclusively linked to its pKa, which is low (4.19), but rather to its aromatic structure and hydrophobicity. In fact, as stated by Freese (1978), the mechanism of action of lipophilic acids is due their coefficient of partition lipid/water and to the ability of the molecules to delocalize the negative charge of the ions and thus increase membrane mobility and permeability, as well as to their pKa (Freese 1978). In the Principal Component Analysis of OA antimicrobial properties conducted by Hsiao and Siebert (1999), four principal components were identified to be responsible of 93% of the total variance, and one of the principal component determining an organic acid efficacy (PC2) was almost entirely related to the number of carbon-carbon bonds and the conjugated double bonds; this feature collocated benzoic acid and sorbic acid in a group apart from other acids. Medium chain fatty acids, and, again, sorbic acid, had high values of principal components number one (PC1), which alone accounted for almost 30% of the total variance, and that was strongly influenced by pKa, therefore confirming the importance of pKa in determining the organic acid efficacy at intestinal level. In conclusion, the efficacy of lactic acid and di-tricaboxylic acids may be strongly affected by the medium pH, making them more effective in acidic environments or at stomach level, whereas other acids, such as sorbic or MCFA can have broader application ranges. Batch fermentations allow a gross investigation of microbial metabolism and, even if to a limited extent, they can also predict the behavior of an additive in vivo. Clearly, as all in vitro systems, it is only an extrapolation of what happens in vivo, which instead is the final result of the complex interplay among host immunitary system, microbes, mucosa and diet. Nevertheless, it is a useful mean to screen different supplementation strategies before they are validated in vivo and in-field. As the antimicrobial activity of OA is not selectively directed toward pathogens, it must be emphasized that OA impact also the microflora in toto and that each acid exerts dramatically different effects in modulating the gastrointestinal ecosystem. Biagi and Piva (2007) reported that among different acids tested at 60, 120, and 240 mM, lactic acid enhanced and not inhibited bacterial fermentation at every concentration; citric acid also strongly increased gas volume when used at 60 and 120 mM, whereas it almost completely inhibited bacterial activity at 240 mM. The gas production was also increased by fumaric acid at 60 mM and α-ketoglutaric acid at 7.5, 60 and 120 mmol/l, thus supporting the double role

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played by lactic acid and TCA cycle acids, that can act as both substrates and end-products of microbial fermentation (Renault et al., 1988; Loubiere et al., 1992; Tran et al., 1997; Tielens and Van Hellemond, 1998 Medina de Figueroa et al., 2000). Sorbic acid showed the strongest antibacterial activity and was the only acid that reduced final gas volume when used at 60 mM; while sorbic, fumaric, citric, and benzoic acid reduced ammonia after 24h of fermentation at any given concentration, other acids, such as formic, acetic, propionic, butyric, and malic, failed to exert an effective control of ammonia levels in the fermentation liquor. The fermentation pattern resulting from this study depicts a scenario in which it is impossible to predict the inhibitory effect of an organic acid in an environmental complexity such the intestine. Even those acids that have an high inhibitory effect against a single strain tested in a MIC assay, which is usually subjected to very few variables, may behave in a different way in vivo, and almost each acid may elicit a different microbial activity in relation to the type and concentration used.

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Multi-Hurdle Technique: The Synergy Between Organic Acids and Natural Compounds The main constraint in using a single approach in food safety, in this case OA, is related to the high amount of substance that is needed to achieve pathogens inhibition (Nazer et a., 2005). Those amounts can be prohibitive costwise, and, sometimes, also from a technologic standpoint. In this context, the combined used of preservative strategies can be a useful approach, and, plant-derivative compounds or permeabilizers seem to be promising (Nazer et al., 2005). Plant extracts contains a very wide number of active substances in variable amounts; they differ for composition and chemical structures, depending on the extraction method and on the plant cultivar. All of the characteristics that influence plant extract composition affect also their chemical properties and effect, and, for this reason, it is generally accepted to work with their pure derived substances, naturally derived or chemically synthetized, essential oils (EO) or nature-identical compounds (NIC).

Figure 2. Proposed mechanism for organic acids and essential oils against microbes (adapted from Russell and Diez-Gonzales, 1998). On-Farm Strategies to Control Foodborne Pathogens, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook Central,

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The antimicrobial properties of essential oils have been reviewed by many authors in the past, but only in recent times attempts to identify the bioactive principles have been made (Dorman and Deans, 2000).Following these studies, a correlation between the antimicrobial activity of a compound and its % in an essential oil, its chemical structure, functional groups, and configuration has been observed (Dorman and Deans, 2000). Considering the large numbers of constituents of EO, it must be supposed that their antimicrobial activities, as for OA, is not related to a specific mechanism of action but rather to several targets in the microbial cell (Skandamis and Nychos., 2001; Carson et al., 2002). It is supposed that EO acts through two different mechanism of action: the first is related to their hydrophobicity which allow them to insert in the phospholipid bilayer of the bacterial cell, and the second is related to the inhibition of bacterial enzymes and receptors through their interaction with specific sites. Through their hydrophobic structure EO are able to disrupt the bacterial membrane and to change its permeability (Knobloch et al., 1989; Sikkema et al., 1994; Oosterhaven et al., 1995; Ultee et al., 2000, 2002); this causes a ion efflux from the inner cell to the external medium (Oosterhaven et al., 1995; Gustafson et al., 1998; Helander et al., 1998; Cox et al., 2000; Lambert et al., 2001). The ion leakage is usually coupled with other cytoplasmic constituents leakage, and until a certain amount of loss it can be tolerated by the bacterial cell without loss of viability, but, if the efflux is prolonged, it will cause cell to collapse. The importance of the hydroxyl group in determining the antimicrobial power has been confirmed, since it has been found that phenolic compounds, such as thymol and carvacrol, have the highest antimicrobial activity (Dorman and Deans, 2000; Ultee et al., 2002; Nazer et al., 2005). Furthermore, it has been established that EO can act on the proteins linked to the cytoplasmic membrane and two possible mechanisms have been suggested to explain the interactions of phenols on such proteins (Knobloch et al., 1989). The first implies that the hydrocarbons can accumulate in the phospholipid membrane and interfere with the lipid-protein interactions; the other is that the lipohilic hydrocarbon can directly interact with hydrophobic parts of the proteins (Juven et al., 1994; Sikkema et al., 1994). Another supposed action of EO is the interference with cells enzymes: Conner and Beuchat (1984) found that the presence of EO in culture media stimulate the growth of pseudomycelia, as a result of incomplete separations of newly formed cells in yeasts, suggesting that EO may be involved in energy regulation or synthesis of structural constituents, whereas Gill and Holley recently communicated an inhibition of the ATP-ases in E. coli and L. monocytogenes mediated by eugenol and carvacrol (Gill and Holley, 2006). The mode of action of carvacrol and its analogue, thymol, two of the maior constituents of oregano and thyme essential oils, has been extensively studied. Carvacrol and thymol are able to disintegrate the outer membrane of gram-negative bacteria, releasing lipopolysaccharides (LPS) and increasing the permeability of the cytoplasmic membrane to ATP. Ultee et al. conducted different studies on B. cereus in order to investigate the mechanism through which carvacrol exerts its antibacterial action, and results showed that carvacrol dissolves in the phospholipid bilayer aligning between the fatty acid chains resulting in conformational changes of the membrane. A disturbance of the van der Waals interactions between the acyl chains in the membrane causes an increase in membrane fluidity, which in turn would increase passive permeability (Ultee et al., 2002). In the same study the difference between cymene, a precursor of carvacrol differing only for the absence of the hydroxyl group, and carvacrol has been investigated; to asses the lipophilicity of the two substances the Po/w , e.g. the partition coefficient octanol/water, has been measured.

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Ester Grilli and Andrea Piva

Since cymene resulted to have greater Po/w values than carvacrol and a lesser antimicrobial power it has been postulated that the disgregation of the membrane could not be the unique mechanism of action of those molecules, and that another factor must be involved. This factor is the presence of the hydroxyl group in the carvacrol molecule. The mechanism of action proposed by Ultee et al., (2002) is that carvacrol act as a transmembrane carrier of monovalent cations. The characteristic feature of a phenolic hydroxyl group is its significantly greater acidity than that of an aliphatic hydroxyl group. Substantially it should acts as an organic acid does: undissociated phenolic compound pass through the cytoplasmic membrane toward the cytoplasm and it dissociates by releasing the proton in the cytoplasm. It re-associates by carrying a K+ ion or any other cation and in that form is able to re-diffuse through the cytoplasmic membrane to the external environment. This hypothesis was supported by the author‘s observed efflux of K+ and influx of H+ in B. cereus during exposure to carvacrol. Similar to carvacrol, thymol contains both a hydroxyl group and a system of delocalized electrons and was found to possess strong antimicrobial activity as well. Juven et al. (1994) examined the activity of thymol against S. typhimurium and S. aureus and hypothesised that thymol binds to membrane proteins hydrophobically and by means of hydrogen bonds, thereby changing the permeability characteristics of the membrane; furthermore thymol was found to be more inhibitive at pH 5.5 than 6.5. At a lower pH thymol would be more undissociated and therefore more hydrophobic, thereby binding better to the hydrophobic areas of proteins and dissolve better in the lipid phase (Juven et al., 1994). Beside the antibacterial activity of nature-identical compounds, there is a new thrilling hypothesis which relies on the antioxidant/anti-inflammatory properties of plant derived compounds. Winter et al. (2010) recently reviewed on Nature that Salmonella uses inflammation products as a mean to outgrowth and outcompete with other microbes in the intestine. More in particular, the reactive oxygen species generated during intestinal inflammation react with tiosulphate, present at intestinal level, and form tetrathionate, which acts like an elettron acceptor to allow Salmonella to ― breathe‖ in an anaerobic environment. Essential oils and their pure-derived NIC, such as carvacrol and thymol, have both antioxidant and anti-inflammatory properties (Alma et al., 2003; Landa et al., 2009), and, as a such, can suppress inflammation at intestinal level and indirectly inhibit the growth of Salmonella (Grilli et al., 2011). As OA and natural compounds have different targets in the microbial cell, it is presumable that they could act in synergy. The proposed mechanism of action is the permeabilizing effect of aromatic compounds, which are more soluble in fat and can dissolve in the phospholipid layer of the bacterial membrane, that would eventually facilitate the entrance of the acid. Synergy is reported when the observed inhibition of a substance in combination with another is larger than the observed inhibition of the substance alone, ie when the combination of two substances is more effective than one substance alone (Nazer et al., 2005). Another definition of synergy is given when the combination of the molecules gives a >1 log10 greater inhibition than the one given by the sum of the inhibition given by the two substances alone (Eliopoulos and Moellering, 1991). In a study conducted by our research group (unpublished data) we evaluated the synergy of a combination of citric or sorbic acid with carvacrol or thymol. According to Ohran et al., we calculated the fractional inhibitory concentration (FIC index) was calculated as follows: FIC = FIC A + FIC B, where FIC A was the ratio between the MIC of substance A in combination and MIC of substance A alone and FIC B was the

On-Farm Strategies to Control Foodborne Pathogens, Nova Science Publishers, Incorporated, 2012. ProQuest Ebook Central,

Organic Acids and Their Role in Reduce Foodborne Pathogens in Food Animals

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ratio between the MIC of the substance B in combination and MIC of substance B alone. Synergy was defined as a FIC  0.5, an additive effect was defined when the FIC is >0.5 to 50 mM, which was the highest tested. Anyhow, sorbic acid 50 mM caused a 96% reduction in optical density at 24 h. Thymol alone was effective at 1.36 mM, which was aligned with results from Nazer et al.(2005), whereas carvacrol alone did not completely inhibit the growth of Salmonella at 1.82 mM even if it caused a growth reduction by 50%. FIC index of combinations indicated that, even though a real synergy, as defined by Ohran et al., (2005), could not have been observed, with the exception of citric acid in combination with thymol at 10 h of incubation, the additive effect was quite strong, as demonstrated by the FIC index, which was always < than 1 (Table 3). If we consider that the lower the FIC index, the higher the synergistic or additive effect, we could assume that in presence of citric, the use of thymol or carvacrol, allowed to reduce the dose of inclusion of the two selected components much more than with sorbic.

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Table 3. Minimal inhibitory concentration (MIC) of sorbic and citric acid combined with thymol (a) or carvacrol (b) against S. typhimurium (broth dilution method; pH = 6.5) a) Acid

h

MIC* Acid alone

Citric

10 24 10 24

250 >250 50 >50

1.36 1.36 1.36 1.36

h

MIC* Acid alone

Carvacrol alone

Sorbic b) Acid

Thymol alone

Acid combined with thymol 31.25 + 0.77 62.5 + 0.77 25 + 0.77 50 + 0.58

FIC index‡ 0.65 1.82 125 + 1.02 1.82 12.5+1.36 50 >1.82 25+1.36