Foodborne Microbial Pathogens.

Food Science Text Series
Arun K. Bhunia
Foodborne Microbial
Pathogens
Mechanisms and Pathogenesis
Second Edition
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Arun K. Bhunia
Foodborne Microbial
Pathogens
Mechanisms and Pathogenesis
Second Edition
Arun K. Bhunia
Molecular Food Microbiology Laboratory
Department of Food Science
Department of Comparative Pathobiology
Purdue University
West Lafayette, IN, USA
ISSN 1572-0330 ISSN 2214-7799 (electronic)

ISBN 978-1-4939-7347-7 ISBN 978-1-4939-7349-1 (eBook)
https://doi.org/10.1007/978-1-4939-7349-1
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Dedicated to my wife Banashri and our son, Arni,
and daughter, Irene
Preface, Second Edition
The first edition of this graduate-level Foodborne Microbial Pathogens:

Mechanisms and Pathogenesis textbook was published in 2008, and it was
also translated into the Russian language in 2014. Ever since, a significant
advancement in the pathogenic mechanism of microbes, in particular those
associated with foodborne diseases, necessitated the second edition. The first
edition had 15 chapters and the new edition is expanded to 20. The five new
chapters cover the description of diseases that are caused by viruses, para-
sites, molds and mycotoxins, fish and shellfish toxins, and opportunistic and
emerging foodborne pathogens. Additionally, several pathogens such as
Nipah virus, Ebola virus, Clostridium difficile, Enterobacter sakazakii,
Brucella abortus, and Aeromonas hydrophila, among others, are added to this
edition. This edition is rich in many more graphic illustrations for easy com-
prehension of the complex mechanistic process.
I am ever so grateful to my doctoral and postdoctoral mentors, Prof. Bibek
Ray and Prof. Michael G. Johnson, for their constant encouragement and for
instilling a deep-rooted positive influence in exploring, learning, and sharing
the knowledge of microbiology. The students whom I interacted throughout
the world and their love for microbiology were a constant inspiration for this
book. Once again, my graduate students and professional colleagues came
forward to review some of the book chapters. My sincerest appreciation goes
to Prof. M.G. Johnson, Dr. Gary Richards, Prof. J.S Virdi, Dr. Peter Feng, Dr.
David Kingsley, Dr. Kristin Burkholder, Rishi Drolia, and Taylor Bailey for
their excellent input and insights of many chapters they reviewed. This text-
book should be an excellent learning tool for graduate and undergraduate
students, academicians, and food microbiology professionals to gain
advanced knowledge and insights of the microbial pathogenesis of foodborne
or zoonotic pathogens that are transmitted to humans through food. This book
is dedicated to all the microbiologists who are passionate about exploring the
intricacies and complexity of the life of a microorganism.
West Lafayette, IN, USA Arun K. Bhunia
vii
Preface, First Edition, 2008
Ever since my days in veterinary school, I was fascinated with the field of
microbiology. I always wondered how such small microscopic organisms
are capable of causing infections in other living organisms, big or small,
young or old, and healthy or immunocompromised. The subject captured my
imagination. Many of the same microorganisms that cause diseases in ani-
mals also infect humans. In recent days, pathogens of animal origin impose
even greater concern with increasing threat of avian influenza to cause pan-
demic and spread of deadly bovine spongiform encephalopathy (mad cow
disease) and many bacterial pathogens such as Listeria, E. coli O157:H7,
Salmonella, Yersinia, and Campylobacter. I am especially intrigued by the
cunning strategy pathogens employ for their survival in a host and their
exploitation of host cellular machinery to promote their own invasion into
the host. Pathogenic mechanism is complex, and unraveling that process
requires great minds. Today, microbiologists, cell biologists, and immunolo-
gists employing many sophisticated molecular tools are unraveling that
secret at a very fast pace. Thus, it requires a great deal of efforts to compile
and update information in a textbook, and it was rather a monumental task.
My goal with this book was to paint a bigger picture of pathogenic mecha-
nism of foodborne pathogens, which are responsible for many of modern-
day outbreaks and diseases worldwide, and narrate the subject with
easy-to-comprehend illustrations. When I began teaching an advanced grad-
uate-level food microbiology course that dealt with pathogenic mechanism
of foodborne pathogens in the mid-1990s, there was hardly any textbook that
covered different foodborne microorganisms and the depth of materials
needed for the course, especially the mechanism of infection for foodborne
pathogens. That necessitated the collection and review of great deal of litera-
ture to provide updated materials to my students. That was the beginning and
was also the inspiration and motivation to write a textbook on the subject. In
the last two decades, there had been a tremendous progress in the area of
food microbiology especially the study of molecular mechanism of patho-
genesis, and a great deal of efforts was placed to compile those information
in the first edition of the current textbook. In this book, an introductory chap-
ter highlights the significance of foodborne pathogens, epidemiology, and
the reason for increasing cases of foodborne illnesses. In Chap. 2, a brief
review of biology of microorganisms and the importance of structural com-
ponents as those related to pathogenesis is provided. In addition, diseases
ix
x Preface, First Edition, 2008
caused by viruses, parasites, mycotoxins, and seafood toxins have been

included. In Chap. 3, a comprehensive review of the digestive system, muco-
sal immunity, and the host immune system has been described. This chapter
provides the basic foundation for the understanding of the complexity of
disease production by foodborne pathogens. First of all, foodborne patho-
gens’ primary site of action is the digestive tract; therefore, one must have
adequate knowledge to understand the interaction of pathogens with host
gastrointestinal tract. Second, host innate and adaptive immune responses
dictate the progression of a disease. Moreover, some pathogens exploit the
host immune system as part of their disease-producing mechanism.
Therefore, it is essential to have some basic understanding of immune sys-
tem in order to understand the disease process. As it is often said, “It takes
two to tango,” or “One needs two hands to clap”; thus, I believe that the
knowledge of biology of a pathogen and the corresponding host immune
response go hand in hand to comprehend the full picture of pathogenesis
process. In Chap. 4, general mechanisms of foodborne pathogens have been
included to provide the overall big picture of mechanism of infection and
intoxication. In Chap. 5, a brief review of the animal and cell culture models
as necessary tools to study pathogenesis is discussed. In Chaps. 6, 7, 8, 9, 10,
11, 12, 13, 14, and 15, sources, biology, pathogenic mechanism, prevention
and control, and detection or diagnostic strategies for individual foodborne
bacterial pathogens are described. In addition to traditional foodborne patho-
gens, descriptions of some of the key pathogens with bioterrorism implica-
tions such as Bacillus anthracis and Yersinia pestis have been included to
provide unique perspective. In this book, I am pleased to generate both digi-
tal and hand-drawn artworks to illustrate the pathogenic process, and I hope
these illustrations will aid in better understanding of the mechanism of
pathogenesis with greater enthusiasm. I also hope this textbook would be a
valuable resource not only for food microbiology graduate or undergraduate
students but also for the medical microbiologists, microbiology profession-
als, and academicians involved in food microbiology and food safety-related
research or teaching. I would like to convey my gratitude to my current and
former postdoctoral research associates and graduate students particularly
Kristin Burkholder, Jennifer Wampler, Pratik Banerjee, and Ok Kyung Koo
for their assistance in collecting literatures and reading the draft chapters.
The comments and inputs provided by the students of FS565 over the years
were extremely helpful in developing the course contents for this book.
Finally, my sincerest and humble gratitude goes to my professional col-
leagues for their generous time and efforts in reviewing the chapters and
providing expert comments and critiques: Chap. 1, Prof. M. Cousin, Purdue
University; Chap. 2, Prof. M.G. Johnson, University of Arkansas; Chap. 3,
Prof. R. Vemulapalli, Purdue University; Chap. 4, K. Burkholder, Purdue
University; Chap. 6, Dr. P. Banada, Purdue University; Chap. 7, Prof.
A. Wong, University of Wisconsin, and Prof. J. Mckillip, Ball State
University; Chap. 8, Dr. G.R. Siragusa, USDA-ARS, Athens, GA, and
Dr. V. Juneja, USDA-ARS, Wyndmoor, PA; Chap. 10, Prof. B. Reuhs,
Purdue University; Chap. 11, Prof. S. Rickie, University of Arkansas, and
Preface, First Edition, 2008 xi
Dr. M. Rostagna, Purdue University; Chap. 12, Dr. R. Nannapaneni,

Mississippi State University; Chap. 13, Prof. J.S. Virdi, University of Delhi
South Campus, India; Chap. 14, Dr. G.B. Nair, Center for Health and
Population Research, Dhaka, Bangladesh; and Chap. 15, Prof. C. Sasakawa,
University of Tokyo, Japan.
West Lafayette, IN, USA Arun K. Bhunia

Contents
1 Introduction to Foodborne Pathogens �� 1

Introduction to Food Microbiology �� 1
What Is a Pathogen?�� 4
What Are the Attributes of Pathogenicity?�� 6
Sources of Foodborne Pathogens�� 6
Meats, Ground Meat, and Organ Meats�� 8
Vacuum-Packaged Meats �� 8
Poultry�� 9
Fish and Shellfish�� 9
Fruits and Vegetables �� 10
Dairy Products �� 11
Delicatessen Foods�� 12
Spices �� 12
Low-Moisture Products�� 12
Foodborne Pathogen Statistics and Socioeconomic Impact�� 12
Why High Incidence of Foodborne Outbreaks?�� 13
Surveillance and Reporting�� 14
Agricultural Practices and the Food Manufacturing�� 15
Consumer Habits�� 16
Increased at-Risk Populations�� 17
Improved Detection Methods and Tracking of Pathogens�� 17
Emerging Pathogens with Improved Survivability
in Stressed Conditions�� 17
Food Safety Authorities and Pathogen Control Acts in the USA�� 17
Global Concerns with Foodborne Pathogens�� 18
Alternate Protein Source�� 19
Climate Change and Global Warming �� 19
Foodborne Outbreaks Associated with Imported Foods�� 20
Travel-Associated Foodborne Infections �� 20
Control of Foodborne Diseases in Economically
Underdeveloped Countries�� 21
Challenges and Concerns with Global Food Safety�� 21
Summary�� 21
Further Readings�� 22
xiii
xiv Contents
2 Biology of Microbial Pathogens�� 25

Introduction�� 25
Bacteria�� 25
Gram-Positive Bacteria�� 28
Cell Wall and Peptidoglycan�� 28
Teichoic Acid and Lipoteichoic Acid�� 30
Cytoplasmic Membrane �� 31
Gram-Negative Bacteria�� 31
Outer Membrane�� 31
Peptidoglycan�� 32
Periplasmic Space�� 32
Protein Secretion Systems�� 32
Accessory Structures in Gram-Positive and Gram-Negative
Bacteria�� 33
Fimbriae (Pili) and Curli�� 34
Flagella�� 34
O (Somatic) Antigen�� 34
Capsule�� 35
Endospore Formation�� 35
Viruses�� 36
Virus Classification/Taxonomy�� 37
Virus Structure �� 37
Viral Replication�� 37
Parasites �� 38
Life Cycle and Growth Characteristics�� 39
Molds and Mycotoxins�� 39
Mold Structure �� 39
Mycotoxins�� 40
Fish and Shellfish Toxins �� 41
Summary�� 41
3 Host Defense Against Foodborne Pathogens �� 43
Innate Immune Response�� 44
Adaptive Immune Response�� 45
Innate Immunity of Intestinal Tract �� 45
Skin�� 46
Mucus Membrane�� 46
Goblet Cells and Mucus�� 47
Antimicrobial Peptides�� 49
Toll-like Receptor�� 50
NOD Proteins�� 50
Resident Microbiota�� 51
Other Components of Innate Immunity �� 53
Immune Response in the Gastrointestinal Tract�� 53
Mouth/Throat/Respiratory Tract�� 53
Stomach�� 53
Contents xv
Small Intestine �� 54

Large Intestine�� 54
Adaptive Immunity�� 54
Characteristics of Adaptive Immune Response �� 55
Phases of Immune Response�� 56
Tissues and Cells of Immune System�� 56
Tissues�� 56
Bone Marrow �� 57
Thymus�� 57
Lymph Node�� 57
Spleen�� 57
Lymphoid Tissue�� 58
Cells of Immune System�� 58
Monocytes and Macrophages�� 58
Dendritic Cells �� 60
Granulocytes�� 60
Lymphoid Tissue�� 60
T Lymphocytes�� 61
Helper T Cells�� 61
Cytotoxic T Lymphocytes�� 63
Natural Killer Cells�� 63
Natural Killer T Cells�� 64
Cytokines �� 64
Properties of Cytokines�� 64
Cytokines in Innate and Adaptive Immunity �� 64
Interferon�� 65
Tumor Necrosis Factor�� 65
Interleukins�� 65
B Lymphocytes and Antibodies �� 66
Structure of Immunoglobulin�� 66
Classes of Immunoglobulins�� 67
Diversity of Antibodies�� 68
Antibody Production�� 69
Function of Antibody�� 69
Antigen�� 70
Types of Antigens�� 71
Epitope or Antigenic Determinant �� 71
Hapten�� 71
Antigen–Antibody Reaction�� 72
The Major Histocompatibility Complex�� 72
Structure of MHC�� 72
Antigen-Presenting Cells �� 72
MHC-Restricted Antigen Processing and Presentation �� 73
Class II-Restricted Antigen Presentation �� 73
Class I-Restricted Antigen Presentation�� 74
Accessory Molecules Involved during MHC-Restricted
T-Cell Activation�� 75
xvi Contents
The Complement System�� 75

The Classical Pathway �� 76
Alternative Pathway �� 76
Function of Complement �� 77
Control of Complement Activation�� 78
Immunity to Microbes�� 79
Extracellular Bacteria�� 79
Inflammation�� 79
Toxins�� 79
Innate Immunity�� 80
Adaptive Immune Response�� 80
Evasion of Immune System by Extracellular Bacteria�� 80
Intracellular Bacteria�� 81
Evasion of Immune System by Intracellular Bacteria �� 82
Immunity to Virus�� 82
Evasion of Immune System by Viruses�� 83
Immunity to Parasites�� 83
Evasion of Immune System by Parasites �� 84
Summary�� 84
4 General Mechanism of Pathogenesis�� 87
Foodborne Infection�� 87
Infectious Dose�� 88
Colonization and Adhesion Factors �� 89
Invasion and Intracellular Residence �� 93
Pathogen Translocation by Epithelial Barrier Disruption�� 96
Iron Acquisition �� 97
Motility and Chemotaxis�� 98
Evasion of Immune System �� 98
Intoxication�� 99
Toxicoinfection�� 99
Toxins�� 99
Endotoxin�� 107
Genetic Regulation and Secretion Systems for Virulence Factors�� 108
Pathogenicity Islands �� 108
Protein Secretion System �� 109
Regulation of Virulence Genes�� 113
Summary�� 114
Contents xvii
5 Animal and Cell Culture Models to Study Foodborne

Pathogens�� 117
Animal Model�� 117
Advantages of animal models�� 119
Limitations and cautions of animal models �� 119
Trangenic animal models �� 120
Organ Culture�� 120
Ligated Intestinal Loop Assay �� 120
Embryonated Chicken Egg Assay�� 120
Zebrafish Embryo�� 121
Cultured Cell Lines�� 121
Advantages and Limitations of Cultured Cell Models�� 122
Co-cultured Cell Model �� 122
Growth and Maintenance of Cultured Cell Lines�� 122
Measurement of Virulence�� 124
Cell Culture Model�� 125
Measurement of Specific Steps in Colonization and Invasion�� 128
Cell Culture Model�� 129
Summary�� 130
6 Foodborne Viral Pathogens and Infective Protein�� 133
Sources and Transmission�� 133
Virus Classification/Taxonomy�� 134
Foodborne Viral Pathogens�� 134
Adenovirus�� 134
Astrovirus�� 134
Rotavirus�� 135
Hepatitis Viruses�� 136
Norovirus �� 139
Zoonotic Viral Pathogens�� 141
Avian Flu Virus�� 141
Nipah Virus�� 143
Ebola Virus�� 145
Prevention and Control of Foodborne Viruses �� 146
Infective Proteins �� 146
Bovine Spongiform Encephalopathy �� 146
Summary�� 148
7 Foodborne Parasites�� 151
Protozoa �� 151
Giardia duodenalis�� 151
Entamoeba histolytica�� 152
Toxoplasma gondii �� 154
xviii Contents
Cryptosporidium parvum �� 156

Cyclospora cayetanensis�� 156
Cystoisospora belli�� 157
Trypanosoma cruzi�� 157
Cestodes (Tapeworms) �� 158
Taenia solium and Taenia saginata�� 158
Echinococcus granulosus�� 159
Nematodes (Roundworm)�� 160
Anisakis simplex�� 160
Trichinella spiralis �� 160
Ascaris lumbricoids �� 162
Trematodes (Flatworm or Fluke) �� 163
Clonorchis sinensis�� 163
Paragonimus Species �� 163
Schistosoma Species�� 164
Prevention and Control of Parasitic Diseases�� 164
Summary�� 165
8 Molds and Mycotoxins�� 167
Biology�� 168
Aflatoxin�� 168
Ochratoxin�� 170
Fumonisins�� 171
Trichothecenes �� 171
Patulin�� 171
Zearalenone�� 172
Citrinin �� 172
Alternaria Toxin �� 172
Emerging Mycotoxins�� 172
Ergot Alkaloids�� 172
Prevention and Control of Mycotoxins�� 173
Summary�� 173
9 Fish and Shellfish Toxins �� 175
Diseases Caused by Fish and Shellfish Toxins�� 175
Scombroid Toxin�� 177
Paralytic Shellfish Poisoning �� 177
Diarrhetic Shellfish Poisoning �� 177
Neurotoxic Shellfish Poisoning�� 178
Ciguatera Fish Poisoning �� 178
Azaspiracid Shellfish Poisoning�� 178
Amnesic Shellfish Poisoning �� 178
Pufferfish Poisoning�� 179
Prevention and Control�� 179
Summary�� 179
Contents xix
10 Staphylococcus aureus�� 181

Classification�� 182
Morphology�� 182
Cultural and Biochemical Characteristics�� 183
Virulence Factors �� 183
Adhesion Proteins�� 183
Toxic Shock Syndrome Toxin-1 (TSST-1)�� 184
Exfoliative Toxin�� 184
Miscellaneous Enzymes and Toxins�� 185
Enterotoxins �� 185
Molecular Regulation of Virulence Gene Expression�� 186
Food Association and Enterotoxin Production�� 187
Mechanism of Pathogenesis�� 187
Emesis and Diarrhea�� 187
Superantigen Activity�� 188
Animal and Cell Culture Models �� 189
Symptoms�� 189
Detection�� 190
Culture Methods�� 190
Nucleic Acid-Based Methods�� 190
Immunoassays�� 191
Other Rapid Methods�� 191
Summary�� 191
11 Bacillus cereus and Bacillus anthracis�� 193
Biology�� 194
Bacillus cereus Group and Other Pathogens�� 196
Bacillus cereus �� 197
Foods Involved�� 197
Regulation of Toxin Biosynthesis�� 199
Detection of Bacillus cereus�� 202
Bacillus anthracis�� 203
Biology�� 203
Virulence Factors and Mechanism of Pathogenesis�� 203
Treatment and Prevention�� 206
Detection of Bacillus anthracis �� 206
Summary�� 206
xx Contents
12 Clostridium botulinum, Clostridium perfringens,

Clostridium difficile�� 209
Classification of Clostridium Species�� 210
Clostridium botulinum �� 210
Biology�� 210
Sources�� 212
Botulism�� 212
Symptoms�� 217
Prevention and Treatment�� 217
Detection of C. botulinum or toxin�� 218
Clostridium perfringens �� 219
Biology�� 219
Sources�� 219
Clostridium perfringens Enterotoxin �� 220
α-Toxin�� 222
β-Toxin �� 222
Epsilon Toxin �� 223
Iota Toxin �� 223
NetB Toxin�� 223
Genetic Regulation of Virulence�� 223
Animal and Mammalian Cell Culture Models�� 223
Symptoms�� 224
Detection of C. perfringens or Toxins�� 224
Clostridium difficile �� 224
Biology�� 224
Source�� 225
Virulence Factors and Pathogenesis�� 225
Symptoms�� 226
Diagnosis and Detection of C. difficile�� 226
Summary�� 226
13 Listeria monocytogenes�� 229
Biology�� 230
Source�� 231
Disease �� 233
Gastrointestinal Form�� 233
Systemic Listeriosis �� 233
Abortion and Neonatal Listeriosis �� 233
Intestinal Phase of Infection and Systemic Spread�� 235
Contents xxi
Attachment�� 238
Listeria Adhesion Protein�� 238
Autolysin Amidase�� 239
P60 �� 240
LapB�� 240
FbpA�� 240
Invasion�� 240
Internalin A�� 241
Internalin B�� 241
Vip�� 242
Lysis of Vacuole (Phagosome)�� 242
Listeriolysin �� 242
Phosphatidylinositol-Specific PLC�� 243
Intracellular Growth�� 243
Cell-to-Cell Spread�� 243
ActA�� 243
Lysis of Double-Membrane Vacuole�� 244
Phosphatidylcholine-Specific PLC�� 244
Immunity to Listeria monocytogenes�� 245
Detection�� 246
Conventional Culturing Method�� 246
Immunoassays and Lateral Flow Assay �� 247
PCR and Whole Genome Sequencing �� 247
Summary�� 247
14 Escherichia coli�� 249
Biology�� 249
Sources�� 250
Serotypes�� 251
Virotypes�� 251
Enterotoxigenic E. coli�� 253
Characteristics�� 253
Symptoms�� 255
Enteropathogenic E. coli�� 256
Virulence Factors and Pathogenesis of EPEC�� 256
LEE and Regulation of Virulence Genes �� 258
Symptoms�� 258
Enterohemorrhagic E. coli �� 258
Food Association and Outbreaks �� 259
EHEC Pathogenesis �� 260
Attachment and Effacement�� 260
xxii Contents
T3SS and Delivery of Effector Proteins�� 261

Shiga Toxin�� 261
Enterohemolysin�� 263
Other Virulence Factors �� 264
Symptoms�� 264
Enteroaggregative E. coli �� 264
Symptoms�� 266
Enteroinvasive E. coli�� 266
Disease and Symptoms�� 266
Diffusely Adhering E. coli �� 266
Animal and Cell Culture Model Used for Diagnosis
of E. coli�� 267
Prevention, Control, and Treatment �� 267
Summary�� 268
15 Salmonella enterica�� 271
Biology�� 271
Source and Transmission �� 272
Salmonellosis and Animal Specificity �� 273
Septicemic Salmonellosis�� 273
Swine Salmonellosis�� 273
Pullorum Disease �� 274
Fowl Typhoid �� 274
Typhoid Fever�� 274
Gastroenteritis�� 274
Animal Models for Salmonellosis �� 276
Pathogenicity Islands and Virulence Factors �� 276
SPI-1�� 277
SPI-2�� 277
SPI-3�� 277
SPI-4�� 278
SPI-5�� 278
SPI-6�� 278
SPI-7 or Major Pathogenicity Island (MPI)�� 278
SPI-8�� 278
SPI-9�� 279
SPI-10�� 279
Salmonella Genomic Island-1 (SGI-1)�� 279
High-Pathogenicity Island (HPI) �� 279
Contents xxiii
Type III Secretion System�� 279

Pathogenic Mechanism�� 279
Adhesion and Colonization�� 279
Invasion and Intracellular Growth�� 281
Phagocytosis by M Cells�� 281
Phagocytosis by Dendritic Cells�� 281
Induced Phagocytosis by Epithelial Cells�� 282
Survival in Phagocytes�� 282
Immunity to Salmonella�� 284
RpoS Regulator�� 285
ATR Response�� 285
Treatment and Prevention of Gastroenteritis �� 285
Detection�� 285
Culture Methods�� 285
Immunological Methods�� 286
Nucleic Acid-Based Assays �� 286
Summary�� 286
16 Campylobacter and Arcobacter�� 289
Campylobacter�� 289
Biology�� 290
Sources�� 291
Antibiotic Resistance �� 292
Intestinal Colonization �� 292
Symptoms�� 295
Arcobacter�� 296
Biology�� 296
Pathogenesis�� 297
Prevention, Control, and Treatment of Campylobacter
and Arcobacter�� 297
Detection of Campylobacter and Arcobacter�� 298
Summary�� 298
17 Yersinia enterocolitica and Yersinia pestis�� 301
Yersinia enterocolitica�� 302
Biology�� 302
Sources�� 302
Chromosome-Linked Virulence Gene Products�� 304
xxiv Contents
Plasmid (pYV)-Linked Virulence Gene Products�� 305

Type III Secretion System�� 306
Pathogenic Mechanism�� 307
Symptoms�� 308
Prevention, Control, and Treatment �� 309
Detection of Yersinia enterocolitica�� 309
Yersinia pestis�� 310
Biology�� 310
Symptoms�� 312
Detection of Yersinia pestis�� 312
Summary�� 312
18 Vibrio cholerae, Vibrio parahaemolyticus,
and Vibrio vulnificus�� 315
Biology�� 315
Vibrio cholerae�� 316
Biology�� 316
Virulence Factors and Pathogenic Mechanism�� 318
Immune Response to Cholera Toxin�� 320
Symptoms of V. cholerae Infection�� 320
Control, Prevention, and Treatment of Vibrio cholerae�� 321
Vibrio parahaemolyticus�� 321
Biology�� 322
Vibrio vulnificus �� 324
Biology�� 324
Virulence Factors and Pathogenic Mechanism�� 324
Adhesion Factors �� 324
Capsular Polysaccharide�� 324
Iron Acquisition �� 325
Flagella and Motility�� 325
Hemolysin�� 325
Metalloprotease�� 326
Septicemia�� 326
Wound Infection�� 326
Symptoms of V. vulnificus Infection�� 326
Prevention and Control of V. parahaemolyticus
and V. vulnificus Infection�� 327
Detection of Vibrio Species�� 327
Contents xxv
Summary�� 328
19 Shigella Species�� 331
Biology�� 331
Invasion�� 334
Intracellular Multiplication�� 336
Bacterial Movement: Inter- and Intracellular Spreading �� 336
Cell Death and Inflammation �� 337
Shiga Toxin and Hemolytic Uremic Syndrome �� 337
Immunity Against Infection �� 338
Symptoms�� 339
Diagnosis and Detection�� 340
Bacterial Culture Methods �� 340
Immunological Methods�� 340
Molecular Techniques�� 340
Summary�� 340
20 Opportunistic and Emerging Foodborne Pathogens:
Aeromonas hydrophila, Plesiomonas shigelloides,
Cronobacter sakazakii, and Brucella abortus �� 343
Aeromonas hydrophila �� 343
Biology�� 343
Source�� 344
Disease �� 344
Symptoms�� 345
Plesiomonas shigelloides �� 345
Biology�� 345
Source�� 345
Pathogenesis and Disease�� 346
Symptoms�� 346
Cronobacter sakazakii �� 346
Biology�� 346
Source�� 346
Pathogenesis and Disease�� 347
Symptoms�� 347
Brucella abortus�� 348
Biology�� 348
xxvi Contents
Source�� 348
Symptoms�� 349
Summary�� 349
Index�� 351
Introduction to Foodborne
Pathogens 1
Introduction to Food Microbiology immune system to provide protection against

chronic metabolic disease (e.g., diabetes), bacte-
Food microbiology is a branch of microbiology rial infection, atherosclerosis, osteoporosis, and
that focuses on the study of microorganisms allergic reactions. Examples of beneficial micro-
associated with food intended for human or ani- organisms are Lactobacillus acidophilus, L.
mal consumption. Microorganisms use food as a rhamnosus, L. casei, L. plantarum, Lactococcus
source of nutrient for survival and growth or a lactis, Streptococcus thermophilus, Pediococcus
vehicle for transmission to the human or animal acidilactici, and Saccharomyces cerevisiae.
host. Food microbiology is subdivided into three These microorganisms generally are safe and do
focus areas that include the study of beneficial, not cause disease in humans, unless the opportu-
spoilage, and disease-causing microorganisms nistic environment such as immunocompromised
(Fig. 1.1). Beneficial microorganisms are those condition or other health complications facilitate
used in food fermentation to produce shelf-stable systemic dissemination of the organisms.
products with unique flavors, tastes, texture, and Food spoilage microorganisms are those,
appearance. These include cheese, fermented which upon growth in food, that produce undesir-
meat (pepperoni), vegetables (pickled cucumber, able texture, odor, and appearance and make food
olives), dairy products (yogurt), and ethnic fer- unsuitable for human consumption. These micro-
mented products such as kefir, sauerkraut, idli, organisms produce protease, lipase, and glyco-
kimchi, and so forth. In fermented products pro- lytic enzymes (amylase, pectinase, etc.) to break
duced by natural or controlled fermentation, down proteins, fats, and carbohydrates, respec-
microorganisms metabolize complex substrates tively, and alter physical (such as slimy texture),
to produce enzymes, flavor compounds (diace- chemical (color change, oxidation) and sensory
tyl), acids (lactic acid, acetic acid, propionic acid, properties. Sometimes uncontrolled growth of
etc.), alcohols, and antimicrobial agents (bacte- many of the natural fermentative microorganisms
riocins, hydrogen peroxide) to improve product can also cause spoilage, such as the growth of
shelf life and to provide characteristic product lactic acid bacteria in ready-to-eat meats or pas-
attributes. Microorganisms also produce teurized milk. It is estimated that 25% of all food
enzymes, which also help the breakdown of indi- produced (postharvest) are lost due to the micro-
gestible compounds to make the product more bial spoilage. Food spoilage is a serious issue in
palatable and easy to digest. In addition, the ben- developing countries because of inadequate pro-
eficial microorganisms also serve as probiotics to cessing and refrigeration facilities. Controlling
impart direct health benefit by modulating the microbial food spoilage will continue to receive
© Springer Science+Business Media, LLC, part of Springer Nature 2018 1

A. K. Bhunia, Foodborne Microbial Pathogens, Food Science Text Series,
https://doi.org/10.1007/978-1-4939-7349-1_1
2 1 Introduction to Foodborne Pathogens
Bacillus species, and some mold species are

known food spoilage organisms, yet they can also
produce toxins and cause disease. Based on the
nature of the disease elicited by pathogens, food-
borne diseases are again classified into three
types: “intoxication,” “toxicoinfection,” and
“infection.” The knowledge pertaining to the
mechanism of pathogenesis and the diseases
caused by foodborne pathogens continues to aug-
ment our understanding of epidemiology and dis-
ease transmission process and to develop novel
detection and diagnostic tool, to design novel
vaccine and drugs, and to help formulate effec-
tive prevention and control strategies (Fig. 1.2).
Intoxication results when the preformed
Fig. 1.1 Three branches of food microbiology. Beneficial
and spoilage microorganisms, and spoilage and pathogens microbial toxin is ingested with food and water
have some overlapping activity (shaded area) resulting in the onset of symptoms very quickly.
Food intoxications are generally caused by tox-
ins produced by Staphylococcus aureus,
increasing attention since food demand is Clostridium botulinum, and Bacillus cereus.
expected to double in 2050 to feed estimated 9.3 Intoxication is also associated with algal toxins
billion people in the world. The current (2018) ingested by fish or shellfish and mycotoxins pro-
world population is about 7.6 billion. Examples duced by fungi. “Toxicoinfection” refers to the
of food spoilage microorganisms are disease caused by the toxins produced inside the
Alicyclobacillus, Brochothrix, Bacillus, host by the live pathogens after ingestion of
Clostridium, Erwinia, Lactobacillus, food. Toxins interact with the host epithelium
Leuconostoc, Pseudomonas, Shewanella, and and result in the gastrointestinal symptoms
some yeasts and molds (Botrytis, Alternaria, including diarrhea and occasional vomiting.
Penicillium, Fusarium, Mucor, Rhizopus). The Examples are Clostridium perfringens, entero-
spoiled food is generally of less concern from a toxigenic Escherichia coli (ETEC), and Vibrio
public health standpoint, since many disease- cholerae. “Foodborne infection” occurs when
causing microorganisms may not be able to sur- the live pathogenic microorganisms, which are
vive in the microenvironment created in a spoiled generally invasive, are ingested and cause severe
food. Moreover, the pathogenic microorganisms tissue damage. “Foodborne infection” is caused
are poor competitors and may not survive in food by bacterial pathogens such as Salmonella
in the presence of a natural microbial community enterica, Campylobacter jejuni, enterohemor-
whose metabolic by-products such as acids, bac- rhagic Escherichia coli (E. coli O157:H7),
teriocin, hydrogen peroxide, and CO2 are inhibi- Shigella spp., Yersinia enterocolitica, and
tory to pathogens. Listeria monocytogenes; viral pathogens includ-
Foodborne pathogenic microorganisms ing Norovirus, hepatitis A virus, and Rotavirus;
(Table 1.1), on the other hand, when present or and parasites including Toxoplasma gondii,
grown in a food may not alter the aesthetic or Cryptosporidium parvum, Cyclospora cayeta-
sensory quality of products, and thus the micro- nensis, Giardia intestinalis, Taenia solium, and
bial safety may not be easy to assess based on Trichinella spiralis.
product appearance alone. Thus, microbiological Among the foodborne pathogens, Norovirus
or analytical testing is required to assess patho- tops all infections with over a million people
gen contamination. Some foodborne pathogens infected each year in the USA. Protozoan infec-
such as proteolytic Clostridium botulinum, tions are mostly associated with water and fresh
Introduction to Food Microbiology 3
Table 1.1 List of foodborne pathogens involved in outbreaks from contaminated food and water
Bacteria Virus Parasite
Aeromonas hydrophila Astrovirus Cryptosporidium parvum
Bacillus anthracis Hepatitis A virus Cyclospora cayetanensis
Bacillus Hepatitis E virus Entamoeba histolytica
cereus/subtilis/licheniformis Norovirus Giardia intestinalis
Brucella Rotavirus Cystoisospora belli
abortus/melitensis/suis Taenia solium/saginata
Campylobacter jejuni/coli Toxoplasma gondii
Clostridium botulinum Trichinella spiralis
Clostridium perfringens Infective proteins Molds and mycotoxins
Cronobacter sakazakii BSE (bovine spongiform Aflatoxin, ochratoxin, fumonisin, patulin,
Escherichia coli encephalopathy) trichothecenes, DON (deoxynivalenol)
Listeria monocytogenes TSE (transmissible
Mycobacterium spongiform
paratuberculosis encephalopathy)
Salmonella enterica Emerging pathogens Seafood toxins
Shigella spp. Clostridium difficile Scombroid toxin, ciguatera fish poisoning, diarrhetic
Staphylococcus aureus Brucella abortus shellfish poisoning, paralytic shellfish poisoning
Vibrio spp. Trypanosoma cruzi (saxitoxin), pufferfish poisoning
V. cholerae non-01 Avian flu virus
V. parahaemolyticus Nipah virus
V. vulnificus Ebola virus
V. fluvialis
Yersinia enterocolitica
Fig. 1.2 The

interrelationship of
foodborne pathogens
and pathogenesis with
other branches of
microbiology
produce such as the fruits and vegetables. A large neurological disorders. Thus, the significance of
volume of these commodities is imported from mycotoxin ingestion is often overlooked. In con-
countries where food production and processing trast, bacterial or viral pathogens affect a large
are performed under inadequate hygienic prac- population resulting in high morbidity and mor-
tices, which may contribute to the increased inci- tality, and their surveillance statistics are updated
dences of contamination. Molds produce routinely. The importance of molds in food has
mycotoxins such as aflatoxin, ochratoxin, rarely peaked consumers’ or regulatory agencies’
fumonisin, trichothecene, and DON (deoxyniva- interest, especially in the USA; however, in
lenol) that are mutagenic, carcinogenic, or hepa- recent years, increased emphasis has been placed
totoxic. Prolonged exposure to mycotoxins may on understanding the properties, synthesis, and
result in serious and sometimes fatal diseases. pathogenesis of foodborne molds and their
The onset of symptoms due to mycotoxin intoxi- mycotoxins.
cation is delayed and is not as dramatic as bacte- Microorganisms are ubiquitous and can sur-
rial- or virus-induced diarrhea, vomiting, or vive and grow in extreme conditions in nature, in
food, and in human or animal hosts. Some are able to adapt well to the harsh environments
bacteria can grow in high temperatures and are of food and maintain their pathogenic traits.
called thermophiles (50–70 °C); some even grow During the transition to the host, pathogens may
in hot geysers such as extremophiles (>70 °C) or express a completely separate set of genes that
extreme cold (−20 °C). Most thermophilic and ensure their survival and disease-producing capa-
extremophilic microorganisms are nonpatho- bilities in the host.
genic to humans. Some microorganisms grow at Food safety is essentially an ongoing problem
refrigeration to ambient room temperatures in rapidly changing and growing food industry,
(1–25 °C), while mesophiles grow at due in part to consumer’s increasing reliance on
25–37 °C. Both psychrotrophs and mesophiles ready-to-eat convenience foods. Food is globally
are capable of causing diseases in humans. sourced and distributed; thus, contamination of a
Based on their response to different pH, food with a pathogen will present a greater eco-
microbes are grouped as aciduric (<pH 7), alkali- nomic and social impact than ever before. Certain
phile (pH > 7.0), halophile (salt-loving), or baro- foods can also become vulnerable to malicious
philes (pressure-loving). Based on the oxygen contamination with infective agents; thus, food
requirements, bacteria are grouped as aerobic, defense is becoming an essential part of our edu-
obligate anaerobic, or facultatively anaerobic. cation. Knowledge and understanding of patho-
Foods are prepared and stored under controlled gens in foods and their survival mechanisms in
environments where residual oxygen concentra- foods as well as in the human and food animal
tions can dictate what type of microorganisms host should be important areas of focus to prevent
will survive and grow. Anaerobes can grow only food-related illnesses and increase the well-being
in the vacuum-packaged or canned foods in of the population.
which oxygen is removed mechanically or by
heating. Aerobes grow on the surface of food
where oxygen is abundant. A similar scenario What Is a Pathogen?
applies for pathogens that cause disease in the
gastrointestinal tract, where oxygen gradients In fact, only a small fraction of all microbes cause
vary from the upper part of the small intestine to disease in humans or animals via the food- or
the lower part of large intestine. The upper part is feed-borne route. A pathogen is an organism that
highly oxygenated, while the lower part is devoid is able to cause cellular damage by establishing
of oxygen. Again, oxygen concentrations vary in tissue, which results in clinical signs with an
from the center of the lumen to the proximity of outcome of either morbidity (defined by general
the epithelial lining where the oxygen concentra- suffering) or mortality (death). More specifically,
tion is higher because of cellular respiration. a pathogen is characterized by its ability to repli-
These environments select the types of pathogens cate in a host, by its continued persistence of
that will colonize or be present in different parts breaching (or destroying) cellular or humoral
of the intestine. Besides microorganisms being barriers that ordinarily restrict it, and by express-
introduced into our body via food, water, or air, ing specific virulence determinants to allow a
microorganisms also exist since birth as com- microbe to establish within a host for transmis-
mensals in the digestive tract, skin, nasal pas- sion to a new susceptible host. Pathogens could
sages, and reproductive and urogenital tract, and be classified as zoonotic, geonotic, or human ori-
these microbes are generally beneficial to the gin based on their transmission patterns and
host. However, under favorable conditions, com- movement among different hosts and vectors.
mensals may behave as opportunistic pathogens. Zoonotic diseases are characterized by transmis-
Food is a complex milieu that contains salts, sion of infective agents such as bacteria, viruses,
acids, ions, aldehydes, flavoring agents, and anti- parasites, and fungi from animals to humans.
microbial preservatives. Pathogenic microorgan- Examples of zoonotic pathogens are Escherichia
isms transmitted through foods, in most cases, coli O157:H7, Staphylococcus aureus,
What Is a Pathogen? 5
Salmonella enterica serovar Typhimurium, S. Table 1.2 Diseases and symptoms caused by foodborne
pathogens
enterica serovar Enteritidis, Campylobacter
jejuni, Yersinia enterocolitica, Mycobacterium Disease or clinical
symptoms Pathogens/toxins involved
tuberculosis, Ebola virus, Nipah virus,
Vomiting, Staphylococcus, Bacillus,
Toxoplasma gondii, and Trichinella spiralis. diarrhea, Cronobacter, Salmonella,
Geonotic diseases are acquired from soil, water, dysentery Shigella, Yersinia, Vibrio,
or decaying plant materials. An example of Norovirus, Rotavirus,
geonotic pathogen is Listeria monocytogenes. Entamoeba, Cryptosporidium,
Cyclospora, Giardia,
Human origins are exclusively transmitted from Cystoisospora, Taenia,
person to person, and pathogens of human origin Trichinella
are Salmonella enterica serovar Typhi, Vibrio Arthritis (reactive Campylobacter, Salmonella,
cholerae, Shigella spp., and hepatitis A. Different arthritis, Reiter’s Shigella, Yersinia
syndrome,
diseases caused by foodborne pathogens are
rheumatoid
summarized in Table 1.2. arthritis)
Poverty, competition for food, crowding, war, Hemorrhagic Shiga toxin-producing E. coli
famine, and natural disaster help pathogens to uremic syndrome (STEC); Shigella spp.
survive and spread in the environment. (HUS), kidney
disease
Domestication of animals also allowed patho-
Hepatitis and Hepatitis A virus (HAV),
gens to come in close contact with humans and, jaundice hepatitis E virus (HEV)
thus, helped foster human acquisition of these Guillain–Barré Campylobacter
pathogens. In recent years, there is a great con- syndrome (GBS),
cern of possible pandemic outbreak of bird flu Miller Fisher
syndrome (MFS)
(avian influenza virus) in humans due to the
CNS disorder: Listeria, bovine spongiform
transmission of the virus to the poultry handlers meningitis, encephalopathy (BSE), Taenia
or people being exposed to the infected flocks. encephalitis spp. (cysticercosis)
Bird flu virus (strain H5N1) was responsible for Miscarriage, Listeria monocytogenes,
several fatal infections in Asia and other parts of stillbirth, neonatal Toxoplasma gondii
infection
the world, often via live poultry markets (see
Paralysis Clostridium botulinum, shellfish
Chap. 6). toxin (algal toxin),
In the past few decades, industrialized coun- Campylobacter
tries have witnessed increased number of out- Malignancies and Mycotoxin (aflatoxin,
breaks of the foodborne pathogen, in part, due to autoimmune deoxynivalenol)
diseases
massive expansion of food industry, inadequate
Allergic response Seafood toxin (scombroid toxin)
hygienic and sanitary practices, and distribution
of a food item to consumers globally (see section
on “Why High Incidence of Foodborne
Outbreaks”). Foodborne outbreaks may be char- orbidity. Foodborne pathogens may not be a
m
acterized as an epidemic when a large population pandemic threat, but global distribution and trad-
of consumers from multiple states is affected due ing of food may be a cause for concern in the
to ingestion of pathogen originating from a single future. Epizootic outbreak refers primarily to ani-
food item. In contrast, sporadic outbreak involves mal diseases and is linked to an infectious disease
incidence of one or two cases in a community. that appears as new cases in a given population of
The pandemic outbreak is defined as the inci- animals. If such infectious agents are found in
dence of disease that is associated with a highly food animals and if there is a potential for trans-
infectious agent affecting people from multiple fer of those infective agents to animal handlers
countries on a global scale with a large number and food producers/processors, it would be a
of populations succumb to high mortality or major cause for concern to humans.
hat Are the Attributes

W tions (homologous recombination), or in-frame
of Pathogenicity? deletion. Genetic complementation is often done
to restore the gene function and to confirm the
Some pathogens are designated as “primary involvement of that gene in pathogenesis.
pathogens,” which regularly cause disease. Some Transposon (Tn) elements are used routinely to
are classified as “opportunistic pathogens” that induce mutation. The major benefit of using
infect primarily immunocompromised or at-risk transposon is that they carry antibiotic markers
populations. Nevertheless, both primary and that help in locating a specific gene on the chro-
opportunistic pathogens share similar attributes: mosome. However, transposon could act as a
entry and survival inside a host, finding a niche transcriptional terminator. If Tn lands on the pro-
for persistence, able to avoid the host’s defense moter or the first gene of an operon, it affects the
(stealth phase), able to replicate to a significant transcription of the genes located downstream. In
number, transmitted to other host with high fre- addition, if the transposon is inserted in a house-
quency, and able to express specialized traits keeping gene, essential for bacterial growth or
within the host. For example, Salmonella enterica survival, that bacterium cannot be selected from
invades host cells when proper levels of O2, pH, that experiment. Transposons carry antibiotic
and osmolarity are maintained. This sends appro- resistance gene(s) and insertion sequences for
priate signals to the PhoP/Q regulon for expres- integration into the host chromosome. They may
sion of specific invasion-associated genes. also carry virulence genes. Transposons are gen-
Several factors affect pathogens growth and erally specific for either Gram-positive (Tn916,
survival inside a host such as O2, CO2, iron, nutri- Tn917, Tn1545) or Gram-negative (Tn5, Tn7,
ents, pH, bile salts, mucus composition, the bal- Tn10) bacteria. In-frame deletion is highly effi-
ance of natural microflora, quorum sensing, and cient and is used widely to create a mutant strain
physiological status such as stress hormone like by employing a method called SOE (splicing by
epinephrine or norepinephrine. overlap extension). Genome sequence of many
Pathogens are clonal and they are generally microorganisms is now available; thus, a series of
derived from a single progenitor. They are often polymerase chain reaction (PCR) methods is
selected by the environmental selective pressures used to selectively remove a target gene by SOE
such as heat, extremes of pH, and antibiotic treat- to create an in-frame deletion mutant. Whole
ments. However, the emergence of a new or a genome sequencing is also a powerful tool for
highly virulent form of pathogens suggests pos- studying pathogenesis. Sequencing of a gene or
sible transfers of virulent genes are occurring its product (amino acid sequence) and subse-
among microorganisms. These novel genetic quent matching with the database will reveal the
variants have arisen due to a point mutation, identity and function of the gene. Genetic cloning
genetic rearrangement in the chromosome, and is also an important strategy for studying patho-
gene transfer between organisms through hori- genesis. In cloning experiments, a suitable vector
zontal (Shigella to E. coli) or vertical (E. coli to is constructed with a gene of interest and, subse-
E. coli) modes (Fig. 1.3). Plasmids, bacterio- quently, transferred to an avirulent strain by elec-
phages, transposons, and pathogenicity islands troporation or conjugation. The function of the
(i.e., a large piece of DNA carrying virulence gene in the new strain is analyzed by in vitro cell
gene cluster (see Chap. 4) can all serve as a vehi- culture or in vivo animal bioassays.
cle for gene transfer.
Involvement of a specific gene or genes in
pathogenesis can be studied in the laboratory by Sources of Foodborne Pathogens
using various molecular tools such as mutation,
genome sequencing, and cloning. In mutation, a Many foodborne pathogens are ubiquitous in
specific gene is disrupted by chemical mutagen- nature and generally found in soil, water, ani-
esis, transposon mutagenesis, site-specific muta- mals, and plants. Pathogens are introduced into a
Sources of Foodborne Pathogens 7
Fig. 1.3 Gene transfer

and acquisition in
bacteria
Fig. 1.4 Food

production and supply
chain network
food processing plant through raw materials quorum sensing, the viable but nonculturable
(Fig. 1.4). Humans, air, water, and equipment state (VBNC), spore formation, stress response/
may also bring the organisms to foods in the pro- adaptation to intrinsic conditions in food such as
cessing plant. Recontamination of processed pH, osmotic stress (salt), acid, temperature, and
food also frequently occurs and contributes to resistance to sanitizers (see Chap. 4).
foodborne outbreaks and illnesses. Some patho- When considering a product safety, it is
gens can survive for a prolonged period on the important to know the type of microorganisms or
inanimate objects and serve as a source. A list of toxins likely to be present, their numbers, and
foodborne microorganisms and their survival on concentrations. In addition, their response to the
inanimate surface is summarized in Table 1.3. heat, pH, salts, and other processing conditions is
Pathogenic microbes use various strategies to important to consider. The numbers and types of
persist on food and in food production facilities. microorganisms present in a finished food prod-
These strategies may include biofilm formation, uct are influenced by the original source of the
Table 1.3 Persistence of pathogens on inanimate hide, or feathers. Ground beef is made from meat,
surfaces
trimmings, and fat and has a large surface area
Organisms Duration of survival that favors the growth of aerobic bacteria. The
Bacterial pathogens knives of a meat grinder, if not properly sanitized
Campylobacter jejuni Up to 6 days
or washed, may be the source of contamination.
Escherichia coli 1.5 h–16 months
Furthermore, one heavily contaminated piece of
Clostridium difficile (spores) 5 months
Listeria species 1 day–several meat may be sufficient to contaminate an entire
months lot of ground meat. Pathogens such as Clostridium
Mycobacterium bovis >2 months perfringens, Bacillus cereus, Listeria monocyto-
Mycobacterium tuberculosis 1 day–4 months genes, and enterohemorrhagic E. coli (EHEC)
Salmonella enterica serovar 6 h–4 weeks are associated with these types of products. The
Typhi
liver, kidney, heart, and tongue may carry Gram-
Salmonella enterica serovar 10 days–4.2 years
Typhimurium
positive cocci, coryneforms, Moraxella, and
Shigella species 2 days–5 months Pseudomonas. Lymph nodes in food animals are
Staphylococcus aureus 7 days–7 months secondary lymphoid tissues in which pathogens
Streptococcus pyogenes 3 days–6.5 months are supposed to be destroyed biologically but if
Vibrio cholerae 1–7 days there is survival can serve as a major source of
Viral pathogens pathogens. Mechanically deboned meat/poultry/
Astrovirus 7–90 days fish generally carry lower microbial loads
Adenovirus 7 days–3 months because of less handling and minimal human
Norovirus 8 h–7 days
interventions. In addition, electrical stimulation
Influenza virus 1 day–2 days
converts glycogen to lactic acid, thus resulting in
Rotavirus 6 days–60 days
Hepatitis A virus 2 h–60 days lowered pH, which suppresses bacterial loads.
Adapted from Kramer et al. (2006). BMC Infect. Dis.
During rigor mortis, the release of cathepsin
6:130 facilitates muscle tenderization, and lysozyme
reduces the bacterial counts.
food, its microbiological quality in the raw or
unprocessed state, the sanitary conditions under
which the product was handled or processed, and Vacuum-Packaged Meats
the conditions for subsequent packaging, han-
dling, storage, and distribution (Fig. 1.4). In a In products vacuum packaged in oxygen imper-
raw agricultural product, generally, the maxi- meable films, air is removed to increase the shelf
mum microbial load is on the food surfaces, life. Generally, the shelf life of vacuum-packaged
whereas generally there is negligible to none meats is about 15 weeks. In another strategy, ini-
inside. A list of foods involved in major con- tially, an oxygen permeable packaging film
firmed outbreaks since the year 2000 is summa- allows the growth of indigenous Pseudomonas
rized in Table 1.4. spp., but lactic acid bacteria (Lactobacillus,
Leuconostoc, and Carnobacterium) become pre-
dominant after the Pseudomonas spp. remove
eats, Ground Meat, and Organ
M oxygen and increase the CO2 level inside the
Meats package, which in turn favors the growth of fac-
ultative anaerobes (lactic acid bacteria) and
Raw beef carries a large number of E. coli since anaerobic microbes such as Clostridium species
they are natural inhabitants of intestines of mam- (Clostridium botulinum, C. perfringens). Later,
mals; therefore, during slaughter the carcass may lactic acid bacteria such as Lactobacillus
be contaminated with fecal bacteria. Fresh meats and Brochothrix grow as well as pathogenic bac-
also can be contaminated with Salmonella and teria such as Yersinia enterocolitica and
Staphylococcus aureus originating from the skin, Staphylococcus aureus.
Table 1.4 Foods and pathogens involved in select outbreaks from 2000–2016
Year Foods involved Place Pathogen No. of illnesses No. of deaths
2016 Live poultry and backyard USA Salmonella (multiple 895 3
flock serovars)
2016 Raw scallops (sushi) USA Hepatitis A virus 292 2
2016 Frozen vegetables USA L. monocytogenes 9 3
2015 Ice cream USA L. monocytogenes 10 3
2014 Chicken USA Salmonella Heidelberg 634 –
2014–2015 Apple USA L. monocytogenes 35 7 deaths, 1
miscarriage
2012 Tuna fish from India USA Salmonella Bareilly 258 –
2011 Lettuce USA E. coli O157:H7 60 –
2011 Strawberry USA E. coli O157:H7 15 1
2011 Cantaloupe USA L. monocytogenes 148 33
2011 Sprouts Germany E. coli O104:H4 3911 48
2011 Papaya USA Salmonella 106 –
2011 Alfalfa sprouts USA Salmonella 140 –
2010 Celery USA L. monocytogenes 10 5
2010 Lettuce USA E. coli O145 26
2010 Eggs USA S. Enteritidis 1939 –
2009–2010 Black and red pepper USA S. Montevideo 272
2009 Alfalfa sprouts USA Salmonella 235
2009 Frozen Raspberries Finland Norovirus 200
2009 Peanut butter USA S. Typhimurium 714 9
2008 Mexican-grown peppers USA S. Saintpaul 1017 2
2007 Chicken and turkey pot USA Salmonella 401 –
pies
2007 Peanut butter USA Salmonella 425 –
2007 Frozen ground beef patties USA E. coli O157:H7 40 –
2008 Ground beef USA E. coli O157:H7 45 –
2007 Milk or milk-related USA L. monocytogenes 5 3
products
2006 Green onions USA E. coli O157:H7 67 –
2006 Spinach USA E. coli O157:H7 199 3
2002 Hamburger USA E. coli O157:H7 18 –
2002 Processed chicken USA L. monocytogenes 46 7
2000 Bean sprouts USA Salmonella 23 –
2000 Dairy farms USA E. coli O157:H7 56 –
2000 Delicatessen turkey USA L. monocytogenes 30 4 deaths, 3
miscarriages
2000 Homemade Mexican-style USA L. monocytogenes 12 5 miscarriages
cheese
Poultry be the source of Pseudomonas, coryneforms, and

yeasts. Ground turkey also may carry fecal
Salmonella enterica serovars Enteritidis, streptococci.
Typhimurium, Infantis, Reading, Blockley, and
Kentucky, Clostridium perfringens,
Campylobacter jejuni, and E. coli are associated Fish and Shellfish
with intact and ground raw poultry. Workers may
also be the source of other Salmonella serovars Microbial loads in shrimps, oysters, and clams
like – Sandiego and Anatum. Fresh poultry may depend on the quality of the water from which
they are harvested and the months of the year. If cuts, wounds, roots, insect, and animal bites.
untreated raw sewage is present, the microbial These pathogens residing inside the plant tissue
quality deteriorates. During handling, fecal coli- will not stimulate plant defense system because
forms, fecal streptococci, and Staphylococcus they are not harming living tissues. In the absence
aureus may be incorporated into the product. of plant defense, these pathogens will not be
Salmonella also is found in oysters possibly due exposed to plant antimicrobials since these are
to contaminated water. Fish and shellfish also are compartmentalized in specialized cells.
the source for Pseudomonas spp., Clostridium Additionally, vacuum and hydrocooling such as
perfringens, Listeria monocytogenes, Vibrio showering or washing warm produce with chilled
parahaemolyticus, Vibrio vulnificus, Salmonella water may help create a partial internal vacuum,
enterica serovar Enteritidis and Typhimurium, facilitating internalization of pathogens into
Campylobacter jejuni, Yersinia enterocolitica, produce.
and enteroviruses (hepatitis A). Smoked salmon Bacterial species commonly found on plant
and shrimp may carry pathogenic L. monocyto- surfaces are Enterobacter agglomerans,
genes. Seafood may also be a major source of Pseudomonas syringae, and Pseudomonas fluo-
algal toxins or neurotoxins originating from sea- rescens. These microorganisms can coexist with
sonal feeding on dinoflagellate algal blooms. the normal microflora on the fresh produce that is
Certain allergic diseases such as scombroid poi- contaminated from the field. Plant pathogens and
soning are also associated with fish and shellfish spoilage microorganisms such as Agrobacterium
(see Chap. 9). spp., Erwinia spp., Clostridium spp., Bacillus
spp., Klebsiella spp., Pseudomonas spp.,
Ralstonia spp., Xanthomonas spp., and Xylella
Fruits and Vegetables spp. can initiate interactions with the plant to
have successful colonization on the plant. Human
Sources of human pathogen contamination in pathogens such as E. coli O157:H7, Salmonella
fruits and vegetables at preharvest stage include enterica, Clostridium perfringens, C. botulinum,
soil, irrigation water, inadequately composted and Listeria monocytogenes from soil, manure,
animal manure, dust, wild and domestic animals, irrigation water, wild animals, and insect vectors
human handling, and water used for pesticide can attach to the plant surface through secondary
spray, foliar treatments, and growth hormones colonization. Protozoan species such as Giardia
(Fig. 1.5). The sources of contamination at post- intestinalis, Entamoeba histolytica,
harvest stage include human handling (workers, Cystoisospora belli, and Cyclospora cayetanen-
consumers), harvesting equipment, transport sis are also associated with produce and herbs.
containers, wild and domestic animals, pests, Examples of outbreaks associated with fruits and
dust, wash and rinse water, sorting, packing, cut- vegetables are E. coli O157:H7 in apple cider,
ting and further processing equipment, cross- spinach, lettuce, and sprouts; E. coli O104:H4 in
contamination, transportation, and sales. fenugreek sprouts; Salmonella enterica in canta-
Human pathogens interact with fresh produce loupes, watermelons, cilantro, tomatoes, sprouts,
not merely in a physical manner but attach to the and almonds; Cyclospora cayetanensis in rasp-
surface by employing fimbriae, pili, and flagella berries; hepatitis A in strawberries and green
involving biochemical signals and multiple com- onion (scallion); and L. monocytogenes in cab-
pounds for such adherence. Furthermore, patho- bage, celery, cantaloupe, and apples. These
gens on the produce surface can be attached as events emphasize the need for further sanitation
aggregates, partially buried in the surface wax or and processing or improved quality assurance to
in cracks in the cuticle, and are protected against assure the safety of these minimally processed
environmental stress and surface disinfectants. products. Postharvest and external contamination
Furthermore, human pathogens can also enter has been considered the major concerns in pro-
into tissues (xylem) of fresh produce, through duce safety; however, recent outbreaks with
Fig. 1.5 Mode of transmission of foodborne enteric pathogens in fruits and vegetables (Adapted and redrawn from
Brandl 2006. Annu. Rev. Phytopathol. 44, 367–392)
lettuce and spinach suggest that these foods can borne outbreaks have occurred due to consump-
take up pathogens internally on the field prior to tion of raw milk, homemade ice cream containing
harvest (Fig. 1.5). Indeed, in the 2006 spinach fresh eggs, or cheese made with unpasteurized
outbreak with E. coli O157:H7, feral pigs and cow’s milk. In 1980–1981, 538 cases of
cattle from the nearby ranch were responsible for Salmonella infection occurred with cheddar
the transmission of this pathogen. Experimental cheese and raw milk and certified raw milk. In
evidence showed that some pathogens like E. coli 1980–1982, 172 cases of Campylobacter infec-
O157:H7 and Salmonella are able to survive and tion occurred with raw milk and certified raw
grow inside the veins of the above two plant tis- milk. In 1995, a Salmonella outbreak occurred
sues and tomatoes due to the abundant supplies with ice cream when a tanker carrying raw liquid
of carbohydrates and moisture. egg also transported pasteurized milk without
proper cleaning and sanitization between prod-
ucts. In 1985, 2000, and 2006, L. monocytogenes
Dairy Products infection occurred due to consumption of
Mexican-style soft cheese (queso fresco). This
Cow’s udder, hide, and milking utensils may ethnic product is made from unpasteurized milk
carry predominantly Gram-positive bacteria and has a relatively non-acidic pH. In 2013,
such as aerobic spore formers (Bacillus spp.), Listeria monocytogenes outbreak associated
psychrotrophic Pseudomonas spp., and others with cheese caused six illnesses and one death,
including Mycobacterium and Clostridium spe- and in 2015, outbreak associated with a nation-
cies. Historically, cows with mastitis could pro- ally distributed brand of ice cream resulted in ten
duce raw milk infected with Staphylococcus illnesses and three deaths. In 2016–2017, soft
aureus which if improperly pasteurized can raw milk cheese was responsible for six illnesses
cause foodborne illness. More recently, food- and two deaths in the USA. Yersinia enteroco-
litica outbreaks also were associated with pas- Low-Moisture Products

teurized and unpasteurized milk.
These include chocolate, powdered infant for-
mula, raw almonds, toasted oats breakfast cereal,
Delicatessen Foods dry seasonings, paprika-seasoned potato chips,
dried coconut, infant cereals, peanut butter, and
Microbial load and type of pathogens in delica- children’s snacks made of puffed rice and corn.
tessen foods such as salads, sandwiches, and deli Salmonella enterica has been the primary patho-
foods depends on the ingredients, meats, and gen associated with many of these products.
vegetables and spices used to make them. Food Additionally, Cronobacter sakazakii has been
handler’s direct contact also can lead to increased associated with powdered infant formula. Even
incidences of Staphylococcus aureus via work- though these pathogens do not grow in these
ers’ nasal passages and hands. While considered dried products, but a surviving low infectious
to be relatively low-moisture ingredients, spices dose allows their transmission to host causing
(see below) may carry spores of Clostridium, disease when these foods are rehydrated.
Bacillus, and molds which are able to survive in
the presence of 8–12% moisture. Delicatessen
foods also carry psychrotrophic Listeria monocy- oodborne Pathogen Statistics
F
togenes. Dehydrated foods such as soups of and Socioeconomic Impact
chicken noodle, chicken rice, beef noodle, vege-
table, mushroom, and pea may carry as high as Worldwide, foodborne pathogens cause numer-
7.3 log10 g−1 ml−1 of Clostridium perfringens as ous illnesses and deaths. The World Health
well as coliforms. Organization (WHO) estimated that worldwide,
in 2005, 1.4 million people died from diarrheal
diseases due to contaminated water and food. In
Spices 2012, the estimated global mortality rates due to
foodborne disease ranged from 0.26 to 15.65
Spices constitute seed, flower, leaf, bark, roots, or deaths per 100,000 populations. Globally, among
bulb and are used whole or ground or singly or the foodborne pathogens, Norovirus caused 125
mixed to enhance taste and flavor of food. Spices million cases, while Campylobacter species
are an integral part of many ethnic cuisines and caused 96 million cases. In Africa, Asia, and
used in a variety of dishes. Spices include dehy- Latin America, there are about 1 billion cases of
drated onion, garlic, mustard seed, red pepper, gastroenteritis per year in children under the age
sesame seed, black pepper, paprika, cinnamon, of 5, leading to 5 million deaths. In Mexico and
cumin seed, white pepper, oregano, poppy seed, Thailand, half of the children aged 0–4 years suf-
ginger, allspice, anise, basil, bay leaves, cloves, fer from the Campylobacter-induced enteritis. In
fennel seed, sage, thyme, and turmeric. Spices are Europe, 50,000 cases per million population suf-
considered low-moisture ingredients, and they fer from acute gastroenteritis. In the Netherlands,
contain about 8–12% moisture. Some spices may about 300,000 cases per million population occur
contain microorganisms as high as 106–7 cfu g−1 yearly. In Northern Ireland and the Republic
which include the spores of molds, Aspergillus, Ireland, about 3.2 million episodes of gastroen-
Fusarium, Alternaria, Penicillium, Rhizopus, teritis are reported each year. In Australia, 5.4
Cladosporium, and Trichoderma, and bacteria, million cases of foodborne gastroenteritis occur
Bacillus and Clostridium spp. In addition, each year. In England, 20% of the population,
Salmonella enterica and Staphylococcus aureus i.e., 9.4 million people, suffer from acute gastro-
have been found. In 2010, Salmonella enterica enteritis each year, and the primary contributing
serovar Montevideo outbreak was associated with microorganisms are identified as Norovirus,
salami and contaminated black pepper as an Campylobacter species, rotavirus, and nonty-
ingredient was responsible for transmission. phoidal Salmonella species.
Why High Incidence of Foodborne Outbreaks? 13
In the USA, there are an estimated 48 million billion. Besides acute gastroenteritis, the sequelae
cases with 128,000 hospitalizations and 3000 of the foodborne infections result in chronic
deaths associated with foodborne infections each rheumatoid conditions; ankylosing spondylitis–
year. Of the 48 million cases, 9.4 million cases autoimmune disease (HLA); hemolytic uremic
are caused by 31 known pathogens of bacterial syndrome (HUS) due to Shiga toxin (Stx) from
(3.6 million cases, 39%), viral (5.5 million cases, EHEC; atherosclerosis due to lipid deposition in
59%), and parasitic (0.2 million or 2%) origin. arteries; Guillain–Barré syndrome and Miller
The majority of illnesses were caused by norovi- Fisher syndrome from Campylobacter infec-
rus (58% illnesses), followed by nontyphoidal tions; reactive arthritis from Salmonella, Shigella,
Salmonella (11%), Clostridium perfringens and Campylobacter infections; autoimmune dis-
(10%), and Campylobacter spp. (9%). The lead- ease such as allergic encephalitis; and autoim-
ing causes for hospitalizations were Salmonella mune polyneuritis. Foodborne infections also
spp. (35%), Norovirus (26%), Campylobacter vary between countries due to eating habits of the
(15%), and Toxoplasma gondii (8%), while the population. In Japan, high Vibrio parahaemolyti-
most deaths were associated with nontyphoidal cus cases are seen due to consumption of raw
Salmonella (28%), Toxoplasma gondii (24%), fish. Scandinavians and people from m iddle/east-
Listeria monocytogenes (19%), and Norovirus ern countries sometimes suffer from botulism
(11%). Foodborne diseases of microbial origin due to consumption of improperly processed fish,
have become the number one food safety concern meat, and vegetables.
among the US consumers and regulatory agen-
cies, and this trend is probably true in most coun-
tries (Table 1.5). Why High Incidence of Foodborne
Food animals and poultry are the most impor- Outbreaks?
tant reservoirs for many of the foodborne patho-
gens. Therefore, meat, milk, or egg products may It is believed that some new pathogens are emerg-
carry Salmonella enterica, Campylobacter jejuni, ing, which are responsible for increased inci-
Listeria monocytogenes, Yersinia enterocolitica, dences of foodborne diseases (Table 1.1). Some
or E. coli O157:H7. Control of pathogens in raw are recognized recently whose ancestors probably
unprocessed products is now receiving major caused foodborne illnesses for many thousands of
emphasis to reduce pathogen loads before arrival years. An example is E. coli O157:H7, a new
at a processing plant. On-farm, pathogen- strain first reported on 1982, which is responsible
controlling strategies will help achieve that goal. for numerous outbreaks in recent years. This bac-
However, the presence of pathogens in ready-to- terium has evolved relatively recently from an
eat (RTE) raw produce products is a serious con- enteropathogenic E. coli (EPEC) progenitor.
cern since those products generally do not receive Likewise, E. coli O104:H4, an enteroaggregative
any treatments lethal to microbes before con- bacterium associated with the sprout outbreak in
sumption. Also, many recent foodborne out- 2011 in Germany, has acquired bacteriophage-
breaks resulted from consumption of undercooked encoded stx2 gene. It is thought to be a recently
or processed RTE meats (hotdogs, sliced lun- evolved serovar. In addition, many of the older
cheon meats, and salami), dairy products (soft pathogens are reemerging and contributing to the
cheeses made with unpasteurized milk, ice overall foodborne outbreak statistics. Besides,
cream, butter, etc.), in addition to minimally pro- human sufferings and fatalities, the high number
cessed fruits (apple cider, strawberries, canta- of foodborne outbreaks in recent years have had
loupe, etc.) and vegetables (sprouts, lettuce, devastating economic impacts on food producers
spinach, etc.). and processors. It has been a challenging task for
Annual economic losses in the USA result scientists to figure out the reasons for the greater
from deaths, illnesses, loss of work, loss of man- numbers of outbreaks in recent years. Some fac-
power, and product loss account for about $78 tors (Table 1.6) are discussed below which may
Table 1.5 Estimated yearly cases of foodborne diseases, related deaths, and associated cost in the USA
Pathogens Cases Hospitalizations Deaths (%) Cost (million dollars)
Bacteria
Bacillus cereus 63,400 20 0 15
Brucella spp. 839 55 1 (0.3) 18
Campylobacter spp. 845,024 8463 76 (5.5) 6879
Clostridium perfringens 965,958 438 26 (0.4) 93
Clostridium botulinum 55 42 17.3 (0.2) 466
Shigatoxigenic E. coli (STEC), O157 63,153 2138 20 (2.9) 635
STEC, non O157 112,752 271 20 154
Enterotoxigenic E. coli (ETEC) 17,894 12 0 24
Listeria monocytogenes 1591 1455 255 (15.9) 2040
Salmonella spp. (nontyphoidal) 1,027,561 19,336 378 (0.5) 11,391
Shigella spp. 131,254 1456 10 (0.1%) 1254
Staphylococcus aureus 241,148 1064 6 (0.1) 168
Streptococcus group A 11,217 1 0 24
Vibrio parahaemolyticus 34,664 100 4 88
Other Vibrio 17,564 83 8 (1.7) 88
Vibrio cholerae 84 2 0 0.2
Vibrio vulnificus 96 93 36 (1) 268
Yersinia enterocolitica 97,656 533 29 (0.1) 1107
Parasites
Giardia intestinalis 78,840 225 2 (0.1) 282
Toxoplasma gondii 86,686 4428 327 (20.7) 3456
Cryptosporidium parvum 57,616 210 4 (0.4) 168
Cyclospora cayetanensis 11,407 11 0 17
Trichinella spiralis 156 6 0 2
Viruses
Norovirus 5461 14,663 149 (6.9) 3.677
Rotavirus 15,443 348 0 18
Astrovirus 15,443 87 0 19
Hepatitis A 1566 99 7 58
Total 77,671
(known = 32,462 + unknown = 45,208)
Adapted from Scallan et al. (2011). Emerg. Infect. Dis. 17, 7–15
explain the plausible reasons for increased inci- their doctors, and sporadic cases were not
dence: (1) increased surveillance and reporting, reported routinely. In addition, the causative
(2) changes in the food manufacturing and agri- agents were not always identified because of lack
cultural practices, (3) changes in consumer’s hab- of better methodologies. Sensitive detection
its, (4) increased at-risk populations, (5) improved methods are now available, and the epidemiolog-
detection methods, and (6) emerging pathogens ical survey has been improved. Computer-based
with survivability in stressed conditions. databases such as FoodNet and PulseNet in the
USA, EC Enter-Net for Salmonella species and
E. coli O157:H7 in Europe, and WHO Global
Surveillance and Reporting Salm-Surv are now available to assess the trends,
changes in expected numbers, and types from
In the past, foodborne incidence reporting was historical data (Table 1.7). These following data-
poor or underreported. Sometimes, persons suf- bases are used as an alert mechanism for future
fering from illness also did not always consult outbreaks.
Why High Incidence of Foodborne Outbreaks? 15
Table 1.6 Factors affecting the emergence of increased gricultural Practices and the Food
A
foodborne illnesses from food
Manufacturing
1. Increased surveillance and reporting
2. The food manufacturing and agricultural practices Agricultural practices affect the incidence of
Centralized production facility
microorganisms in the intestinal tract and on the
Distribution to multiple states/other countries
surfaces of the food animal. High ambient tem-
Intense agricultural practices
Minimal processing (produce and fruits) perature and moisture can encourage salmonellae
Increased importation of fresh produce growth in animal feed. Use of antibiotics in feeds
3. Consumer habits generally kills the certain population of microor-
Eating more meals outside the home ganisms including pathogens but encourage other
Increased popularity of fresh fruits and vegetables resistant ones to grow. Transportation to the
4. Increased at-risk populations slaughterhouse in crowded trucks, in extreme
(immunocompromised, elderly)
weather conditions, creates stress and weakened
5. Improved detection methods and tracking of
pathogens
immune systems, thus favoring microbial growth.
6. Emerging pathogens with improved survivability in Feed withdrawal practice in poultry, if exceeds
stressed conditions more than 12 h before slaughtering, may cause
leaky or thinned gut, vulnerable to rupture during
evisceration and possibly the transfer of patho-
gens to other tissues. High-speed slaughter and
Table 1.7 List of surveillance programs currently used evisceration also can result in the product con-
in the USA and other countries
tamination due to damaged gut. Intensive farm-
Program Purpose ing allows faster growth of pathogens, and
US surveillance and monitoring programs
recycling of animal waste products and animal
FoodNet Foodborne Diseases Active
Surveillance Network (FoodNet): by-products results in increased opportunities for
routine surveillance of select transmission of pathogens to humans. Feeding
foodborne pathogens in ten states in animal products to another species allows one
the USA (see Table 1.5) pathogen (e.g., a prion) to adapt and transmit to
PulseNet DNA fingerprints of pathogens based
another host (see Chap. 6). Since 1997, the US
on pulsed-field gel electrophoresis
CalciNet Fingerprints of calciviruses
government and several European countries have
EHS-Net Environmental health and cause of banned the use of animal products (MBM, meat
foodborne diseases bone meal) as an animal feed ingredient to con-
CAERS CFSAN Adverse Event Reporting trol the spread of the prion agent that causes
System for foods, cosmetics, dietary bovine spongiform encephalopathy (BSE) (see
supplements
Chap. 6).
eFORS Electronic Foodborne Outbreak
Reporting Systems of CDC (Center Changes in food manufacture and consump-
for Disease Control and Prevention) tion practices are also contributing factors in
eLEXNET Electronic Laboratory Exchange increased foodborne diseases. Consumption of
Network (data from FDA, USDA, preprepared foods at home and outside the home,
DOD) from all 50 states
consumption of chilled and frozen foods, and
Global food industries and air travel
EC Enter-Net European Commission Enter
increased consumption of poultry and fish as part
Networks for Salmonella and E. coli of a healthy diet may increase the incidence of
O157:H7 salmonellosis, Campylobacter-induced enteritis,
OzFoodNet Australian foodborne disease and Vibrio parahaemolyticus-induced gastroen-
information for risk assessment and teritis. Cross-contamination of raw foods with
policy, training for foodborne disease
investigation cooked/processed foods and undercooking of
WHO Global Surveillance resources and training in meat and holding at a higher temperature also are
Salm-Surv foodborne disease for participating contributing factors. Reduced use of salt, less use
countries of food preservatives (sorbate and benzoate) for
health reasons, and demand for more natural, 199 illnesses with 3 fatalities in 26 states in the
fresher, healthier, and convenience meals may USA. In 2006–2007, Salmonella enterica serovar
also serve as contributing factors. These types of Tennessee contaminated several peanut butter
food require greater care during production, stor- containing products resulting in 628 cases in 47
age, and distribution to ensure pathogen growth states in the USA. In 2011, 3911 people were
does not occur in these products. Tightening or infected and 47 deaths were associated with E.
enforcing strict hygienic measures in the food coli O104:H4 contamination of fenugreek sprouts
manufacturing facilities is needed to reduce the in Germany and France. In 2010, S. enterica
incidence of pathogens. Improved sanitary prac- serovar Enteritidis in eggs were responsible for
tices during cleaning of processing plant equip- 1939 illnesses; S. enterica serovar Montevideo
ment with the rotation of sanitizers can help outbreak with salami was responsible for 230 ill-
avoid the emergence of resistant microbes. nesses and 43 hospitalizations. L. monocytogenes
Ever larger centralized food processing facili- was responsible for several outbreaks between
ties are thought to be a major contributing factor 2010 and 2015: 57 cases with 22 fatalities in
in recent years. The products produced by such Canada due to consumption of processed RTE
processors are distributed widely to multiple meat products, 27 cases with 8 fatalities from
states or many countries worldwide. Thus, such Quargel sour milk curd cheese in Europe, 146
products if contaminated can affect large popula- cases with 32 deaths from cantaloupe, 32 cases
tions with devastating consequences. For exam- with 7 deaths from caramel apple, and 10 cases
ple, in 1985 in a pasteurization plant in Chicago, with 3 deaths from a national distributed brand of
raw unpasteurized milk contaminated finished ice cream in the USA. The cantaloupe outbreak
pasteurized milk at packaging resulting in an led the FDA to press criminal charges against the
estimated 15,000 cases of illnesses from salmo- grower/owners of the farms and packing sheds
nellosis. In January 1993, in a major fast-food from which this fruit was shipped.
restaurant chain, undercooking of hamburger
meat resulted in E. coli O157:H7 outbreaks in
which more than 600 persons were infected Consumer Habits
including many children. Several were hospital-
ized, 35 showed hemolytic uremic syndrome, Consumption of food outside the home also
and 3 died. The FDA then declared raw ground increases the chance of foodborne illnesses. Meal
beef contaminated with this pathogen to be prepared and eaten at home also could cause dis-
“adulterated,” necessitating that it be recalled and ease because of poor hygienic practices during
destroyed. In 1997, E. coli O157:H7-tainted storage and preparation of food. Persons with
ground beef resulted in 25,000 pounds of ground underlying conditions also serve as the contribut-
beef recall. In 1998–1999, an outbreak of L. ing factors: liver disease patients are susceptible
monocytogenes occurred due to consumption of to Vibrio vulnificus infection; therefore, these
hotdogs/lunchmeats resulted in 79 cases with 16 patients are advised not to eat raw oysters; and
deaths and 3 miscarriages. In 2000–2001, con- immunocompromised and pregnant women
sumption of Mexican-style soft cheese resulted should avoid RTE meats, pâté, deli, or prepared
in 12 cases of listeriosis in the USA. In 2002, meals and cheeses made with unpasteurized milk
consumption of sliced turkey meat caused a mul- because of possible Listeria monocytogenes con-
tistate outbreak of L. monocytogenes with 50 tamination. If outbreaks occur in public institu-
cases, 7 deaths, and 3 abortions. In 2003, raw tions such as restaurants, hotels, hospitals, cruise
milk cheese was responsible for an outbreak in lines, manufacturing facilities, institutions, and
Texas, and in 2005, a multistate outbreak involv- so forth, the consequences are devastating
ing consumption of turkey deli meat affected because large numbers of people are at risk. This
nine states and caused 12 illnesses. In 2006, spin- happens because of ignorance, poor manage-
ach outbreak with E. coli O157:H7 resulted in ment, sloppy practices, and lack of education or
Food Safety Authorities and Pathogen Control Acts in the USA 17
understanding principles and practices needed consumption of raw milk, ice cream contami-
for safe handling of foods. nated with raw liquid eggs, consumer demands
for fresher foods, and increased consumption of
chicken that may be undercooked results in high
Increased at-Risk Populations numbers of enteritis cases. The emergence of
antibiotic or preservative-resistant organisms
Populations that are susceptible to infections are also is a contributing factor. Antibiotic and acid-
increasing and people are living longer. Young, resistant Salmonella enterica and E. coli and also
old, pregnant, and immunocompromised (YOPI) heat-resistant Salmonella and Listeria can sur-
people are susceptible to various foodborne dis- vive certain sublethal processing conditions and
eases. In addition, diabetes, cancer patients persist in the product. Microorganisms acquiring
receiving chemotherapy, patients with organ virulent genes through vertical or horizontal
transplants, and AIDS (acquired immune defi- transfer may become a new pathogen with highly
ciency syndrome) patients are vulnerable to virulent gene sets. For example, acquisition of stx
foodborne illnesses. Pathogen-free food may genes by E. coli O157:H7 and E. coli O104:H4
lead to increased susceptibility to diseases strains has been shown. The viable but noncultur-
because subclinical infection may strengthen able organisms are also problematic such as
immunity. “Delhi belly,” “Montezuma’s revenge,” Norovirus, Campylobacter jejuni, and Vibrio spe-
or “traveler’s diarrhea” affects only travelers and cies that are difficult to detect.
not the indigenous populations. Nutritional fac-
tors, physiological status, and concurrent or
recent infection of intestinal tract also can favor ood Safety Authorities
F
increased infection. and Pathogen Control Acts
in the USA
I mproved Detection Methods Federal food safety authorities in the USA

and Tracking of Pathogens include the US Department of Agriculture
(USDA), Food and Drug Administration (FDA),
Improved detection methods are now capable of Environmental Protection Agency (EPA),
detecting low numbers of pathogens in products. Department of Justice (DOJ), and Department of
Biosensors, PCR, immunoassays, mass Defense (DOD) (Table 1.8). President Lincoln
spectrometry, and whole genome sequencing
founded the USDA in 1862. The Pure Food and
(WGS) techniques are very sensitive, and now Drug Act and the Federal Meat Inspection Act
low numbers of pathogens can be detected from (FMIA) were passed in 1906. The USDA’s
products, which would normally give negative Bureau of Chemistry and Bureau of Animal
results with earlier less sensitive detection Industry (BAI) were responsible for enforcing
technologies. FMIA. In 1927, the USDA’s Bureau of Chemistry
was recognized, and it was renamed in 1931 as
the Food and Drug Administration (FDA). In
merging Pathogens with Improved
E 1940, the FDA was separated from the USDA. In
Survivability in Stressed Conditions 1953, the FDA became a part of the Department
of Health, Education, and Welfare and now the
Newly identified and emerging pathogens are Department of Health and Human Services
also a major contributing factor. Clinical, epide- (HHS).
miological, and laboratory studies have identified In 1938, the Federal Food, Drug, and Cosmetic
a number of so-called emerging and new patho- Act was passed, and the FDA holds the authority
gens (Table 1.1). Emergence due to unsafe food to establish food safety standards. In 1957, the
handling practices, undercooking of hamburger, Poultry Products Inspection Act; in 1967, the
Table 1.8 Food safety authorities in the USA Risk-Based Preventive Controls) principles that
Agency Responsibility are scientifically and technically sound should
US Department of Meat, poultry, and liquid be an integral part of prevention. Furthermore,
Agriculture-Food Safety egg products the FDA assumes the authority to prevent inten-
Inspection Service
(USDA-FSIS) tional contamination of products. (2) Inspection
Food and Drug Fruits and vegetables, milk and compliance involve mandatory inspection
Administration (FDA) and dairy products, shell and laboratory testing, mandated inspection fre-
eggs, nuts, flours, spices, quency, access to records, and testing by accred-
canned low-acid foods
ited laboratories. (3) In response category, if a
Environmental Waterborne diseases
Protection Agency production facility is implicated in an outbreak,
(EPA) the FDA has the authority for mandatory recall,
Animal and Plant Zoonotic diseases, animal suspension of registration of the food production
Health Inspection health facility, enhanced product tracing abilities, and
Service (APHIS)
additional record keeping for high-risk foods.
Department of Defense Intentional contamination
(DOD) and Department of products with pathogens, (4) Imports: the imported food must meet the US
of Justice (DOJ) biothreat agents standard; third-party certification and the FDA
have the authority to deny entry of food from a
foreign producer who fails to comply with the
Wholesome Meat Act; in 1968, the Wholesome US standard. (5) FSMA also requires the
Poultry Act; and in 1970, the Egg Products enhanced partnership and collaboration among
Inspection Act (EPIA) were passed. The FDA all food safety agencies domestic or foreign for
was responsible for inspection of whole egg and public health safety.
egg products, and since 1995, the FSIS (Food
Safety Inspection Service) is the responsible
agency for inspection of pasteurized liquid, fro- lobal Concerns with Foodborne
G
zen, or dried egg products, and the FDA assumed Pathogens
responsibility for shell egg safety. In 1996, the
FSIS included Pathogen Reduction/HACCP In 2018, human population in the world is about
(Hazard Analysis Critical Control Points) as a 7.6 billion. Worldwide, about 852 million people,
landmark rule to control and prevent microbial one-sixth of the world population, are chronically
pathogens. hungry due to an extreme poverty. According to
In 2011, the Food Safety Modernization Act the Food and Agriculture Organization (FAO) of
(FSMA) was passed to prevent foodborne ill- WHO, up to 2 billion people lack food security
nesses and deaths. The FSMA enables the FDA intermittently due to varying degrees of poverty.
to focus more on preventing foodborne illnesses Each year about 6 million children die of hunger,
rather than reactive response after an outbreak i.e., about 17,000 per day. By 2050, the world
happens. The key elements of the FSMA are to population is expected to be 9.3 billion, and food
supply safe food to consumers by implementing demand is anticipated to double as compared to
critical preventive measures through five broad today’s demand. Besides population growth, cli-
focus areas: prevention, inspection and compli- mate change (such as drought, high temperature,
ance, response, imports, and enhanced partner- flooding), conflict and war, and social unrest will
ship. (1) Prevention strategy is intended for also impact farming and food production. In
science-based decision-making process to con- many economically impoverished countries, lack
trol pathogens across the food supply chain of infrastructure for food transportation, process-
including food facilities, production and packaging, unsanitary and unhygienic food production
ing system, and products. Incorporation of and preparation practices, insects, uncontrolled/
HACCP and HARPC (Hazard Analysis and unregulated use of pesticides, chemicals, and
Global Concerns with Foodborne Pathogens 19
antibiotics also affect food production and supply

of nutritious safe food to masses. Case Study
The health of food animals is also critical in Seventeen days in June 2008 (29 Air France
food security since the food animal account for flights from Central and West Africa arrived
large quantities of protein (meat, milk, egg) in the at Paris), 134 people were searched, 9 car-
daily diet. The Animal Production and Health rying bush meat and 83 had livestock or
Division (AGA) of FAO reports that globally sev- fish. Total amounts of product seized were
eral diseases could be problematic including rin- 414 pounds. One passenger had 112 pounds
derpest, avian influenza (H5N1), African swine of bush meat and no other luggage. Most of
fever, contagious bovine pleuropneumonia, foot- the bush meats were smoked and dried car-
and-mouth disease (FMD), Rift Valley fever, casses. Some were identifiable; others were
rabies, and African trypanosomiasis. not, so those animals were boiled to expose
The field of food microbiology has a critical their skeleton for identification. Eleven
role to play in securing foods to the masses, espe- types of bush meats were confiscated: mon-
cially by preventing food losses from contamina- keys, large rats, crocodiles, small ante-
tion of food spoilage and pathogenic lopes, and pangolins (anteaters). Forty
microorganisms. Beneficial microorganisms, on percent of which are listed as endangered
the other hand, will help protect the food supply species. It is estimated that about 5 tons of
by converting nutritionally poor raw materials to bush meat arrive in Paris each week.
rich nutritious products through active fermenta- Likewise, bush meats are also imported to
tion. Microbial fermentation processes can also the USA. Kristine Smith, a wildlife veteri-
help preserve seasonal foods to yield self-stable narian in the US Customs office said, “We
products for year-round supply. Single-cell pro- get these big boxes of meat, sometimes you
teins from algae and yeasts will also be able to see primate heads or hands in there.”
supplement protein in the diet to curtail food R. Ruggiero of the US Fish and Wildlife
shortage. Service said, “In Africa today, many wild-
life populations are being eaten to extinc-
tion. The greatest impact to wildlife
Alternate Protein Source populations in Africa is the bush meat
trade.”
Consumption of meat from wildlife, often
referred to as “bush meat,” has increased in
Central and West Africa and is perceived as a fac- Climate Change and Global Warming
tor that can alleviate poverty. Wild animals such
as elephant, gorilla, chimpanzee and other pri- Water is called the environmental common since
mates, crocodile, forest antelope (duikers), por- it links environment, agriculture, animals, and
cupine, pig, cane rat, pangolin, monitor lizard, humans. Without water, life cannot be sustained.
guinea fowl, and anteaters have been smoked, Yet, it can serve as an efficient vehicle for rapid
dried, and often exported to other countries. This transportation of pathogens from a local source
rate of harvesting would endanger wildlife popu- to a global scale through tributaries, rivers, and
lation to extinction. Moreover, there is a concern oceans. The scarcity of water leads to drought,
for infectious disease transmission such as mon- and that can cause a rise in many pathogens such
key poxvirus, Ebola virus, HIV-like virus, simian as molds and mycotoxins. Global warming and
foamy virus, and Nipah virus to humans (see climate change leading to temperature rise can
Chap. 6). have an impact on the persistence and occurrence
of bacteria, viruses, parasites, and fungi and the Table 1.9 Global concern of food and water associated
organisms
corresponding foodborne diseases. Global warm-
ing can affect microbial ecology and growth, Source Pathogens of concern
plant and animal physiology, and host suscepti- Water Shigella, Vibrio, protozoa,
nematodes
bility resulting in the emergence of plant and ani-
Fresh fruits and Protozoan species (Giardia,
mal diseases and insect infestations, all of which vegetables Cyclospora), Shigella, nematodes
could influence foodborne diseases and zoono- Dairy products Campylobacter, Staphylococcus,
ses. The rise in ocean temperature can increase (milk) Listeria
toxic algal blooms, yielding high levels of bio- Rice and pasta Bacillus cereus (Bacillus spp.)
toxins in marine waters and then their bioaccu- Meats E. coli, Salmonella enterica
Fresh water fish Vibrio, Aeromonas
mulation in animals and fish. Hurricane (cyclone/
and sea foods
tornado) and flooding increase waterborne dis- Street foods Staphylococcus, Bacillus,
eases such as cholera and dysentery and fish and hepatitis A, enteric viruses
shellfish poisoning. Flash floods can cause Powdered infant Cronobacter sakazakii
increased runoff of soil nutrients such as nitrogen formula
and phosphates from agriculture field into rivers, Ingredients (spices Molds and mycotoxins,
and herbs): Salmonella, Bacillus, and
lakes, and oceans promoting increased toxic algal Clostridium spores
bloom which can contaminate potable water and
fish and shellfish growing in that water.
Table 1.10 Travel-associated enteric pathogen infection
in returning Americans from abroad between 2004 and
oodborne Outbreaks Associated
F 2009
with Imported Foods Travel-associated cases in
Pathogen the USA (2004–2009) %
According to the Economic Research Service Campylobacter 3445 41.7
(ERS) of the USDA, overall, 16 percent of all Salmonella 3034 36.7
foods eaten in the US is imported with 85% of the Shigella 1071 13.0
seafood and up to 60% of fresh produce being Cryptosporidium 317 3.8
imported. Increased outbreaks are associated STEC 257 3.1
Cyclospora 54 0.7
with imported foods in the USA. According to
Vibrio 44 0.5
CDC, between 2005 and 2010, there were 39 out-
Yersinia 32 0.4
breaks linked to imported foods, fish, and spices, Listeria 16 0.2
resulting in 2348 illnesses involving foods from Total 8270 100
15 countries (Table 1.9). Fish was the most com- Adapted from Kendall et al. (2012). Clin Infect Dis 54,
mon commodity (17 outbreaks) followed by S480–S487
spices (6 outbreaks). Import from Asia was
responsible for about 45% of the outbreaks.
E. coli (ETEC, responsible for traveler’s diar-
rhea) cases after returning to the USA. Forty-two
Travel-Associated Foodborne percent cases were due to Campylobacter, 32%
Infections due to Salmonella, and 13% due to Shigella
(Table 1.10). The greatest risks were in Africa
The WHO estimates that about 15–20 million (~76 cases per 100,000), Asia (~23 per 100,000),
travelers to developing countries experience diar- and Latin America and Caribbean countries (20
rhea annually. The CDC reported that between per 100,000). During this reporting period, the
2004 and 2009, travel-associated enteric infec- top travel destinations were Mexico, India, Peru,
tion cases were 8270 excluding enterotoxigenic and the Dominican Republic.
Summary 21
Fig. 1.6 Street food

safety
ontrol of Foodborne Diseases

C Summary
in Economically Underdeveloped
Countries Food microbiology is a branch of microbiology
that focuses on the study of microorganisms that
Several factors should be considered to improve are associated with food intended for human or
food safety in economically poor countries: animal consumption. Microorganisms use food
improvement in farming practices, infrastructure, as a source of nutrient for survival and growth or
and transportation of food products to market; a vehicle of transmission to the human or animal
availability of clean and potable water; improved host. Food microbiology is broadly classified into
food processing facility, hygienic practices during three focus areas: beneficial microorganisms,
food preparation, refrigeration, and storage; per- spoilage microorganisms, and pathogenic micro-
sonal hygiene; insect control; street food safety organisms. Beneficial microorganisms are those
(Fig. 1.6); food safety training and education; and used for making traditional or ethnic fermented
implementation of HACCP in food production. products, and as probiotics, which are gaining
increased popularity because of their health-
beneficial effects. Spoilage microorganisms, on
hallenges and Concerns with Global
C the other hand, are responsible for product spoil-
Food Safety age and place an economic burden on the produc-
ers, processors, and retail store owners for
Foodborne disease statistics and surveillance product losses. This is a serious issue in develop-
data are scanty. Underreporting of foodborne dis- ing countries because of inadequate processing
eases, misdiagnoses, large susceptible popula- and refrigeration facilities. Foodborne pathogen
tions, poverty, and lack of infrastructure hinder contamination in foods presents a serious chal-
data collection by the public health sector. This lenge which may result in severe diseases such as
lack of public health data can limit policymaking food intoxication, toxicoinfection, and infection.
and the formation of more effective guidelines Mortality, morbidity, and product recalls are seri-
for proper farming, processing, cooking, distribu- ous consequences of outbreaks caused by food-
tion, and food safety practices. Malnourishment, borne pathogens. Most foodborne pathogens
undernourishment, immunological health, and grow in the mesophilic range and a few in the
poor education and communication can also hin- psychrophilic range and their growth does not
der progress. usually alter the aesthetic quality of foods. Some
pathogenic traits are sometimes acquired through 2. Bhunia, A.K. (2014) One day to one hour: how
quickly can foodborne pathogens be detected? Future
plasmids, transposons, bacteriophages, or Microbiol 9, 935–946.
through pathogenicity islands. Foodborne patho- 3. Brandl, M.T. (2006) Fitness of human enteric patho-
gens can be zoonotic, geonotic, or human origin, gens on plants and implications for food safety. Annu
and consumption of contaminated foods results Rev Phytopathol 44, 367–392.
4. Callejón, R.M., Rodríguez-Naranjo, M.I., Ubeda,
in foodborne diseases. In order for a foodborne C., Hornedo-Ortega, R., Garcia-Parrilla, M.C. and
pathogen to cause disease, the microbe must be Troncoso, A.M. (2015) Reported foodborne out-
able to survive in food and, when transferred to breaks due to fresh produce in the United States
human hosts, find niches, multiply, and express and European Union: Trends and causes. Foodborne
Pathog Dis 12, 32–38.
virulence factors to cause host cell damage. 5. Crim, S.M., Griffin, P.M., Tauxe, R., Marder, E.P.,
Worldwide, foodborne pathogens are responsible Gilliss, D., Cronquist, A.B., Cartter, M., Tobin-
for large numbers of outbreaks, illnesses, and D'Angelo, M., Blythe, D., Smith, K., Lathrop, S.,
mortalities. Foodborne pathogens are a serious Zansky, S., Cieslak, P.R., Dunn, J., Holt, K.G.,
Wolpert, B. and Henao, O.L. (2015) Preliminary inci-
public health concern, and outbreaks are attrib- dence and trends of infection with pathogens trans-
uted to the emergence of new pathogens and mitted commonly through food - Foodborne Diseases
reemergence of some old pathogens. The routine Active Surveillance Network, 10 US Sites, 2006-
epidemiological and food product surveys are 2014. MMWR Morb Mortal Wkly Rep 64, 495–499.
6. Dewaal, C.S. and Plunkett, D.W. (2013) The Food
introduced by many countries in order to provide Safety Modernization Act - a series on what is essen-
an accurate picture of global distribution and tial for a food professional to know. Food Protect
occurrence of foodborne diseases. The reasons Trends 33, 44–49.
for the emergence of increased foodborne dis- 7. Falkow, S. (1997) What is a pathogen? ASM News 63,
359–365.
eases have been investigated. Several factors are 8. Flint, J.A., Duynhoven, Y.T.V., Angulo, F.J., DeLong,
thought to be responsible: improved survey sys- S.M., Braun, P., Kirk, M., Scallan, E., Fitzgerald, M.,
tem and the creation of the database for various Adak, G.K., Sockett, P., Ellis, A., Hall, G., Gargouri,
pathogens; changes in agricultural and food man- N., Walke, H. and Braam, P. (2005) Estimating the
burden of acute gastroenteritis, foodborne disease,
ufacturing practices; consumer’s habits of food and pathogens commonly transmitted by food: An
consumption and preparation; the increased pop- international review. Clin Infect Dis 41, 698–704.
ulation of the susceptible group; improved sur- 9. Kendall, M.E., Crim, S., Fullerton, K., Han, P.V.,
vival and adaptation of pathogens in harsh food Cronquist, A.B., Shiferaw, B., Ingram, L.A., Rounds,
J., Mintz, E.D. and Mahon, B.E. (2012) Travel-
environments; and improved detection methods. associated enteric infections diagnosed after return to
To control foodborne pathogen-related illnesses the United States, foodborne diseases active surveil-
and deaths, the US government has passed the lance network (FoodNet), 2004–2009. Clin Infect Dis
Food Safety Modernization Act (FSMA) in 2011 54, S480–S487.
10. Kirk, M.D., Pires, S.M., Black, R.E., Caipo, M.,
as a science-based proactive preventive strategy Crump, J.A., Devleesschauwer, B., Doepfer, D., Fazil,
rather than a reactive passive approach to the A., Fischer-Walker, C.L., Hald, T., Hall, A.J., Keddy,
food safety. Globally, food safety is a major con- K.H., Lake, R.J., Lanata, C.F., Torgerson, P.R.,
cern due to increasing numbers of food- and Havelaar, A.H. and Angulo, F.J. (2015) World Health
Organization estimates of the global and regional dis-
water-associated illnesses and mortality, the ease burden of 22 foodborne bacterial, protozoal, and
emergence of highly infectious diseases from viral diseases, 2010: A data synthesis. PLoS Med 12,
bush meats derived from wild animals, travel- e1001921.
associated intercontinental transfer of pathogens, 11. Kramer, A., Schwebke, I. and Kampf, G. (2006) How
long do nosocomial pathogens persist on inanimate
and globalization of food supplies. surfaces? A systematic review. BMC Infect Dis 6.
12. Methot, P.-O. and Alizon, S. (2014) What is a patho-
gen? Toward a process view of host-parasite interac-
Further Readings tions. Virulence 5, 775–785.
13. Ray, B. and Bhunia, A. (2014) Fundamental Food
Microbiology. Fifth edition. Boca Raton, FL: CRC
1. Baird-Parker, A. (1994) Foods and microbiological
Press, Taylor and Francis Group.
risks. Microbiology 140, 687–695.
Further Readings 23
14. Scallan, E., Hoekstra, R.M., Angulo, F.J., Tauxe, R.V., foodborne infections. Int J Food Microbiol 139,
Widdowson, M.A., Roy, S.L., Jones, J.L. and Griffin, S16–S28.
P.M. (2011) Foodborne illness acquired in the United 18. Tirado, M.C., Clarke, R., Jaykus, L.A., McQuatters-
States—major pathogens. Emerg Infect Dis 17, 7–15. Gollop, A. and Frank, J.M. (2010) Climate change and
15. Scharff, R. (2012) Economic burden from health food safety: A review. Food Res Int 43, 1745–1765.
losses due to foodborne illness in the United States. 19. Trevejo, R.T., Barr, M.C. and Robinson, R.A. (2005)
J Food Prot 75, 123–131. Important emerging bacterial zoonotic infections
16. Silk, B.J., Mahon, B.E., Griffin, P.M., Gould, L.H., affecting the immunocompromised. Vet Res 36,
Tauxe, R.V., Crim, S.M., Jackson, K.A., Gerner- 493–506.
Smidt, P., Herman, K.M. and Henao, O.L. (2013) 20. Walsh, K.A., Bennett, S.D., Mahovic, M. and Gould,
Vital signs: Listeria illnesses, deaths, and outbreaks - L.H. (2014) Outbreaks associated with cantaloupe,
United States, 2009-2011. MMWR Morb Mortal Wkly watermelon, and honeydew in the United States,
Rep 62, 448–452. 1973–2011. Foodborne Pathog Dis 11, 945–952.
17. Tauxe, R.V., Doyle, M.P., Kuchenmueller, T., 21. Zweifel, C. and Stephan, R. (2012) Spices and herbs
Schlundt, J. and Stein, C.E. (2010) Evolving pub- as source of Salmonella-related foodborne diseases.
lic health approaches to the global challenge of Food Res Int 45, 765–769.
Biology of Microbial Pathogens
2
Introduction environments or physiological conditions or

some may exist in singlet or doublet. Some cells
All organisms belong to one of the three domains are motile and may display unique motility such
of life: Bacteria, Archaea and Eukarya (Fig. 2.1). as tumbling, corkscrew rotation, swimming, and
The phylogenetic classification of the organisms swarming.
is achieved based on the sequence comparison of Nutrients, temperature, and gaseous composi-
small subunit rRNA gene. The classical taxo- tion of the environment influence bacterial
nomic ranks are a domain, phylum, class, order, growth and metabolism. The aerobic bacteria
family, genus, species, serovar, and strain. An require oxygen for respiration and energy, while
example for each rank is provided for a strain of the anaerobic bacteria grow in the absence of
Escherichia coli (Fig. 2.2). In this chapter, gen- oxygen. Obligate or strict anaerobes cannot with-
eral properties of microorganisms including bac- stand traces of oxygen, while aerotolerant anaer-
teria, viruses, molds, parasites, and algae (the obes have tolerance for some amounts of
source of most seafood-associated toxins) that oxygen.
are responsible for foodborne diseases are Microbes are also classified based on their
reviewed. In addition, the morphological and temperature requirements. Some microorganisms
structural characteristics of microorganisms in grow at refrigeration to ambient room tempera-
relation to their pathogenesis are discussed so tures (1–25 °C), and they are called psychro-
that this background knowledge will aid in under- trophs (cold tolerant). Their optimal growth
standing the mechanism of pathogenesis and the temperature is above 15 °C and maximum is
host response to pathogens in the subsequent above 20 °C. Psychrophiles (cold loving) can
chapters. grow and reproduce at −20 °C to +20 °C with an
optimal temperature of growth at 15 °C.
Psychrophiles are also found in the polar ice,
Bacteria permafrost, and at the bottom of an ocean.

Mesophiles grow at 25–37 °C and both psychro-
Bacterial genera associated with foodborne infec- trophs and mesophiles are capable of causing dis-
tions or food intoxications are listed in Table 2.1. eases in humans. Some bacteria grow at high
Morphologically, bacterial cells are rods temperatures and are called thermophiles (50–
(1–10 μm in length and 0.5–1 μm in diameter), 70 °C); some even grow in hot geysers such as
spherical, curved, or spiral (Fig. 2.3). Some cells extremophiles (>70 °C). Maintenance of mem-
may form long chains depending on the growth brane fluidity is critical for bacterial survival and

26 2 Biology of Microbial Pathogens
Fig. 2.1 Universal

phylogenetic tree based
on a comparison of
small-subunit rRNA
gene sequences
(Adapted and redrawn
from Eckburg et al.
2003. Infect. Immun.
71:591–596)
Fig. 2.2 A flow diagram showing the taxonomic classification of bacteria

Bacteria 27
Table 2.1 Bacterial genera associated with foodborne growth under extreme conditions (both low and
infections or food intoxication
high temperatures) so that the enzyme activity,
Gram-positive Gram-negative electron transport, ion pump, and nutrient uptake
Bacillus Aeromonas are possible. Altogether, only a very small frac-
Clostridium Arcobacter
tion of all microbes is capable of causing diseases
Listeria Brucella
in humans or animals. Most of the pathogenic
Mycobacterium Campylobacter
Staphylococcus Cronobacter (Enterobacter) bacteria are mesophilic and a few are psychro-
Escherichia philic, while thermophiles are rarely pathogenic.
Salmonella Based on the microbial response to acidity (pH),
Shigella bacteria are also grouped as aciduric (<pH 7) or
Vibrio alkaliphilic (pH 8.5–11 with an optimum of pH
Yersinia 10.0). Thiobacillus ferrooxidans is an example of
Fig. 2.3 Bacterial cells: (a) Morphology, (b) Structural microscopic cross section of Gram-positive Listeria
differences in Gram-positive and Gram-negative bacteria, monocytogenes and Staphylococcus aureus cells
(c) Schematics and original (d) transmission electron
extremophile aciduric bacterium that can grow at Yersinia, and Vibrio; and the acid-fast bacteria
pH of ~1.5. Some bacteria also require salt for are Mycobacterium tuberculosis and M. bovis.
their growth, can tolerate moderate to high levels The outermost layers of Gram-positive bacte-
of salt (1.7–30%), and are called halophiles ria contain a thick rigid cell wall or peptidogly-
(examples are Vibrio vulnificus, Staphylococcus can (PGN) structure. Cell wall also contains
aureus). Some bacteria that can thrive at high proteins and teichoic acid (TA) or wall teichoic
pressure are called barophiles or piezophiles such acid (WTA), teichuronic acid, lipoteichoic acids
as those found in deep sea or ocean floor and can (LTA), lipoglycan, and polysaccharides. The
withstand pressure exceeding 38 MPa. inner layer is a porous cytoplasmic membrane
Bacteria are divided into two groups based on (CM) which consists of a lipid bilayer (Fig. 2.4).
their cell wall structures and staining characteris- Gram-negative bacteria, on the other hand,
tics: Gram-positive and Gram-negative, named have an outer membrane (OM) layer, a thin pep-
after the Danish bacteriologist, Hans Christian tidoglycan layer, and an inner cytoplasmic mem-
Gram (1853–1938), who developed the Gram brane (Fig. 2.5). The OM consists of a lipid
staining method in 1884. Gram-positive bacterial bilayer, in which the lipopolysaccharide (LPS) is
cell envelope consists of a thick rigid peptidogly- located on the outer leaflet of the bilayer. The
can polymer and retains crystal violet–iodine major components of LPS are lipid A, which is a
complex to appear purple to blue after Gram glycophospholipid consisting of β-1,6-d-
staining, while the Gram-negative bacterial cell glucosamine disaccharide. Phosphate and car-
wall is porous and does not retain this stain. boxylate groups of the lipid A provide a strong
Counter staining of the Gram-negative cells with negative charge to the outer surface.
safranin (carbol fuchsin) stains the cells pink to
red. Acid-fast bacteria such as mycobacteria have
mycolic acid and lipid on their cell envelope, Gram-Positive Bacteria
rendering them unstainable with Gram stain.

Mycobacterium retains carbol fuchsin after Cell Wall and Peptidoglycan
acid-fast staining also known as Ziehl–Neelsen
staining. Examples of Gram-positive bacteria The cell wall consists of a large number of mol-
are Listeria, Staphylococcus, Streptococcus, ecules that have a multitude of functions. In addi-
and Clostridium; Gram-negative examples are tion, the cell wall protects cells from mechanical
Salmonella, Escherichia, Campylobacter, damage or osmotic lysis. The major component
Fig. 2.4 A schematic

cross section of the
Gram-positive bacterial
cell wall
Gram-Positive Bacteria 29
Fig. 2.5 A schematic cross section of Gram-negative bacterial cell wall and membranes
Fig. 2.6 The structure of cell wall (peptidoglycan) from N-acetylmuramic acid (Adapted and redrawn from
a typical Gram-positive bacterial species (Staphylococcus Navarre and Schneewind. 1999. Microbiol. Mol. Biol.
aureus). Enzymes are marked by an oval circle showing Rev. 63:174–229)
their site of action. NAG N-acetyl-glucosamine, NAM
of the cell wall is peptidoglycan (about 20–80 nm with disaccharide GlcNAc-(β1–4)-MurNAc mol-
thick) and is known as murein, which consists of ecules and provides stability to the peptidoglycan
peptides and sugar moieties (Figs. 2.4 and 2.6). backbone. In Staphylococcus aureus, the tetra-
PGN is highly complex and dynamic structure, peptide, consisting of Ala, Glu, Lys, and Ala,
which contains a disaccharide N-acetyl-d- forms a bridge with the pentaglycine (Gly5) pep-
glucosamine (NAG) and N-acetylmuramic acid tide. In Listeria monocytogenes, pentaglycine is
(NAM) and linked by β-1,4-glycosidic linkage absent, but the crossbridge is formed by an amide
(GlcNAc-(β1–4)-MurNAc) and pentapeptide bond between the ε-amino group of a meso-
(Fig. 2.6). The enzyme transpeptidase (e.g., sor- diaminopimelic acid (m-Dpm) and the d-Ala of
tase) helps in the formation of peptide cross-link the adjacent cell wall (Fig. 2.6). Cell wall
p eptidoglycans with a low degree of cross-link- antigen. PGN also initiates complement activa-
ing are much more susceptible to the degradation tion through the alternative pathway and sup-
by the cell wall hydrolases than are those with a presses appetite by inducing increased tumor
high degree of cross-linkers. Penicillin or other necrosis factor (TNF-α) production (see Chap.
β-lactam antibiotics inhibit transpeptidases used 3). During innate immunity against bacteria, toll-
for cell wall formation, hence affecting the bacte- like receptor (TLR) of immune cells such as the
rial cell wall growth in Gram-positive bacteria. In macrophage binds to PGN for recognition.
addition, β-lactam can activate autolysin, which TLR-2 was thought to interact with PGN; how-
degrades peptidoglycan. A carbohydrate-ever, it was later determined that the nucleotide-
hydrolyzing enzyme, lysozyme (Mr. 14.4 kDa), binding oligomerization domain protein (Nod)-1
breaks down peptidoglycan. Lysozyme present in and Nod-2 serve as mammalian pattern recogni-
body fluids, such as saliva and tears, and in the tion receptor for PGN (see Chap. 3). PGN can be
avian egg white is also known as β-1,4-N- cytotoxic to some host cells.
acetylmuramidase and cleaves the glycosidic
bond between the C-1 of NAM and the C-4 of
NAG in the peptidoglycan (GlcNAc-(β1–4)- Teichoic Acid and Lipoteichoic Acid
MurNAc). Lipoteichoic acid, teichoic acid, and
surface proteins can prevent lysozyme action and Teichoic acid is a polyanionic polymer and has a
protect the bacterium from lysis. Other enzymes polysaccharide backbone, which consists of
that hydrolyze PGN are glucosaminidase, endo- glycerol or ribitol linked by phosphodiester
peptidase (e.g., lysostaphin from S. aureus), bonds, i.e., sugar–alcohol–phosphate, and is bur-
muramidase, amidase, and carboxypeptidase ied in the peptidoglycan backbone (Fig. 2.4). Cell
(Fig. 2.6). Cell wall peptidoglycan helps main- wall TA or WTA is uniformly distributed over the
tain the structural integrity and the shape of cells; entire peptidoglycan exoskeleton. The function
therefore, when the PGN is totally removed, the of TA is not fully known, but it is thought that the
bacterium forms a structure called protoplast negatively charged TA captures divalent cations
while the bacterial cell wall with some remnants or provides a biophysical barrier that prevents the
of PGN called a spheroplast. diffusion of substances and binds enzymes that
Cell wall also carries several surface proteins hydrolyze peptidoglycan. TA or WTA also plays
containing an amino acid sequence motif an important role in bacterial physiology, cell
consisting of L (Leucine), P (Proline), X (any), T division, ion homeostasis, biofilm formation,
(Threonine), and G (Glycine) in the C-terminal bacterial pathogenesis, and host immune response
end, where X could be any amino acid. This including complement activation and phagocyto-
LPXTG motif helps bacterial surface proteins to sis (opsonization) (see Chap. 3).
anchor to the peptidoglycan backbone. Examples Lipoteichoic acid being a polyanionic poly-
of proteins that contain LPXTG motif are intern- mer is inserted into the lipid portion of the outer
alin in Listeria monocytogenes, the M protein in leaflet of the cytoplasmic membrane (CM), trav-
Streptococcus pyogenes, protein A in els through the peptidoglycan, and is exported
Staphylococcus aureus, and fibrinogen-binding outside the cell wall (Fig. 2.4). Both WTA and
protein in S. aureus and S. epidermidis. These LTA are unique for Gram-positive bacteria and
proteins serve as adhesion factors or binding are absent in Gram-negative bacteria. The func-
molecules for the host cell receptors. An immune tion of LTA is unknown, but it serves as species-
response against Gram-positive bacterial patho- specific decorations of the peptidoglycan
gens often targets PGN as an antigen. exoskeleton. LTA and the surface proteins pro-
PGN performs multiple functions in a host. vide unique serotype characteristics of a bacte-
PGN is a strong vaccine adjuvant and activates rium, for serological classification. LTA with
dendritic or macrophages and monocytes for different sugar molecules determines the sero-
improved antigen presentation, cytokine produc- type of the bacteria. This antigenic classification
tion, and strong immune response against an is called somatic antigen or O antigen.
Gram-Negative Bacteria 31
Cytoplasmic Membrane
The cytoplasmic membrane consists of a lipid

bilayer and carries transport proteins, which bind
specific substrate and transport unidirectionally
toward the cytoplasm. The proteins are responsi-
ble for secretion of periplasmic extracellular pro-
teins, energy metabolism (electron transport
system, ATPase), and cell wall synthesis
Gram-Negative Bacteria
Outer Membrane
The cell envelope of Gram-negative bacteria is

surrounded by an outer membrane (OM), which
is about 7.5–10 nm thick and consists of phos-
pholipids, proteins, and lipopolysaccharide
(LPS) (Fig. 2.5). The OM has a lipid bilayer
arrangement; the outer leaflet consists of LPS
and the inner leaflet has phospholipid. The LPS
monomer has a strong lateral interaction with
other LPS in the OM thus providing rigidity and
gel-like appearance. LPS consists of three dis- Fig. 2.7 Structure of lipopolysaccharide (LPS) from
Salmonella enterica serovar Typhimurium. It has three
tinct regions: O side chain, core oligosaccharide, regions: O side chain, core oligosaccharide, and the lipid
and lipid A (Fig. 2.7). The O side chain consists A. Abe abequose, Man mannose, Rha rhamnose, Gal
of repeating polysaccharide subunits of mannose, galactose, Glc glucose, GlcNAc N-acetylglucosamine,
rhamnose, and galactose and is the most variable Hep heptulose, KDO 2-keto-3-deoxyoctonate, GlcN glu-
cosamine, P phosphate, EtN ethanolamine
part of the LPS and can even show variability in
strains within a species. The core oligosaccharide
of LPS is covalently linked to the lipid A through enzymes, such as protease and phospholipase.
the acidic sugar, 2-keto-3-deoxyoctanate (KDO). The OMPs serve as the adhesin for interaction
The lipid A is the most conserved region in the with mammalian cells. The most abundant pro-
LPS and it secures LPS in the OM architecture. teins in OM are porins, which are trimeric
Lipid A is very toxic, and it is one of the most β-barrels forming channels. The pore diameter in
important molecules involved in bacterial patho- porins varies from 6Å to 15Å, and porins serve as
genesis and immune modulation. In Gram- a selective transport system for small molecules
negative bacterial pathogens when LPS is (<600 Da). Porins are filled with water, and they
sloughed off, it is referred to as endotoxin or help in the passive diffusion of small solutes
pyrogen. LPS induces host cytokine (TNF-α, without binding them. Nutrients can diffuse
IL-1) production by macrophages and increased through porins, but if the concentration is very
body temperature (fever). low, transport can occur via a substrate-specific
The OM is also embedded with many proteins active transporter system. Some porins may be
and is collectively called OMPs (outer membrane substrate-specific such as the sucrose-specific
proteins). The OMPs serve as a transporter of porin, ScrY in Salmonella, and
nutrients and ions across the OM to the periplasm maltooligosaccharide-specific maltoporin, LamB
and are residence sites for membrane-bound in E. coli. Porins also play an important role in
bacterial pathogenesis by activating an immune Protein Secretion Systems

response, signaling pathway, and antibiotic resis-
tance. Porins can activate both classical and alter- Several nanomachine channels also known as
native complement pathways and induce an secretion systems exist in both Gram-positive
inflammatory response. and Gram-negative bacteria to transport proteins,
DNA, and certain virulence factors across the
bacterial cell envelope (Table 2.2). In Gram-
Peptidoglycan negative bacteria, six secretion systems facilitate
protein export from the cytosol across the outer
The peptidoglycan structure of Gram-negative membrane. Those systems are type I secretory
bacteria is similar to that seen in Gram-positive system (T1SS), type II (T2SS), type III (T3SS),
bacteria, but it is much thinner (about 5–10 nm) type IV (T4SS), type V (T5SS), and type VI
and is less defined. It is attached to the OM by a (T6SS). T1SS, T2SS, T3SS, T4SS, and T6SS
lipoprotein, wherein the lipid moiety remains span from cytoplasmic membrane (CM) to the
embedded in the inner leaflet of the OM. PGN is outer membrane (OM), while T5SS spans only
sensitive to lysozyme, but this enzyme is too the OM. T1SS exports a large variety of protein
large to pass through the porin in Gram-negative substrates from cytosol to the extracellular milieu
bacteria, which are thus resistant to lysozyme. in both pathogenic and nonpathogenic Gram-
negative bacteria. Examples of type I exporter are
those proteins belonging to ATP-binding cassette
Periplasmic Space (ABC), iron-binding proteins, and hemolysins.
T2SS is also found in a wide range of pathogenic
Periplasmic space (PS) is the area or the compart- and nonpathogenic Gram-negative bacteria and
ment located between peptidoglycan and the helps in secretion of the protein from periplasm
cytoplasmic membrane. PS contains a concen- into the extracellular milieu. Examples are
trated gel-like matrix called periplasm and serves pseudolysin of Pseudomonas aeruginosa and
as the storage space for numerous enzymes (e.g., cholera toxin of Vibrio cholera. T3SS is primarily
phosphatases), proteins that bind nutrients (sug- found in pathogenic bacteria and exports effector
ars, amino acids, vitamins, ions) and direct them proteins (virulence proteins) directly into the
to the cytoplasmic membrane for transport, oli- cytoplasm or the plasma membrane of target
gosaccharides (prevent changes in osmolarity), eukaryotic cells. T3SS is present in Gram-
toxins, and some peptidoglycans which cross- negative bacterial genera of Salmonella, Shigella,
linked into gel. Gram-negative bacteria also con- Pseudomonas, and Yersinia and enteropatho-
stantly release membrane vesicles (MVs) from genic (EPEC) and enterohemorrhagic (EHEC)
OM. These MVs contain an OMP, LPS, phospho- Escherichia coli. It is also known as a “molecular
lipids, and a periplasm containing protease, phos- syringe,” which directly delivers virulence pro-
pholipase C, alkaline phosphatase, and autolysin. teins across the cytoplasmic membrane of the
MVs are released from both planktonic and bio- host without exposing the proteins to extracellu-
film forming cells and attack other vulnerable lar milieu. T4SS is present in both Gram-positive
bacterial clones under nutrient starvation condi- and Gram-negative bacteria and exports proteins
tions providing selective growth or colonization and DNA–protein complexes from the bacterial
advantages. MVs are thought to play a role in cell into the cytoplasm of the recipient cell (both
pathogenesis by delivering virulence factors such prokaryote and eukaryote). This system is ubiq-
as phospholipase C, protease, and hemolysins uitous and promiscuous and is capable of trans-
directly to the host cells without being degraded porting DNA–protein complex to other bacteria,
by inactivating environmental enzymes. In many archaea, yeasts, molds, and plants. For example,
bacteria such as Pseudomonas, Proteus, Serratia, VirB in Agrobacterium tumefaciens allows the
Vibrio, Haemophilus, Neisseria, Escherichia and transfer of DNA–protein complex in plant tissues
others, these MVs participate in pathogenesis. causing cancerous growth. Other examples of
Accessory Structures in Gram-Positive and Gram-Negative Bacteria 33
Table 2.2 Protein secretion systems in bacteria

Name Source Function
Type I secretion Gram-negative Exports a large variety of protein substrates from cytosol to
system (T1SS) extracellular milieu in both pathogenic and nonpathogenic bacteria.
Examples: proteins with ATP-binding cassette (ABC), iron-binding
proteins, and hemolysins
Type II secretion Gram-negative Secretes protein from periplasm into extracellular milieu such as
system (T2SS) pseudolysin of Pseudomonas aeruginosa and cholera toxin of Vibrio
cholera
Type III secretion Gram-negative Exports effector proteins (virulence proteins) directly into the
system (T3SS) cytoplasm or the plasma membrane of target eukaryotic cells
Type IV secretion Gram-negative and Exports proteins and DNA–protein complexes from the bacterial cell
system (T4SS) Gram-positive into the cytoplasm of the recipient prokaryote and eukaryote cell
Type V secretion Gram-negative Secretes virulence factors and involves in cell-to-cell adhesion and
system (T5SS) biofilm formation
Type VI secretion Gram-negative Translocates toxic effector proteins into both prokaryotes and
system (T6SS) (Proteobacteria) eukaryotes
Type VII secretion Mycobacterium A specialized secretory system for export of virulence proteins in
system (T7SS) (Gram-positive Mycobacterium tuberculosis and M. bovis
SecA, SecA2 Gram-negative Export virulence proteins and enzymes
Gram-positive
type IV system include CAG and ComB system complex comprised of SecY, SecE, and SecG and
in Helicobacter pylori, Ptl system in Bordetella are mediated by ATPase SecA. Precursor proteins
pertussis, and Dot in Legionella pneumophila. acquire their folded structure after being translo-
T4SS is also responsible for the transfer of cated out of the cytosol by either SecA1 or
plasmid-borne antibiotic resistance gene. T5SS is SecA2. SecA2 is also present in Gram-negative
a single membrane-spanning secretion system bacteria. Most proteins are exported by SecA1,
and is an autotransporter. It secretes virulence while only limited proteins including some viru-
factors and is involved in cell-to-cell adhesion lence proteins are translocated by SecA2. Sec-
and biofilm formation. T6SS is widely distributed dependent proteins contain a classical Sec signal
in Proteobacteria, and it translocates toxic effec- sequence at the amino terminus; however, SecA2
tor proteins in both prokaryotes and eukaryotes. can translocate proteins bearing no classical Sec
It plays an important role in bacterial pathogene- signal sequence. The virulence factors that are
sis and competition. An additional secretory translocated by SecA2 are superoxide dismutase
system, T7SS, was originally isolated from myco- (SodA) in L. monocytogenes and Mycobacterium
bacteria and is a specialized secretory system for and LAP (Listeria adhesion protein) in L.
export of virulence proteins in Mycobacterium monocytogenes.
tuberculosis and M. bovis. Mycobacteria have
mycomembrane consisting of free lipids and
mycolic acid linked to the peptidoglycan via ara- ccessory Structures in Gram-
A
binogalactan, and T7SS translocates protein from Positive and Gram-Negative
CM to the extracellular milieu. Interestingly, Bacteria
T7SS gene clusters are also found in some non-
acid-fast bacteria such as Listeria monocyto- The accessory extracellular structures including
genes, Bacillus subtilis, and Staphylococcus fimbriae, pili, flagella, and capsules provide struc-
aureus. tural integrity and facilitate bacterial colonization,
In Gram-positive bacteria, most secreted pro- motility, exchange of genetic materials, and sur-
teins are translocated across the cell envelope by vival in in vitro and in vivo environments. These
Sec pathway consisting of membrane protein structures also serve as important virulence
f actors for pathogenic bacteria by promoting bac-

terial adhesion, colonization, motility, and eva-
sion of host immune system (see Chap. 4).
Mutations in the genes encoding these accessory
structures provide antigenic shift thus allowing
pathogens to overcome host immune defense sys-
tem. Furthermore, genetic and phenotypic proper-
ties of the accessory structures allow bacterial
classification. The following key antigens are
used as a target for serotyping or serogrouping.
Fimbriae (Pili) and Curli
Gram-negative bacteria carry cilia-like long

appendages composed of glycoproteins, which Fig. 2.8 Flagellar arrangements in bacteria
can be used by bacteria to exchange genetic
materials between organisms or used for attach- The flagellum, a complex self-assembling
ment to a different substrate including the host nanomachine, consists of a woven structure of
cell. Fimbrial antigens are also called coloniza- flagellin proteins. There are three main struc-
tion factor antigens (CFA) and are important in tures: an engine, a propeller, and a universal joint
bacterial adhesion, motility, and inter-bacterial that connects them to form the bacterial flagel-
communication (see Chap. 4 for details). Curli lum. The engine includes a rotor and a stator,
are extracellular amyloid-like protein fibers and which are embedded in the cytoplasmic mem-
are involved in bacterial adhesion, surface colo- brane, and a rod that acts as a drive shaft, which
nization, and biofilm formation. extends from the rotor through the peptidoglycan
layer to the outer membrane. The helical filament
acts as a propeller for locomotion and movement.
Flagella The proteins involved in the flagellar assembly
are similar to the proteins seen in T3SS in Gram-
The flagellar antigen is designated “H,” which negative bacteria. In some bacteria, flagella are
stands for “Hauch” in German. Both Gram- considered important virulence factors aiding in
positive and Gram-negative bacteria may carry adhesion and invasion by bacterial pathogens
one or more filamentous whiplike structures such as Campylobacter jejuni, Legionella pneu-
extending outward from their cell wall, called fla- mophila, Clostridium difficile, Salmonella
gella. Flagella aid in bacterial motility or loco- enterica serovar Typhi, and Vibrio cholera. In
motion and reaching the nutrient-rich some other pathogens, flagella are also involved
environment. Flagella may exist as either single in secretion of virulence factors.
polar flagellum or multiple flagella surrounding
the bacterium. They are designated as monotri-
chous, a single polar flagellum such as in Vibrio O (Somatic) Antigen
cholera; amphitrichous, one on each pole, for
example, are Alcaligenes faecalis and In Gram-negative bacteria, O antigen represents
Campylobacter jejuni; lophotrichous, multiple O side chain, core oligosaccharide of the LPS
flagella in each pole, for example, Vibrio fischeri; located in the outer membrane (Fig. 2.8).
and peritrichous, multiple flagella surrounding O-polysaccharide is conserved, but it may be
the bacterial cell, for example, Salmonella shared among different genera of Gram-negative
enterica and E. coli (Fig. 2.8). bacteria. For example, E. coli O8 antigen is
Accessory Structures in Gram-Positive and Gram-Negative Bacteria 35
shared by Klebsiella pneumoniae and Serratia Endospore Formation

marcescens; E. coli O157 antigen is shared by
Citrobacter freundii and Salmonella enterica. In Some bacterial species forms spores as a survival
addition, proteins in OM may contribute to O mechanism. Only a few bacterial genera, namely,
serotyping profile. In Gram-positive bacteria, the Gram-positive Bacillus, Alicyclobacillus,
O-antigenic profile is shared by peptidoglycan, Clostridium, Sporolactobacillus, and
TA or WTA, LTA, and surface proteins. Sporosarcina and the Gram-negative
Desulfotomaculum, are capable of forming
spores. Among these bacteria, Bacillus (B.
Capsule cereus, B. anthracis) and Clostridium (C. per-
fringens, C. botulinum) cause foodborne diseases
The capsule is a thick layer of a gel-like structure in humans. Bacterial spores are called endospores
composed of complex polysaccharides (i.e., since they are produced inside a cell and there is
sialic acid, N-acetylglucosamine, glucuronic only one spore per cell. Endospores may be
acid, glucose, galactose, fucose) and located out- located terminal, central, or off-center, causing
side the cell envelope. In some bacteria, the cap- bulging of the cell. Under a phase-contrast micro-
sule may be thin and is called glycocalyx. The scope at 1000 × magnification, spores appear
capsule is generally visualized by negative stain- refractile spheroid or oval. The surface of a spore
ing (generally stained with India ink). It is also is negatively charged and hydrophobic; hence, it
known as the K antigen and helps protect the is sticky. Spores are much more resistant to phys-
cells against desiccation. The capsule acts as a ical and chemical antimicrobial treatments as
virulence factor and it helps bacterial adhesion to compared to vegetative cells. This is because the
the host cells. It protects cells from engulfment specific structure of bacterial spores is quite
by macrophages and other phagocytic cells. The different from that of vegetative cells from which
Gram-positive bacteria that produce capsules they are formed (Fig. 2.9). In the core of the
include Bacillus cereus, Bacillus anthracis, spore, protoplasm contains DNA, RNA, enzymes,
Streptococcus mutans, Streptococcus pyogenes, dipicolinic acid (DPN), divalent cations, and very
and S. pneumoniae, while the Gram-negative little water. The core is surrounded by an inner
bacteria are Haemophilus influenza, Pseudomonas membrane, which is the predecessor of the cell
aeruginosa, Escherichia coli, Agrobacterium cytoplasmic membrane. The germ cell wall sur-
tumefaciens, and Acetobacter xylinum. The latter rounds the inner membrane, which is the prede-
two are not normally pathogenic to man. cessor of the cell wall in the emerging vegetative
Fig. 2.9 Drawing of an

electron microscopic
image of a clostridial
bacterial spore,
exosporium, coat, outer
membrane, cortex, germ
cell wall and inner
membrane, and core
cell. The cortex located surrounding the cell wall

is composed of peptides, glycan, and an outer
forespore membrane. The spore coats located
outside the cortex and the membrane are com-
posed of layers of proteins such as small acid-
soluble proteins (SASP), which protect spore
DNA from thermal denaturation and other harsh
treatments. Spores of some species may have a
structure called exosporium located outside the
spore coat. During germination and outgrowth of
spore, the cortex is hydrolyzed, and outer fore-
spore membrane and spore coats are removed by
the emerging vegetative cell.
The spores are metabolically inactive or dor-
mant, and can survive for years, but are capable
of emerging as vegetative cells (one cell per
spore) under a suitable environment. The life
cycle of spore-forming bacteria has a vegetative
Fig. 2.10 Schematic presentation of the cycles of cell
cycle (by binary fission) and a spore cycle, which division and endospore formation, germination, and out-
goes through several stages in sequence, during growth of spore forming bacteria (Adapted and redrawn
which a cell sporulates and a vegetative cell from Foster 1994. J. Appl. Bacteriol. 76:S25–S39)
emerges from a spore (Fig. 2.10). These stages
are genetically controlled and influenced by dif- Conversely, Clostridia usually require the pres-
ferent environmental parameters and biochemi- ence of carbon and nitrogen substrates to produce
cal events, which are briefly discussed here. mature heat-resistant spores.
Sporulation in bacteria is triggered by the changes
in the environmental factors such as reduction in
nutrient availability (particularly carbon, nitro- Viruses
gen, and phosphorous sources) and changes in
the optimum growth temperature and pH. The Virus means “poison” in Latin. Food virology
transition from the cell division cycle to sporula- includes the study of viruses that are transmitted
tion is genetically controlled, involving many via food and are commonly known as enteric.
genes. A cell initiates sporulation only at the end Enteric viruses are transferred via fecal–oral or
of completion of DNA replication and synthesis fecal–water–oral routes and are highly infectious.
of adenosine bis-triphosphate (Abt) by spore- Viruses are shed in large numbers (109 particles
formers under carbon or phosphorous depletion. per gram) from infected patients through feces
Sporulation events have several stages: (1) termi- and a slightly lower numbers by vomitus. Person-
nation of DNA replication, alignment of chromo- to-person or fecal–oral transmission is a common
some in axial filament, and formation of mechanism for viral infection. The infectious
mesosome; (2) invagination of cell membrane dose is about 10–100 particles. Infection is also
near one end and completion of the septum; (3) referred as “stomach flu” characterized by watery
engulfment of prespore or forespore; (4) forma- diarrhea, nausea, and vomiting and resembles
tion of germ cell wall and cortex, accumulation bacterial food poisoning. Viruses are obligate
of Ca2+, and synthesis of DPN; and (5) deposition intracellular parasites, i.e., they require a living
of spore coats. In general, Gram-positive spore- host for replication and cannot grow outside their
formers in the Bacillus group can form mature specific host nor in the food. Viruses are generally
spores in the absence of carbon and nitrogen “host-specific,” i.e., a virus can be a plant, animal,
sources on a substrate such as soil extract agar. or human, or bacteria-specific and generally do
Viruses 37
not show cross-species infection. However, some small particles generally ranging from 20 to
zoonotic viruses are able to infect both humans 300 nm. The smallest virus is picornavirus (20–
and animals, and some viruses undergo genetic 30 nm), a causative agent of foot-and-mouth dis-
modifications to adapt themselves to different ease (FMD). Some of the large viruses are the
hosts. Viruses are metabolically dependent on the poxvirus or vaccinia virus (~300 nm), Nipah
host. Animal cell culture or chick embryos are virus (500 nm), and Ebola virus (1200 nm). Virus
used for viral growth, replication, and isolation. structure varies – picornavirus has an icosahedral
Viruses replicate rapidly to yield prodigious titers symmetry, tobacco mosaic virus (TMV) is heli-
in the host cells. Though there are a few antiviral cal, and herpesvirus, vaccinia virus, and poliovi-
drugs, viruses generally do not respond well to the rus are spherical. The nucleic acid core is
conventional antibiotics. Hence, prevention strat- surrounded by a “protein coat” called nucleocap-
egies involve appropriate vaccination and proper sid, which consists of capsomere. In some cases,
hygienic practices. protein coat is surrounded by an envelope made
up of a lipid bilayer and accessory protein mole-
cules. The envelope is sensitive to solvents. This
Virus Classification/Taxonomy envelope carries specific surface molecules that
aid viral interaction with the host cell receptors.
Enteric viruses are generally nonenveloped RNA For example, hemagglutinin (H) and neuramini-
viruses and belong to the family of Adenoviridae, dase (N) in human influenza virus or bird flu
Caliciviridae, Hepeviridae, Picornaviridae, and virus bind to glycoside receptors on the host epi-
Reoviridae. Viruses are classified based on the thelial cells (Fig. 2.11).
size, shape, structure, and nucleic acid content.
Viruses contain either DNA or RNA. The nucleic
acid may be single- or double-stranded nucleic Viral Replication
acid molecules. DNA viruses generally have
double-stranded DNA, and RNA viruses have Environmental survival is critical for viral persis-
single-stranded RNA molecule. Enteric viruses tence and transmission and enteric viruses can
are found in the gastrointestinal tract, and small survive in the stomach acid. Viruses are obligate
round structured viruses are called “SRSVs,” intracellular parasites and require a living host
which are found in feces. Based on the genetic for their replication. The virus life cycle has
elements, Dr. David Baltimore has classified seven steps: (1) attachment to the host receptor,
viruses into seven types (1) dsDNA, (2) ssDNA, (2) penetration into the cell, (3) uncoating of
(3) dsRNA, (4) (+) sense ssRNA, (5) (−) sense DNA/RNA, (4) transcription and/or translation,
ssRNA, (6) RNA reverse transcribing viruses, (5) nucleic acid replication, (6) assembly of viral
and (7) DNA reverse transcribing viruses. Later, nucleic acid and coat proteins, and (7) release of
Baltimore received the Nobel Prize for discover- matured virus particles (Fig. 2.12). RNA viruses
ing that the RNA viruses reproduce by using a encode genes for RNA-dependent RNA poly-
novel enzyme, RNA reverse transcriptase. merase, a nonstructural enzyme (replicase),
which is needed for RNA replication inside the
host. On the other hand, DNA-dependent RNA
Virus Structure polymerase is also called RNA transcriptase
responsible for transcription of RNA from DNA
Common particle shape of a virus may be cubical leading to structural protein synthesis. The struc-
or icosahedral, i.e., a polyhedron with 20 triangu- tural proteins such as capsid proteins are needed
lar faces, 12 corners, and spherical or helical for viral packaging.
shape. The size of the virus is determined by an For gastroenteritis caused by Norovirus, the
electron microscopy or virus’s ability to pass virus enters, replicates, and destroys mature
through a defined membrane filter. Viruses are enterocytes, triggering decreased absorption/
Fig. 2.11 Schematic

viral structure (influenza
virus)
immature cells are unable to perform a normal

physiological function. Viruses are shed in high
numbers and are stable outside the host environ-
ment. Norovirus and Rotavirus are true gut
inhabitants, while poliovirus, hepatitis A virus
(HAV), and hepatitis E virus (HEV) are found in
nonenteric locations. An elaborate description of
several key enteric viral diseases is described in
Chap. 6.
Parasites
Parasites are another type of organisms that

require living host cells in which to grow and
reproduce by deriving nourishment and protec-
tion from the host. Parasites are unicellular or
multicellular and can be transmitted by vectors
such as flies, mosquitos, ticks, or food. Parasites
may be ectoparasite such as ticks and mites,
endoparasite such as Trichinella, and epiparasite
that feeds on another parasite. Foodborne para-
sites (Table 2.3) are considered endoparasites
Fig. 2.12 Viral replication in a host cell: (1) attachment including protozoa, cestodes (tapeworms), nema-
to the host cell receptor; (2) penetration, (3) uncoating, (4) todes (roundworm), and trematodes (flatworms
transcription and/or translation, (5) nucleic acid replica- or flukes). Some endoparasites are unicellular
tion, (6) assembly of viral proteins and nucleic acids, and
(7) release of matured virus particles
(protozoa) and some are multicellular (metazoa).
A majority of parasitic diseases are considered
reabsorption resulting in epithelial inflammation zoonotic. Immunocompromised people are
and diarrheal symptoms. Even though the dam- highly susceptible to the intracellular protozoan
aged enterocytes are replaced by the immature parasites. Contamination of food products
enterocytes, gastroenteritis continues since these (generally fresh produce) with parasites often

Molds and Mycotoxins 39
indicates inadequate sanitary hygienic practices erate in food; therefore, they are only detected by
employed during production and processing. direct means using microscopy or molecular and
Water, fresh fruits, and vegetables are known to antibody-based assays. Parasites cannot be cul-
be the major source of parasites. Globalization of tured even in a rich growth medium but require a
food supply presents a favorable condition for living host. Details of individual foodborne para-
spread and distribution of parasites thus present- sitic diseases are presented in Chap. 7.
ing a major challenge in prevention and control
of foodborne parasites.
Molds and Mycotoxins
Life Cycle and Growth Characteristics Molds or fungi are ubiquitous in nature and cause
diseases in humans, animals, and plants. Molds
Parasites are significantly larger than the bacteria also cause significant food spoilage affecting
and vary in their sizes. Many are intracellular. grains, nuts, fruits, and vegetables. Some molds
The general life cycle consists of a stage in which produce mycotoxins, which may be carcino-
parasites are maintained as a cyst or oocyst (egg) genic, teratogenic, and/or retard growth of the
in the environment (feces, soil, water, produce) infected host (Table 2.4).
and can contaminate food products. Parasites can
be also transmitted through meat if the meat
animal harbors the parasite. Oocysts are then
Mold Structure
transformed into the larvae stage called trophozo-
ites, bradyzoites, or merozoites, which then Molds are multicellular microorganisms, and a
mature into adults. The larval stage is the most typical mold possesses hyphae, conidiophore –
infective stage. Parasites often require more than consisting of stalk, vesicle, sterigmata, and
one animal host, i.e., an intermediate host to conidia (spores) (Fig. 2.13). Often the identifica-
complete their life cycle. The parasites that com- tion of a mold species is done by examining the
plete their life cycle in a single host are known as morphological shape and size of the spores under
homoxenous (Example, Giardia lamblia, a light microscope. The cell wall of a mold
Trichinella spiralis). Parasites that require more (hypha) consists of a polymer of hexose chitin
than one host to complete their life cycle are and N-acetylglucosamine (NAG). Mold spores or
called heteroxenous (Toxoplasma gondii, Taenia hyphae may be allergenic to some humans, and
solium, Taenia saginata). Parasites do not prolif- mold hyphae may proliferate in damp environ-
ments such as in buildings and walk-in coolers
Table 2.3 Foodborne parasites
with high humidity and poor air circulation.
Group Genus/species
Protozoa Giardia lamblia
Entamoeba histolytica Table 2.4 Foodborne molds and mycotoxins
Cyclospora cayetanensis
Cryptosporidium parvum Mold Mycotoxin
Cystoisospora belli Aspergillus flavus Aflatoxin
Toxoplasma gondii Aspergillus parasiticus
Trypanosoma cruzi Aspergillus ochraceus Ochratoxin
Cestodes (tapeworms) Taenia saginata, T. solium Penicillium verrucosum Ochratoxin
Echinococcus spp. Aspergillus carbonarius Ochratoxin
Nematode Anisakis simplex Fusarium verticillioides Fumonisin
(roundworms) Ascaris lumbricoides Fusarium verticillioides Deoxynivalenol-DON
Trichinella spiralis Fusarium graminearum Zearalenone
Trematodes (flatworms Schistosoma mansoni Penicillium expansum Patulin
or flukes) Fasciola hepatica Claviceps purpurea Ergot (alkaloid)
Fig. 2.13 Schematic structure of molds: (a) Aspergillus species and (b) Penicillium species. Panels (c) and (d) are the
light microscopic photograph of Aspergillus niger (c) and Penicillium citrinum (d) (magnification 400×)
Mycotoxins optimal condition for mycotoxin production is at a

water activity (Aw) of 0.85–0.99 and a temperature
Toxigenic molds produce a variety of mycotoxins range of 10–30 °C. Some of the same mycotoxins
as secondary metabolites (Table 2.4), and the can be produced by different mold species.
Summary 41
The toxigenic molds are a major problem in n eurotoxic shellfish poisoning (NSP), ciguatera
agriculture commodities such as the grains, fish poisoning (CFP), azaspiracid shellfish poi-
cereals, nuts, and fruits. Mycotoxins produced in soning (AZP), and amnesic shellfish poisoning
these products can cause severe health problems (ASP). These toxins are neurotoxins and are
in both humans and animals. Mycotoxins are known as Na channel blockers. The symptoms
produced as secondary metabolites that are low vary from a tingling sensation in the lips, prostra-
molecular weight molecules. Mycotoxins are tion, and a reduced heart rate, and depending on
highly stable and are difficult to destroy by tradi- the severity of the disease, patients may suffer
tional food processing conditions. Some myco- from paralysis and succumb to death. In addition,
toxins emit fluorescence when exposed to the the scombroid toxin or histamine food poisoning
ultraviolet (UV) light; thus, UV can be used for is associated with scombroid fish such as tuna
screening of contaminated food/feedstuff. and mackerel due to the microbial metabolic
Mycotoxins can cause acute disease affecting action. First, microbial proteinases attack fish
the kidney and liver leading to chronic diseases proteins to release free histidine, which is then
such as liver cancer, birth defects, skin irritation, decarboxylated to form biogenic amines: hista-
neurotoxicity, and death. Three general mecha- mine, cadaverine, and saurine. These amines
nisms of mycotoxin action are described: muta- elicit a typical allergic response in humans.
genic, teratogenic, and carcinogenic. During the Details of the fish and shellfish toxins are dis-
mutagenic action, the mycotoxin (e.g., aflatoxin) cussed in Chap. 9.
binds to DNA, especially the liver mitochondrial
DNA resulting in point mutation or frameshift
mutation due to deletion, addition, or substitu- Summary
tion in DNA, and affects liver function (hence
hepatotoxic). The teratogenic action causes All organisms belong to one of the three domains
DNA breakage and leads to birth defects, while of life: Bacteria, Archaea, and Eukarya. As food-
the carcinogenic effect causes irreversible borne pathogens, general properties such as the
defects in cell physiology resulting in abnormal morphological and structural characteristics of
cell growth and metastasis. In recent years, the bacteria, viruses, molds, parasites, and algae are
importance of mycotoxins has been highlighted reviewed in this chapter. The cell walls of Gram-
for their potential use as a weapon for bioterror- positive bacteria contains a thick peptidoglycan
ism. Three major genera of molds, Aspergillus, (PGN), which is highly complex and dynamic
Fusarium, and Penicillium, are of significant containing a disaccharide N-acetyl-d-
interest in food safety for their production of glucosamine (NAG) and N-acetylmuramic acid
mycotoxins. Details of several key mycotoxins (NAM) and linked by β- 1, 4-glycosidic linkage
are presented in Chap. 8. (GlcNAc-(β1–4)-MurNAc) and pentapeptide.
The PGN not only protects the bacterial cells
against mechanical or physical damages but also
Fish and Shellfish Toxins hosts a numerous structural and functional pro-
teins for rigid exoskeleton and for functional
Fish and shellfish toxins are derived from single- attributes such as bacterial pathogenesis and
cell marine plant, algae (marine dinoflagellates), induction of host immune response. The Gram-
bacteria (Cyanobacteria), or microbial actions negative cell envelope consists of the outer mem-
originated from marine, brackish (a mixture of brane (OM) which carries LPS (an endotoxin), a
freshwater and seawater), and freshwater envi- thin PGN layer, and a cytoplasmic membrane.
ronments. In general, the seafood toxins are small Due to the presence of OM, the protein secretion
molecules and are heat-stable. Six classes of tox- system in Gram-negative bacteria is mediated by
ins are significant: paralytic shellfish poisoning several secretory pathways designated as tType
(PSP), diarrhetic shellfish poisoning (DSP), I–Type VI secretory pathways,. Oof which tType
III secretion system is known as the molecular teria: structural and mechanistic insights. Nat Rev
Microbiol 13, 343-359.
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directly to the interior of the host cell. Some bac- Int J Food Microbiol 103, 207.
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long-term survival strategy for the bacteria. (2009) Emerging food-borne parasites. Vet Parasitol
163, 196-206.
Most foodborne viruses cause severe gastro- 7. Eckburg, P.B., Lepp, P.W. and Relman, D.A. (2003)
enteritis and affect a large number of people Archaea and their potential role in human disease.
every year. Foodborne enteric viruses are shed in Infect Immun 71, 591-596.
large numbers (109 particles per gram) from 8. Feltcher, M.E. and Braunstein, M. (2012) Emerging
themes in SecA2-mediated protein export. Nat Rev
infected patients through feces or vomitus. Microbiol 10, 779-789.
Person-to-person or fecal–-oral transmission is a 9. Foster, S.J. (1994) The role and regulation of cell wall
common mechanism for the spread of such viral structural dynamics during differentiation of endo-
infection. Since viruses are highly infectious, spore-forming bacteria. J Appl Bacteriol 76, S25-S39.
10. Galdiero, S., Falanga, A., Cantisani, M., Tarallo, R.,
only a small dose of 10–100 particles is required Pepa, M.E.D., D’Oriano, V. and Galdiero, M. (2012)
to infect a person. Protozoan parasites are Microbe-Host Interactions: Structure and Role of
increasingly becoming a major concern due to Gram-Negative Bacterial Porins. Curr Protein &
their spread through fresh vegetables and fruits. Peptide Sci 13, 843-854.
11. Kalaitzis, J.A., Chau, R., Kohli, G.S., Murray, S.A.
Immunocompromised people are highly suscep- and Neilan, B.A. (2010) Biosynthesis of toxic natu-
tible to the intracellular protozoan parasites. rally-occurring seafood contaminants. Toxicon 56,
Water and soil tainted with feces generally serve 244-258.
as the major contamination sources, and the pres- 12. Keates, S., Hitti, Y.S., Upton, M. and Kelly, C.P. (1997)
Helicobacter pylori infection activates NF-kappa
ence of these pathogens indicatesing breaches in B in gastric epithelial cells. Gastroenterology 113,
the hygienic or sanitary practices during food 1099-1109.
production and harvest. Mycotoxins are small 13. Murphy, P.A., Hendrich, S., Landgren, C. and Bryant,
molecules produced as secondary metabolites by C.M. (2006) Food mycotoxins: An update. J Food Sci
71, R51-R65.
the toxigenic molds. Mycotoxins may exert car- 14. Navarre, W.W. and Schneewind, O. (1999) Surface
cinogenic, mutagenic, or teratogenic activities. Proteins of Gram-Positive Bacteria and Mechanisms
Seafood toxins are generally associated with fish of Their Targeting to the Cell Wall Envelope.
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15. Rodriguez-Lazaro, D., Cook, N., Ruggeri, F.M.,
algae or are due to the bacterial enzymatic activi- Sellwood, J., Nasser, A., Nascimento, M.S.,
ties on fish proteins. D’Agostino, M., Santos, R., Saiz, J.C., Rzezutka, A.,
Bosch, A., Girones, R., Carducci, A., Muscillo, M.,
Kovac, K., Diez-Valcarce, M., Vantarakis, A., von
Bonsdorff, C.H., Husman, A.M.D., Hernandez, M.
Further Readings and van der Poel, W.H.M. (2012) Virus hazards from
food, water and other contaminated environments.
1. Beveridge, T.J. (1999) Structures of Gram-negative FEMS Microbiol Rev 36, 786-814.
cell walls and their derived membrane vesicles. J 16. Schneewind, O. and Missiakas, D. (2014) Sec-
Bacteriol 181, 4725-4733. secretion and sortase-mediated anchoring of proteins
2. Boneca, I.G. (2005) The role of peptidoglycan in in Gram-positive bacteria. Biochem Biophys Acta -
pathogenesis. Curr Opin Microbiol 8, 46-53. Mol Cell Res 1843, 1687-1697.
3. Conlan, J.V., Sripa, B., Attwood, S. and Newton, P.N. 17. Setlow, P. (2014) Germination of spores of Bacillus
(2011) A review of parasitic zoonoses in a changing species: What we know and do not know. J Bacteriol
Southeast Asia. Vet Parasitol 182, 22-40. 196, 1297-1305.
4. Costa, T.R.D., Felisberto-Rodrigues, C., Meir, A., 18. Weidenmaier, C. and Peschel, A. (2008) Teichoic
Prevost, M.S., Redzej, A., Trokter, M. and Waksman, acids and related cell-wall glycopolymers in Gram-
G. (2015) Secretion systems in Gram-negative bac- positive physiology and host interactions. Nat Rev
Microbiol 6 (4), 276-287.
Host Defense Against Foodborne
Pathogens 3
The ability of the human body to protect

Introduction against microbial infections is continuously
evolving because of continued exposure to differ-
Interaction of pathogenic microbes with their ent microbes and their components. Ironically,
hosts leads to two consequences: a full-blown microbes are also evolving in the same manner so
disease or a subclinical or no infection at all. that they can adapt themselves to the host. The
Pathogen dominance results in either morbidity human body has a rich source of nutrients, miner-
or mortality, while a successful protective host als, vitamins, and trace elements that support the
response averts disease. The immune system growth of both pathogens and commensal
plays a key role in both of these outcomes. In microbes. Though the pathogens are the primary
1798, Edward Jenner (1749–1823), a British sci- disease-causing organisms, the commensals can
entist, first noticed that the milkmaids recovering also cause disease but only under a favorable
from cowpox (vaccinia virus infection) never condition; hence, these are referred to as opportu-
contracted smallpox. Later, he injected the con- nistic pathogens. In most situations, the immune
tent from a cowpox pustule (pox lesion) into an system restricts the spread of an infective agent;
8-year-old boy, who subsequently never devel- however, a breach in the immune system or an
oped smallpox after a challenge with the patho- overt response toward the pathogen results in the
gen. He termed this phenomenon as “vaccine,” a onset of clinical symptoms. Therefore, to under-
Latin word derived from “vaccinus” meaning stand the disease process, one has to have a
“from cow.” This work is regarded as the founda- deeper knowledge of the host immune system
tion of immunology. Later, Louis Pasteur (1822– and the underlying defense mechanism. The host
1895) developed vaccines against anthrax and immune system can be compared to “an impene-
rabies. In addition, the pioneering works of Paul trable fort” impervious to attack by enemies and
Ehrlich (1854–1915) and Emil von Behring that is guarded by the soldiers like immune cells,
(1854–1917) demonstrated the importance of armed with deadly weapons such as cytokines,
passive vaccination or acquired immunity in dis- toxins, antimicrobial peptides, complement pro-
ease defense. At the same time, Elie Metchnikoff teins, and antibodies to stay at an “alert” position
(1845–1916) discovered that the innate immunity to combat sudden or deliberate attack by unsus-
plays an important role against an infection. pected enemies as pathogens.
Collectively these early pioneering works laid the Foodborne pathogens enter the body primarily
foundation for our understanding of the modern- through oral route; therefore, the immune
day immunology and vaccinology. response in the gastrointestinal (GI) tract is

44 3 Host Defense Against Foodborne Pathogens
Fig. 3.1 Schematic drawing showing the gastrointestinal goblet cells (b) and (c). Transmission electron microscopy
tract, various immunologic organs, and tissues in human. images of mouse ileum showing goblet cells and mucus
(a) Light microscopy image of mouse ileal section (H&E secretion (Courtesy of Rishi Drolia and Arun Bhunia)
stained) showing villi, crypt, mucus, and mucus-secreting
c ritical in preventing localized or systemic infec-by subsequent exposure to invading agents. The
tion. Adult human GI tract is over 23 ft. long major site of action of a foodborne pathogen or
starting from the mouth to the anus, and the dif- their toxins is the gastrointestinal tract; therefore,
ferent parts, the mouth, esophagus, stomach, the onset of clinical symptoms such as nausea and
small intestine (duodenum, jejunum, and ileum), vomiting could start as early as 30 min to 1 h from
and large intestine (cecum, colon, and rectum), ingestion. The natural enteric defense is the most
each possess unique physiological and immuno- crucial against intoxication or infection. Enteric
logical defense barriers against enteric pathogens host defense is multifaceted, and several factors
(Fig. 3.1). Immunological defense is of two work in concert to achieve protection against a
types, innate and adaptive, which are described pathogen in the intestinal tract. These include
below (Table 3.1). intestinal epithelial barrier, mucus, complement
proteins, antimicrobial peptides (AMPs), resident
microbiota, and pattern recognition receptor of
Innate Immune Response pathogens or pathogen-associated molecular pat-
tern (PAMP) such as a toll-like receptor (TLR)
Innate immune response is naturally present in the and nucleotide-binding oligomerization domain
body and is the first line of defense against a (NOD) protein. PAMP, TLR, and NOD can initi-
pathogen. It is constitutive but not antigen- ate signaling events for enhanced phagocytosis to
specific. It can be activated but cannot be induced eliminate pathogens from the host.
Innate Immunity of Intestinal Tract 45
Table 3.1 The major differences between the innate and adaptive immunity
Characteristics Innate Adaptive
Specificity Lack specificity (fewer shared antigens) High (microbial and
nonmicrobial antigens)
Diversity Limited (narrow) Very large
Memory None Yes
Cellular/chemical Skin, mucosal epithelial, antimicrobials Lymphocytes in epithelia,
barriers secreted antibodies on the surface
Proteins Complement, PAMP, TLR, cytokines, antimicrobial peptides Antibodies, cytokines
Cells Phagocytes (macrophages, neutrophils, eosinophils, Lymphocytes
keratinocytes, Langerhans cells, dendritic cells), natural
killer (NK) cells
Action Immediate Slow to develop, takes at least
4–7 days
In the intestinal architecture, immediately kines. The adaptive immune response is slow to
below the mucus layer, an epithelial cell lining develop and typically requires 4–7 days. During
provides protection against luminal microbes/ foodborne pathogen infection, the adaptive
pathogens from entering into the host. The epi- immune response may have a significant role
thelial lining consists of four cell lineages: only against a limited set of pathogens that
absorptive epithelial cells, hormone-producing require a long incubation period and cause sys-
enteroendocrine cells, mucus-secreting goblet temic infection. These include Listeria monocy-
cells, and antimicrobial peptide-producing togenes, Shigella species, Salmonella enterica,
Paneth cells (Fig. 3.1). Lamina propria (LP) is Yersinia enterocolitica, hepatitis A virus,
located immediately beneath the epithelium, Toxoplasma gondii, Trichinella species, and
home to monocytes and macrophages, dendritic some additional viral and parasitic pathogens.
cells (DCs), and lymphocytes, which play an Adaptive immune response to some of the food-
important role in innate and adaptive immunity. borne bacterial pathogens may also lead to
DCs extend their appendages in between the indi- adverse outcomes such as reactive arthritis and
vidual epithelial cells and samples the luminal Guillain–Barré syndrome for life.
content to initiate an innate response. DC also
presents antigens to T and B lymphocytes for a
subsequent adaptive immune response. Peyer’s Innate Immunity of Intestinal Tract
patch and lymphoid follicles located in the sub-
mucosal layer are part of the adaptive immune The intestinal environment is a complex ecosys-
system and provide specific immune response tem and consists of gastrointestinal epithelium,
against pathogens in the intestine. immune cells, antimicrobial molecules, oxygen
deficiency, and resident microbes. Pathogens
overcome these barriers to cause infection.
Adaptive Immune Response Pathogens first adhere and colonize the gut and
then activate the signaling pathways to induce
Adaptive immunity is also referred to as acquired pathological effects. Pathogen colonization may
immune response. It is induced or stimulated by be transient or long-term. Some may also use
the pathogen, and the response is generally host cell cytoskeleton as a target to gain entry
pathogen-specific. The response increases in (see Chap. 4). The intestinal environment is also
magnitude with successive exposure to an anti- conducive for pathogen survival and growth.
gen, and the response is mediated by immune Roles of each innate or natural immune compo-
effector cells or molecules: cytotoxic T lympho- nent in protection against pathogens are dis-
cytes (CTL), antibodies, cytokines, and chemo- cussed below.
Skin The epithelial layer environment is moist,

warm, and suitable for bacterial colonization and
The skin is the first line of defense against many growth. Epithelial cells are tightly bonded to form
pathogens including the foodborne pathogens a barrier against pathogens or microbes in the
since food handlers may encounter pathogens intestine. They are highly selective in translocat-
during handling. Human skin is made up of epi- ing molecules across the membrane. This con-
dermis and dermis layers. The epidermis con- trasts to the endothelial cells, located in the blood
sists of stratum corneum, stratum lucidum, vessels, that are loosely bound and allow move-
stratum granulosum, stratum mucosum, and stra- ment of blood cells and bacterial cells. The junc-
tum germinativum. The dermis layer contains tion between two epithelial cells has four areas:
glands, adipose tissues, and lymphoid tissues. tight junction (TJ), adherens junction (AJ), des-
The skin has a slightly acidic pH (pH 5.0) and mosome (Des), and gap junction (Gap) (Fig. 3.2).
acts as a natural barrier for pathogen entry. The TJ consists of about 40 proteins including zona
epidermis is dry; thus, it prevents bacterial occludin (ZO-1, ZO-2, and ZO-3), membrane-
growth since bacteria require moisture for active associated guanylate kinase family, occludin,
replication. In addition, the resident microflora claudin, cingulin, and phosphoproteins. TJ pro-
on the skin prevents colonization of harmful bac- teins are regulated by classical signal transduction
teria. Lysozyme secreted from the sweat glands pathways such as heterotrimeric G proteins, Ca2+,
in the hair follicle has antibacterial effect by dis- protein kinase C, and raft-like membrane micro-
rupting the peptidoglycan architecture of bacte- domains. AJ consists predominantly of cadherin–
rial cell wall. Skin-associated lymphoid tissue catenin complex (E-cadherin, α-catenin, and
(SALT) is located in the dermis and provides β-catenin), vinculin, actin, and so forth.
specific immune response against infective Desmosome architecture is shaped by desmopla-
agents. Several phagocytic and antigen-present- kin, plakophilin and plakoglobin, desmoglein,
ing cells (APC), Langerhans cells (localized and desmocollin. TJ and AJ maintain cell polar-
phagocytic cells), dendritic cells, and keratino- ization; however, certain pathogens or toxins such
cytes, are also located in the skin and may help as Clostridium perfringens enterotoxin (CPE) and
eliminate the pathogen. The major APC of the Vibrio cholerae ZOT toxin target TJ domain to
epidermis are keratinocytes, which maintain the modulate intestinal epithelial permeability.
acidic environment, produce cytokines, and Epithelial cells also carry several receptor
ingest and kill skin-associated bacteria such as molecules for interaction with microorganisms.
Pseudomonas aeruginosa and Staphylococcus Those are called (1) pattern recognition receptor
aureus. (PRR), (2) toll-like receptor (TLR), (3)
nucleotide- binding oligomerization domain
(NOD) protein, and (4) pathogen-associated
Mucus Membrane molecular pattern (PAMP), which bind to bacte-
rial lipopolysaccharide (LPS), peptidoglycan
The mucus membrane (Fig. 3.1) forms the inner (PGN), and lipoteichoic acid (LTA).
layer of the gastrointestinal tract, respiratory Paneth cells located in the crypt, or folded
tract, and urogenital tract. Mucus membranes area, of the mucosal layer are phagocytic cells and
consist of epithelial cells, and depending on the produce lysozyme and cryptdin, a 35-amino acid-
location, the shape and organization of the cells long antimicrobial peptide. Cryptdin is inhibitory
may vary. Cells may appear as stratified, squa- to Listeria monocytogenes, Escherichia coli,
mous, cuboidal, or columnar. There are four epi- Salmonella enterica, and other pathogens. Goblet
thelial cell lineages in the gastrointestinal tract: cells are located in between the epithelial cells.
epithelial cell, Paneth cells, goblet cell, and They secrete mucus and play a major role in the
enteroendocrine cell. intestinal natural immunity as discussed below.
Fig. 3.2 Schematics of intestinal villi, epithelial cell lining, and cell junctions (TEM image; Courtesy of Rishi Drolia
and Arun Bhunia)
Goblet Cells and Mucus increases progressively distally along the

intestine. Mucus is produced by the goblet cells,
Mucus is an important natural protective barrier which are derived from the stem cells located in
against microbial colonization and infection. The the crypt. As they mature, they ascend upward to
thickness of mucus layer varies throughout the the villus (Figs. 3.1 and 3.2). They are polarized
gut. The stomach and colon have two layers of cells containing apical and basolateral domains.
mucus while the remaining gut has one layer. Brush border (BB), also known as microvillus, is
Mucus thickness is greater in the large intestine located on the apical side of the goblet cells. BB
than in the small intestine, and the thickness consists of actin filaments and actin-bundling
proteins (villin and fimbrin), and it is responsible exocytosis, and (2) delayed release, i.e., mucin is
for vesicle trafficking. Mucin, after synthesis, is first stored in a large vesicle after synthesis and
packaged in the secretory granule and is released then is released. The release is regulated by spe-
into the lumen after 5–7 days during goblet cell cific stimuli involving activation of signaling
exfoliation (death). Goblet cell proliferation and pathways. The activators may include acetylcho-
differentiation is regulated by Wnt and Hedgehog line, vasoactive peptides, neurotensin, IL-1, nitric
signaling pathways. Exfoliation occurs near the oxide (NO), and cholera toxin. In contrast,
tip of the villi, which is initiated by a cell death Clostridium difficile toxin A downregulates exo-
program called “anoikis.” Anoikis is mediated by cytosis thus favoring bacterial colonization and
focal adhesion kinase or β1-integrin-related survival in the intestine.
events such as protein kinase signaling pathways: Mucin binds small molecules and proteins.
PI-3-kinase/Akt, mitogen-activated protein The complex architecture of mucin performs sev-
kinase, stress-activated protein kinase/Jun amino- eral functions: (i) binds water (hygroscopic), (ii)
terminal kinase, and Bcl-2. Goblet cell function selective transport of molecules toward the mem-
and the composition of mucus are affected by brane due to gel-filtration effect, (iii) ion-
numerous factors including changes in the resi- exchange molecule, and (iv) sequestration
dent microbial flora, the presence of harmful function due to its ability to serve as a receptor
enteric pathogens, and nutritional deficiencies. for selectins, lectins, adhesion molecules, and
Mucus consists of mucin protein and polysac- microorganisms.
charide. The polysaccharide makes mucus sticky. Mucus acts as a barrier network for patho-
Mucin is a glycoprotein. The core protein moiety, gens. Mucus contains sIgA, lactoferrin, lyso-
apomucin, is cross-linked with carbohydrate zyme, and lactoperoxidase. Lactoferrin binds
chains or O-linked oligosaccharides (O-glycans) iron intimately, thus sequestering it from bacte-
that are attached to the serine and threonine resi- rial utilization. Lysozyme breaks down peptido-
dues by a glycoside bond. Mucin forms a high- glycan of bacterial cell wall, and lactoperoxidase
order structure through polymerization of several produces superoxide “O” radical, which is toxic
molecules resulting in a large molecule with to bacteria. Ciliated cells in the mucus mem-
molecular weights in millions of daltons. There brane also propel or sweep away invading patho-
are three subfamilies of mucin: gel forming, sol- gens. Mucus gel is a source of energy and
uble, and membrane bound. Eighteen genes nutrients for resident microflora and promotes
encode for mucin (MUC) synthesis, and the microbial colonization. Mucus also protects the
mucins are designated as MUC1, MUC2, epithelial cells against inflammation and injury
MUC3A, MUC3B, MUC4, MUC5B, MUC5AC, from pathogens or toxins. For example, mucin
MUC6–MUC13, and MUC15–MUC17. Among inhibits the adhesion of enteropathogenic E. coli
them, the MUC2, MUC5AC, MUC5B, and (EPEC), Yersinia enterocolitica, Shigella, and
MUC6 exist in the secreted form and assemble rotavirus. However, some pathogens regulate
via interchain disulfide bridges. Mucins on the mucin secretion and favor bacterial colonization.
membrane are found either as membrane-bound For example, Helicobacter pylori colonizes
or the secreted form. Membrane-bound mucin is mucus and reduces mucin exocytosis. L. mono-
highly adherent and remains associated tightly cytogenes induces exocytosis of mucin through
with the membrane, while the secreted form is listeriolysin O (LLO) and inhibits bacterial
loosely attached. The secreted form remains entry. Probiotic b acteria such as Lactobacillus
associated with the membrane-bound mucus by spp. induce mucin production and prevent patho-
forming covalent and noncovalent bonds. gen attachment. Mucus also protects and lubri-
Mucin secretion occurs by two processes: (1) cates epithelial surface. It helps in fetal
immediate exocytosis, i.e., the mucin is produced development, epithelial renewal, carcinogenesis,
and is released immediately from the cells by and metastasis.
In the mucus membrane, M (microfold) cells Antimicrobial Peptides

located in the intestine as part of the gut-
associated lymphoid tissue (GALT) or Peyer’s Antimicrobial peptides (AMPs) are produced by
patches deliver pathogens to the local lymphoid many vertebrates including fish, frogs, and mam-
tissue by transporting them across the mucosa mals. AMPs have broad-spectrum antimicrobial
(Fig. 3.3). M cell is naturally phagocytic and activity and are associated with the airways, gin-
serves as the gateway for translocation of intra- gival epithelia, and corneas. AMPs are also pres-
cellular bacteria such as L. monocytogenes, ent in the reproductive, the urinary, and the GI
Salmonella, Yersinia, and Shigella across the epi- tract. They play a major role in innate immunity
thelial barrier. M cells constitute only less than and assist in pathogen elimination. AMPs are
0.1% of the epithelial cells in the lining of the small peptides of 20–40 amino acids long and
intestine and are present in the follicle-associated have α-helix and β-sheets. Intrachain disulfide
solitary lymphoid structure throughout the intes- bonds formed between cysteine residues stabilize
tine. Mucus-associated lymphoid tissue (MALT) the structure. AMPs are produced as inactive pre-
is synonymous to GALT and is a secondary lym- propeptides and are activated after removal of the
phoid tissue, which responds selectively to the N-terminal signal sequence. Their antimicrobial
pathogen. activity is nonspecific. Peptides are inserted into
Fig. 3.3 Specific immunity in the mucosal surface. and present them to the T cells for the production of anti-
Bacteria are engulfed by M (microfold) cells and translo- body (sIgA) by B cells. sIgA (secretory IgA) travels
cated across the mucosal barrier. Resident macrophages through the epithelial cells and is secreted into the lumen
or dendritic cells can engulf the bacteria, process antigens,
the membrane to form pores by “barrel-stave,” was thought to recognize peptidoglycan, but it
“carpet-like,” or “toroidal pore” mechanism. was shown that it interacts with lipoprotein or
They induce leakage of ions from cells and cause lipoteichoic acid often present in the peptidogly-
the collapse of the proton-motive force (PMF). In can preparations as a contaminant. Peptidoglycan
addition, the peptides may affect the cytoplasmic interacts with intracellular NOD-1 and NOD-2
membrane, septum, cell wall, nucleic acid and receptors in mammalian cells. TLR-3 recognizes
protein, and enzyme activity in microbes. They viral dsRNA; TLR-7 and TLR-8 recognize
are effective against bacteria, and enveloped single-stranded viral RNA and small synthetic
viruses, but not against the nonenveloped viruses. compounds and TLR-9 bacterial DNA. Following
Pathogens may develop resistance by expelling recognition of the microbes by TLR on macro-
AMPs by energy-dependent pumps, altering phage or other immune cells, a cascade of signal-
membrane fluidity, and cleaving the AMPs with ing pathways is activated to allow transcription of
proteases. proinflammatory immune response genes for
AMPs are produced by epithelial cells, Paneth IL-1, TNF, TGF-β, IL-6, IL-8, IL-12, IL-17,
cells, neutrophils, and macrophages. Examples IL-18, and a variety of other chemokines. The
of AMPs are lysozyme, phospholipase A2, transcription factor NF-κB regulates the expres-
α1-antitrypsin, defensin, cryptdin, angiogenin 4, sion of these cytokines. These cytokines eventu-
and cathelicidins. Human β-defensins 1, 2, and 3 ally help eliminate the microbes through lysis of
are cationic and are cysteine-rich peptides of target cells or by inducing an adaptive immune
3.5–4 kDa and are produced by intestinal epithe- response. For example, IL-1 and TNF activate T
lial cells. Human α-defensins 5 and 6 are pro- cells, IL-6 activates B cells, and IL-12 activates
duced by the Paneth cells. NK cells to provide protective immunity.
PML polymorphonuclear leukocytes, DC den-
dritic cells, NK natural killer cells, NOD
Toll-like Receptor nucleotide-binding oligomerization domain pro-
tein, dsDNA double-stranded DNA, ssRNA
Toll-like receptors (TLRs) are transmembrane single-stranded RNA.
proteins known as signaling molecules and were
originally discovered in Drosophila (fruit fly).
These evolutionarily conserved pattern recogni- NOD Proteins
tion molecules are typically expressed in intesti-
nal epithelial cells and immunocompetent cells in Nucleotide-binding oligomerization domain
the lamina propria. They are present on the cell (NOD) proteins are also another class of PAMP-
surface and in endosomal membranes (Fig. 3.4). recognizing molecules that play an important
Several TLRs are now identified, and they play a role during innate immune response. The
major role in innate immunity through activation nucleotide-binding domain leucine-rich repeat-
of NF-κB, cytokine production, and recruitment containing receptors (NLRs) serve as innate
of inflammatory cells. For example, TLRs are immune receptors. The first identified NLRs are
present in macrophages and are known to form a NOD-1 and NOD-2. NOD-1 expressed in intesti-
“microbial recognition” system. Toll-like mole- nal epithelial cells binds to peptidoglycan (DAP,
cules are analogous to the IL-1 receptor. Different diaminopimelic acid) of the invasive Gram-
TLRs are activated by different types of micro- negative pathogen and other commensal bacteria.
bial factors (Table 3.2). For example, TLR-1 rec- NOD-1 is thought to be highly active in intestinal
ognizes bacterial lipoprotein and unconventional cells in Crohn’s disease patients and is responsi-
LPS; TLR-4 is activated by bacterial lipoarabino- ble for an uncontrolled inflammatory response.
mannan (LAM), bacterial lipoprotein (BLP), and NOD-2 is highly expressed in the Paneth cells
yeast cell wall particles (zymosan). TLR-4/ and monocytes, and it recognizes muramyl
TLR-6 are activated by LPS and LTA, TLR-5 by dipeptide (MDP) of PGN of both Gram-positive
bacterial flagella, and TLR-11 by profilin. TLR-2 and Gram-negative bacteria. NOD-2 promotes
Fig. 3.4 Schematic of

toll-like receptor (TLR)
location in a cell and
their corresponding
ligands. DAP,
diaminopimelic acid;
MDP, muramyl
dipeptide; PGN,
peptidoglycan; LPS,
lipopolysaccharide;
NOD, nuclear
oligomerization domain;
LAM,
lipoarabinomannan
Table 3.2 Toll-like receptors (TLRs) and the corresponding ligands

Receptor Cell type Ligand
TLR1 Ubiquitous Bacterial (triacyl) lipoproteins
TLR2 PML, DC, monocytes Lipoteichoic acid from G(+) bacteria
TLR3 DC, NK dsRNA
TLR4 Macrophages, DC, endothelial cell LPS
TLR5 Monocytes, immature DC, epithelial cells, NK, Flagellin
and T cells
TLR6 High in B cells, lower in monocytes and NK cells Bacterial (diacyl) lipoproteins
TLR7 B cells, DC ssRNA recognition on endosomes (mouse)
TLR8 Monocytes, low in NK and T cells ssRNA recognition on endosomes (human)
TLR9 DC, monocyte, macrophages, PML, NK, CpG (unmethylated DNA)
microglial cells
NOD-1 DC, macrophages, epithelial cells Diaminopimelic acid (DAP) of peptidoglycan from
Gram-negative bacteria
NOD-2 DC, monocytes, macrophages, paneth cells, Muramyl dipeptide from peptidoglycan of both
epithelial cells Gram + and Gram- bacteria
Paneth cell activation for increased cryptdin pro- birth, and eventually, every surface exposed to
duction, while DC and macrophage activation the external environment becomes inhabited by
result in proinflammatory cytokine production commensal bacteria. Microbes are aerobic, facul-
and augmented bacterial killing. tative anaerobic, and anaerobic. In the GI tract,
the facultative anaerobes are associated close to
the epithelial layer for oxygen. The proportion of
Resident Microbiota anaerobic bacteria increases from proximal to
the distal part of the intestine. Contents of the
All mammals are born without any microorgan- stomach and duodenum contain approximately
ism in their system; however, they begin to 101–103 cfu g−1, of the proximal small bowel 104–
acquire microbes during and immediately after 108 cfu g−1, of the terminal ileum to colon 1010
Table 3.3 The major bacteria phyla in the gut

Firmicutes Proteobacteria Bacteroidetes
Approx. 255 genera; 2475 species Approx. 366 genera; 1644 species Approx. 20 genera; 130 species
Lactobacillus Enterobacteriaceae family: Bacteroides genus (major)
Bacillus Escherichia
Bifidobacterium Salmonella
Clostridium Shigella
Streptomyces Proteus
Mycoplasma Enterobacter
Klebsiella
Serratia
Non-Enterobacteriaceae family
Vibrio and nitrogen-fixing bacteria
cfu g−1, and of the colon to rectum 1013 cfu g−1. epithelial cells secrete IL-1 and IL-6, DCs
The major phyla present in the gut are Firmicutes, secrete IL-12 and IL-23, Th1 and intraepithelial
Proteobacteria, and Bacteroidetes (Table 3.3). lymphocyte cells (IEC) release IFN-γ, Th17
The Firmicutes represent approximately 255 secretes IL-17A, macrophages produce TNF-α
genera and 2475 species, and the major genera and IL-12, and B cells secrete commensal-spe-
include Lactobacillus, Bacillus, Clostridium, cific IgG. Epithelial barrier function is also com-
Streptomyces, and Mycoplasma. The promised during dysbiosis. While the pathogens
Proteobacteria represent approximately 366 induce a proinflammatory response by activating
genera and 1644 species, and the major genera NF-κB, the natural microbiota (example,
belong to the Enterobacteriaceae (EB) family Lactobacillus spp.) suppress unnecessary inflam-
comprising Escherichia, Salmonella, Shigella, matory response and help maintain immune
Proteus, Enterobacter, Klebsiella, and Serratia homeostasis and prevent epithelial barrier
and non-EB such as Vibrio and nitrogen-fixing dysfunction.
bacteria. Phylum Bacteroidetes comprises Microflora or their components such as LPS,
approximately 20 genera and 130 species, and PGN, and FMLP (formylated chemotactic oligo-
Bacteroides is the major genus related to human peptide) translocate actively through the mucosal
gut microbiota. barrier (mucus and epithelium) and are found in
Interestingly, the host requires the coloniza- the lamina propria. There they activate macro-
tion by commensal microbiota for its develop- phages, dendritic cells, neutrophils, NK cells,
ment and maintenance of health. Indigenous and T cells (Fig. 3.3). Microflora together with
(autochthonous) bacteria provide essential nutri- the immune system regulate intestinal motility,
ents and help in the metabolism of indigestible secretion, proliferation, villous length, and the
compounds by synthesizing necessary enzymes. crypt depth. Furthermore, the amount of IgA pro-
Microbiota also influence the release of biologi- duced is directly dependent on the number of gut
cally active gastrointestinal peptides, regulate flora. For example, germ-free animals have low
intestinal endocrine cells, and contribute toward levels of plasma cells, IgA, and a decreased num-
the development of intestinal architecture. They ber of immune cells. Overall, the natural micro-
also influence nutrient absorption, mucosal bar- flora help in the development of the immune
rier fortification, xenobiotic metabolism, angio- system. Ironically, in some individuals, the
genesis, and postnatal intestinal maturation. immune response against natural microflora may
They also prevent colonization by opportunistic lead to the onset of inflammatory bowel’s disease
pathogens and participate in innate and the adap- (IBD): Crohn’s disease (CD) and ulcerative coli-
tive immune response. The imbalance between tis (UC).
microbiota and the pathogens may lead to Natural microflora in the gut act as probiotics
dysbiosis or a disease state. During dysbiosis, and prevent invasion of enteric pathogens. For
Immune Response in the Gastrointestinal Tract 53
Table 3.4 Role of intestinal flora in intestinal function ther Components of Innate
O
and immune response
Immunity
Influence the release of biologically active peptides,
regulate intestinal endocrine cells and epithelial
structure
Transferrin is a glycoprotein abundant in the liver
Nutrient absorption, mucosal barrier fortification, that can prevent bacterial growth by sequestering
xenobiotic metabolism, angiogenesis, postnatal iron. Complement proteins, especially those
intestinal maturation activated in the alternative pathway by bacterial
Participate in innate and adaptive immune system LPS or LTA or by viral proteins, are present in
Pathogens induce proinflammatory response by the blood circulation (see subsection on
activating NF-κB and by production of IL-1β, TGF-β,
TNF-α, IL-6, IL-8, IL-12, IL-17, IL-18, IL-23, and Complement). The complement activation prod-
other chemokines uct “C3b” acts as an opsonin and facilitates
Natural microbiota (e.g., Lactobacillus spp.) suppress enhanced phagocytosis by macrophages, and the
unnecessary inflammatory response – help maintain “membrane attack complex” (MAC) directly
immune homeostasis
destroys microbes by exerting in the cytoplasmic
Microflora or components, i.e., LPS, PGN, and FMLP,
translocate actively through mucosal barrier (mucus membrane by forming pores. Macrophages, neu-
and epithelium) and found in lamina propria trophils, and NK cells also eliminate pathogens
Activate macrophages, dendritic cells, neutrophils, nonspecifically during the early phase of infec-
NK, T cells tion. Macrophage-derived cytokines, α and β
Microflora plus immune system regulate intestinal interferons (IFN), and TNF are important during
motility, secretion, proliferation, villous length, and
crypt depth innate immune response. Interferons inhibit viral
Amount of IgA produced is directly dependent on the replication, and TNF initiates inflammation to
gut flora protect against bacteria-induced damage.
Microbiota activate plasma cells to produce IgA
Decreased diversity of flora leads to decreased number
and activation of immune cells
Immune Response
Bottom line: natural microflora help develop immune
system
in the Gastrointestinal Tract
Mouth/Throat/Respiratory Tract
example, microcin produced by E. coli prevents Saliva in the mouth contains lysozyme, lactofer-
invasion of Salmonella, Shigella, and Listeria. rin, and sIgA and provides protection against
Lactobacillus and Bifidobacterium, which remain pathogens. Resident bacteria compete for
stable throughout life, can inhibit Salmonella nutrients and the colonization sites with invading
invasion. Lactobacillus may reduce the pathogen- pathogens. Ciliated epithelial cells sweep away
induced loss of epithelial integrity. Bacteriocins pathogens, and the mucus ball helps remove
produced by some natural flora can kill certain pathogens. Epithelial cells are sloughed off peri-
pathogens in the gut. In summary, the AMPs and odically to dispose of colonized bacterial cells on
the microbiota together provide frontline defense them as a strategy to eliminate pathogens. Large
by inducing mucus secretion and activating particles are sometimes expelled by coughing.
immune response thereby protecting the host Alveolar macrophages in the respiratory tract or
against invading pathogens (Table 3.4). nasopharynx also remove pathogens.
Gut microbiota also play an important role
outside the gut. A balanced gut microbiota is
essential for brain development and psychologi- Stomach
cal well-being and prevents the onset of many
chronic diseases and disorders including obesity, Gastric juice has a low pH (2–2.5) due to the
type I diabetes, autoimmune disease, allergy and secretion of HCl from parietal cells, and most
asthma, autism, atherosclerosis, and cancer. foodborne pathogens are inhibited by acid. Food
can neutralize the pH of stomach juice to pH 4– is highly adherent and rich in sIgA and AMPs,
pH 5; thus, acid-resistant or acid-tolerant patho- while the loosely attached outer layer carries resi-
gens are unaffected and can pass through the dent microbes. The large intestine is home to an
stomach to the small intestine where the pH is abundant resident microflora (1010–14 cfu g−1), 97%
about 6.8. In addition, liquid food like milk, soup, of which are anaerobic. The microbial genera pres-
and beverages can rapidly transfer bacteria ent in the large intestine include Bifidobacterium,
through the stomach thus preventing longer expo- Lactobacillus, Escherichia, Clostridium,
sure to acid. Psychosomatic disease or physiolog- Bacteroides, Streptococcus, Peptostreptococcus,
ical abnormalities, such as achlorhydria, may also Ruminococcus, Fusobacterium, Pseudomonas,
raise the stomach pH making the individuals vul- Veillonella, Proteus, yeast, and protozoa. These
nerable to a foodborne infection. The stomach microbes prevent colonization of pathogens in the
also secretes some proteolytic enzymes that can gut by physical hindrance and by producing meta-
destroy pathogens. Natural microflora such as bolic by-products such as short-chain fatty acids
Lactobacillus, Streptococcus, and some yeast are (SCFA: formic acid, acetic acid, butyric acid, pro-
present at 101–103 cfu ml−1 in the stomach and pionic acids, and valeric acid) and bacteriocins that
may contribute toward the innate immunity. are inhibitory to the transient pathogens. Epithelial
cell sloughing also occurs in the colon and, together
with mucus, can prevent colonization and attach-
Small Intestine ment of pathogens.
The small intestine has three regions: duodenum,

jejunum, and ileum. In the small intestine, bile Adaptive Immunity
salts (sodium taurocholate and sodium glyco-
cholate) are secreted from the gall bladder, and Adaptive immunity is characterized by specific
their detergent-like action helps destroy the cell response to a particular antigen resulting in the
walls of Gram-positive bacteria. There is a fast production of specific antibody or cytokines. The
flow of mucus and cell sloughing, and peristaltic most important characteristics of adaptive
movement of the intestine may eliminate patho- immune response are that it remembers each
gens from the gut. As mentioned earlier, the encounter, i.e., a memory response, and that the
Paneth cells located in the crypt of the mucosal responses are amplified with each successive
layer (Fig. 3.2) in the small intestine also provide encounter with the pathogen (Fig. 3.5). Specific
protection against enteric pathogens. These cells immunity can be active or passive based on the
possess phagocytic activity and produce lyso- form of immune response it generates. Active
zyme and cryptdin that inhibit foodborne patho- immunity is developed when the host immune
gens. GALT also removes bacteria by inducing a system is stimulated by an antigen. For example,
specific immune response. Natural microbiota immunization with the tetanus toxoid, an inactive
present in the jejunum and ileum are in the range form of tetanus toxin, develops an antibody
of 104–108 cfu ml−1. The major genera present response that is capable of neutralizing tetanus
in this part of intestine are Lactobacillus, toxin in the subsequent exposure. Passive immu-
Enterobacter, Streptococcus, Bacteroides, nity is achieved when the serum or cells from a
Bifidobacterium, and Fusobacterium; they pro- previously immunized host are introduced to a
vide protection against pathogen colonization. recipient to confer immediate protection against
the pathogen. Examples of passive immunity are
anti-botulinum serum against botulism and anti-
Large Intestine venom serum given after a snakebite.
Adaptive immunity is also classified based on
The large intestine has three segments: cecum, the components of the immune system that pro-
colon, and rectum. The epithelial lining is covered vide the response, i.e., humoral or cell mediated.
with inner and outer mucus layers. The inner layer Humoral immunity involves the production of
Adaptive Immunity 55
Fig. 3.5 A graph

showing the primary and
secondary immune
response against
hypothetical antigens A
and B
antigen-specific antibodies by B lymphocytes. 3. Specific immune response remembers the first

Generally, extracellular antigens such as protein encounter of an antigen, and the response is
toxins or soluble protein antigens induce humoral enhanced during the subsequent exposure due
immunity. Cell-mediated immunity (CMI) to the presence of memory cells, which sur-
involves activation of T-lymphocyte subsets that vive for prolonged period even in the absence
are involved in the elimination of pathogens. of an antigen. Memory cells respond to a very
Generally, intracellular pathogens such as bacte- low concentration of antigen with increased
ria, viruses, or parasites induce CMI. immune response and with higher affinity.
Memory response is larger, more rapid, and
qualitatively different from the first.
Characteristics of Adaptive Immune 4. The specific immune response is self-
Response regulated, and it does not produce antibodies
or cytokines in the absence of an antigen.
The adaptive (specific) immune response is Lymphocytes perform the initial response
highly specific, diverse, and self-regulated, function for a brief period and then convert to
remembers the first antigen encounter (memory the memory cell. Feedback mechanisms also
response), and is able to discriminate self- control the immune response with the help of
antigens from nonself. Properties of specific a blocking antibody or increased concentra-
immune response are: tion of cytokines. Antigen–antibody complex
also regulates T-cell response and alter cyto-
1. The specific immune response is generally kine cascades to control the immune response.
raised against structural components of com- 5. The specific immune response can discrimi-
plex proteins or polysaccharides. Antigenic nate self from nonself. It is an important fea-
determinants or epitope-specific lymphocyte ture of the specific immune response to be able
clones are selected during an immune to distinguish self-antigens from a foreign
response, and these clones continue to pro- antigen. T or B cells that are stimulated by
duce specific antibodies or cytokines during self-antigen are generally eliminated from the
their life spans. body. Self-antigens are also known as tolero-
2. The specific immune response can respond to gens since no immune response is developed
a large number (~10 ) of antigenic determi-
9
against them. Immunologic unresponsiveness
nants due to the broad “lymphocyte reper- is called “anergy.” Foreign antigens may act as
toire” in a host. tolerogens or immunogens depending on the
Fig. 3.6 Phases of

immune response
consist of activation,
proliferation, and
differentiation into
antibody-secreting
plasma cells or memory
cells. Similar events also
occur for T
lymphocytes. Activation
of a clone is
antigen-specific
physicochemical state of the antigen, the dos- where membrane-bound IgM and IgD serve as
age, and the route of administration. An anti- receptors. In the activation phase, the clones are
gen administered orally or intravenously may activated and enlarged and begin to proliferate
not elicit a response; however, if the same anti- and differentiate into the effector and the mem-
gen is administered subcutaneously or intra- ory cells. The effector cells produce antibodies or
peritoneally, it may induce an immune cytokines to initiate effector functions through
response. If an immune response is generated complement activation or activation of phago-
against a self-antigen, it leads to autoimmune cytes to neutralize antigens, while the memory
disease. For example, systemic lupus erythe- cells remain dormant, circulate in the body, and
matosus (SLE) is a condition where the anti- are involved in the immune surveillance. When
body is produced against cellular nucleic acids the memory cells encounter an antigen, they con-
resulting in glomerular nephritis and vasculi- vert into effector cells and respond to the antigen
tis. In myasthenia gravis, the antibody is devel- with vigor. Memory cells can survive for a pro-
oped against the acetylcholine receptor and longed period depending on the antigen type and
thus interferes with the transmission of nerve the frequency of exposure. The most effective
impulses causing muscular weakness. vaccine against an infectious agent is the one that
is capable of generating a robust long-lasting
memory cell response.
Phases of Immune Response
The introduction of an antigen initiates a cascade Tissues and Cells of Immune System
of events leading to the immunologic response
and the elimination of the pathogen from a host Tissues
(Fig. 3.6). Each antigen has the capacity to acti-
vate separate existing clones, and each clone can The primary lymphoid organs are bone marrow
proliferate to produce a specific effector response. and the thymus in mammals. In avian species, the
In the cognitive phase, initially an antigen binds bursa of Fabricius, a specialized gland located in
to the membrane receptor of resting lymphocyte cloaca, is equivalent to the mammalian bone mar-
Tissues and Cells of Immune System 57
row. The secondary lymphoid organs are lymph Lymph Node

nodes, spleen, and lymphoid tissues such as gut-
associated lymphoid tissue (GALT, Peyer’s Lymph nodes are secondary lymphoid tissue and
patch) (Figs. 3.1 and 3.3), mucus-associated lym- are part of the lymphatic system in mammals but
phoid tissue (MALT), and skin-associated lym- are absent in birds. An adult human body may
phoid tissue (SALT). have 400–450 lymph nodes distributed through-
out the body. Examples are cervical lymph nodes
(neck area), axillary lymph nodes (armpit area),
Bone Marrow bronchial lymph nodes (bronchus), mesenteric
lymph nodes (intestinal tract associated), inguinal
Bone marrow contains stem cells which are lymph nodes (groin area), and so forth. For food-
responsible for hematopoiesis, i.e., all blood cells borne diseases, mesenteric lymph nodes (MLN)
are originated from these stem cells. Bone mar- play an important role during infection, and they
row also serves as the site for B-cell growth and serve as a firewall to prevent live organisms from
the early phase of maturation. Growth factors or the systemic spread. In humans, there are about
colony-stimulating factors for B cells such as 100–150 MLN distributed in the mesentery.
granulocyte–monocyte colony-stimulating factor Structurally, a lymph node has three regions: an
(GM-CSF) and for macrophages such as granulo- outer cortex, paracortex, and inner medulla. The
cyte–colony-stimulating factor (G-CSF), mono- cortex has lymphoid follicles containing a distinct
cyte colony-stimulating factor (M-CSF), IL-1, germinal center in each follicle. Germinal centers,
IL-2, and IL-7 are produced in the bone marrow. also called B-cell zones, are where B cells prolif-
In birds, the bursa is the site for B-cell growth erate and differentiate into the antibody-secreting
and maturation. plasma cells. In the cortex, macrophages, den-
dritic cells, and interdigitating reticular cells are
localized and can process and present antigens to
Thymus B cells. The paracortex has the T-cell zone and is
primarily occupied by the helper T cells (Th or
The thymus is located adjacent to the larynx in CD4+). Cytotoxic T cells (TC or CD8+) are also
the upper part of the thoracic cavity and provides found in this area. The medulla is home to the
the microenvironment for T-lymphocyte matura- matured antibody-secreting B cells (plasma cells),
tion and growth. It is a bilobed organ containing Th cells, macrophages, and dendritic cells.
multiple lobules. The cortex of the lobule is the
resident site for thymocytes (T lymphocytes) that
are found at various stages of their maturation. Spleen
Immature T cells do not carry any antigen recep-
tors. Mature T cells migrate from the cortex to The spleen is a secondary lymphoid organ and is
medulla and encounter “self-antigen” for the first the largest in the lymphatic system, present in
time. Depending on the type of antigens they virtually all vertebrates. It is located in the left
encounter, these T cells differentiate into either upper quadrant just above the stomach. The
CD4+ or CD8+ T cells and express co-receptor spleen is brownish in color. It filters blood and
molecules, CD4 or CD8, respectively. In the helps remove dead blood cells from the circula-
medulla, the epithelioid cells, also known as tory system. It has two regions of interest: white
giant macrophages, and the resident macrophages pulp and red pulp. White pulp has periarteriolar
present “self-antigens” to the T cells. During lymphoid sheaths that contain lymphoid follicles.
development in the thymus, only T cells that do Lymphoid follicles contain germinal centers
not react to “self-antigens” are allowed to mature which are home to B cells. White pulp also
and enter the blood circulation. contains CD4+ and CD8+ T cells. The red pulp
Fig. 3.7 Diagram

showing the origin of
various immune cells
from the stem cells
contains macrophages, dendritic cells, and the myeloid lineage and the lymphoid lineage
plasma cells, and the antigen-specific interaction (Fig. 3.7). Cells originated from the myeloid lin-
takes place on this site. eage are granulocytes; neutrophil, basophil,
eosinophil, and phagocytes; monocyte, and mac-
rophage. The lymphoid lineage comprises T lym-
Lymphoid Tissue phocytes, B lymphocytes, and natural killer (NK)
and natural killer T (NKT) cells.
The mucosal immune system is a regional lym-
phoid tissue such as gut-associated lymphoid tis-
sue or Peyer’s patch or solitary intestinal Monocytes and Macrophages
lymphoid tissue (SILT). It contains “M” cells that
engulf bacteria and transport them to the subepi- Monocytes are originated from the bone marrow
thelial region in the lamina propria. Localized and then migrate to blood circulation. Matured
dendritic cells present antigens to T cells, which monocytes differentiate into macrophages and
in turn activate plasma cells for production of reside in various tissues. The tissue-specific mac-
immunoglobulins. Dendritic cells also transport rophages are known as histiocytes, epithelioid
the pathogens to the deeper tissues such as the cells (skin), or multinucleated (polykaryon fused
liver, spleen, and lymph nodes. The primary cells) giant cells. Macrophages are also present in
immunoglobulin type found in the gut is secre- various organs and differentiate into alveolar
tory IgA (sIgA), which is transported to the macrophages (lungs), Kupffer cells (liver),
lumen for neutralization of the antigen. splenic macrophages (spleen), mesangial cells
(kidney), synovial A cells (joints), microglial
cells (brain), and Langerhans cell (skin).
Cells of Immune System The cytoplasm of macrophages appears gran-
ular due to phagocytic vacuoles (phagosomes)
Cells that are important in immune response are and lysosomes. Lysosomal contents have low pH
originated from pluripotent stem cells in the bone and contain proteolytic enzymes, reactive “O”
marrow and belong to two distinct lineages: radicals, NO (nitric oxide), prostaglandin,
Cells of Immune System 59
defensin, and lysozyme. After phagocytic contents. The phagocytosis process involves sev-
engulfment, the antigen is trapped inside the eral steps. First, the macrophage recognizes an
phagosome. The phagosome then fuses with the opsonin-coated target with or without using a
lysosome creating the phagolysosome. specific surface receptor and then forms a
Lysosomal antimicrobial components inactivate pseudopod to trap it inside the phagosome. The
and subsequently degrade the pathogen. phagosome fuses with the lysosome forming the
Macrophages also possess surface receptors for phagolysosome. Lysosomal contents aid in the
antibody (FcR), complement, IL-2, and transfer- pathogen destruction and degradation. The
rin. During the oxidative burst, NO is produced vesicle-containing degraded products are eventu-
by the macrophage and attacks metalloenzymes ally transported outside the cell by exocytosis.
of bacteria to produce the superoxide peroxyni-
trite (OONO−) which can also cause collateral Antigen Presentation
damage to the host. Macrophages have three dis- Macrophages are rich in the major histocompati-
tinct functions: professional phagocytosis, anti- bility complex (MHC) class II protein; hence, it
gen presentation, and production of cytokines. is considered an efficient antigen-presenting cell
(APC). Macrophages process the antigen (patho-
Professional Phagocytosis gen) by using the lysosomal proteolytic enzymes
As the first line of defense, macrophages engulf and then present the peptide fragments using
pathogens and destroy them (Fig. 3.8). MHC class II molecules to T or B cells for effec-
Macrophages can readily recognize antibody- tor function. Macrophages found in the skin,
coated pathogen using the Fc (fragment crystal- lymph nodes, spleen, and thymus are involved in
line) part of an antibody binding receptor (FcR) antigen presentation in these sites.
for elimination. Antibody, complement, or lectin-
like molecules that help macrophages to recog- Production of Cytokines
nize antigens are called “opsonins,” and the After encountering an antigen, macrophage
phagocytosis process is known as “opsonization.” releases soluble effector molecules called cyto-
Macrophages remove dead or injured self-cells, kines that recruit inflammatory cells such as
tissues, tumor cells, and apoptotic cells. Thus, neutrophils, eosinophils, basophils, monocytes
they prevent inflammation which may be trig- and other macrophages, and lymphocytes.
gered by the dead or injured cells and their Macrophages also produce growth factors for
Fig. 3.8 Steps showing

phagocytosis and
destruction of a
pathogen by a
macrophage
fibroblasts and vascular endothelium to repair Eosinophil

injured tissues resulting from inflammation. The Eosinophil, a polymorphonuclear leukocyte,
cytokines produced by macrophage are IL-1α, responds during inflammation. The cytoplasmic
IL-1β, TNF-α, IL-6, IL-10, and IL-12. granules (lysosome) stain intensely with an
acidic red dye, eosin, hence the name eosinophil
(Fig. 3.9). Granules contain acid phosphatase,
Dendritic Cells peroxidase, cationic protein, and reactive oxygen
species (ROS). They carry receptors for IgE
Dendritic cells (DCs) have a starlike appearance (FcRε) and other immunoglobulins. Eosinophils
resembling the dendrites of a neuron. DCs are are also professional phagocytes and assist in
phagocytic and known antigen-presenting cells chemotaxis of other immune cells. They are
similar to macrophages. DCs originate from the effective against parasitic infection such as proto-
bone marrow from the progenitor cells (stem zoa and helminths, which are often resistant to
cell). The immature DCs circulate throughout the the degradation by lysosomal enzymes of neutro-
body and convert into matured DCs in the pres- phils and macrophages. Eosinophil counts in the
ence of antigens and cytokines. They migrate to blood are high in patients showing immediate
different organs and tissues including the skin, hypersensitivity (allergic) reactions or suffering
airways, intestine, and lymphoid organs (spleen from parasitic infections.
and lymph nodes). They play important roles in
both innate immunity and adaptive immunity. Basophil
They are professional antigen-presenting cells; The cytoplasmic granules of some leukocytes
they phagocytose antigens, process them, and stain intensely with the basophilic dye, hematox-
present them to T and B cells using MHC class II ylin, and hence are given the name basophil
molecules on their surface. DCs express mem- (Fig. 3.9). Basophils are circulating counterpart
brane markers CD40, CD70, and CD86, and they of mast cells located in tissue, and the granules
secrete IL-12 and interferons. During enteric contain vasoactive amines such as histamine and
pathogen infection, they are known to carry intra- serotonin. They also carry receptor (FcR) for
cellular pathogens such as Salmonella enterica IgE. Binding of IgE–antigen complex to the
and Listeria monocytogenes from submucosal receptor of basophils or mast cells triggers a sig-
locations to extraintestinal sites. naling cascade resulting in the release of vasoac-
tive amines, which are key mediators of
immediate (type I) hypersensitivity reactions.
Granulocytes Basophils are primarily effective in removing
allergens from the system by increasing the
Neutrophil membrane permeability due to the action of vaso-
Neutrophils are polymorphonuclear (PMN) leu- active amines and allowing other cells such as
kocytes and contain granules and multilobed NK and macrophages to reach to the sites. They
nuclei (Fig. 3.9). Granules known as lysosomes are nonphagocytic in nature.
contain protease, myeloperoxidase, lysozyme,
elastase, defensins (cysteine-rich cationic pep-
tides), and acid hydrolases, such as Lymphoid Tissue
β-glucuronidase and cathepsin B. Neutrophils
also contain a receptor for antibody (FcR) and The lymphoid lineage consists of two popula-
complement. They are attracted to the site of tions of lymphocytes: B lymphocytes and T lym-
infection during inflammation where they act as phocytes (Fig. 3.10). B lymphocytes are derived
professional phagocytes and aid in chemotaxis of from the bursa of Fabricius in birds and in an
other immune cells. Neutrophils are primarily equivalent organ, bone marrow, in mammals. B
effective against bacterial infection. lymphocytes differentiate into plasma cells which
Fig. 3.9 Granulocytes have a multilobulated nucleus and the destruction of microbes or induction of hypersensitiv-
contain numerous granules. The granular contents are ity reaction
proteolytic enzymes, “O” radical, or amines, which aid in
Fig. 3.10 T- and

B-lymphocyte lineages:
origin, differentiation,
and maturation
produce antibodies. T lymphocytes undergo their have receptors for immunoglobulins (Ig). They
maturation phases in the thymus and produce carry specific surface markers which are desig-
interleukins, interferons, and cytokines. T lym- nated as CD (cluster of differentiation) followed
phocytes differentiate into T-cell subsets: cyto- by a number. For example, the CD2 molecule is
toxic T cells (CTL or Tc), helper T cells (Th), and expressed on all T cells and binds to sheep red
regulatory T cells (Treg). blood cell (RBC) forming a rosette-like appear-
ance. Numerous CD molecules are identified so
far, and T-cell subsets are classified based on the
T Lymphocytes presence of different surface CD markers
(Table 3.5).
T lymphocytes originate in the bone marrow.
They migrate to the thymus where they differen-
tiate into Th and CTL. They produce a plethora Helper T Cells
of cytokines. They also express MHC (classes I
and II) molecules on their surface and recognize Helper T cells are also designated as Th or CD4+
peptide antigens presented in association with T cells because of the presence of the unique
MHC molecules with the T-cell receptor (TCR). CD4 surface marker. Th cells are classified into
They do not respond to soluble antigens. They Th1, Th2, Th17, and regulatory T cells (Treg).
Helper T cells recognize antigens when presented developed during helminth infection and with
in association with an MHC class II molecule and exposure to common environmental allergens,
subsequently secrete cytokines which activate B and they are responsible for removal of extracel-
cells and macrophages. lular infective agents. IL-4 production by other
cell types favors the development of Th2 cell.
Th1 In general, Th1 cells induce a cellular Th2 cells secrete IL-3, IL-4, IL-5 (eosinophil
inflammatory type of response. Th1 cells respond activation factor), IL-6, IL-10, IL-13, and
to antigens when presented by macrophages, and GM-CSF. They stimulate B-cell proliferation and
in turn, they further activate macrophages isotype switching for IgE, IgM, IgG1, and
(Fig. 3.11). They secrete IL-2, IL-3, interferon-γ IgA. IL-4 promotes IgE and IgG1 production by
(IFN-γ), lymphotoxin, GM-CSF, TNF-β, and B cells (plasma cells).
lymphokines that promote B-cell proliferation.
Th1 cells help B cells to synthesize IgG2a. Th1 Th17 The Th17 cell lineage is characterized by
cells are not stimulated by IL-1 (macrophage its capacity to produce IL-17, but not IFN-γ or
derived). Th1 cells help in cell-mediated immu- IL-4. However, the development and the expan-
nity and delayed (type IV) hypersensitivity reac- sion of Th17 cells are dependent on IL-23. Th17
tions. Th1 cells are preferentially developed cells and the Treg cells share common develop-
during intracellular bacterial and viral infections. mental pathways. Th17 cells carry a transcription
IL-12 and IFN-γ produced by macrophage and factor, RORγt (RAR-related orphan receptor
NK cell support the development of Th1 cell. gamma), while Treg carries FoxP3 (forkhead box
P3) protein, also known as scurfin. TGF-β is
Th2 Th2 cells respond to the antigens presented required for the development of Th17 and Treg
by B cells and play an important role during cells from naïve T cells. In the presence of TGF-β
humoral immune response (Fig. 3.11). Th2 or TGF-β plus IL-6, T cells can differentiate into
expresses membrane CD30, a member of TNF FoxP3+ Treg cells or RORγt+ Th17 cells,
receptor superfamily. The Th2 population is respectively.
Th17 cells are a key pathological marker for
Table 3.5 Selected surface markers for T lymphocytes many human diseases. Th17 cells regulate inflam-
mation by producing IL-17 and its family mem-
Cluster designation Distribution
CD1 Thymus
bers: IL-17A, IL-17B, IL-17C, IL-17D, and
CD2 All T cells; rosette formation IL-17E. IL-17E is also referred to as IL-25. In
CD3 Mature T cells addition, Th17 cells produce TNF-α, IL-6, IL-21,
CD4 Helper T cells (Th1, Th2, Th17) IL-22, and IL-26. IL-17 has diverse biological
CD8 Cytotoxic T cells functions: it recruits neutrophils, activates mac-
CD4+/CD25+/Foxp3+ Regulatory T cells (Treg) rophages, and stimulates the production of
Fig. 3.11 Differences

in antigen presentation
by helper Th1 and Th2
cells and their respective
effector functions
p roinflammatory cytokines (IL-1α, IL-1β, IL-6,

TNF-α, IFN-γ) and AMPs from a variety of
immune and nonimmune cells. IL-17 can also
enhance the capacity of CD4+ T cells to produce
IL-2 and promotes the proliferation of both con-
ventional T cells and Treg cells. IL-17 is necessary
for protective immunity against Escherichia coli,
Salmonella enterica, Klebsiella pneumoniae,
Bordetella pertussis, and other infective agents.
Th17 cells are also involved in the pathology of
many autoimmune diseases such as allergy, rheu-
matoid arthritis, multiple sclerosis, and inflam-
matory bowel disease.
Treg A subpopulation of T lymphocytes carrying

the transcription factor FoxP3 is now recognized
as the regulatory T lymphocyte (Treg), which con- Fig. 3.12 Cytotoxic T-lymphocyte (CTL)-mediated kill-
ing of target cells
trols the immune response in a specific manner.
Treg cells maintain tolerance to self-antigens and
immune homeostasis. Treg cells originate from out affecting the neighboring cells (Fig. 3.12).
thymus, and they are mostly CD4+ T cells and The cytotoxin or perforin (pore-forming toxin)
express CD25; hence, they are designated CD4+/ produced by the CTL induces apoptosis in the
CD25+/Foxp3+ T lymphocytes. They can sup- target cells. CTL also produce granulysin, which
press the activation, proliferation, and effector kills host cells infected with intracellular bacteria
functions of CD4+ and CD8+ T lymphocytes, NK such as Listeria monocytogenes. CTL-mediated
cells, B cells, and APC and can control autoim- killing is antigen-specific and requires cell-to-
mune disease, immunopathology, allergic cell contact; however, the CTL itself is not injured
response, tolerance to tissue grafts, and fetal during this interaction. They secrete large
maintenance during pregnancy. They release amounts of IFN-γ which in turn increases expres-
IL-10 and TGF-β. Treg cell numbers increase in sion of MHC class I molecules by the infected
blood and other tissues in cancer patients, thus cells for enhanced killing.
favoring tumor progression. Chemotherapeutic
drugs such as dacarbazine prevent tumor prolif-
eration and thus have an immunosuppressive Natural Killer Cells
effect on T-lymphocyte populations including the
Treg cells. Natural killer (NK) cells are prototypical mem-
ber of the innate lymphoid cell (ILC) family and
classified as group 1 ILC. NK cell function and
Cytotoxic T Lymphocytes development are mediated by the transcription
factor T-bet, but it is not strictly dependent on
Cytotoxic T cells (CTL), also known as CD8+ T T-bet. NK are called large granular lymphocytes
cells, provide immunity against viral and intra- (LGL) because they carry numerous large cyto-
cellular bacterial infections and cancer. They are plasmic granules containing cytotoxic perforin
responsible for graft rejection and are the primary and granzymes to cause programmed cell death
effector cells in tumor immunity (antitumor of targets. They are called non-T and non-B cells
activity). They recognize antigens through T-cell because of lack of characteristic T-cell receptor-
receptor (TCR) when presented in association like CD3 or B-cell receptor-like immunoglobu-
with the MHC class I molecule and selectively lin. NK cells express CD56 and depending on the
kill the target cell with the help of cytotoxin with- stage of the development, some may express
lymphocytes are called lymphokines, and those

produced by leukocytes are called interleukins
(IL). Cytokines play important roles in both
natural immunity and adaptive immunity and
regulate lymphocyte activation, growth, and
differentiation. Cytokines also activate nonspe-
cific inflammatory cells and stimulate immature
leukocyte growth and differentiation.
Properties of Cytokines
Fig. 3.13 Specific destruction of virus-infected cell by
the natural killer (NK) cell with the help of virus-specific 1. Cytokines are produced during the effector
antibody. This event is also called antibody-dependent
phase of natural and specific immunity. For
cell cytotoxicity (ADCC)
example, LPS activates macrophages which
produce IL-1 during natural immunity. IL-1
CD16 and hence are designated CD3+/CD56+/ stimulates the thermoregulatory center that
CD16+ NK cell. They are also known as increases body temperature (fever) to inhibit
lymphokine-activated killer (LAK) cells and are bacterial growth.
activated by IL-12 for enhanced cytotoxic effect. 2. Cytokine secretion is brief and self-limiting
NK cells secrete IFN-γ and IL-2 and are effective because the cytokine mRNA is unstable.
against tumor cells or virus-infected cells. The 3. Many individual cytokines are produced by
target cell killing is generally nonspecific, i.e., multiple diverse cell types. For example, IL-2
not restricted to antigen–MHC presentation; is produced by T and NK cells, while IL-6 is
however, specific target cell destruction is produced by B cells, T cells, macrophages,
achieved by using immunoglobulin and is termed fibroblasts, and endothelial cells.
antibody-dependent cell cytotoxicity (ADCC) 4. Cytokines act upon different cells. Cytokine
(Fig. 3.13). action is redundant, and many cytokines have
a similar function.
5. Cytokines often influence the synthesis of
Natural Killer T Cells other cytokines and can have antagonistic or
synergistic action.
Natural killer T (NKT) cells share properties of 6. Cytokine action is mediated by binding to its
both NK and T cells and express variants of TCR, receptor. Cytokine action can be autocrine
CD3, and CD4. They carry the nonclassical MHC (the action is on the secreting cell), paracrine
class I molecule CD1d and are activated in the (the action is on nearby cell), or endocrine
presence of microbial lipids including those of (systemic circulation allows action on dis-
Mycobacterium and Leishmania. NKT cells tantly located cells) similar to hormones.
secrete IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-13, 7. Cytokines regulate cell division. For example,
and GM-CSF. NKT cells contribute to the innate GM-CSF helps granulocyte–monocyte cell
and preadaptive immune response. division and growth.
Cytokines ytokines in Innate and Adaptive

C
Immunity
Cytokines are protein hormones produced by
immune cells. Cytokines produced by mono- Two major cytokines are produced during innate
nuclear phagocytes (monocytes) are called immunity: Type I interferon (IFN) and
monokines. Cytokines produced by activated T TNF. During adpative immune response, Type II
Cytokines in Innate and Adaptive Immunity 65
interferon and interleukins are predominant. Tumor Necrosis Factor

Cytokines promote growth, differentiation, and
activation of immune cells and can initiate Tumor necrosis factor (TNF) is one of the major
inflammatory reactions that protect against cytokines in innate immunity and is responsible
microbial infections. for immune cell regulation and cell signaling.
Originally, it was discovered for its activity to
induce cell death and suppress tumor growth.
Interferon Two TNF have been described: TNF-α and
TNF-β (lymphotoxin). TNF-α consists of three
Interferons (IFN) are of two types: type I and subunits of 17 kDa each with a total molecular
type II. The type I interferon is again divided mass of 51 kDa. It is produced primarily by mac-
into two groups depending on the molecular rophages but also by other cell types such as
weights and the type of cells that produce them: CD4+ T cells, NK cells, eosinophils, neutrophils,
IFN-α and IFN-β. IFN-α is an 18 kDa polypep- mast cells, fibroblasts, epithelial and endothelial
tide and is called leukocyte interferon since it is cells, and neuron. TNF-α is produced largely in
produced by the mononuclear phagocytes. IFN-β response to bacterial infections primarily due to
is a 20 kDa polypeptide and is called fibroblast the release of elevated levels of LPS from Gram-
interferon since it is produced by fibroblast cells. negative bacteria and peptidoglycan and other
Type I is involved in the natural immunity and components of Gram-positive bacteria. TNF
inhibits the viral replication by establishing the induces inflammation and activates NF-κB and
“antiviral state” in infected cells. Type I IFN MAPK (mitogen-activated protein kinase) path-
stimulates the production of oligoadenylate syn- ways to produce inflammatory cytokines for
thetase, which acts on adenosine triphosphate immune cell recruitment, differentiation, and
(ATP) to produce adenine trinucleotide (AT). AT proliferation. TNF-α also stimulates macro-
activates RNase L (endoribonuclease) which phages to produce IL-1. TNF-α, together with
cleaves the viral mRNA, thus inhibiting viral IL-1, raises body temperature (fever) through a
protein synthesis resulting in “antiviral state.” prostaglandin-mediated pathway to inhibit bacte-
Interferon action is host species-specific but not rial growth. TNF-α blocks lipoprotein lipase
virus-specific. action; therefore, the fatty acids are not released
An example of type II interferon is IFN-γ from lipoprotein for use by the body cells. As a
which has both immunostimulatory and immuno- result, the patient loses muscle mass and the body
modulatory effects and plays a key role in adap- weight leading to cachexia (a wasting condition
tive immunity. IFN-γ is a 21–24 kDa polypeptide characterized by loss of body mass). The cachec-
and is produced by NK and NKT cells during tic condition is also mediated by TNF-induced
innate immune response and by Th1 CD4+ and appetite suppression in the host. Large amounts
CD8+ cells during the adaptive immune response. of TNF-α production affects heart muscle con-
IFN-γ upregulates both MHC class I and class II traction and vascular smooth muscle tone result-
molecules. It activates mononuclear phagocytes, ing in lowered blood pressure and ultimately
increases MHC class II expression, and increases septic shock.
phagocytosis and antigen presentation. It also
aids in T- and B-cell differentiation. IFN-γ plays
an important role in clearance of intracellular Interleukins
pathogens including bacterial (Salmonella,
Listeria), viral, and protozoan (Toxoplasma, Interleukins (ILs) are produced by leukocytes in
Leishmania) diseases. IFN-γ is also involved in response to antigens and serve as effector mole-
tumor immunity. cules in innate and adaptive immune response by
Table 3.6 Summary of interleukins and their target cells

Name Produced by Target cells
IL-1α Macrophages T, B
IL-1β Macrophages T, B
IL-2 T, NK T (Th, CTL), B, NK, macrophages
IL-3 T Hematopoietic stem cells, B, mast, macrophages
IL-4 Th2 Stem cell, T, B, macrophages
IL-5 Th2 T, B, eosinophil
IL-6 T, B, macrophage, fibroblast, endothelial B, T, stem cells, neurons, hepatocytes, macrophages
cells
IL-7 Bone marrow stromal cells Pre-B, pre-T, and T cells (immature lymphocytes)
IL-8 Macrophages, lymphocytes, hepatocytes, T, neutrophil, basophil (chemoattractant)
endothelial cells
IL-9 CD4+ T Th cells, none on CTL
IL-10 Macrophages, Treg cells Macrophages, DC
IL-11 Stromal cell B cells, platelet production
IL-12 NK, macrophage Th1, NK
IL-13 CD4+ T B cell, epithelial, fibroblast, macrophages
IL-15 Macrophage NK, T cells
IL-16 T, mast, epithelial, eosinophils CD4 + T, monocytes, eosinophils
IL-17A/B/C/D/F Th17 Mucosal tissues, epithelial and endothelial cells
IL-18 Monocytes, macrophage, DC NK, Th1, monocyte, neutrophils
IL-19 Monocytes, macrophages Keratinocytes, macrophages
IL-20 Monocytes Keratinocytes
IL-21 Th2, Th17 Th17, B cell, NK, DC
IL-22 Th1, Th17, NK Fibroblasts, epithelial cells, hepatocytes
IL-23 Macrophage, DC Th17 differentiation
IL-24 Monocytes, CD4+ T Keratinocytes
IL-25 (IL-17E) Th2, mast cells, eosinophils, macrophages NK?
IL-26 Activated T cells, monocytes Unknown
IL-27 Activated DC, macrophages T cells, NK
IL-28/IL-29 DC? Unknown
IL-31 Activated T cells, Th2 Myeloid progenitor, keratinocytes
IL-33 Smooth muscle cells, keratinocytes, Th2, mast cells
fibroblasts
IL-35 Treg Effector T cells
activating other cells (NK, T, B, and hematopoi- mammals and avian species, and there are subtle
etic stem cells). Several interleukins are identi- differences among them, which are described
fied and their source and functions are below.
summarized in Table 3.6.
Structure of Immunoglobulin
B Lymphocytes and Antibodies
A typical immunoglobulin (Fig. 3.14) consists
B lymphocytes express surface receptors that of two identical heavy chains and two light
interact with antigens. The B-cell receptors are in chains. Two heavy chains (55 or 70 kDa) are
fact membrane-bound antibody. Antibodies joined by two disulfide bridges in the hinge
secreted from B cells are present in the γ-globulin region. Heavy chains have one variable domain
fraction of the serum and hence called immuno- called VH and three constant domains designated
globulin (Ig). Immunoglobulins are produced by CH1, CH2, and CH3. The variable domain (VH)
B Lymphocytes and Antibodies 67
Fig. 3.14 Structure of

immunoglobulin and the
products of proteolytic
(papain and pepsin)
digestion
has a hypervariable region, which is also called Classes of Immunoglobulins

complementarity- determining region (CDR).
The CDR again has three subregions, CDR1, IgA It consists of a heavy chain type α1 or α2
CDR2, and CDR3, of which CDR3 is the most and exists either as a monomer or a dimer. In the
variable and is located close to the N-terminal dimeric form, a joining chain (15 kDa) holds two
end. Two light chains κ and λ (24 kDa each) con- monomers together. IgA is found in the serum at
tain one variable (VL) and one constant (CL) low concentrations (3 mg ml−1). Dimeric IgA
domain. Light chains are linked to the heavy also exists as a secretory form (sIgA) where a
chain by disulfide bonds. Each structurally dis- secretory molecule of 70 kDa is attached that
tinct domain (such as CH1, CH2, and CH3) in protects sIgA from proteolytic degradation. sIgA
each chain consists of 110 amino acids, and is about 400 kDa and is abundant in saliva,
each domain is joined by disulfide bridges. mucus, and other body fluids (bile, synovial flu-
Based on the heavy chain types, immunoglobu- ids, respiratory and intestinal tract secretions).
lins are classified as IgA, IgG, IgM, IgE, and
IgD (Fig. 3.15). IgA is made with α-heavy IgE It is about 190 kDa and contains an addi-
chain, IgG with γ, IgE with ε, IgD with δ, and tional domain in Fc region. Generally IgE level is
IgM with μ-heavy chain (Table 3.7). very low in serum (0.05 mg ml−1), but its concen-
The antigen-binding end, which is also the tration increases during an allergic response or
N-terminal end, is called Fab, and the C-terminal during parasitic infection such as helminth
end is Fc (fragment crystalline). It is the infection.
C-terminal end that binds Fc receptors present on
various cell types. The Fc region contains carbo- IgD It is about 180 kDa and remains mostly
hydrate molecules and a complement-binding membrane bound. Thus, it is present in serum in
site. Proteolytic enzymes are used to cleave Fab trace amounts. IgD acts as a receptor for antigens
from Fc region. Papain cleaves immunoglobulin when B cells serve as an APC.
molecule in the hinge region resulting in two
separate Fab molecules and 1 Fc molecule, while IgM It is present in pentameric form (950 kDa)
pepsin cleaves the hinge region below the disul- where 5 units are joined by a joining chain
fide bond to produce an intact F(ab)2 appearing (J-chain). This is the predominant antibody in the
as the letter “V” and fragmented Fc. blood (1.5 mg ml−1) during primary immune
Fig. 3.15
Immunoglobulin
structures of different
classes of
immunoglobulin
Table 3.7 Classes and subclasses of immunoglobulins Avian (chicken) antibodies consist of three
Heavy Molecular weight immunoglobulin subclasses: IgA, IgM, and IgY.
Class chain type Subclass (kDa)
IgA and IgM are similar to mammalian IgA and
IgA α1, α2 IgA1, IgA2, 150, 300, or 400
sIgA
IgM, while IgY is equivalent to mammalian IgG.
IgD δ None 180 These antibodies are found in serum as well as in
IgE ε None 190 eggs. In eggs, IgA and IgM are present in albu-
IgG γ Mouse: IgG1, About 150 each men in low concentrations (0.15 and 0.7 mg ml−1,
IgG2a, IgG2b, respectively), while IgY is found in yolk in a
IgG3 large amount (~25 mg ml−1). Structurally, IgY
Human: IgG1, (180 kDa) is larger than the mammalian IgG
IgG2, IgG3,
IgG4 (150 kDa), and it can be readily harvested in
IgM μ None 950 (pentamer) large quantities from eggs. The H-chain in IgY is
68 kDa and consists of four constant domains
response when challenged with an antigen. IgM (Cv1–Cv4) instead of three for IgG. IgG constant
is very unstable and rapidly loses its activity if it domains Cγ1, Cγ2, and Cγ3 are equivalent to IgY
is subjected to temperature abuse. Cv1, Cv3, and Cv4, respectively. IgY lacks a
hinge region (Fig. 3.15).
IgG The molecular mass of IgG is 150 kDa.
Subclasses of IgG vary between mice and humans
depending on the heavy chain type. In mice, sub- Diversity of Antibodies
classes are IgG1, IgG2a, IgG2b, and IgG3; in
humans, subclasses are IgG1, IgG2, IgG3, and A total number of antibody specificities that an
IgG4. IgG concentration in blood is very high individual can produce is called the “antibody
(IgG1, 9 mg ml−1; IgG2, 3 mg ml−1; IgG3, repertoire.” There are about 107–109 different
1 mg ml−1; IgG4, 0.05 mg ml−1) and is the pre- antibody molecules with unique amino acid
dominant immunoglobulin during secondary sequences in the antigen-binding site. This gener-
immune response. IgGs are very stable. ates significant diversity in a host. The variable
B Lymphocytes and Antibodies 69
Fig. 3.16 B-cell

maturation, growth
phases, and synthesis of
immunoglobulin
Fig. 3.17 Steps

showing isotype
switching of
immunoglobulins during
humoral immune
response
region has a hypervariable region or CDR. The cells can produce antibody subclass of IgM, IgG,
unique determinant of the CDR varies from anti- IgA, or IgE (Fig. 3.17).
body to antibody and is called idiotope, while the
collection of idiotopes on a particular antibody
molecule constitute idiotype. Function of Antibody
Acts as B-Cell Receptor Membrane-bound

Antibody Production antibody binds antigens which allow B cells to
process and present antigenic peptides to T cells
The sequence of events that takes place for B via MHC class II molecules. Hence, B cells are
cells to produce antibody molecules for a specific efficient antigen-presenting cells.
antigen begins in the stem cell (Fig. 3.16). B cells
originate from stem cell and give rise to pre-B, Neutralization of Antigen Antibody neutral-
immature B, and mature B cells, sequentially. izes toxins, viruses, or bacteria. It binds to the
Immature and mature B cells leave bone marrow antigenic determinant and prevents the antigen
and migrate to secondary lymphoid organs such from interacting with the host cell by “steric
as the lymph nodes and spleen where they hindrance.”
transform into antibody-secreting B cells.

Immunoglobulin isotype switching takes place at Activation of Complement IgG or IgM after
this stage and depending on the antigen type B forming a complex with an antigen activates
complement cascade via the classical pathway to Mucosal Immunity by IgA Secretory IgA
produce complement by-products such as C3b (sIgA) is abundant in mucus and other bodily
and membrane attack complex (MAC) that facili- fluids and provides specific or nonspecific

tate lysis of the microorganism. immunity at the mucosal surface.
Opsonization The antibody also serves as an Neonatal Immunity-Mediated by

opsonin, a molecule that facilitates the recogni- IgG Colostrum, milk produced by mother
tion of target microorganisms by phagocytic cells immediately after the birth of the fetus, contains
(e.g., macrophages) for elimination. Antibody a high level of maternal IgG, which protects neo-
first forms a complex with microbes, and then nate against pathogens in the early part of its life.
the Fc domain of the antibody binds to the Fc
receptor on the phagocytic cells (macrophage, Feedback Inhibition of Immune Response Is
neutrophil or eosinophil) for engulfment and Mediated by IgG Binding of excess circulating
destruction. IgG to Fcγ receptor on the B-cell surface inhibits
further activation of B cells. This process is called
Antibody-Dependent Cell Cytotoxicity antibody feedback inhibitions.
(ADCC) NK, neutrophil, and eosinophil recog-
nize and specifically destroy target cells when
coated with antibodies (IgG, IgE, and IgA). A Antigen
target cell coated with antibody binds to the Fc
receptor (CD16) of the phagocytic cell for spe- A foreign molecule that is capable of stimulating
cific recognition and destruction of virus or tumor immune system is called an antigen or immuno-
infecting a target cell. gen. Most effective antigens are large, rigid, sta-
ble, and chemically complex. Factors that
Immediate Hypersensitivity Reaction by influence antigenicity are:
IgE Basophils or mast cells have Fc receptor
(FcεR1) for IgE. IgE forms a complex with the Molecular Size of the Antigen Large molecules
antigen (allergen) and binds to the Fc receptor of are more antigenic than the small molecules. For
the mast cell. This interaction prompts the release example, 14.4 kDa lysozyme is better antigen
of vasoactive amines such as histamine, than 1 kDa angiotensin, the 69 kDa albumin is
leukotrienes, or prostaglandins that are responsi- better antigen than lysozyme, γ-globulin
ble for immediate hypersensitivity reaction (156 kDa) is better than albumin, fibrinogen
(Fig. 3.18). Examples are hay fever, asthma, and (~340 kDa) is better than γ globulin, and IgM
food allergy. (900 kDa) is better than the fibrinogen.
Fig. 3.18 Allergen-

mediated activation of
basophil and mast cell
for secretion of
vasoactive amines in the
IgE-mediated
hypersensitivity
response
Antigen 71
Complexity of Antigen LPS and proteins are antigens are called T-dependent antigen, i.e.,
complex, whereas lipid, carbohydrate, and these antigens activate T cells for effector func-
nucleic acids as polymers of repetitive units are tion, whereas lipopolysaccharide (LPS) or pepti-
considered poor antigens. doglycans (PGN) consisting of carbohydrates are
called T-independent antigens which activate B
Structural Stability of Antigen Immune cells. However, some zwitterionic polysaccha-
response recognizes shape and stability. Flexible rides that carry both positive and negative charges
molecules have no shape and thus are considered such as polysaccharide A from Bacteroides fragi-
poor antigens. For example, gelatin is wobbly lis can activate T cells (Th1 cells).
and is a poor antigen. Similarly, flagella are struc-
turally unstable and thus are considered poor
antigens. Epitope or Antigenic Determinant
Degradability of Antigen Antigens should be Antigenic determinant, or epitope, is the most

degraded relatively easily for processing and pre- immunodominant structure in an antigen and
sentation by APC. Stainless steel pins and plastic binds to the antibody. The number of epitopes in a
joints are made of large inert organic or inorganic molecule depends on the size of the antigen; larger
polymers, which the immune system cannot antigens have multiple epitopes. Epitopes can be
degrade. Thus, they are recognized as poor anti- either linear, conformational, or neoantigenic.
gens. Proteins made of d-amino acid isomeric Linear antigenic determinants remain intact even
forms are poor antigens because immune cells after denaturation by the action of physical or
cannot degrade them. Only L-amino acid iso- chemical treatments. Conformational epitopes are
forms are used by cells. destroyed by denaturation and are unable to bind
to the antibody. In this case, an antibody only rec-
Foreignness The immune system does not ognizes the natural conformation of the antigen.
respond to self-antigens, and the immune cells Neoantigenic determinants are unraveled only
which respond to self-antigens are eliminated or after treatment of the antigen with an enzyme or
turned off. The degree of foreignness also deter- heat that allows binding to an antibody.
mines the antigenicity.
Hapten
Types of Antigens
Hapten means “to grasp” or “fasten.” Small mol-
Microbial and nonmicrobial antigens are diverse. ecules that are unable to induce an immune
Bacterial antigens include cell wall or somatic response by themselves are called haptens.
antigen (O), capsule (K), pili or fimbriae (F), fla- Examples are penicillin, allergens, and peptides.
gella (H), cell membrane, proteins, ribonucleo- They need carrier molecules to induce antibody
protein, enzymes, and toxins (endotoxins and production. Carrier molecules such as KLH (key-
exotoxins). hole limpet hemocyanin) and BSA (bovine serum
Viral antigens include envelope (lipoprotein, albumen) are large and immunogenic and are
glycoprotein), capsomere (a subunit of capsid), used as carriers for experimental production of
and receptor proteins. Cell surface antigens of antibodies against haptens. Naturally, in some
mammalian cells include (1) blood group anti- individuals, penicillin or allergens, though small,
gens, ABO, Rh, MN, Kell, Duffy, Luthern, and can interact with serum proteins in the blood
Lewis; (2) histocompatibility antigens (MHC forming a larger complex that is capable of induc-
class I, class II); (3) cluster differentiation (CD) ing an immune response. Generally, an immune
or lymphocyte surface antigens; and (4) autoanti- response against penicillin leads to a hypersensi-
gens such as hormones, myelin, and DNA. Protein tive, or allergenic, response.
Antigen–Antibody Reaction molecular mass of α-chain in human is 44 kDa

and in mouse 47 kDa. The α-chain has three
Antigen–antibody reactions are specific and domains, α1, α2, and α3, which resemble the
reversible. Binding forces involved during anti- constant regions of an immunoglobulin. The
gen–antibody reactions are (1) Vander Waal force, β-chain (12 kDa; a β2-microglobulin), a non-
where transient dipole interactions allow weak MHC coded gene product, noncovalently inter-
bonding between the antigen and antibody, (2) acts with the α-chain to provide stability to the
hydrogen bond [O]–H–[O] or [N]–H–[N] which MHC class I molecule. An antigen-binding
forms between molecules in an antigen and anti- pocket is formed by the α1 and α2 domains,
body, (3) electrostatic bond ([+]–[−]) which forms which can accommodate a small peptide (pro-
by positive and negative charges contributed by cessed antigen) consisting of 8–10 amino acid
both antigen and antibody, and (4) hydrophobic residues. MHC class I molecules are expressed
bond which forms by removing water molecules by most nucleated cells in the body and present
from the binding sites of antigen and antibody. antigen to CD8+ T cells (CTL) for effector func-
Binding strength between an antigen and anti- tion (Fig. 3.19). In other words, during infection
body molecule is expressed as affinity. Affinity is the with intracellular pathogens (bacteria, virus, and
strength of binding between a single complemen- protozoa), pathogen-specific antigens are pre-
tary site of the antibody and an epitope of an anti- sented on the surface of infected host cells by
gen. The avidity of an antibody defines the strength MHC class I for targeted destruction by CTL
of attachment of combining sites of all available cells.
epitopes, i.e., the overall strength of binding.
HC Class II
M
The MHC class II antigen consists of two nonco-
he Major Histocompatibility
T valently associated polypeptide chains
Complex (Fig. 3.19). The α-chain is about 32–34 kDa,
while the β-chain is 29–32 kDa. The peptide-
The major histocompatibility complex (MHC) binding pocket is made up of both α1 and β1
molecules, or MHC antigens, are expressed on the domains that hold a peptide of 13–25 amino
surface of a variety of cells. They are essential for acids. Both α- and β-chains are encoded by two
antigen presentation to T cells. They are also separate MHC genes and are expressed only in
responsible for graft rejection. During organ or few cells: macrophages, dendritic cells,
graft transplant, if the host receives tissues or Langerhans cells, B cells, some T cells in humans
organs that are expressing the same MHC, the and rats but not in mice, and some endothelial or
graft is accepted, but if the MHC are different, the epithelial cells after induction by IFN-γ. CD4+ T
graft is rejected. There are two classes of MHC cells (T helper cells) respond to the antigen when
antigens: class I and class II. T cell recognizes for- presented by class II molecules (Fig. 3.20).
eign antigen only when presented within MHC
class I or class II. Exogenous peptide fragments
always bind to the class II (example, bacterial exo- Antigen-Presenting Cells
toxins or other surface proteins), whereas endoge-
nously synthesized peptides are presented with Antigen-presenting cells (APC) are defined as
class I (example, viral proteins or tumor proteins). those which after internalization of an antigen
(either by phagocytosis or by the active invasion
of intracellular microbes) process and present
Structure of MHC antigens on their surface either in association
with MHC class I or in class II molecules for rec-
HC Class I
M ognition by T or B cells. Most cells in the body
MHC class I proteins consist of a single α-chain when infected by intracellular pathogens (virus,
(also known as a heavy chain) (Fig. 3.19). The intracellular bacteria, protozoa) or tumors can
The Major Histocompatibility Complex 73
Fig. 3.19 Structure of MHC class I and class II molecules
express class I molecule, while only a limited by IFN-γ. Note: In general, IFN-γ can induce
number of specialized cells can express class II class II expression in any APC cells.
molecules and present the antigen. Cells that use 6. B lymphocytes: They are considered highly
MHC class II for antigen presentation are: efficient antigen-presenting cells because they
carry surface immunoglobulin (IgD or IgM),
1. Mononuclear phagocytes: Macrophages which serves as a receptor for a protein anti-
actively phagocytose large particles and infec- gen, and they present antigens to Th cells. B
tious organisms such as extracellular bacteria cells bind antigen efficiently even at a low con-
and parasites and degrade them, and the centration with high affinity. They are impor-
immunodominant peptide is presented in tant for T-cell-dependent antibody production.
association with class II molecules to the Th
or B cells.
2. Dendritic cells: DCs are originated from bone HC-Restricted Antigen Processing
M
marrow. They reside in the spleen, lymph and Presentation
nodes, and submucosa. They process and
present antigen to T cells. Endogenously synthesized antigens are restricted
3. Langerhans cells: These cells are located in to class I-mediated presentation, while the exog-
the skin. They contain characteristic Birbeck enously synthesized antigens are restricted to the
granule (rod shaped or tennis racket shaped) class II-mediated presentation.
in their cytoplasm, express class II molecules,
and serve as APC of the skin.
4. Venular endothelial cells: These cells also lass II-Restricted Antigen
C
express class II molecule and present antigen Presentation
to T cells.
5. Epithelial cells: They also can express class II Class II-restricted antigen processing takes place
molecules and serve as APC when activated with an exogenous protein antigen. The sequence
Fig. 3.20 MHC class I- and class II-restricted exogenous and endogenously synthesized antigen processing and
presentation
of events that take place for antigen processing and peptide fragments (13–25 amino acids).
presentations are summarized below (Fig. 3.20): Unbound peptides are further degraded to
amino acids and discarded from the cells by
1. Initially, binding and internalization of protein exocytosis.
antigens or native proteins occur by phagocy- 4. In the subsequent step, a fusion of vesicles
tosis, endocytosis, or pinocytosis, and the containing MHC class II with the bound pep-
antigens are trapped in the endosome or tide and the cytoplasmic membrane occurs.
phagosome. Through exocytosis (reverse phagocytosis),
2. In the processing step, a fusion of the phago- the MHC with bound peptide is displayed out-
some with the lysosome (carries degradative side for recognition by CD4+ T cells.
enzymes) results in the proteolytic digestion
of proteins in the acidic environment and the
generation of immunogenic peptides. lass I-Restricted Antigen
C
3. Next, the fusion of endosome containing pep- Presentation
tides with the MHC class II proteins containing
vesicle takes place. In this stage, class II mole- Intracellular bacteria, viruses, parasites, or tumor
cule binds to the appropriate immunodominant antigens are capable of infecting different types
The Complement System 75
of host cells and are processed and presented by

MHC class I molecule on the surface of infected
cells also known as “target cells.” After the inva-
sion, intracellular pathogens escape from the
phagosome and multiply inside the host cyto-
plasm. Thereafter, the sequence of events are
somewhat similar to class II-mediated pathway;
however, in this case, the pathogen-specific pro-
teins are synthesized inside the host cell through
active gene transcription and translation process
(Fig. 3.20). (1) In the first step, the bacterial DNA
or viral DNA or RNA is transcribed to synthesize
proteins. At the same time, DNA for MHC class I
is also transcribed to synthesize class I molecules.
(2) Pathogen-derived proteins are synthesized in Fig. 3.21 Involvement of accessory molecules during
the cytoplasm and then are degraded by host cell antigen presentation to T cells
proteasome into small peptides. (3) The peptides
(eight to ten amino acids) are transported into the The Complement System
lumen of the rough endoplasmic reticulum and
bind to MHC class I molecules. The peptide- The complement proteins are synthesized in the
bound MHC complex is transported to the Golgi liver and are present in serum. They play impor-
for further processing. (4) Vesicles containing tant roles during both innate and adaptive immune
MHC class I with bound peptides are fused with response. Jules Border in his classical experiment
cytoplasmic membrane and are transported to the demonstrated that if an antibacterial antibody is
exterior of the cells through exocytosis and pre- mixed with fresh serum at 37°C, it caused lysis of
sented to the CD8+ T (CTL) cells. the bacteria; however, when the serum was heated
to 56°C or higher prior to mixing, lysis did not
occur. Bacterial lysis was reestablished when the
ccessory Molecules Involved
A fresh serum was added, suggesting the involve-
during MHC-Restricted T-Cell ment of a heat-labile component, which can com-
Activation plement the antibody function, and hence was
given the name “complement.” Complement con-
During the antigen presentation by MHC class I sists of a series of proteins designated C1 to C9,
or class II molecules to T cells, several other mol- each of which remains in serum in an inactive
ecules are also involved (Fig. 3.21). T-cell recep- form. Complement cascade can be activated
tor (TCR) consisting of αβ- or γδ-chains are the sequentially by proteolytic enzymes through
primary component that recognizes the MHC– three mechanisms: (1) classical pathway, (2)
peptide complex on the surface of APC. The CD3 alternative pathway, and (3) lectin pathway
and CD4/CD8 present on T cells also bind to the (Fig. 3.22). The classical pathway is activated by
MHC–peptide complex and stabilize the struc- an antigen–antibody (IgG or IgM) complex dur-
ture. In addition, the CD2 molecule on the sur- ing the adaptive immune response. An infectious
face of T cell interacts with the LFA-3 agent and a lectin-like glycoprotein can activate
(lymphocyte function-associated antigen) of alternative and lectin pathways, respectively,
APC, and LFA-1 of T cell interacts with ICAM-1 generally during innate immune response. The
(intracellular adhesion molecule) of APC. These primary and the most abundant component of the
accessory molecules increase the strength of the complement cascade is C3, which is converted to
adhesion between T and APC or target cells, C3a and C3b by C3 convertase. Individuals with
serve as surface markers, and transduce the bio- complement deficiency are susceptible to many
chemical signal to the interior of the cell. infective agents.
Fig. 3.22 Activation of

complement by the
classical, alternative, and
lectin pathways
The Classical Pathway forms a donut-shaped hole on the surface of the

target pathogen. The complement activation by-
The classical pathway is activated during the products C3a, C4a, and C5a are collectively
specific immune response (Fig. 3.23). Circulating called anaphylatoxins, which are a chemoattrac-
IgG or IgM in the blood form a complex with the tant for immune cells and are known to induce
specific pathogen or antigen and activates the hypersensitivity response.
inactive form of the protein C1. C1 is a large
multimeric protein consisting of three subunits:
C1q, C1r, and C1s. The active protein complex Alternative Pathway
C1qrs then catalyzes inactive C4 to produce
active C4b and a soluble by-product, C4a. C4 During the innate immune response, the comple-
can also be activated by mannose-binding lectin ment system plays a major role in protecting the
(MBL), which is a pattern recognition receptor- host against invading pathogens in the absence of
specific to bacterial carbohydrates. C2 catalyzes antibodies. Viral proteins, bacterial LPS, peptido-
the C4b to form C4b2. C1qrs complex can also glycan, teichoic acid (TA), lipoteichoic acid
activate C4b2 to form the C4b2a, which is also (LTA), and microbial surface polysaccharide can
called C3 convertase. The C3 convertase cata- activate complement directly via the alternative
lyzes the C3 to form C3a and C3b, a major com- pathway. The resulting complement by-products
ponent of the complement activation pathway. play important roles in the host natural immunity.
C3b also forms a complex with C4b2a forming In the alternative pathway (Fig. 3.24), several
the C4b2a3b complex, which is known as C5 serum factors including B, D, H, I, and properdin
convertase. The C5 convertase catalyzes C5 to systems are involved. Initially, microbial factors
form C5a and C5b. C5b forms a complex, activate C3 to form C3b. In the presence of factor
C5b6789, also known as membrane attack com- B, factor D catalyzes C3b to form C3bBb. The
plex (MAC) through a sequential reaction of C3bBb complex is called C3 convertase and is
C5b with C6, C7, C8, and C9 components. MAC very unstable. Factor I rapidly degrades this pro-
The Complement System 77
Fig. 3.23 Complement

activation through
classical pathway
tease. Properdin proteins, however, bind and sta- (Fig. 3.25); however, a majority of the host
bilize C3 convertase. C3 convertase catalyzes C3 cells are protected by CD59, a membrane-
to form C3b and a small molecule, C3a. C3b bound glycoprotein that inhibits MAC forma-
forms a complex with C3bBb to produce tion by blocking the aggregation of C9.
C3bBb3b, a C5-convertase, which converts C5 to 2. Complement protein C3b aids in the opso-
form C5b and C5a. Sequential catalysis of C6, nization (phagocytosis) process by serving as
C7, C8, and C9 results in the formation of an opsonin, which binds to microbes.
C5b6789 complex, called the MAC, similar to a Phagocytes (macrophages and neutrophils)
by-product generated in the classical pathway. express the C3b receptor (CD35) which binds
to the opsonin-coated microbes for engulf-
ment. The opsonization process can be
Function of Complement enhanced several- fold when both antibody
and C3b coat the target pathogen as an
The complement system plays a major part in the opsonin.
defense against microbes in both innate and 3. Complement protein by-products C3a, C4a,
adaptive immune responses. and C5a also serve as anaphylatoxins. They
induce the release of soluble inflammatory
1. Complement-mediated bacterial lysis is mediators such as histamine to increase mem-
accomplished by MAC, which is produced in brane permeability, smooth muscle contrac-
both classical and alternative pathways. MAC tion, and diffusion of inflammatory cells such
also can attack host cells by inserting into the as neutrophils and macrophages. C5a acts as a
plasma membrane causing collateral damage chemoattractant for neutrophils at the site of
Fig. 3.24 Complement

activation through
alternative pathway
Fig. 3.25 Diagram

showing the insertion of
MAC (membrane attack
complex) into the
plasma membrane
inflammation thus facilitating increased a ntigen–antibody complex is rapidly cleared

clearance of the pathogens from the site of from the body by phagocytes, thus preventing
infection. ill effects of the antigen–antibody complex.
4. Complement proteins also aid in the phago-
cytic clearance of immune complexes.
Antigen–antibody complexes formed during Control of Complement Activation
immune response may be trapped in a variety
of organs/tissues including the kidneys result- Activation of complement and resulting by-
ing in pathological consequences such as products are able to eliminate pathogens from the
glomerulonephritis. Since the complement
host; however, activation can also cause c ollateral
system enhances phagocytic activity, the damage to the nearby host cells. If C3b binds to
Immunity to Microbes 79
the host cells, those cells become the target of Clostridium, Staphylococcus, Bacillus,
phagocytic cells causing cell/tissue damage. In Streptococcus, Escherichia coli, and Vibrio spe-
addition, the MAC can cause host cell damage. As cies. Pathogens cause disease by inducing inflam-
a protective measure, the host cells carry surface mation or by the direct action of toxins or
proteins which prevent C3b from binding to host enzymes that they produce.
cells. For example, the C3bH formed during the
alternative pathway of activation allows degrada-
tion of C3b by protein I that in turn prevents the Inflammation
formation of C3bBb. In addition, the host cells
contain sialic acids, and C3b does not bind to sialic Bacterial cells provoke inflammation that results
acid. However, some bacteria take advantage of in tissue destruction and an influx of neutrophils
the situation by producing a capsule that contains and macrophages at the site of infection. During
sialic acid, thus preventing C3b from binding to inflammation, complement activation takes
self and phagocytic killing. Mannose-binding pro- place, and cytokines are produced by macro-
tein also prevents activation of complement. Some phages. Enzymes and toxins released from the
bacteria express mannose on their surface, thus dead neutrophils and macrophages at the site can
preventing complement activation. Macrophages directly cause cell injury and tissue damage
produce IL-6, which can activate liver cells to pro- resulting in suppurative infection characterized
duce mannose-binding protein, which binds to by pus formation. Three cardinal signs of inflam-
mannose on the bacterial surface. This alters the mation are redness, swelling, and pain: (1)
configuration and can activate complement. increased blood supply to the site of infection
causes “redness,” (2) increased capillary perme-
ability and fluid accumulation result in swelling,
Immunity to Microbes and (3) recruitment of neutrophils and macro-
phages by chemoattractant C5a results in tissue
The principal physiological function of the injury that invokes pain.
immune system is to protect the host against
pathogenic microbes such as extracellular bacte-
ria, intracellular bacteria, viruses, and parasites. Toxins
Important characteristics of immune response to
microbes are as follows. (1) Defense against Exotoxins are protein toxins that are secreted by
microbes is mediated by both innate and adaptive the microbes and cause diverse pathological
immunity. (2) Different types of microbes stimu- effects resulting in cell injury or cell death (see
late distinct populations and subpopulations of Chap. 4). Shiga toxin and diphtheria toxin inhibit
lymphocytes. (3) Survival of microbes in the host protein synthesis. Botulinum toxin blocks neu-
or progression of the disease depends on their rotransmitter release. Cholera toxin stimulates
ability to evade the immune system. (4) Tissue cAMP synthesis resulting in Cl− secretion and
injury and disease related to infection are caused H2O loss. Endotoxins are bacterial components,
primarily by the overt host response rather than such as LPS and PGN, and are called pyrogens
the microbe itself. because they induce fever in the host. Pyrogens
stimulate the release of cytokines, IL-1 and TNF-
α, activate B cells, and act as an adjuvant to stim-
Extracellular Bacteria ulate macrophages to produce more cytokines.
These cytokines induce fever, decrease smooth
Extracellular bacteria replicate outside the host muscle contraction, increase membrane
cells, i.e., in circulation, interstitial space, and the permeability, lower blood pressure, and under
lumen of the respiratory and the intestinal tracts. severe conditions can prompt septic shock and
Examples of extracellular bacteria are death.
Innate Immunity MHC–SEB complex interacts with T-cell

receptor and activates CD4+ T cells to produce
The innate immune response against extracellu- copious amounts of IFN-γ, which in turn
lar bacteria is spontaneous and is mediated by induces and enhances MHC expression on
immune cells and cytokines. (1) Neutrophils, many cell types, antigen (SEB) presentation,
monocytes, and tissue macrophages actively and cytokine production by macrophages
phagocytose bacteria. (2) Complement is acti- leading to toxic shock syndrome.
vated via the alternative pathway or mannose lec- 3. Antibodies (IgG and IgM) help in the opso-
tin pathway by bacterial peptidoglycan or nization of pathogens, neutralization of tox-
mannose-binding lectin. The complement activa- ins, and activation of complement via the
tion by-product C3b enhances opsonization, classical pathway. The activation by-products
while MAC induces cell lysis. (3) LPS or PGN C3b and MAC cause increased phagocytosis
activates macrophages to produce inflammatory and cell lysis, respectively.
cytokines TNF-α, IL-1, and IL-6, which in turn
activate neutrophils and macrophages for
enhanced bacterial clearance. These cytokines vasion of Immune System
E
also induce a fever to retard bacterial growth or by Extracellular Bacteria
may induce “septic shock” or “endotoxin shock”
leading to fatal consequences. In order to cause a successful infection, bacteria
must be able to evade the immune system. Indeed,
they have developed various strategies to achieve
Adaptive Immune Response that.
The adaptive or specific immune responses 1. Antigenic variation is an important strategy

against extracellular bacteria are mediated by cell for some microorganisms such as Salmonella
wall components and the exotoxins, which are enterica, Haemophilus influenzae, and E. coli.
summarized below: Bacteria use surface molecules such as pili,
fimbriae, flagella, or other surface proteins for
1. In the specific immune response, cell compo- adhesion and colonization; however, genetic
nents (i.e., LPS and PGN) act as T-independent variation in bacterial surface molecules may
antigens and activate B cells to produce spe- preclude recognition by the antibodies previ-
cific IgM. Antigen-mediated specific T-cell ously produced against the same organism.
activation results in specific cytokine release, 2. Some bacteria (Bacillus anthracis, B. cereus,
which is responsible for isotype switching to and Pneumococcus spp.) exhibit antiphago-
produce specific immunoglobulin class (IgG, cytic mechanisms. They express capsules
IgA, IgE). made of sialic acid and hyaluronic acid, both
2. Exotoxins are T-dependent antigens and are of which are also present in host cell mem-
processed by APC (macrophages, B cells) and brane and prevent binding of C3b. As a result,
presented via MHC class II molecules to acti- these bacterial cells are not recognized by
vate CD4+ T cells and B cells for cytokine and macrophages for destruction.
antibody production, respectively. Certain 3. Sialic acid component of the capsule also
toxins act as superantigens, which induce T inhibits complement activation and helps
cells to produce increased cytokines in the evade pathogen inactivation.
absence of antigen processing and classical 4. Some bacteria (Staphylococcus aureus,
MHC presentation leading to toxic shock syn- Streptococcus pyogenes) cover themselves
drome (TSS). For example, staphylococcal with host antibody leaving the immune sys-
enterotoxin B (e.g., SEB) binds to MHC class tem unable to recognize them as foreign. S.
II molecule on the macrophages directly. This aureus produces protein A, and S. pyogenes
Intracellular Bacteria 81
produces protein G which binds IgG at the Fc tive protection. However, it is important to point
domain masking the bacteria from macro- out that despite the lack of protection by humoral
phages even though they carry a receptor for immunity, antibodies against bacterial antigens
Fc. are found in the serum, thus indicating that the
5. Some bacteria such as Streptococcus pneu- antigens are processed and presented by MHC
moniae, Haemophilus influenzae, Neisseria class II pathways for response by CD4+ T cells
gonorrhoeae, and Neisseria meningitides pro- and B cells. Furthermore, the bacterial antigen–
duce IgA-specific proteases that degrade these antibody complex can activate complement
antibodies. through the classical pathway and can contribute
toward adaptive immunity. The properties of
adaptive immunity are as follows:
Intracellular Bacteria
1. Cell-mediated immunity (CMI) is the major
Several foodborne pathogens maintain an intra- protective immune response against intracel-
cellular lifestyle as part of their infection strat- lular bacteria, where CD8+ T cell acts as the
egy. After binding to a specific host cell receptor, primary effector cell. These cells recognize
intracellular bacteria modulate signaling events the infected target cells expressing antigens
resulting in cytoskeletal rearrangement and facil- on their surface in association with MHC
itating their own entry (induced phagocytosis). class I molecule.
Bacteria trapped inside a phagosome either 2. Macrophages activated by T-cell-derived
escape by lysing the vacuolar membrane or pro- cytokines such as IFN-γ also play an impor-
duce effector molecules that sustain their intra- tant role by promoting active phagocytosis of
cellular life cycle. They either spread from cell to bacteria.
cell or induce apoptosis leading to host cell lysis 3. In some cases, the host induces granuloma
as part of their pathogenic mechanism. Examples (nodule or polyp) formation to contain the
of intracellular foodborne bacterial pathogens are infective agent from spreading, which is seen
Listeria monocytogenes, Yersinia enterocolitica, in chronic infections caused by
Shigella spp., and Salmonella enterica. Mycobacterium, Histoplasma, and some mold
species. There is an onset of a delayed type of
hypersensitivity reaction (DTH) to these
Innate Immunity infections. Both CD4+ and CD8+ T cells
respond to the soluble protein antigens and
Innate immunity including gastric acid, bile, anti- intracellular bacterial antigens, respectively.
microbial peptides, mucins, and natural micro- Following activation, these cells secrete TNF
flora provide some protection; however, many and IFN-γ, which activate vascular endothe-
pathogens are resistant to those. Therefore, the lial cells, which in turn recruit neutrophils,
innate immunity is less effective. Furthermore, lymphocytes, and monocytes. IFN-γ also acti-
phagocytes (macrophages and neutrophils) are vates macrophages, which convert them into
also less effective since bacteria are resistant to epithelioid or giant cells for enhanced elimi-
degradation by lysosomal degradative enzymes. nation of pathogens. If the antigen stimulation
continues to persist, macrophages are chroni-
cally activated to secrete additional cytokines
Adaptive Immunity and growth factors, which help recruit fibro-
blast cells to encase the bacteria-containing
The humoral immune response has limited con- cells to form a protective nodule called a
tribution toward protection since the bacteria are “granuloma.” These nodules of various sizes
mostly localized inside the host cell; thus, the are characteristic for many diseases including
cell-mediated immunity provides the most effec- tuberculosis and histoplasmosis.
vasion of Immune System

E infect neighboring cells (see Chap. 6). Viral pro-
by Intracellular Bacteria teins also block host cell protein synthesis and
thus may induce host cell death showing charac-
Intracellular bacteria utilize various strategies teristic cytopathic effects. Many viruses infect
that allow them to maintain intracellular immune cells as their primary target. For exam-
lifestyle. ple, HIV-I binds to CD4 molecule on helper T
cells and then enter the cell eventually leading to
1. Some bacteria (example, Mycobacterium) acquired immune deficiency syndrome (AIDS)
inhibit phagolysosomal fusion, thus protect- when the infection has resulted in substantial loss
ing the bacteria from toxic lysosomal to the Th population. Epstein–Barr virus (EBV)
contents. binds to type 2 complement receptor (CR2) on B
2. Escape from phagosome: Listeria monocyto- cells and causes infectious mononucleosis. It is
genes secretes a hemolysin that forms pores in also associated with Burkitt lymphoma and other
the phagosome to help bacteria escape into the carcinomas. Rhinovirus binds to intercellular
cytoplasm before phagolysosomal fusion adhesion molecule (ICAM) and causes inflamma-
takes place, thus averting degradation by lyso- tion of the nasal passages. SV40 (simian virus)
somal contents. Furthermore, hemolysin also binds to MHC class I to enter cells and causes
blocks antigen processing, and Listeria anti- cancer in the monkey.
gens are not presented on the surface by MHC
class I molecule for recognition by T cells.
3. Intracellular bacteria such as Listeria monocy- Innate Immunity
togenes, Salmonella enterica, Yersinia spp.,
and Shigella spp. are capable of scavenging Innate immunity against virus infection is multi-
reactive oxygen species to avert toxic effects faceted. (1) Virus infection stimulates the produc-
of oxygen radicals. Superoxide dismutase tion of type I interferon (both IFN-α and IFN-β)
(SOD) inactivates toxic “O” radicals. Bacterial from infected cells, which inhibits viral replica-
catalase breaks down toxic H2O2. tion by creating the “antiviral state” discussed
4. Intracellular bacteria have antiphagocytic earlier. (2) NK cells can kill or lyse a variety of
activity. For example, Yersinia enterocolitica virally infected cells. Moreover, the type I inter-
outer membrane proteins such as YopE has feron enhances NK activity. (3) Viral protein can
antitoxin activity, YopH has tyrosine phospha- activate complement cascade in the alternative
tase activity, and YpkA blocks signaling path- pathway and the complement by-product, C3b,
way that is required for phagocytosis. can enhance phagocytosis.
5. Intracellular localization also prevents the
bacteria from being seen by the immune
system. Adaptive Immunity
Both humoral and cell-mediated immunity (CMI)

Immunity to Virus are important in protecting the host against viral
infection. During the humoral immune response,
Viruses are obligatory intracellular pathogens, the antibody is produced against the viral enve-
which upon entry replicate inside the host cell. lope or capsid proteins, which are responsible for
Viral surface proteins first bind to a host cell binding to host cell receptor. Antibodies are gen-
receptor leading to entry into the cell. Inside the erally very effective in the initial stage of viral
cell, viral nucleic acid replication, protein synthe- infection. Circulating antibodies bind viral sur-
sis, nucleic acid packaging, and matured virion face proteins and block the viral interaction and
synthesis take place. Upon host cell lysis, newly entry into the host cells. On the mucus mem-
developed virions are released and proceed to brane, sIgA plays an important role since they are
Immunity to Parasites 83
abundant in the mucus of respiratory and the some viruses directly infect immune cells and
digestive tract, which serves as a primary portal cause immune suppression. For example, influ-
for viral entry. Antibody-dependent immune enza and rhinovirus suppress the immune system,
responses are as follows. (1) Antibody–viral par- HIV-I diminishes CD4+ T cell populations, and
ticle complex can help enhance phagocytic clear- EBV inhibits production of IL-2 and IFN-γ.
ance by macrophages from the blood circulation
by opsonization. (2) Antiviral antibody forming a
complex with a viral antigen can activate comple- Immunity to Parasites
ment through the classical pathway and promote
phagocytosis. (3) Humoral immunity against Many of the foodborne protozoan species (see
viral antigens is used as a prophylactic vaccine to Chap. 7) cause chronic disease where the natural
protect the host from future infections. Attenuated immunity is weak and less effective. Protozoan
or killed virus generates circulating antibodies in species such as Giardia, Entamoeba,
serum that prevent viral binding to host cells dur- Cryptosporidium, and Cyclospora invade intesti-
ing a later exposure. Current vaccination strategy nal cells and cause massive damage to the site of
embraces the induction of mucosal immunity infection leading to diarrhea, which may be
since many foodborne viruses (hepatitis A, noro- bloody and mucoid. Helminths such as Trichinella
virus, and rotavirus) use intestinal mucosa as the and Taenia species (tapeworms) invade liver and
site of entry to initiate infection. muscle tissues and cause inflammation resulting
Though the antibodies are important protec- in chronic infection. During chronic infection,
tive components of immunity to viruses, they the clinicopathologic consequences are mostly
may not be fully effective since viruses hide due to the host response to the parasites.
inside the cells. Therefore, CMI provides the
most protection. The principal mechanism of
specific immunity against viral infection is the Innate Immunity
generation of cytotoxic T cells (CD8+ T cells).
These cells recognize endogenously synthesized Acid, mucus, bile, antimicrobial inhibitors, resi-
viral proteins in association with MHC class I dent microbiota in the gastrointestinal tract, com-
molecules on the surface of target cells and plement, and phagocytic cells play important
destroy them by producing pore-forming cytoly- roles in antiparasitic immunity. Complement is
sins (perforin and granulysin). activated through the alternative pathway and
results in the formation of MAC, which causes
lysis of parasites. Many parasites are resistant to
Evasion of Immune System by Viruses phagocytic killing since they are capable of repli-
cating inside the macrophage. Parasites also
Viruses use several strategies to overcome induce inflammation and recruit polymorphonu-
immune system. The most important of all is clear cells, macrophages, dendritic cells, and
their intracellular life cycle and utilization of host neutrophils.
cell machinery for replication and spread, in
which immune effector cells are unable to find
them. Antigenic variation is also an important Adaptive Immunity
characteristic. There are a large number of sero-
logically distinct strains present which show Adaptive, or specific, immunity to parasites is
huge antigenic variations in their proteins. effective and is mediated by both humoral and
Therefore, protective antibodies or vaccine is CMI. In humoral response, IgE plays an impor-
ineffective. This characteristic is most important tant role. Parasitic antigens are presented on the
with influenza virus (flu virus) in which the surface of APC and are recognized by CD4+ T
genetic variation is very common. In addition, cell, which produces IL-4 and IL-5. IL-4 also
promotes IgE production from B cells, and IL-5 adaptive, that are in place to protect the host
recruits eosinophils. The IgE-bound parasite against infective agents. Microbial dominance
complex is then recognized by eosinophils via results in the disease, while successful host
receptor FcεR, and consequently, the parasite is response averts the full-blown infection.
destroyed by toxins (peroxidase, cationic protein, Foodborne pathogens affect primarily the diges-
reactive oxygen species) produced by tive system; therefore, the natural immunity of
eosinophil. the gastrointestinal tract is the most important
Parasites also induce granuloma formation, protective immune response. Moreover, the onset
which is seen in infection caused by Trichinella. of symptoms for some diseases (i.e., intoxica-
During infection, both CD4+ and CD8+ T cells tion) is very fast – appearing within 30 min to an
are activated, and they produce cytokines that hour. Thus, protection by adaptive immune
recruit macrophages and fibroblasts. Fibroblast response would have little impact since it is slow
cells help encase the infective agents resulting in to develop, typically requiring 4–7 days.
the formation of granulomas. CMI also induces However, the adaptive immune response is essen-
DTH response. Trichinella (roundworm) eggs tial for foodborne pathogens that have a pro-
cause liver cirrhosis when deposited in the hepa- longed incubation time and are responsible for
tocytes. Schistosoma mansoni, a waterborne par- systemic infection such as Listeria monocyto-
asite (flatworm), form granulomatous polyps in genes, Shigella spp., Campylobacter spp.,
the nasal passage in herbivores and anal polyps in Salmonella enterica, Yersinia spp., hepatitis A
humans. Cytotoxic T cells (CD8+) destroy host virus, Toxoplasma gondii, Trichinella species,
cells that carry intracellular protozoa and serve as and so forth. In innate immunity, gastric acids,
the primary effector component of the CMI. bile, mucus, antimicrobial peptides, natural
microflora, macrophages, neutrophils, NK cells,
interferons, and complement proteins play a criti-
vasion of Immune System
E cal role. In the adaptive immune response, T and
by Parasites B lymphocytes produce cytokines and antibod-
ies, respectively. CD4+ T cells are most important
Parasites also use numerous strategies to evade for protection against extracellular bacterial
the immune system. (i) Protozoa grow intracel- infections and their exotoxins, while CD8+ T
lularly leaving them inaccessible to immune cells are involved in the elimination of intracel-
cells. This strategy is called “anatomic conceal- lular bacterial, viral, and parasitic infective
ment.” Some parasites develop cysts (example, agents. Antibodies neutralize pathogens or toxins
Trichinella) that are not detected by immune by preventing them from binding to the host cell
cells. (ii) Some parasites mask themselves with a receptors, so they become the target for elimina-
coat of host cell proteins on the surface. For tion by macrophages and neutrophils. An anti-
example, Schistosoma mansoni larvae coat them- body–antigen complex also activates complement
selves with ABO blood group antigens and MHC for inactivation of pathogens via opsonization.
molecules before they reach the lungs masking However, the immune system sometimes fails to
them from host immune cells. (iii) Some para- protect the host. It is because pathogens have
sites (e.g., Entamoeba histolytica) spontaneously developed strategies to overcome immune
shed antigen coats after binding to the antibody defense by producing virulence factors that
and thus are not recognized by phagocytic cells. ensure their invasion, survival, replication, and
spread inside the tissues. It is important to recog-
nize that the pathogenic action by foodborne exo-
Summary toxins is very quick and the host has literally no
time to mount any immune response; thus, a
To understand the pathogenic mechanism of majority suffers from this form of food poisoning
microbial infection, one has to have a clear (intoxication) irrespective of their health status or
knowledge of immune response, both innate and immune response.
Further Readings 85
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microbiota shapes intestinal immune responses dur-
Cellular and Molecular Immunology. Philadelphia,
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PA: WB Saunders.
12. Sakaguchi, S., Miyara, M., Costantino, C.M. and
2. Amalaradjou, M.A.R. and Bhunia, A.K. (2012)
Hafler, D.A. (2010) FOXP3+ regulatory T cells in
Modern approaches in probiotics research to control
the human immune system. Nat Rev Immunol 10,
foodborne pathogens. Adv Food Nutr Res 67, 185-239.
490-500.
3. Bevins, C.L. and Salzman, N.H. (2011) Paneth cells,
13. Santaolalla, R. and Abreu, M.T. (2012) Innate immu-
antimicrobial peptides and maintenance of intestinal
nity in the small intestine. Curr Opin Gastroenterol
homeostasis. Nat Rev Microbiol 9, 356-368.
28, 124-129.
4. Blaser, M.J. and Falkow, S. (2009) What are the con-
14. Sekirov, I., Russell, S.L., Antunes, L.C.M. and Finlay,
sequences of the disappearing human microbiota? Nat
B.B. (2010) Gut microbiota in health and disease.
Rev Microbiol 7, 887-894.
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5. Brandtzaeg, P. (2003) Role of secretory antibodies in
15. Shao, L., Serrano, D. and Mayer, L. (2001) The role
the defence against infections. Int J Med Microbiol
of epithelial cells in immune regulation in the gut.
293, 3-15.
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6. Hansson, G.C. (2012) Role of mucus layers in gut
16. Spits, H., Bernink, J.H. and Lanier, L. (2016) NK
infection and inflammation. Curr Opin Microbiol 15,
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17. Tscharke, D.C., Croft, N.P., Doherty, P.C. and La
and mucosal immune responses. Int J Med Microbiol
Gruta, N.L. (2015) Sizing up the key determinants
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8. Janssens, S. and Beyaert, R. (2003) Role of toll-like
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18. Turner, J.R. (2009) Intestinal mucosal barrier function
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in health and disease. Nat Rev Immunol 9, 799-809.
9. Kovacs-Nolan, J., Phillips, M. and Mine, Y. (2005)
19. Ryan, V. and Bhunia, A.K. (2017) Mitigation of
Advances in the value of eggs and egg components
foodborne illnesses by probiotics. In Foodborne
for human health. J Agric Food Chem 53, 8421-8431.
Pathogens: Virulence Factors and Host Susceptibility.
10. Lievin-Le Moal, V. and Servin, A.L. (2006) The front
Edited by Joshua Gurtler, Michael Doyle, and Jeffrey
line of enteric host defense against unwelcome intru-
Kornacki. pp 603-634, Springer.
sion of harmful microorganisms: mucins, antimicro-
General Mechanism
of Pathogenesis 4
Introduction toward the liver, which is rich in iron bound to the

transferrin protein. Bacterial cell envelope con-
The diseases caused by foodborne pathogens are sisting of the capsule can help bacterial survival
classified into foodborne infection, foodborne in the hostile environment, as the capsule protects
intoxication, and foodborne toxicoinfection, and the bacterium from being engulfed by profes-
each is described in detail below (Fig. 4.1). The sional phagocytic cells. In addition, bacterial tox-
primary vehicle of transmission of foodborne ins and enzymes protect the cells from elimination
pathogens is food and water. The oral route is the by the host immune system. The presence of
main portal, and the primary site of action is the commensal bacteria in the site of infection can
gastrointestinal (GI) tract. The GI mucosa com- also assist the invading bacterium to find a niche.
prises of epithelium, lamina propria, and a thin For example, in the case of “wound botulism,”
layer of smooth muscle, and it is the major site of aerobic bacteria first enter the wound, grow and
interaction for foodborne pathogens. Most food- multiply, and utilize oxygen to create an anaero-
borne microorganisms cause localized infection bic microenvironment. As the wound closes from
and tissue damage, while others spread to the the action of blood clotting and fibroblast cell
deeper tissues to induce systemic infection. For a accumulation, Clostridium botulinum, an obli-
successful enteric infection, several factors must gate anaerobe transmitted to the wound through
work cooperatively in a host. Due to the presence sharp object or dust, now has a favorable niche
of multiple protective barriers in the GI tract, for growth and toxin (botulinum) production.
generally, a high dosage is required to cause dis- Patients show a typical sign of botulism, a neuro-
ease compared to the other routes of infection, paralytic disorder. Pathogens also damage the
such as intravenous, intranasal, or intraperito- host tissues and cells by using exotoxins, endo-
neal. Besides, pathogens can transfer via direct toxins, or enzymes that induce cell death by
contact with an animal or a human and from envi- apoptosis or necrosis and promote bacterial sur-
ronments (soil, air) or from an arthropod vector. vival, multiplication, and propagation.
Once inside the host, the pathogen must survive
in the changing environment, multiply and prop-
agate, and avoid the host immune defense. Foodborne Infection
Pathogens must find a suitable niche for coloni-
zation, which is facilitated by adhesion factors, Foodborne infection is committed by intact living
invasion factors, and chemotaxis. For example, microorganisms, which must enter the host to
bacterial affinity for iron propels the organism cause infection. Following ingestion of food or

88 4 General Mechanism of Pathogenesis
Fig. 4.1 Flow diagram showing the various forms of foodborne diseases
water, microorganisms pass through the acidic Often, the prolonged infection is perpetuated by a
stomach environment and move to the intestine, strong immune response mounted by the host
where they attach and colonize using adhesins, rather than the infective agent itself, such as seen
fimbriae (pili) or colonization factor antigens in chronic shigellosis cases leading to chronic
(CFA), curli, and flagella. In some pathogens, the bacillary dysentery. Patients recovering from a
ability to form biofilm may aid in pathogen colo- foodborne infection may shed the organism for a
nization in the intestinal tissues. Invasive patho- prolonged period – observed in salmonellosis, in
gens can cross the epithelial barrier through hepatitis A, and in Norovirus infection. Some
phagocytic M cells located in the lymphoid fol- foodborne infections may lead to chronic debili-
licles or Peyer’s patches – a passive entry mecha- tating sequelae such as Reiter’s syndrome, reac-
nism (Fig. 4.2). Invasive pathogens also can tive arthritis, Guillain–Barré syndrome and
translocate across the intestinal barrier by an Miller Fisher syndrome.
active invasion process through intracellular
route and/or disrupted epithelial tight junction
barrier through paracellular route. Some micro- Infectious Dose
organisms cause local tissue damage and induce
inflammation, while others spread to the lym- The infectious dose of a pathogen or toxin varies
phoid system including mesenteric lymph nodes depending on the immunological health status of
(MLN) and other extraintestinal sites such as the the host and the natural infectivity of the microor-
liver, spleen, gall bladder, kidney, brain, and pla- ganism (Table 4.1). The infectious dose decreases
centa. Foodborne infection can be acute or if the pathogen is consumed with liquid food
chronic. In acute infection, the onset of a disease (milk, soup and beverages) that traverses the
is quick and lasts only for a short duration due to stomach rapidly or food (milk, cheese, etc.) that
a rapid immunological clearance of the microor- neutralizes the stomach acid. Persons with high
ganism. In chronic infection, the disease is pro- gastric pH (achlorhydria) or patients undergoing
longed and the immune clearance is not effective. antibiotic therapy for other ailments are also
Foodborne Infection 89
Fig. 4.2 Schematic of intestinal villus structure and bac- Microbiol. 10, 131–144; and Ribet and Cossart 2015.
terial crossing of the epithelial barrier and subcellular Microbes Infect. 17, 173–183)
translocation (Adapted from Abreu 2010. Nat. Rev.
s usceptible to foodborne infections because anti- intestine or adhere to the mucus or epithelial sur-
biotics reduce the natural microbiota load in the face before exerting their pathogenic actions.
intestine, which renders the host more suscepti- Several adhesion or colonization factors are used
ble to foodborne infections. by pathogens, and general description of each
factor is presented below.
Colonization and Adhesion Factors ili, Fimbriae, and Flagella

P
Pili, also known as fimbriae, are rodlike surface
The gastrointestinal tract, starting with the mouth, adhesin structures mostly found in Gram-negative
esophagus, stomach, small intestine, and large bacteria but also present in Gram-positive bacte-
intestine, is very dynamic and active as the epi- ria such as Lactobacillus rhamnosus GG. Pili
thelial cell surface is constantly washed by mucus consist of pilin protein (20 kDa) and have a long
and fluids. The GI tract uses peristaltic move- helical–cylindrical structure. Pili are located on
ment, mucus, and epithelial ciliary sweeping the surface of bacteria and have a specialized pro-
action to expel pathogens. In addition, the bile, tein tip that helps the bacterium to attach to the
proteolytic enzymes, sIgA, and resident micro- host cell receptors composed of glycolipids,
biota and their metabolic by-products (short- glycoproteins (α-d-galactopyranosyl-β-d-
chain fatty acids: acetic, propionic, butyric acids) galactopyranoside), or other mannose-containing
can also prevent pathogens from colonization. receptors or mucus. Bacteria growing inside the
Foodborne pathogens that are involved in either host constantly lose and reform pili. The host
toxicoinfection or infection must colonize the immune system produces antibodies against pilus
Table 4.1 Infectious dose and incubation periods of common foodborne pathogens.
Pathogens Infectious dose Incubation period
Bacteria
Escherichia coli O157:H7 and other Shiga 10–100 cfu 3–4 days (1–10 days)
toxin-producing E. coli (STEC)
Listeria monocytogenes 102–103 cfu 7–14 days or even longer
Salmonella enterica 1 to 109 cfu 6–24 h
Shigella spp. 10–100 cfu 12 h–7 days, but generally in
1–3 days
Vibrio cholerae/parahaemolyticus/vulnificus 104–1010 cfu 6 h–5 days
Staphylococcus aureus cells 105–108 cfu –
Staphylococcal enterotoxin 1 ng/g of food 1–6 h
Bacillus cereus 105–108 cfu or spores/g 1–6 h (vomiting); 8–12 h (diarrhea)
Bacillus anthracis (inhalation anthrax) 8 × 103–104 spores 2–5 days
Clostridium botulinum neurotoxin 0.9–0.15 μg (i.v. or i.m. route) 12–36 h; 2 h when large quantities
and 70 μg (oral route) are ingested
Clostridium perfringens 107–109 cfu 8–12 h
Campylobacter jejuni 5 × 102–104 cfu 1–7 days (Avg 24–48 h)
Yersinia enterocolitica 107–109 cfu 24–30 h and lasts for 2–3 days
Virus
Norovirus ~10 particles 24–48 h
Hepatitis A 10–100 particles 15–45 days, average 28–30 days
Protozoa
Entamoeba histolytica >1000 cysts (as low as 1 cyst) 1–4 weeks
Giardia duodenalis (lamblia) 10–100 cysts 1–2 weeks
Cryptosporidium parvum 10 oocysts 2–10 days
Toxoplasma gondii 10 oocysts 2–3 days or longer
Table 4.2 Pili/fimbriae of Gram-negative bacteria.

Types of pili Classification Property Present in
Type I CFA/II, CFA/IV Flexible Escherichia coli, Haemophilus influenzae; Yersinia pestis
Type IV – Rigid Pseudomonas, Vibrio, enteropathogenic E. coli (EPEC),
and Neisseria meningitides
P pili CFA/I Rigid E. coli, Haemophilus influenzae, Yersinia pestis
BFP (bundle- CFA/III Flexible EPEC
forming pili)
Curli Flexible Salmonella enterica, E. coli
that can prevent bacterial adhesion to the host IV pili are found in Pseudomonas, Vibrio, entero-
cells by physical hindrance or neutralization. pathogenic E. coli (EPEC), and Neisseria. BFP is
There are four types of pili: type I, type IV, P found in EPEC. Pili are encoded by pap gene in
pili, and bundle-forming pili (BFP) (Table 4.2). E. coli and the pap operon consists of papD,
Type 1 pili are also known as colonization fim- papC, papA, papH, papE, papF, papG, and papK
briae antigen (CFA: CFA/II, CFA/IV), and they genes.
are flexible. P pili (CFA/I) and type IV are rigid, Flagella consist of several thousands of sub-
whereas BFP (CFA/III) is flexible. Type I and P units of flagellin protein and interact with mucus
pili are found in E. coli, Haemophilus influenzae, to aid in bacterial adhesion. EPEC, enterohemor-
and Yersinia pestis. ETEC fimbrial adhesins K88 rhagic E. coli (EHEC), Clostridium difficile, and
(F4), K99 (F5), F41, and F17 attach to the host Campylobacter spp. use flagella to adhere to the
mucus and cause diarrhea in swine (piglet). Type mucus layer of the intestine.
Curli Adhesion Proteins

Curli is a thin-coiled fimbriae-like extracellular Adhesion proteins promote tighter binding of
protein fiber of 6–12 nm wide, produced by bac- pathogens to the host cells and are important for
teria from Enterobacteriaceae family. They were attachment and invasion (Table 4.3). For exam-
first discovered in the 1980s from E. coli that ple, Yersinia enterocolitica YadA adhesin binds
were responsible for bovine mastitis. Since then to fibronectin, laminin, collagen, and β1-integrin
it has been isolated from Salmonella spp. and on host epithelial cells while Yersinia invasin
other pathogenic E. coli, including serovar binds to β1-integrin. In EPEC and EHEC, the
O157:H7. Curli is considered an amyloid fiber attachment and effacement (EAE) protein, inti-
and such fiber formation is typical for many min binds to the translocated intimin receptor
human diseases, such as Alzheimer’s, (TIR) and aids in the formation of a pedestal.
Huntington’s, and prion diseases. The major curli Campylobacter spp. use CadF to interact with
subunit is CsgA (15 kDa), which is capable of fibronectin-binding protein on the host cells. In
self-polymerization forming a β-sheet-rich amy- Listeria monocytogenes, surface protein intern-
loid fiber. In bacteria, curli promote bacterial alin A (InlA) binds to E-cadherin located in the
adhesion to the host cell extracellular matrices adherens junction of the epithelial cell junction;
such as fibronectin, laminin, and type 1 collagen. internalin B (InlB) binds to the tyrosine kinase
It also promotes cell aggregation and biofilm for- Met receptor, a hepatocyte growth factor; LAP
mation. Curli can also induce a strong inflamma- (Listeria adhesion protein) binds to the heat-
tory response in the host. It is typically produced shock protein 60 (Hsp60); and fibronectin-
under stressful conditions, such as suboptimal binding protein binds to host fibronectin. Other
growth temperature (below 30 °C), osmolarity factors such as autolysin amidase (AMI) and LTA
and nutrient-limiting environments, and in the (lipoteichoic acid) are also involved in Listeria
stationary phase of growth. adhesion. Vibrio cholerae uses toxin-coregulated
Table 4.3 Adhesion factors and corresponding host receptors for select pathogens
Pathogens Adhesion factors Host receptor
Listeria monocytogenes Internalin A (80 kDa) E-cadherin
Internalin B (63 kDa) c-Met, gC1q-R/p32
Vip (virulence invasion protein) (90 kDa) Gp90
LAP (Listeria adhesion protein) (104 kDa) Hsp60
Campylobacter spp. CadF (37 kDa) Fibronectin
Arcobacter Hemagglutinin (20 kDa) Glycan receptor
Enteropathogenic and Intimin (94 kDa) Translocated intimin receptor (TIR)
enterohemorrhagic E. coli
(EPEC, EHEC)
Yersinia enterocolitica YadA (160–240 kDa) Collagen/fibronectin/laminin/β1-integrin
Invasin (92 kDa) β1-integrin
Staphylococcus aureus Fibronectin-binding protein (FnBP) Fibronectin
Vibrio cholerae Toxin-coregulated pili (TCP) Glycoprotein
Mannose–fucose-resistant cell-associated Glycoprotein?
hemagglutinin (MSHA), mannose-
sensitive hemagglutinin (MSHA)
Influenza virus Hemagglutinin (HA) Sialic acids attached to galactose through
α2,3-linkage (bird) or α2,6-linkage
(human)
Norovirus Viral capsid protein (VP1) Histo-blood group antigen (HBGA)
Hepatitis A virus Capsid protein Hepatitis A virus cell receptor 1
(HAVCR1)
Fig. 4.3 Biofilm life cycle (Adapted from Ray and Bhunia, Fundamental Food Microbiology, 5th edition, 2014, CRC
Press)
pili (TCP), mannose-sensitive hemagglutinin Biofilm Formation

(MSHA), fucose-sensitive hemagglutinin (FHA), Biofilm formation facilitates bacterial coloniza-
and/or mannose–fucose-resistant hemagglutinin tion in the host tissues (biotic surface) as well as
(MFRHA) for adhesion and colonization in the on inert food processing or medical equipment
intestine. Hemagglutinin (HA) expressed on the (abiotic surface). Some bacteria produce extra-
viral envelope helps avian influenza virus (H5N1) cellular polymeric substances (EPS) consisting
to bind to sialic acids attached to galactose of polysaccharides, proteins, phospholipids, tei-
through α2,3-linkage on the host cell surface, choic acids, nucleic acids, and other polymeric
while human influenza virus binds to 2,6-linked substances. The polysaccharide is made of poly-
sialic acid located in mucosal epithelial cells in β-1,6-N-acetylglucosamine. Bacteria can form
the respiratory tract. dense, multi-organism layers on food contact
surfaces, in the mouth, teeth, or intestinal lining.
Histo-blood Group Antigen Adhesins The first layer of the biofilm attaches directly to
Campylobacter jejuni, Helicobacter pylori, and the host cell surface. Additional layers attach to
Norovirus bind to histo-blood group antigens the basal layer with a polysaccharide matrix. The
(HBGA) expressed on the gut mucosa and on EPS provides protection against biocides, desic-
epithelial cells. H. pylori use blood group- cation, antibiotics, and toxins.
binding adhesin (BabA) and sialic acid-binding Biofilm formation is comprised of several
adhesin (SabA) to bind to mucus in the stomach, stages: (1) attachment, (2) microcolony forma-
in which the bacterium finds its niche to cause tion, (3) maturation, and (4) detachment or
gastric ulcer and cancer. C. jejuni is highly dispersion (Fig. 4.3). Biofilm can be made of a
motile and uses flagella to colonize the mucosa; single organism or multi-organisms. In the bio-
however, HBGA lectins are also implicated in film, microorganisms express fimbriae, curli, fla-
adhesion to mucus. Norovirus interacts with gella, adhesion proteins, and capsules to attach
HBGA found on the epithelial cells in the gastro- firmly to the surface. Microorganisms grow in
intestinal tract and causes severe gastroenteritis. close proximity and can communicate inter- and
The carboxyl-terminal protruding (P1) domain intraspecies and are called “quorum sensing
present in the Norovirus capsid is directly (QS)”, as described below. As the microcolony
involved in binding to HBGA. continues to grow, cells accumulate and form a
matured biofilm with a three-dimensional scaf- survival, growth, density, virulence, and resis-
fold. Loose cells are then sloughed off from the tance to antimicrobials.
matured biofilm and convert into planktonic There are four major categories of AIs: (1)
cells, which again attach to a new surface precon- Autoinducer-1 (AI-1), for example, N-acyl homo-
ditioned by food particles or substrates complet- serine lactone (AHL), alkyl quinolone,
ing the life cycle of a biofilm. In addition to α-hydroxyketones, and a diffusible signal factor, is
exopolysaccharides, proteins are found to play a produced by Gram-negative bacteria and is used
critical role in biofilm formation. A biofilm- for intraspecies communication. (2) Autoinducer-2
associated protein (Bap) and the Bap family of (AI-2) is produced by both Gram- positive and
proteins are involved in biofilm formation in Gram-negative bacteria and is used as a universal
many pathogens: Staphylococcus aureus, S. epi- signaling molecule. (3) Autoinducer-3 (AI-3) is
dermidis, Enterococcus faecalis, Streptococcus produced by EHEC during infection. (4)
pyogenes, Salmonella enterica serovar Autoinducing peptides (AIPs) are produced and
Typhimurium, and E. coli. In addition, type IV used by Gram-positive bacteria.
pili in Vibrio promotes biofilm formation while
curli in Salmonella and E. coli.
Biofilms are important in human diseases, Invasion and Intracellular Residence
including dental plaque caused by Streptococcus
mutans, mastitis by S. aureus, intestinal coloniza- Some bacteria maintain intracellular lifestyle and
tion by Salmonella, endocarditis by Enterococcus they enter cells by two mechanisms: (1) passive
faecalis, and lung infection by Pseudomonas entry into the host through uptake by natural
aeruginosa in cystic fibrosis patients. phagocytic cells such as M (microfold) cells in
the intestinal lining and macrophages and den-
Quorum Sensing dritic cells and (2) active invasion via induced
Bacterial quorum sensing (QS) plays a major role phagocytosis in the nonprofessional phagocytic
during the bacterial response to a changing envi- cells such as epithelial, endothelial, and fibro-
ronment in food or in the host. QS is known as a blast cells. M-cell-mediated entrance is used by
bacterial cell-to-cell communication system, many pathogens including Shigella spp., Listeria
mediated by small signaling molecules and is monocytogenes, and Salmonella enterica. Forced
identified in both Gram-negative and Gram- or induced phagocytosis is used by L. monocyto-
positive bacteria. Microbes use both interspecies genes, Salmonella, Yersinia, and Shigella, which
and intraspecies communication system to use invasin proteins to promote bacterial uptake
develop strategies for the population to adapt to by the host cells. Listeria and Yersinia also use
the harsh environment encountered during colo- caveolae and clathrin-coated vesicles to gain
nization in the host or in other environments. QS entrance. Some pathogens such as uropathogenic
is cell density-dependent: when bacteria reach to and diarrheagenic E. coli and viruses such as
a certain cell mass, bacteria secrete small diffus- hepatitis A virus (HAV) and foot-and-mouth
ible signaling molecules known as autoinducer disease (FMD) virus use sphingolipid–choles-
(AI). Under stressful conditions such as during terol raft that forms invaginations during pinocy-
starvation and high temperature, in the presence tosis for host cell entry. Toxoplasma gondii and
of antimicrobial agents, acids, or other microbes, Cryptosporidium parvum exhibit gliding motility
bacteria secrete AI. AI binds to the receptor on (1–10 μm/s and counterclockwise), which helps
the cell surface and activates gene expression to parasite entry into the enterocytes. The gliding
alter behavior, i.e., adaptation to the harsh envi- motility is an active process for the parasite, but
ronment, tolerance to desiccation, and morpho- the host cell does not play an active role in the
logical transformation leading to biofilm invasion, as invasion does not alter host cell actin
formation or production of spores. AI also regu- cytoskeleton or induce phosphorylation of tyro-
lates gene expression to facilitate microbial sine residues.
To enter host cells, the virus first binds to the Invasin-Mediated Induced
host cell receptor and is then taken up by a pro- Phagocytosis
cess known as endocytosis (see Chap. 6). The Intracellular pathogens may provoke phagocytic
virus may remain trapped inside the endocytic ingestion or induced phagocytosis using invasin
vesicle until a signal is triggered for the nucleic molecules after interaction with the cellular
acid release into the cytoplasm. In the case of receptors (Table 4.3). Induced phagocytosis
enveloped virus, membrane fusion is needed to involves coordinated interaction of sophisti-
penetrate a cell. Viral envelope (membrane) fuses cated quorum sensing and signals transduction
with the cytoplasmic membrane, and the capsid mechanisms. During this process, cytoskeletal
containing the viral genome is released into the proteins undergo rearrangement and accommo-
cytoplasm. In non-enveloped virus, viral genome date bacterial entry. Induction of actin polymer-
is incorporated into a protein shell in the cyto- ization is a crucial event, which aids in
plasm and then is assembled, and viruses are cytoskeletal rearrangements to accommodate
released by cell lysis. bacterial entry. Two distinct mechanisms are
identified for induced phagocytosis: (1) zipper-
Natural Phagocytosis like mechanism and (2) trigger mechanism.
M cells are naturally phagocytic and are present Yersinia pseudotuberculosis and Listeria mono-
throughout the intestine in follicle-associated cytogenes use the zipper mechanism, while
lymphoid tissue such as Peyer’s patch (Fig. 4.2). Salmonella enterica and Shigella spp. use the
Their primary function is to transport intact par- trigger mechanism (Fig. 4.4).
ticles across the epithelial membrane without In the zipper-like mechanism, a relatively
degrading or processing them. Some pathogens modest cytoskeletal rearrangement and mem-
such as Salmonella, Shigella, and Listeria use M brane extension occur following binding of bac-
cells to reach the subepithelial layer. In contrast, terial invasin proteins to a host cell receptor.
professional phagocytic cells such as macro- Ligand–receptor interaction initiates a signaling
phages, dendritic cells, and neutrophils spontane- cascade, which in turn promote recruitment of
ously phagocytose bacteria. Intracellular effector molecules such as actin to form a cuplike
pathogens may survive inside the phagocytic structure that accommodates the bacterium inside
cells and resist killing by using specialized viru- the cup to complete invasion-mediated induced
lence factors such as hemolysin, superoxide dis- phagocytosis. The zipper-like mechanism is a
mutase (SOD), and catalase. Hemolysin forms three-step process: (1) contact and adherence, (2)
pore in the phagosome membrane, SOD inacti- phagocytic cup formation, and (3) phagocytic
vates toxic oxygen radicals, and the catalase cup closure. (1) Contact and adherence happen
breaks down hydrogen peroxide. Salmonella, independent of the actin cytoskeletal rearrange-
Legionella, Mycobacterium, and Brucella block ment but involve ligand and receptor interaction.
or alter the maturation of phagosome, therefore For example, L. monocytogenes uses InlA to
allowing these pathogens to survive inside the interact with E-cadherin, located in the adherens
phagosome. While Listeria monocytogenes and junction of the polarized epithelial cell; L. mono-
Shigella spp. escape the phagosome with the help cytogenes also uses InlB to interact with c-Met, a
of hemolysin before phagolysosomal fusion transmembrane receptor tyrosine kinase. Yersinia
takes place. Bacteria multiply in the cytoplasm, invasin protein interacts with β1-integrin located
induce actin polymerization for intracellular on the basolateral side of the polarized epithelial
motility, and infect neighboring cells. Both cells. (2) The phagocytic cup is formed by mem-
pathogens also avoid autophagosomal degrada- brane extension, which is initiated by the signal-
tion (autophagy) using specialized virulence fac- ing events and induction of actin polymerization
tors. Pathogens such as Shigella, Yersinia, and through the Arp2/3 pathway. (3) Phagocytic cups
Salmonella may induce apoptosis in macro- are then sealed off trapping the bacterium inside,
phages/dendritic cells and persist in subepithelial and this process is facilitated by “membrane
regions in the intestine. retraction” and “actin depolymerization.”
Fig. 4.4 Schematic drawing showing Listeria monocyto- III secretion (T3SS) apparatus directly into host cell cyto-
genes-induced bacterial entry into the cell by zipper mech- sol and induces massive actin polymerization. Shigella
anism and Shigella entry by a trigger mechanism. In zipper injects IpaC, VirA, IpgD and more proteins into the host
mechanism, listerial InlB interacts with Met receptor, cytosol. IpaC activates small GTPases, Cd42, and Rac and
which autophosphorylates and recruits protein adapters: promotes actin polymerization by Arp2/3. VirA inhibits
Gab1, Cbl, and Shc. These proteins activate phosphati- microtubule formation, and IpgD hydrolyzes phosphati-
dylinositol 3-kinase (PI 3 kinase) and small Rho GTPase- dylinositol (4,5) biphosphate (PIP2) to phosphatidylinosi-
kinase, Rac, which in turn promotes actin polymerization tol (5) phosphate (PIP) and disconnects actin from the
via Arp2/3. In zipper mechanism, Shigella bypass the cell membrane (Adapted and redrawn from Veiga and Cossart
adhesion to the receptor but inject effector proteins by type 2006. Trends Cell Biol. 16, 499–504)
The trigger mechanism comprises of four Once the bacterium enters the cell, it employs
steps: (1) pre-interaction stage, (2) interaction adaptation strategies to promote intracellular sur-
stage, (3) macropinocytic pocket formation, and vival, effectively avoiding lysosomal enzymes,
(4) actin depolymerization and closure of the antibacterial peptides, low pH, reactive oxygen
macropinocytic pocket. In the pre-interaction radicals, and low nutrient concentrations. Some
stage, the bacterium synthesizes necessary pro- bacteria also induce actin polymerization for
teins in preparation for initiating an infection. In inter- and intracellular movement. When the bac-
the interaction stage, the bacterium injects dedi- terium reaches the plasma membrane of the adja-
cated bacterial effector proteins through the type cent cell, it forms a protrusion, which is
III secretion system (T3SS). In Salmonella, the endocytosed by the neighboring cell, and traps
effector proteins are SipB and SipC and in the bacterium inside a vacuole. The bacterium
Shigella, IpaB and IpaC. During formation of the then lyses the double membrane of the vacuole
macropinocytic pocket, bacteria initiate signaling using hemolysin and phospholipase and contin-
events that trigger massive actin polymerization ues the infection process. Some bacteria use a
and extension to form the entry foci, known as sugar uptake strategy to garner energy from the
membrane ruffling. In the final stage, the actin host cells. There are several advantages of intra-
depolymerization occurs and invasion proceeds. cellular residence: avoidance of killing by pha-
Fig. 4.5 Transmission electron microscopy picture of epithelial cell–cell junction in polarized Caco-2 cells. TJ, tight
junction; AJ, adherens junction; Des, desmososme (Image courtesy of Rishi Drolia and Arun Bhunia)
golysosomal degradative enzymes, access to an translocation from the lumen to the submucosal
abundance of nutrients in the cytoplasm, and location. However, the enteric pathogens have
protection from antibiotics, antibodies, and
developed diverse fascinating strategies to dis-
complement-mediated cell lysis. rupt the epithelial barrier for the localized or sys-
temic spread. Pathogens induce the production of
inflammatory cytokines: TNF-α and IL-6,
Pathogen Translocation by Epithelial through activation of NF-κB, a central regulator
Barrier Disruption of the epithelial innate immune response. The
relationship between the cytokine (TNF-α and
Some pathogens disrupt epithelial tight junction IL-1β)-induced NF-κB activation and the TJ bar-
integrity as a strategy to cross the epithelial barrier disruption is well documented (Fig. 4.6).
rier in the gut. Epithelial barrier architecture con- Many enteroinvasive pathogens modulate NF-κB
sists of four regions: tight junction (TJ), adherens signaling to establish a successful infection.
junction (AJ), desmosome (Des), and gap junc- EHEC, EPEC, Helicobacter pylori,
tion (Fig. 4.5). In TJ, claudin and occludin are the Campylobacter jejuni, and Yersinia pseudotuber-
major membrane proteins that are stabilized by culosis activate NF-κB, and the resulting inflam-
zona occludin (ZO), F-actin, myosin, and the matory cytokines dysregulate the junctional
myosin light-chain kinase (MLCK). In the AJ, proteins to allow bacterial passage across the epi-
E-cadherin is the major protein accompanied by thelial barrier. Yersinia pseudotuberculosis
two other structural proteins: α-catenin and secretes Yersinia outer protein J (YopJ) to induce
β-catenin. In the desmosome, desmoglein, des- IL-1β production in the gut to disrupt the intesti-
mocollin, and desmoplakin are the dominant pro- nal epithelial barrier – both paracellular and tran-
teins. The cytoskeletal proteins are highly scellular permeabilities through the activation of
regulated permitting nutrient and small molecule MLCK. Helicobacter pylori also induces the
Fig. 4.6 Schematic showing bacteria or bacterial components mediated epithelial tight junction disruption and enteric
bacterial paracellular translocation
paracellular permeability by generating cell- by activating NF-κB and MLCK to facilitate bac-
signaling events that counteract the normal func- terial paracellular translocation across the epithe-
tion of protein kinase C (PKC). Salmonella lium. Streptococcus pneumonia and Haemophilus
enterica serovar Typhimurium induces increased influenzae exploit toll-like receptor (TLR)-
paracellular permeability associated with the mediated downregulation of TJ protein to facili-
mislocalization of occludin and ZO-1. tate translocation across the epithelium.
Campylobacter jejuni also increases paracellular
permeability by activating phosphoinositide
3-kinase (PI3-K). Clostridium difficile toxin B Iron Acquisition
increases PKC-dependent RhoA glycosylation
and actin depolymerization to increase paracel- Iron (Fe3+) is essential for metabolic process, sur-
lular permeability. Shigella flexneri also increases vival, and growth in all living organisms. It is
paracellular permeability by regulating tight required for electron transport, metalloenzyme
junction-associated proteins. In Listeria monocy- activity (oxidoreductases, cytochromes, and non-
togenes, LAP disrupts intestinal barrier functions heme oxidases), and oxygen transport (higher
animals). Pathogenic microbes must acquire iron the intestine is constantly washed with fast-
from the host during infection. However, a major- moving fluids, the ciliary action of microvilli, and
ity of iron is present as insoluble ferric oxide/ the antimicrobials. Microbes display swimming,
hydroxide in the environment. In the human swarming, and gliding movement on surfaces.
body, free iron concentration is very low because Motile microbes have flagella and have a complex
iron is bound to proteins in the form of lactofer- sensory system that allows them to move by
rin, transferrin, ferritin, and hemoglobin. Three swimming and swarming motions in the direction
different ways bacteria can sequester iron from a of nutrients (sugars and amino acids) and oxygen.
host: (1) use of siderophore (from the Greek: Microbes move directionally toward the mucosal
“iron carriers”), (2) direct binding of bacteria to membrane, which provides a greater chance for
the host cells, and (3) killing of the host cells. colonization. For example, Vibrio expresses long
(1) Siderophores are low molecular weight filamentous pili called TCP (toxin-coregulated
organic compounds that are produced by micro- pili), which promote bacterial motility and coloni-
organisms and chelate iron with very high affin- zation of epithelial cells. Campylobacter jejuni
ity. Examples of siderophores are aerobactin, also uses flagella to colonize and invade the gut
enterobactin, catechol, hydroxamate, and so epithelial cells. Salmonella, Clostridium, Yersinia,
forth. After binding of siderophore to the ferric Serratia, Proteus, and Escherichia use flagella for
iron, the siderophore–iron complex is taken up swarming motility and for surface colonization.
by the cell following interaction with the sidero- Quorum-sensing molecules or chemosensors
phore receptor located on the bacterial cell sur- facilitate bacterial colonization through the action
face. Inside the cell, the complex is degraded and of swarming movement and by forming biofilms.
the iron is released. Siderophores are generally In addition, peptides and amino acids act as a che-
pathogen-specific: Salmonella produces entero- motactic factor for Proteus sp. while polysaccha-
bactin, a cyclic trimer of dihydroxybenzoic acid; rides for Salmonella Typhimurium and E. coli.
Yersinia spp. produce yersiniabactin, a catechol-
type siderophore; Vibrio cholerae produces vib-
riobactin; and E. coli produces aerobactin. (2) Evasion of Immune System
Pathogenic microbes also sequester iron by
directly binding to the host cells that carry iron Microbial ability to evade the host immune sys-
on their surface. (3) Some bacteria produce exo- tem is an important strategy for a pathogen to
toxin such as hemolysin that lyses the iron (heme cause disease. Certain bacteria such as
or ferritin)-bearing cells, such as the red blood Streptococcus pyogenes, S. pneumoniae, Bacillus
cell (RBC), when the iron level is low. The bacte- species, and Yersinia pestis express capsules,
rium scavenges the iron from lysed cells. Many which exert anti-phagocytic action. The capsule
pathogens produce hemolysin such as listerioly- prevents serum protein B from binding to the
sin O (LLO) by Listeria monocytogenes, α-lysine complement protein, C3b, but helps protein H to
by Staphylococcus aureus, perfringolysin O bind to C3b forming the C3bH complex, which is
(PFO) by Clostridium perfringens O, streptolysin easily degraded by protein I. As a result, no
O (SLO) by Streptococcus pneumoniae, and so C3bBb, also known as C3 convertase, is formed.
forth. This prevents the production of C3b and the com-
plement cascade from forming the membrane
attack complex (MAC). As a result, the microbes
Motility and Chemotaxis are protected from complement-mediated killing.
Sialic acid and the hyaluronic acid, components
Microbial motility is very important for survival of the capsule, are also present in the mammalian
and existence in nature and in a host. For enteric cell membrane, thus avert binding of the C3b to
pathogens to colonize and persist in the gut, bacterial capsule preventing macrophage recog-
microbes must overcome many obstacles, since nition and phagocytosis. Binding of bacterial
Toxicoinfection 99
lipopolysaccharide to C3b also prevents the for- intoxication cases, the microbial cells in the food
mation of C3 convertase and prevents the forma- matrix transit through the digestive system with-
tion of MAC and complement-mediated killing. out causing any harm. The general mechanism of
Intracellular pathogens also evade the immune pathogenesis of toxins involved in intoxication is
system and survive phagocytosis by producing described below.
hemolysin, superoxide dismutase that destroys
“O” radical, and catalase that breaks down H2O2.
Some pathogens evade the host antibody Toxicoinfection
response by shifting the antigenic structure, such
as structural variations in pili as well as in other Some bacteria are responsible for causing toxi-
surface proteins through mutation in correspond- coinfection, which happens when the ingested
ing genes. The antigenic variation disguises the bacteria first colonize the mucosal surface and
pathogen and fools the immune system from rec- then produce exotoxins in the GI tract. Similar to
ognition. In addition, some pathogens evade the intoxication, the toxins induce toxic effects on
immune system by shrouding themselves with the local cells or tissues, and in some cases, tox-
the host proteins. For example, Streptococcus ins enter the bloodstream and cause disease.
species cover themselves with host fibronectin, Toxicoinfection-causing organisms are V. chol-
and Staphylococcus aureus and Streptococcus erae that produce cholera toxin, enterotoxigenic
pyogenes coat themselves with host IgG using E. coli (ETEC) that produce heat-labile (LT) and
the surface-expressed protein A and protein G, heat-stable (ST) toxins, and Clostridium perfrin-
respectively. Protein A and protein G bind to the gens that produce clostridium perfringens entero-
Fc part (as opposed to the antigen-binding Fab toxin (CPE). In some cases, toxins kill
part) of the IgG, therefore preventing macro- polymorphonuclear leukocytes (PMNL) and aid
phages from recognizing the antigen–antibody bacterial growth and spread as seen in patients
complex. Many pathogens such as Streptococcus suffering from gas gangrene and myonecrosis
pneumoniae, Haemophilus influenzae, Neisseria caused by Clostridium spp. In addition,
gonorrhoeae, and Neisseria meningitides pro- Clostridium spp. produce hydrolytic enzymes
duce immunoglobulin (sIgA)-specific proteases such as lecithinase to break down lecithin, hyal-
to cleave IgA in the hinge region to make it inef- uronidase and protease to disrupt extracellular
fective for antibody-dependent inactivation. matrix and tissue structure, and DNase to reduce
the viscosity of debris from dead host cells, to aid
in bacterial propagation.
Intoxication
Ingestion of preformed exotoxins such as staphy- Toxins

lococcal enterotoxin, botulinum toxin, Bacillus
cereus emetic toxin, mycotoxin, and seafood tox- Broadly, two types of toxins are produced by
ins cause food poisoning or intoxication microbes: exotoxin and endotoxin. Exotoxins are
(Table 4.4). Bacteria present in foods grow under usually excreted outside in the extracellular
a favorable condition and produce toxins. milieu through an active transport system or
Following ingestion of food, toxins are absorbed remained cell associated until they are released
through the gastrointestinal epithelial lining and from the cell after lysis. Examples of exotoxins
cause local tissue damage, induce inflammation, are a botulinum toxin, cholera toxin, Shiga toxin,
and promote vomiting and diarrhea. In some diphtheria toxin, and so forth, while the endotox-
cases, toxins are disseminated through the blood ins are part of the cell structure, such as lipopoly-
or lymphatics to distant organs or tissues such as saccharide (LPS) in Gram-negative bacteria and
the liver, kidney, or peripheral or central nervous peptidoglycan (PGN) and lipoteichoic acid (LTA)
system where they can cause damage. In most in Gram-positive bacteria. Some toxins are
100
Table 4.4 Bacterial toxins and their characteristics

Toxin type Toxins Host cell receptor Producing bacteria Mode of action Target
Membrane damaging Hemolysin Cholesterol E. coli Pore formation Plasma membrane
toxin Listeriolysin O (LLO) Cholesterol L. monocytogenes Pore formation Cholesterol
Perfringolysin O (PFO) C. perfringens Pore formation Cholesterol
α-toxin Cholesterol S. aureus Pore formation Plasma membrane
Streptolysin O Cholesterol S. pyogenes Pore formation Cholesterol
Inhibit protein Shiga toxin or Shiga-like toxin Gb3 (globotriaosylceramide), Gb4 Shigella spp. E. coli N-glycosidase 28S rRNA
synthesis (A–B type) (globotetraosylceramide)
Diphtheria toxin A growth factor receptor C. diphtheriae ADP-ribosylation Elongation factor-2
Anthrax protective antigen ATR (anthrax toxin receptor) B. anthracis Translocates anthrax LF Clathrin-coated pit
(PA) and EF
Activate second Heat-labile toxin (LT) Ganglioside (GM1) E. coli ADP-ribosyltransferase G-proteins
messenger pathways Heat-stable toxin (ST): STa STa: Guanylate cyclase C E. coli Stimulates guanylate Guanylate cyclase
(A–B type) and STb STb: Sulfatide (Glycosphingolipid) cyclase
Cholera toxin Ganglioside (GM1) V. cholerae ADP-ribosyltransferase G-protein
Activate immune Enterotoxins, toxic shock MHC class II S. aureus Superantigen TCR and MHC II
response toxins
4
Protease action Lethal factor (LF) MAPK B. anthracis Metalloprotease MAPK1

MAPK2
Edema factor (EF) ATP B. anthracis Adenylate cyclase ATP
Botulinum neurotoxin Ganglioside C. botulinum Zinc metalloprotease Synaptobrevin,
SNAP-25, syntaxin
Apoptosis-inducing IpaB Membrane Shigella spp. Apoptosis
toxins LLO Cholesterol L. monocytogenes Apoptosis
Adapted and modified from Schmitt et al. 1999. Emerg. Infect. Dis. 5, 224–234
General Mechanism of Pathogenesis
Toxicoinfection 101
Fig. 4.7 Diagram

showing the mechanism
of action of an A–B-type
toxin
d esignated enterotoxins such as cholera toxin and while the B domain has the receptor binding
E. coli LT toxin because they are responsible for function and has the tropism for a specific host
gastroenteritis. Some toxins are also called cyto- cell. The sequence of events for A–B-type toxins
toxins because these are either cell- or tissue- is binding to the receptor, internalization, mem-
specific: neurotoxin affects nerve cell, leukotoxin brane translocation, and enzymatic activity and
attacks leukocyte, hepatotoxin injures liver cell, target modifications (Fig. 4.7). Examples of
and cardiotoxin damages cardiac tissue. Toxin A–B-type toxins are Shiga toxin, cholera toxin,
designation is occasionally based on the bacterial botulinum toxin, and ST (heat satble) and LT
species that produces them: cholera toxin is pro- (heat labile) toxins from E. coli. (2) Membrane-
duced by Vibrio cholerae, Shiga toxin by Shigella disrupting toxins either insert into the cell mem-
species or Shiga-toxigenic E. coli, diphtheria brane to cause pore formation (e.g., hemolysin)
toxin by Corynebacterium diphtheriae, tetanus or remove lipid head groups to destabilize the
toxin by Clostridium tetani, and botulinum toxin lipid bilayer membrane (e.g., phospholipases).
by Clostridium botulinum. Sometimes the toxin (3) Toxin-mediated activation of secondary mes-
is named based on the action it exerts such as senger pathway: Toxins like E. coli LT and ST or
adenylate cyclase produced by Bordetella pertus- cholera toxin interfere with cellular signal trans-
sis and lecithinase by Clostridium perfringens duction pathways. (4) Superantigens activate the
and Listeria monocytogenes. Toxins may also be immune system to produce excess amounts of
designated by a letter such as staphylococcal proinflammatory cytokines, which exert their
enterotoxin A (SEA) or SEB or SEC produced by deleterious effects on the host cells, provoking
Staphylococcus aureus. toxic shock syndrome or septic shock. The exam-
ples of superantigens are staphylococcal entero-
tructure and Function of Exotoxins
S toxin (SE), exfoliative toxin, and toxic shock
There are six types of exotoxins, and they are syndrome toxin (TSST). (5) Proteases: Some
grouped based on the structure and the mode of toxins such as botulinum neurotoxin inactivate
action they exert on the host (Table 4.4; Fig. 4.7): metalloproteases (zinc metalloprotease) action,
(1) A–B-type toxin is a single protein or an oligo- thereby interfering with the propagation of nerve
meric protein complex organized with A and B impulse. (6) Toxin-induced apoptosis or necrosis:
domain structure–function organization. The A Some toxins kill cells by inducing programmed
domain has the enzymatic or catalytic activity, cell death (apoptosis or necrosis).
Fig. 4.8 Diagram showing the mechanism of action of pore-forming toxins such as (a) hemolysin and (b)
phospholipase
A–B Toxins theria toxin (DT) and Pseudomonas exotoxin-A

The A–B-type toxin is a single protein or an cause ADP-ribosylation of EF2 and consequently
oligomeric protein complex organized with A lead to cell death. In contrast, cholera toxin and
and B domain structure–function organization. E. coli LT cause ADP-ribosylation of the
The A domain has a catalytic activity, while the B G-protein, a signal transduction protein, which
domain has receptor binding function and has the controls the adenylate cyclase activity, which in
tropism for specific host cells. The A and B turn increases the cAMP levels. The increased
domains are linked by a disulfide bond or by a cAMP controls ion (Na+, K+, Cl−) pumps and
noncovalent bond. B domain can be a single fluid movement in cells (see Chap. 14 and 18).
monomeric form (B) or an oligomeric form (B5). Stx displays a different type of action – it has
The B domain binds to a specific receptor such as adenine glycohydrolase activity. It removes a
a glycoprotein or glycolipid on the cell surface single adenine residue from the 28S rRNA and
and binding is very specific. If the receptor is blocks protein synthesis (see Chap. 14).
found only on the neuron, B domain will bind to
the neuron only. After interaction of A–B toxin Membrane-Disrupting Toxins
with the receptor, the entire complex is internal- A large group of bacterial pore-forming toxins
ized by endocytosis. The B domain also has the (PFT) is reported. These are protein toxins and
capacity to help translocate the A domain across form pores in the membrane of bacteria, mam-
a lipid bilayer, either at the plasma membrane or malian cells, and plant cells resulting in increased
within the endosome by forming a pore or chan- membrane permeability and ionic imbalance
nel. Through a cellular process, the A domain is (Fig. 4.8). As water enters the cell, it swells and
separated from the B and exerts its enzymatic or ruptures the cell. The soluble PFT toxin produced
catalytic activity; however, the function of A is by bacteria inserts into the target membrane
not specific for a cell. If A is delivered experi- forming a stable multimeric structure. Depending
mentally into a cell, it will exert its function inde- on the type of multimeric structure the PFT
pendent of the B domain. In some toxins, the A forms, it is classified into α-PFT and β-PFT.
domain has ADP-ribosyltransferase activity. This α-PFT forms α-helix structure when forming a
removes ADP-ribosyl group from NAD+ and pore in the membrane while the β-PFT forms
attaches it to a host cell protein, such as elonga- β-sheet during membrane pore formation. The
tion factor 2 (EF2) in a process called ADP- best-described α-PFT is colicin, a bacteriocin
ribosylation of EF2. Modified EF2 is unable to (ColE) produced by E. coli, and it causes lysis of
help in host protein synthesis. For example, diph- other E. coli cells.
Toxicoinfection 103
β-PFTs are hemolysins that oligomerize and accumulation within the lumen of the intestine
form stable multimeric structures in the mamma- and diarrhea.
lian cell membrane. The amphipathic β-hairpins
on a monomeric subunit help insertion of the Superantigens
toxin into the hydrophobic membrane to form Superantigens are a group of protein toxins that
pores, and the size of pores ranges from 2 to show unusual activation of the immune system
50 nm. The largest group of β-PFTs are resulting in septic shock. Superantigens are pro-
cholesterol-dependent cytolysins (CDC) produced by Staphylococcus aureus, Streptococcus
duced by Gram-positive bacteria such as listerio- spp., Mycoplasma arithiditis, and Yersinia pseu-
lysin O (LLO) by L. monocytogenes, streptolysin dotuberculosis. Staphylococcal enterotoxins
O (SLO) by Streptococcus pyogenes, pneumoly- (SEA, SEB, SEC, SEE, etc), exfoliative toxins,
sin O (PLO) by S. pneumoniae, and perfringoly- and toxic shock syndrome toxins (TSST) are
sin O (PFO) by C. perfringens. The CDC pore well-studied superantigens. Unlike other protein
complex formation occurs before the protein antigens, superantigens are not processed (i.e.,
insertion into the membrane. The steps include proteolytic digestion) by the antigen-presenting
lateral diffusion of monomers with the mem- cells (APC). Instead, these toxins bind directly to
brane, pre-pore monomer oligomerization, pore the MHC class II molecules of APC and activate
formation, and insertion of the oligomer into the T cells carrying a T-cell receptor (TCR) com-
membrane forming a channel. Phospholipases posed of the Vβ chain (Fig. 4.9). Members of the
are also considered hemolysins since they remove superantigens possess different structures that
(
the charged phosphate head group PO3+ from ) have an affinity for MHC class II molecules.
the lipid (diacylglycerol) portion of the phospho- Activated T cells then produce a large quantity of
lipid, which destabilizes the membrane structure IL-2, which in turn activates macrophages for
to cause cell lysis (Fig. 4.8). increased production of IL-1, IL-6, TNF-α, and
IFN-γ. IFN-γ also induces increased expression
Induction of Second Messenger Pathways of class II molecules on professional phagocytic
Some toxins alter signal transduction pathways, and nonprofessional phagocytic cells such as epi-
which are required for various cellular functions. thelial cells and subsequently enhanced presenta-
For example, E. coli CNF (cytotoxic necrotizing tion of superantigens to T cells. Production of
factor) 1 or 2 and LT, Clostridium botulinum large quantities of IL-2 and TNF-α also induces
toxin and C3 toxin, and Vibrio cholerae cholera nausea, vomiting, malaise, fever, erythematosus
toxin inactivate or modify a small GTP-binding lesion, and toxic shock.
signal transduction protein, Rho, which regulates
actin cytoskeleton formation. Destabilized epi- Protease
thelial cell architecture increases membrane per- Some toxins also possess enzymatic activities
meability and fluid loss. and cleave target proteins thus interfere with cel-
E. coli STa binds to the membrane guanylate lular function. For example, botulinum neuro-
cyclase C receptor on epithelial cells in the small toxin with zinc metalloprotease activity cleaves
intestine and colon. STa binding to guanylate SNARE (soluble N-ethylmaleimide-sensitive
cyclase C activates protein kinase G (PKG) and factor attachment protein receptor) proteins,
protein kinase C (PKC), increasing IP3-mediated which consists of synaptobrevin, SNAP-25
calcium to increase intracellular cyclic GMP (synaptosomal-associated protein-25), and syn-
(cGMP). Increased cGMP activates calcium ion taxin. The SNARE complex is responsible for
channel and cystic fibrosis transmembrane regu- acetylcholine release from the synaptic vesicle at
lator (CFTR) and increases the concentration of the neuromuscular junction. As a result, neu-
Cl− ions in the extracellular space. As a result, rotransmitter release is impaired causing paraly-
electrolyte imbalance in the bowel leads to fluid sis (see Chap. 12 for details).
Fig. 4.9 Diagram showing the mechanism of action of a superantigen. APC, antigen-presenting cell, TCR, T-cell
receptor, MHC, the major histocompatibility complex
Cells Death in this type of cell death. Necrosis is considered a

Host cell death is a defensive strategy for the host passive event described as “accidental or uncon-
against microbial infection thus helping maintain trolled death” inducing a strong inflammatory
immune homeostasis and host defense. In response. In recent years, however, programmed
response to an infection, the host cell may cell death by necrosis has been proposed as “pro-
undergo programmed cell death for its own ben- grammed necrosis,” “regulated necrosis,” or
efit: eliminate the pathogen during the early “necroptosis.” Pyroptosis is considered a non-
phase of infection without any inflammatory sig- apoptotic cell death but is dependent on caspase-1
nals and activate dendritic cells to remove apop- enzyme activity and induction of inflammatory
totic bodies containing pathogen to induce response. The cell death is characterized by the
MHC-mediated antigen presentation and protec- loss of membrane integrity, cytoplasmic content
tive immunity. Cell death also benefits the patho- release, and inflammatory response (IL-1β and
gen – successful exit from the infected cell to IL-18).
infect neighboring cells, access to nutrients,
evade immune system, and persistence in a host. Apoptosis Microbial toxins induce apoptosis as
However, some intracellular pathogens have part of their infection strategy. The dead cells are
developed strategies to prevent host cell death then removed by phagocytes, thus avoiding the
during infection for their replication, escape, and onset of an inflammatory response. The typical
dissemination of new host cells. Nevertheless, signs of apoptosis (Fig. 4.11) are as follows:
the cell death is a highly controlled and regulated
event that plays a significant role during micro-
bial pathogenesis. There are three types of cell (a) Cell shrinkage – condensation of cell cyto-
deaths: apoptosis, necrosis, and pyroptosis plasm and nucleus.
(Fig. 4.10). Apoptosis is also known as pro- (b) Entrance of cells into the zeiosis stage – cell
grammed cell death referred to as cell “suicide.” morphology alters and shrinkage continues.
It is an active, programmed process of autono- (c) Chromatin condensation/margination –
mous cellular destruction without evoking an crescent-
shaped nucleus localizes to the
inflammatory response. Caspase-1 is not involved nuclear membrane.
Toxicoinfection 105
Fig. 4.10 Depiction of four types of cell death: apopto- species; RIP, receptor-interacting kinase; PAMPs, patho-
sis, pyroptosis, necrosis, and necroptosis. Casp, caspase; gen-associated molecular patterns; DAMPs, danger-asso-
FADD, Fas-associated death domain; TRADD, TNF ciated molecular patterns; NLRP, NOD-like receptor
receptor-associated death domain; ROS, reactive oxygen protein
(d) DNA fragmentation – cysteine proteases (f) Clearance of apoptotic bodies by phagocytic
(caspases) and nucleases are activated which cells – macrophages/dendritic cells engulf
cleave DNA in the factor of ~168 bp frag- apoptotic cells/bodies to prevent
ments resulting in the laddering of DNA inflammation.
bands, which can be detected by gel
electrophoresis. The recognition of apoptotic cells by macro-
(e) Membrane blebbing of apoptotic bodies con- phages is mediated by binding to the phosphati-
taining DNA fragments occurs which can be dylserine (PS) displayed on the lipid bilayer
seen under a microscope. membrane. Normally, PS is located in the inner
Fig. 4.11 (A) Schematics of different stages of apoptotic (b) zeiosis stage, cell morphology alters and shrinkage
cell death and (B) fluorescence photographs of human continues; (c) chromatin condensation/margination, cres-
hybridoma B cells infected with Listeria monocytogenes cent-shaped nucleus; (d) DNA fragmentation; (e) mem-
and stained with ethidium bromide and acridine orange at brane blebbing of apoptotic bodies; and (f) clearance of
different stages of apoptotic cell death: (a) cell shrinkage; apoptotic bodies by phagocytic cells
leaflet of the cytoplasmic lipid bilayer mem- Procaspase-9 activates caspase-3, caspase-6, and
brane; however, when cells are undergoing apop- caspase-7 (known as executioner caspase), which
totic cell death, the PS translocates from the inner break down nuclear proteins histone, actin/lam-
leaflet of the membrane to the outer leaflet and ins, and poly-ADP-ribose polymerase (PARP)
becomes a target for macrophage-mediated leading to apoptosis. C-Myc and p53 proteins
clearance. induce programmed cell death, while the Bcl-2
Apoptosis is a highly regulated process of proprotein family, normally present in the malignant
grammed cell death. Apoptotic stimuli such as cells, suppresses apoptosis.
bacterial toxins activate death ligands FADD Shiga toxin and IpaB protein in Shigella,
(Fas-associated death domain) and TRADD LLO in L. monocytogenes, cholera toxin in V.
(TNF receptor-associated death domain), which cholerae, and Clostridium perfringens entero-
in turn activate procaspase-8. Procaspase-8 phos- toxin (CPE) are some examples, which induce
phorylates Bcl-2-associated death domain programmed cell death. In contrast, some tox-
(BAD), which consequently activates DNase and ins such as LLO and CPE at high concentra-
procaspase-9 (cysteine protease). Procaspase-8 tions induce oncosis, a form of necrosis by
and procaspase-9 are known as initiator caspase. causing physical trauma that leads to cell
Toxicoinfection 107
s welling, lysis, random DNA shearing, and the min-N domain forms a pore in the plasma mem-
release of toxic intracellular contents, which brane to induce cell swelling and osmotic lysis.
induce inflammation. Pathogen-associated molecular patterns (PAMPs)
or danger-associated molecular patterns
Necrosis Necrosis is called non-apoptotic or (DAMPs) are recognized by inflammasomes
accidental cell death characterized by membrane (e.g., NLRP, NOD-like receptor protein), which
lysis, organelle swelling, and the release of cel- activate caspase-1and trigger pyroptosis.
lular contents, which induce strong inflamma- Pyroptosis is characterized by DNA fragmenta-
tion. Necrosis occurs independently of caspase tion, loss of membrane integrity, cytoplasmic
enzyme involvement in the cell death process. content release, and inflammatory response.
Necrosis can be triggered by reactive oxygen
species (ROS) production or danger signals, such
as depletion of ATP, lysosomal destabilization Endotoxin
resulting from bacterial infection, or physical
damage. However, some forms of necrotic deaths Bacterial structural components, such as LPS in
are genetically programmed and termed “pro- Gram-negative bacteria and PGN, WTA and LTA
grammed necrosis.” Programmed necrosis is in Gram-positive bacteria, are called endotoxin
similar to the conventional necrosis process but or pyrogen. They are released after cellular
involves activation through intrinsic and extrinsic destruction or lysis. LPS consists of the hydro-
factors resulting in the activation of signaling phobic lipid A component which is highly toxic
cascades leading to cell death referred to as “pro- (endotoxin), a nonrepeating “core” oligosaccha-
grammed necrosis,” “regulated necrosis,” or ride, and a distal polysaccharide (or O-antigen).
“necroptosis.” It can be initiated by death ligands, A high level of pyrogen in blood circulation is
TLR (toll-like receptor), FADD, TRADD, NLR referred to septicemia and is responsible for the
(NOD-like receptor) and microbial infection. In rise in body temperature (fever). LPS forms a
programmed necrosis, the signal from death complex with LPS-binding protein (LBP) and
receptor such as TNF receptor activates the then interacts with the cellular membrane recep-
receptor-interacting kinase 1 (RIP1) and 3 tor, TLR4, and CD14 on macrophages/mono-
(RIP3), which consequently activate downstream cytes, and through MyD88-dependent signaling
targets, leading to calcium and sodium influx, pathway, it activates NF-κB to produce inflam-
membrane lysis, and programmed necrosis. matory cytokines, IL-1β and TNF-α, which in
Programmed necrosis is a strong antiviral and turn cause the hypothalamus to release prosta-
antibacterial defense strategies and seen against glandins (Fig. 4.12). Increased prostaglandin
vaccinia virus, reovirus, Mycobacterium, level causes the body temperature to rise resulting
Salmonella, Yersinia infection, and C. perfrin- in fever development. In principle, this defense
gens toxins. strategy is designed to suppress bacterial growth.
Aspirin blocks prostaglandin release and thus
lowers the body temperature.
Pyroptosis Pyroptosis is a non-apoptotic cell LPS at low concentration stimulates macro-
death, but it depends on the activity of caspase-1 phages to produce IL-1 and acts as a polyclonal
enzyme, which promotes the production of proin- activator of B cells. LPS at a higher concentration
flammatory cytokines, IL-1β and IL-18, leading can trigger a massive inflammatory response
to a strong inflammatory response. In addition, leading to septicemia and septic shock. LPS acti-
caspase-4, caspase-5, and caspase-11 are also vates monocytes and macrophages to release
involved in pyroptosis. The pyroptosis execu- IL-1, IL-6, IL-8, and TNF-α, which induce pros-
tioner molecule, gasdermin D, serves as a sub- taglandin and leukotriene production, leading to
strate of caspase-1, caspase-4, caspase-5, and the damage of endothelial cells, tissue injury,
caspase-11, and the breakdown product gasder- and disseminated (widespread) intravascular
Fig. 4.12 Lipopolysaccharide (LPS)-mediated inflammation and septic shock
coagulation (DIC) allowing neutrophils, lympho- enetic Regulation and Secretion

G
cytes, and monocytes to stick to the endothelial Systems for Virulence Factors
surfaces (Fig. 4.12). As a result, blood pressure
drops, the patient enters into a shock (referred to Pathogenicity Islands
as septic shock), and death ensues. During septic
shock, the circulatory system collapses, blood A genomic segment that carries a cluster of viru-
pressure drops, fever increases, heart rate lence genes is called pathogenicity island (PAI), a
increases, multiple organs fail, and death follows. term that was first introduced by Prof. Jorg
Prolonged exposure to LPS invokes cachexia, Hacker in the late 1980s. It was first discovered in
characterized by wasting of the muscle and fat an uropathogenic E. coli strain 536, and it was
cells. LPS is also responsible for appetite sup- demonstrated that the deletion of the PAI resulted
pression resulting in weight loss. Endotoxins also in a nonpathogenic phenotype. The PAI encodes
activate the complement pathway (alternative genes for iron acquisition, adhesions, pore-
pathway) to form C3a and C5a, which also cause forming toxins, proteins responsible for apopto-
severe endothelial cell damage. A membrane sis, secondary messenger pathway toxins,
attack complex (MAC) forms and causes cell superantigens, lipases, proteases, O antigens, and
lysis. protein secretion systems such as type I, type II,
Genetic Regulation and Secretion Systems for Virulence Factors 109
type III, type IV, type V, type VI, and type VII Protein Secretion System
secretion systems.
Specific characteristics of a PAI are as fol- Bacteria have developed highly specialized mac-
lows. (1) PAI carries one or more virulence genes. romolecular structures to secrete a wide range of
(2) It is present in pathogenic species but absent molecules, including small molecules, proteins,
in nonpathogenic species. (3) The size range of and DNA. These secreted molecules perform a
PAI is 10–200 kb. (4) The G + C content of the variety of tasks, including pathogenicity, cell
PAI genome (40–60%) is lower than that of the envelope assembly, metabolism, and interaction
core genome (25–75%). (5) PAI are located close with the host cells. In Gram-negative bacteria, the
to tRNA genes, indicating that the tRNA gene secretion is mediated by a different mechanism
serves as an anchor point for insertion of foreign than that of the Gram-positive bacteria because
genes. Note: some bacteriophages also use tRNA of the presence of an outer membrane (OM)
as the insertion points in the host genome. (6) structure (see Chap. 2). In Gram-negative bacte-
PAI may be unstable and deleted from the genome ria, the secreted molecules are translocated either
with a frequency higher than the normal rate of to the outer membrane, to the extracellular
mutation. (7) PAIs are frequently associated with milieu, or to the eukaryotic cells during host cell
mobile genetic elements and are flanked by the interaction. Therefore, different types of secre-
direct repeats (DR). DRs are 16–20 bp long tory machinery are required to achieve the same
sequence with perfect or near-perfect sequence goal. The secretion systems in Gram-negative
repetitions, which serve as integration sites for bacteria are classified as type I secretion system
bacteriophages. PAIs also carry genes for inte- (T1SS), T2SS, T3SS, T4SS, T5SS, T6SS and
grases or transposes, probably acquired from the T7SS (Fig. 4.13). Structural organization shows
bacteriophage. The insertion sequence (IS) ele- that the T1SS, T3SS, T4SS, and T6SS span from
ments are also found in PAI. In addition, PAI can the inner cytoplasmic membrane (CM) to the
represent integrated plasmids, conjugative trans- outer membrane (OM) and deliver molecules
posons, and bacteriophages. directly from cytoplasm to the extracellular loca-
Fig. 4.13 The depiction of protein secretion system in Gram-negative bacteria (Adapted and redrawn from Costa et al.
2012. Nat. Rev. Microbiol. 13, 343–359). OM, outer membrane; CW, cell wall; CM, cytoplasmic membrane
tion, while T2SS and T5SS span only to the OM directly from bacteria to the eukaryotic mem-
and protein secretion involves a two-step process, brane or cytoplasm during interaction with the
first from cytosol to the periplasmic space facili- host cell. It is called a molecular syringe (injecti-
tated by SecYEG translocon or Tat translocon some) and its assembly requires the function of
located in the cytoplasmic membrane and second several genes. In Salmonella enterica serovar
from periplasmic space to the extracellular loca- Typhimurium, it involves about 25 genes and a
tion using a dedicated OM spanning secretion multiprotein (25 proteins) complex of 3.5 MDa.
system. T7SS is found only in Mycobacterium A typical T3SS apparatus has four major parts:
spp., since it has a specialized envelope similar to inner rings, neck, outer rings, and a needle
a Gram-negative bacterium. (Fig. 4.14). Assembly of the T3SS is similar to
that of the flagellum assembly machinery. The
ype I Secretion System
T T3SS is involved in pathogenesis. During inti-
The type I secretion system (T1SS) has an assem- mate contact with the host cells, bacteria use the
bly of an ATP-binding cassette (ABC) transporter T3SS to inject virulence proteins into the host
protein located within the inner membrane, a cell to modulate specific host cell function to pro-
periplasmic membrane, and an outer membrane. mote colonization, invasion, actin-based intra-
T1SS transports a variety of proteins of different and intercellular motility, to induce apoptosis,
sizes and functions from cytoplasm to extracel- and to interfere with intracellular transport pro-
lular milieu including hemolysins. T1SS is cess. The protein translocation is an ATPase-
equivalent to the RND (resistance– nodulation– dependent process. T3SSs are present in various
division) family of the multidrug efflux pump. pathogenic Gram-negative bacterial genera such
RND pump secretes small molecules including as Salmonella, Shigella, Yersinia, Pseudomonas,
antimicrobial compounds out of the cell, thus EPEC, and EHEC. Genes required for T3SS for-
contributing to the antibiotic resistance. mation are encoded in plasmids or in PAI. Genes
for T3SS in Salmonella enterica are located in
ype II Secretion System
T SPI-1 and SPI-2, in the locus of enterocyte
The type II secretion system (T2SS) consists of effacement (LEE) of EPEC and EHEC, and in
12–15 components that are located in the inner PAI in a large plasmid of Shigella.
membrane, the periplasm, and the outer mem-
brane. It is responsible for secretion of folded pro- ype IV Secretion System
T
teins from the periplasm to the extracellular The type IV secretion system (T4SS) is similar
environment across the outer membrane of both to T3SS, but in addition to proteins, it can also
pathogenic and nonpathogenic Gram-negative translocate DNA or DNA–protein complex into
bacterial species that are important for bacterial the eukaryotic host. It is the most ubiquitous
survival and growth. T2SS transports proteins, secretion system in nature and found in both
such as pseudolysin, a hydrolyzing enzyme of Gram-positive and Gram-negative bacteria and
Pseudomonas aeruginosa; pullulanase, an amylo- in some Archaea. Since it translocates plasmids,
lytic enzyme of Klebsiella pneumoniae; and chol- it contributes to the spread of antibiotic resis-
era toxin of V. cholerae. The T2SS substrates are tance. It is also involved in bacterial pathogene-
transported as unfolded proteins from the cytosis and helps translocation of toxins and other
plasm to periplasm by using SecYEG translocon effector molecules to sustain an intracellular
located in the cytoplasmic membrane. The equiv- lifestyle. T4SS plays an important function in
alent of T2SS in Gram-positive bacteria is called the pathogenesis of human pathogens, Bordetella
the Sec (secretory) system (see below). pertussis, Legionella pneumophila, Brucella
spp., and Helicobacter pylori. The best-studied
ype III Secretion System
T T4SS is a plant pathogen, Agrobacterium tume-
The primary function of the type III secretion faciens, where it mediates the translocation of
system (T3SS) is to translocate effector proteins DNA–protein into the plant cells to induce tumor
Fig. 4.14 A schematic

model of the type III
secretion system (T3SS)
of Salmonella enterica
(Adapted and redrawn
from Galan and
Wolf-Watz 2006. Nature
444, 567–573; and
Galan and Collmer
1999. Science 284,
1322–1328)
formation. T4SS is a complex structure com- Type VI Secretion System

posed of about 12 proteins and is encoded by Ti The T6SS is composed of 13 components and
plasmid of A. tumefaciens. spans from the cytoplasm to the outer membrane
and is distributed among Proteobacteria. It has
ype V Secretion System
T two main complexes: the cytoplasmic membrane
The type V secretion system (T5SS) is also proteins similar to T4SS and the tail complex
known as an autotransporter. It is a single related to the contractile bacteriophage tail. T6SS
membrane-spanning secretion system. The secre- translocates toxic effector proteins into eukary-
tion system and the substrates are synthesized as otic and prokaryotic cells and plays important
preproproteins and are released into the peri- role in pathogenesis and bacterial competition.
plasm via the SecYEG translocon. After proteo-
lytic cleavage of the N-terminal leader peptide, Type VII Secretion System
the transporter domain of the proprotein is oligo- The type VII secretion system (T7SS) is a spe-
merized to form a β-barrel structure in the outer cialized secretion system found in Mycobacterium
membrane, which allows the “passenger” domain tuberculosis and is responsible for secretion of
to be excreted outside. The T5SS secretes pro- virulence proteins. The cell envelope of
teins involved in pathogenesis, cell-to-cell adhe- Mycobacterium consists of a cytoplasmic mem-
sion, and biofilm formation. In pathogenic E. brane, a periplasmic space that contains peptido-
coli, T5SS is encoded by genes located on EspC glycan, and a thick complex waxy outer
PAI and in Salmonella enterica; it is encoded by membrane called mycomembrane consisting of
SPI-3. arabinogalactan, mycolic acids, and free lipids.
Fig. 4.15 The Sec machinery in Gram-positive bacteria (Adapted and redrawn from Feltcher and Braunstein
for protein transport. (a) Canonical SecA-mediated pro- 2012. Nat. Rev. Microbiol. 10, 779–789)
tein transport system, (b) SecA2-dependent system
T7SS is a 1.5 MDa protein complex and the gene of three proteins: SecY, SecE, and SecG. It trans-
clusters have been identified in Listeria monocy- locates primarily the unfolded precursor proteins
togenes, Bacillus subtilis, and Staphylococcus across the CM. Most, but not all, Sec-dependent
aureus. proteins contain a classical Sec signal sequence
at the amino acid terminus, except for a few pro-
SecA System teins whose translocation and/or secretion are
All bacteria possess a conserved general secre- dependent on SecA. SecA, a cytosolic motor pro-
tion pathway (Sec) to translocate proteins across tein, binds to preprotein and drives its transloca-
the cytoplasmic membrane (CM) (Fig. 4.15). The tion through the SecYEG channel. During this
Sec pathway or the SecYEG translocon consists event, the Sec signal peptide sequence remained
associated with SecYEG channel. There are two Transcription consists of three steps: initiation,
SecA proteins, SecA1 and SecA2, and both are elongation, and termination of RNA synthesis, in
present in all mycobacteria and a small subset of which the RNA polymerase is involved in the
Gram-positive bacteria including Staphylococcus, synthesis of mRNA from the DNA. The core
Listeria, and Streptococcus spp. SecA1 is respon- RNA polymerase (RNAP) has five subunits: α1,
sible for protein transport via a canonical Sec α2, β, β′, and ω. For the RNAP to bind a specific
pathway as described above and is essential. promoter, another subunit known as sigma factor
SecA2 is present in some species of Gram- (σ) is required. The sigma factor greatly increases
positive bacteria but is absent in Gram-negative the specificity of binding of RNAP and helps in
bacteria and is dispensable. SecA2 contributes to the separation of DNA strands during the initia-
virulence and may have a specialized function for tion of the transcription process. The sigma fac-
proteins, which cannot be efficiently translocated tors detach from the DNA after the initiation
by the canonical Sec system. For example, SecA2 process of transcription has begun.
is required for optimal secretion of superoxide Prokaryotic sigma factors are classified into
dismutase (SodA) in L. monocytogenes and SodA three structurally unrelated families: σN, σ54, and
and catalase–peroxidase in Mycobacterium and σ70. The σN is mostly involved in nitrogen metab-
therefore contributes to bacterial virulence by olism and is also involved in a variety of meta-
countering the oxidative defenses of the host bolic processes. The σ70 family is larger and more
cells. SecA2 is also important in the pathogenesis diverse than σ54 and is divided into four groups
of Streptococcus gordonii, Streptococcus para- (group I–IV) based on their primary amino acid
sanguinis, Staphylococcus aureus, sequences and structures. The group I sigma pro-
Corynebacterium glutamicum, Clostridium diffi- tein/the primary sigma factor (e.g., σA of Bacillus
cile, and Listeria monocytogenes by facilitating subtilis) is also known as “housekeeping” sigma
secretion of many virulence proteins. factor because it regulates expression of “house-
keeping” genes or genes responsible for basic
metabolic processes and cell functions. The other
Regulation of Virulence Genes groups are known as alternate sigma factors and
they regulate specific physiological processes
Foodborne pathogens encounter a variety of such as survival during stationary phase and dur-
harsh environments in food products and during ing exposure to various stressful environments.
transit through the gastrointestinal tract. Food- Another intriguing aspect is that alternate sigma
associated environments may include acids, salts, factors are now shown to regulate virulence genes
preservatives, peroxides, antimicrobial chemi- or virulence-associated genes required for bacte-
cals, flavoring agents, sugars, and storage tem- rial pathogenesis. Some σ70 family members like
peratures. While in the GI tract, pathogens the σS of Gram-negative bacteria (group II), σB of
encounter gastric acid, bile salts, mucus, lyso- Gram-positive bacteria (group III), and
zyme, and natural microbiota. Although these extracytoplasmic functioning sigma factors
environments are stressful, the pathogens must (group IV) contribute to the bacterial stress
be able to express necessary colonization and responses. A few of the alternate sigma factors
invasion factors for their survival and multiplica- also play a role in the bacterial virulence as these
tion. A complex regulatory element is thought to respond to the host environment, which is critical
play important roles during maintenance of sap- in the infection process during the intestinal
rophytic lifestyle and subsequent interaction with phase of infection.
the host system. Synthesis of stress proteins depends on the
Protein synthesis in prokaryotes consists of expression of stress-related genes, some are
two major steps: transcription and translation. inducible whereas others are constitutive but
During transcription, the instructions stored in expressed at low levels when the cells are not
the DNA are transferred to the mRNA. under the stress. As some of the gene systems are
global, gene expression by one stress can also c onsisting of several mild treatments simultane-
help cells to adapt to other related stresses. ously could effectively inactivate the pathogens.
Expression of stress-related genes is initiated by
specific polypeptides or sigma factor (σ) synthe-
sized by specific genes. Some of these, such as σB Summary
or σ37 (encoded by gene sigB), helps cope with
general stress in Gram-positive bacteria; σ32 Foodborne pathogens cause three forms of dis-
(encoded by rpoH gene) and σ24 (encoded by ease: foodborne infection, foodborne intoxica-
rpoE gene) help cope with heat response; and σ38 tion, and foodborne toxicoinfection. The
or σs (encoded by rpoS gene) helps cope with prinicipal route of infection/intoxication for
general stress and starvation in Gram-negative foodborne pathogens is oral and the primary site
bacteria (i.e., Salmonella). Under a specific stressof action is the gastrointestinal tract. The infec-
(such as heat stress), rpoH is turned on and pro- tious dose of foodborne pathogens varies and
motes synthesis of RpoH or σ32 protein in high depends on the type of organisms or toxin as
amounts. This sigma factor (also called a regu- well as the type of food (liquid vs. solid) ingested.
lon) then combines with the core RNA poly- The pathogens that are responsible for infection
merase, consisting of four subunits, ααββ, to colonize the gut by producing various adhesion
form the complete RNA polymerase enzyme or factors including fimbriae, curli, adhesin pro-
holoenzyme. This holoenzyme then binds to the teins, and extracellular matrices that allow bio-
promoter of a heat-shock gene family, leading to film formation. Invading pathogens have
the synthesis of heat-shock proteins, which then developed strategies to cross the epithelial bar-
protect the structural and functional units of rier. Some use M cells to reach the subcellular
stressed cells susceptible to heat damage (e.g., location or some pathogens actively penetrate
DNA and proteins). Sigma factors can also pro- epithelial cells by rearranging the host cell cyto-
tect against other stresses, such as cold, heat, lowskeletal structure. Pathogens localized in the
pH, and UV. subcellular locations multiply, move from cell-
to-cell, and induce inflammation and elicit cell
Pathogens Survival in Acidic pH damage to induce diarrhea and gastroenteritis.
The enteric pathogens, especially the Gram- Some intracellular pathogens induce apoptosis
negative bacteria are susceptible to low pH and or necrosis in macrophages, dendritic cells, neu-
die off rapidly in the stomach and high-acid foods trophils, and other cells, thus ensuring their sur-
(pH ≤ 4.5). Also, at low pH, normal cells are sus- vival in host tissues. Pathogens may also
ceptible to other antimicrobial treatments, such translocate to deeper tissues including the liver,
as hydrostatic pressure, pasteurization tempera- lymph nodes, spleen, brain, and placenta.
ture, or preservatives at a much lower level. Foodborne intoxication is mediated by exotoxins
However, if bacteria are first acid-adapted, they produced by pathogens in the food, which
become relatively resistant to low pH and other induces cell damage, fluid and electrolyte losses,
treatments at minimal levels and can survive well and apoptosis or blocks nerve impulse following
in the food and in the GI tract. Recent foodborne consumption of the contaminated food. The
disease outbreaks of Salmonella enterica, E. coli mechanisms of exotoxin action may vary, and
O157:H7, and L. monocytogenes linked to the based on the toxin action, the toxins can be clas-
fruit juice, fermented sausage, and acidified sified as A–B type toxins, membrane-acting tox-
foods helped these pathogens to be acid-adapted. ins, superantigens, proteases, protein synthesis
Acid adaptation possibly helped these pathogens inhibitors, and signal transduction modulators.
to withstand high acidity and thermal processing. The bacterial cell wall or membrane-associated
To overcome such problem, it is necessary to endotoxins (LPS, PGN) are generally associated
avoid exposure to the food containing pathogens with systemic foodborne infection, and these
to mild treatments; instead, hurdle concept toxins modulate the immune system to induce
Further Readings 115
the release of large quantities of cytokines that 12. Fink, S.L. and Cookson, B.T. (2005) Apoptosis,
pyroptosis, and necrosis: Mechanistic description of
promote fever, decrease blood pressure, and
dead and dying eukaryotic cells. Infect Immun 73,
induce septic shock. In most pathogens, viru- 1907–1916.
lence factors encoded genes are located in patho- 13. Galan, J.E. and Wolf-Watz, H. (2006) Protein delivery
genicity islands or islets, which may be found on into eukaryotic cells by type III secretion machines.
Nature 444, 567–573.
plasmids, bacteriophage, or the chromosome. 14. Guttman, J.A. and Finlay, B.B. (2009) Tight junctions
Virulence proteins are exported from the as targets of infectious agents. Biochem Biophys Acta
microbes by various secretory machinary and 1788, 832–841.
those are designated type I–type VII and Sec 15. Harshey, R.M. (2003) Bacterial motility on a surface:
Many ways to a common goal. Annu Rev Microbiol
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gene expression is a complex process that may 16. Hawver, L.A., Jung, S.A. and Ng, W.-L. (2016)
be controlled by different regulatory elements in Specificity and complexity in bacterial quorum-
the food system as well as in the host. Alternate sensing systems. FEMS Microbiol Rev. 40, 738–752.
17. Henkel, J.S., Baldwin, M.R. and Barbieri, J.T. (2010)
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mucus. Trends Microbiol 20, 30–39.
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the intestinal epithelium: how bacterial recognition Hendrixson, D.R. (2011) Motility and chemotaxis
shapes intestinal function. Nat Rev Microbiol 10, in Campylobacter and Helicobacter. Annu Rev
131–144. Microbiol 65, 389–410.
2. Al-Sadi, R., Boivin, M. and Ma, T. (2009) Mechanism 21. Rajkovic, A. (2014) Microbial toxins and low level
of cytokine modulation of epithelial tight junction of foodborne exposure. Trends Food Sci Technol 38,
barrier. Front Biosci 14, 2765–2778. 149–157.
3. Ashida, H., Mimuro, H., Ogawa, M., Kobayashi, T., 22. Ray, B. and Bhunia, A. (2014) Microbial Attachments
Sanada, T., Kim, M. and Sasakawa, C. (2011) Cell and Biofilm Formation. In Fundamental Food
death and infection: A double-edged sword for host Microbiology. pp.73–78. Boca Raton, FL: CRC Press.
and pathogen survival. J Cell Biol 195, 931–942. 23. Ribet, D. and Cossart, P. (2015) How bacterial patho-
4. Barnhart, M.M. and Chapman, M.R. (2006) Curli gens colonize their hosts and invade deeper tissues.
biogenesis and function. Annu Rev Microbiol 60, Microbes Infect 17, 173–183.
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5. Barreau, F. and Hugot, J.P. (2014) Intestinal barrier genesis: A molecular approach. Washington, D.C.:
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in siderophore biosynthesis. Curr Opin Chem Biol 13, 17, 14–56.
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7. Cossart, P. and Sansonetti, P.J. (2004) Bacterial inva- Bacterial toxins: friends or foes? Emerg Infect Dis 5,
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G. (2015) Secretion systems in Gram-negative bac- dispersion and quorum sensing. Curr Opin Microbiol
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themes in SecA2-mediated protein export. Nat Rev cirrhosis - new insights into disease mechanisms. Nat
Microbiol 10, 779–789. Rev Gastroenterol Hepatol 10, 627–636.
Animal and Cell Culture Models
to Study Foodborne Pathogens 5
Introduction cell culture or in an animal model. Therefore,

finding an alternative sensitive cell culture sys-
Our knowledge of the pathogenic mechanism of tem or an animal model is crucial for studying the
foodborne pathogens has stemmed largely from host–pathogen interaction. (4) In some cases,
the use of various mammalian cultured cell lines organ culture such as the ligated intestinal loop or
and animal models. In the early years, animal an embryonated egg model can be used.
models were often used to confirm the patho-
genic attributes of a microbial isolate that was
involved in a disease outbreak. In recent years, Animal Model
animals are still used as a model to study not only
the pathogenic mechanism and the immune Humans are the ideal model to study human
response but also the efficacy of a vaccine. pathogens; however, it is not possible to use
Cultured cell lines are still considered an indis- humans for safety, bioethics, and expense-related
pensable powerful tool in studying the molecular concerns. However, in certain nonfatal diseases,
and cellular mechanisms of pathogenesis. The human volunteers have been used. Animal mod-
knowledge gained from the cell culture model is els are used frequently as a substitute. Among
then substantiated on an animal model, a neces- them, nonhuman primates (monkey, baboon, and
sary step toward understanding the pathogenic chimpanzee) are ideal for mimicking many dis-
mechanism, and associated pathology and clini- eases because of a higher genetic relatedness
cal signs. Thus, one must be familiar with various between primates and humans. However, the pain
cell culture and animal models and their applica- and suffering inflicted on animals during experi-
tions while studying microbial pathogenesis. mentation and expense-related considerations
While studying pathogen–host interaction, may limit their widespread application. Thus,
some guidelines must be followed: (1) it is impor- animal use in research is highly regulated and an
tant to use the same strain of the microbe that alternative to the animal models is highly desir-
caused the disease because of the clonal nature of able. Rodents are the most commonly used ani-
the pathogen. (2) Pathogens may also lose viru- mal models that include mice, rats, rabbits,
lence traits during subculture; therefore, one has hamsters, gerbils, guinea pigs, and ferrets
to avoid multiple subculturing (passage) before (Table 5.1). Pigs or piglets, bovine calves, sheep,
performing the pathogenicity experiment. (3) goat, dogs, cats, chicken, and zebrafish have been
During pathogenicity testing, some strains may used. In addition, insects, such as fruit fly
not exhibit pathognomonic symptoms in either (Drosophila melanogaster) and larvae of the

118
Table 5.1 Common laboratory rodents’ breeding data sheet

Life span Adult body Sexual Gestation period
5
Species Strains (years) weight maturity (days) Average litter size Age at weaning (weeks)
Mouse Balb/C, A/J, C57BL/6 1.5–2.5 20–25 g 6–8 weeks 19–21 6–8 pups (2–12) 3 weeks
Gerbil (desert rats) Great gerbil, 3–4 70 g 8–12 weeks 24–28 4 (1–8) pups 3–4 weeks
Mongolian gerbil
Rat Wistar, Sprague Dawley, 2 250–500 g 5 weeks 21–23 10–13 pups 4 weeks
Fischer 344 Zucker
Guinea pig Dunkin-Hartley 4–5 700–1200 g 4 weeks 63–68 (59–72) 3 (1–6) pups 3 weeks (pups born
fully developed)
Rabbit New Zealand White, Baladi 7–8 2–6 kg 5–7 months 31–32 7–9 kits 5–8 weeks
Animal and Cell Culture Models to Study Foodborne Pathogens
Animal Model 119
greater wax moth (Galleria mellonella), and Researchers must use appropriate personal pro-
nematodes, in particular, Caenorhabditis elegans tective equipment (PPE) that include a lab coat,
(C. elegans), have been used to study microbial mask, hairnet, and gloves before conducting an
pathogenesis. Some animals such as horse, don- animal experiment using pathogens. Animals
key, sheep, goat, camels, llama, and alpacas are also can attack, scratch, and bite during handling.
used for the production of large quantities of Furthermore, there are some fundamental differ-
polyclonal antibodies. Therefore, careful consid- ences in physiology between the rodents and
erations should be given while choosing an ani- humans. Rodents are coprophagy; thus, reinges-
mal model for pathogen–host interaction study: tion of the same bacteria may compromise the
pathogens must infect animals by the same route experimental data especially for an experiment
as humans and exhibit a similar colonization pat- conducted with the foodborne pathogens.
tern, a similar tissue distribution pattern, and the Resident microflora population and composition
same degree of virulence as in humans. in rodents may be different from that of the
human; thus, rodents may respond differently to
the human pathogens and the outcome of the dis-
Advantages of Animal Models ease may be different. Most importantly, some
animals are not sensitive to pathogens and thus
The major advantages of using an animal model, may not show the same human-type symptoms or
especially the rodent model, are the availability tissue distribution. For example, Salmonella
of an inbred line; therefore, the infection could be enterica serovar Typhimurium do not show gas-
reproduced and the variability between the ani- troenteritis or same tissue distribution as humans
mals could be minimized. Rodents are small, when administered orally into mice. The gastro-
hence, require less housing space compared to enteritis symptom (diarrhea) is achieved only
the primate or other animals, and are less expen- when the mice are pretreated with antibiotic,
sive to house or maintain. Rodents breed rapidly streptomycin, to inhibit resident gut microbiota.
and have a big litter size; thus, a large number of Yet, rodents are being used as prevailing mod-
the same inbred line can be obtained in a short els. Several strategies are employed to overcome
period, for use in an experiment to obtain a statis- shortcomings of rodent models. (1) Related bac-
tically significant result (Table 5.1). In addition, terial strains, which are infective, are used. (2)
genetically engineered rodents such as transgenic Different routes of administration are employed,
mice or rats with targeted gene knockout can elu- i.e., ingestion vs. intraperitoneal route, intranasal
cidate the role of a specific gene product in route, or intravenous route. (3) Neonatal animals
microbial pathogenesis. are used since their immune system has not fully
developed thus exhibiting high susceptibility to a
pathogen. (4) Germ-free gnotobiotic animals
Limitations and Cautions of Animal without any resident microbiota living in or on
Models them may show enhanced susceptibility to a
pathogen. (5) Humanized mouse models that are
Animal models present some limitations thus expressing human cell and tissue transplants or a
requiring careful considerations before use. specific gene for a receptor protein or cytokine
Animal use requires prior institutional approval, can show increased interaction to a pathogen. For
and there are strict guidelines one has to follow to example, Salmonella enterica serovar Typhi
use animals for research purposes. Animals must (causative agent for typhoid fever) does not show
be procured from authentic vendors or sources. typhoid-like symptom in mice, but a human-
Proper housing facility, breeding, handling, and ized mouse can show the symptom. (6)
veterinary care must be provided. Caregivers or Immunocompromised or immunodeficient ani-
animal handlers must take precautions to avoid mals with impaired immune system are highly
accidental exposure to the infective agents. susceptible to foodborne pathogens.
120 5 Animal and Cell Culture Models to Study Foodborne Pathogens
Trangenic Animal Models Organ Culture
Immunocompromised conditions can be induced The animal organ can be used as an alternative to
by exposing animals to gamma irradiation, which the whole animal and is ideal for investigating
destroys immune cells (T cells, B cells, NK cells, certain pathogen–host interaction. Ligated intes-
macrophages) since the immunological stem cells tinal loop and the embryonated eggs are exam-
are susceptible to irradiation. Immunodeficient ples of organs that are routinely used to study
mice are genetically engineered (transgenic mice) infectious agents. The advantages of organs are
to have impaired immune system, and these as follows: they are genetically intact, multiple
include “nude” mice, “SCID” (severe combined cell types are represented, and the cells retain
immunodeficient) mice, and “Rag”-deficient their original shape and configurations.
mice. Nude mice have a homozygous mutation in
Foxn1, which encodes a transcription factor for
thymus development and hair follicle growth thus Ligated Intestinal Loop Assay
making the mouse athymic and hairless. The
absence of the thymus prevents CD4+ and CD8+ T Ligated intestinal loop (LIL) assay has been used
cells from differentiation and maturation, making to study intestinal inflammation and fluid secre-
the nude mice (Foxn1−/−) deficient in functional T tion by diarrheagenic microorganisms and their
cells. The SCID mice have a homozygous muta- toxins such as the species of Salmonella,
tion in Prkdc, which encodes DNA-dependent Clostridium, Bacillus, Vibrio, Shigella, and
protein kinase required for DNA repair. DNA Escherichia. In addition, LIL has been used to
damage occurs during somatic recombination of investigate microbial translocation across the
TCR (T-cell receptor) and Ig (immunoglobulin) intestinal barrier including the involvement of M
gene; thus, Prkdc−/− mice lack both functional T cells in pathogenesis. The LIL from mice, rats,
and B cells. Likewise, Rag−/−-deficient mice do guinea pigs, rabbits, chicken, calves, and piglets
not express Rag1 and Rag2, which are required is used. Animals are deprived of food and water
for somatic recombination of TCR and Ig genes for about 12 h and anesthetized before initiating
resulting in functional T- and B-cell deficiency. the experiment. A small incision is given in the
The immunocompromised and immunodeficient abdomen, and a portion of the small intestine is
animals are expensive to procure and require pulled out from the abdominal cavity. The knots
greater care in their handling and maintenance. are placed in every 2–3 cm intervals using strings
They need pathogen-free environment since they to create loops. Test materials along with proper
are highly susceptible to infective agents and even controls are injected into the loops and the intes-
the commensal microbes. tine is placed back inside, and the skin opening is
Suckling mouse model has been used to study closed by suturing. After a period of 18–24 h, the
the pathogenesis of diarrheagenic microbes intestine is examined for fluid accumulation, typ-
including Vibrio cholerae and enterotoxigenic E. ical “ballooning” due to the action of the diar-
coli (ETEC). In this model, the test microorgan- rheagenic toxin.
isms or the toxin preparations are administered
orally in 2–4-day-old suckling mice, and mice are
sacrificed and examined for fluid accumulation in Embryonated Chicken Egg Assay
the gut after 4–24 h. The ratio of gut weight to the
body weight has been used to determine the The embryonated chicken egg (Fig. 5.1) offers an
enterotoxic effect of the toxins. Suckling mice alternative to investigate microbial pathogenesis.
have also been used to study the translocation of The immunity of the egg is similar to that of
protozoan parasites including Cryptosporidium higher mammals. Twelve- to fourteen-days-old
parvum in the gastrointestinal tract and their embryonated hen’s eggs are injected aseptically
oocyst counts in the intestinal contents. with test organisms in the chorioallantoic
Cultured Cell Lines 121
Cultured Cell Lines
Cells derived from human, animal, or insect tis-

sues are attractive models for studying the patho-
genesis of infective agents. Cultured cells are
also used for cancer research, drug screening and
toxicity testing, viral growth, vaccine develop-
ment, tissue or organ transplant, stem cell ther-
apy, and genetic engineering to produce insulin,
hormones, and antibodies. Microorganisms or
toxins have tropism for different cell types origi-
nating from different organs or tissues; therefore,
the use of a specific cell type can provide insights
Fig. 5.1 Schematic of an embryonated hen egg of the mechanism of infection for the test organ-
ism in vitro. The predominant cell type is epithe-
lial and endothelial cells; however, fibroblast,
embrane (CAM) or directly in the embryos of
m lymphocyte, and neuronal cells are also used.
eggs and incubated for 3–4 days. The death of There are two types of cells, primary and sec-
embryos or characteristic cytopathic effects ondary. Primary cells are harvested directly from
(CPE) is indicative of the infective nature of the the animal tissues or organs. Tissues are homog-
test organism. This model is used for various bac- enized, processed by enzymatic digestion, and
terial pathogens including Listeria monocyto- maintained using appropriate cell culture growth
genes, Yersinia enterocolitica, and Klebsiella media. The primary cells consist of mixed cell
pneumoniae; viruses such as influenza virus, her- types and are short-lived with a maximum lifes-
pes simplex virus, Rous sarcoma virus, and pan of 12–14 days. Secondary cells are immortal
Newcastle disease virus; and molds such as and are derived from a single lineage of the cell
Aspergillus fumigatus and Candida albicans. and have a uniform genetic composition, thus
referred to as a “cell line” (monoculture). The
secondary cells are originated either from tumor
Zebrafish Embryo cells, from transformed cells, or from hybridoma
cells. The transformed cells are generated by
Zebrafish (Danio rerio) embryo is used as a model introducing viral oncogenes such as those from
host to study the function of the vertebrate simian virus 40 (SV40) or Epstein–Barr virus
immune system (both innate and acquired immu- (EBV). This technique is routinely used to con-
nities) during microbial infection. The acquired vert a primary cell line to a transformed immortal
immunity in the embryo is developed only after cell line. The hybridoma cells are made from the
day 30. The embryo is used in the first 7 days after fusion of primary cells and cancer cells such as
the eggs are deposited. The pathogens are injected myeloma cells, and they share properties of both
in different areas by using a microinjection sys- partner cell lines. Hybridoma B cells generated in
tem, and primarily the host innate immunity is this process are immortal and produce monoclo-
studied during the pathogen–host interaction. nal antibodies. Both primary and secondary cells
Since the embryo is transparent, every organ and can grow either in suspension or as adherent cells
tissue are easily visible under a microscope for forming monolayers in tissue culture flasks. Cells
manipulation and imaging. This model has been are typically grown at 37 °C in a humidified incu-
used to study intracellular pathogens Salmonella bator with a supply of CO2 (5–7%) (see below).
enterica serovar Typhimurium and Mycobacterium Examples of commonly used secondary or trans-
marinum. The zebrafish model requires specific formed cultured cell lines are Caco-2 and HT-29
facilities to maintain the fish and the expertise for (colon adenocarcinoma cells), HepG2 (liver
handling and microinjection. cells), HEp-2 (larynx cell), Henle-407 (small
intestine–jejunal), HeLa (cervix), J-774 (macro- consist of only one type of cell; therefore, bacte-
phage), Vero (monkey kidney), and CHO rial interaction with concerted host cell cannot be
(Chinese hamster ovary) (Table 5.2). studied with a monoculture model. In cultured
cells, mucus and other secretory components are
absent, which normally interact with pathogens.
Advantages and Limitations Monoculture system is also unable to provide
of Cultured Cell Models intercellular communication, such as communi-
cation between the antigen-presenting cell and T
There are several advantages of cell culture cell for immune response, and cytokine or growth
model for studying microbial pathogenesis. factor production.
Cultured cell line is a simple and controlled
model to study host–microbe interaction and
easy to run experiments with radioactive materi- Co-cultured Cell Model
als, toxins, or transfection with foreign DNA;
cells multiply rapidly; thus, experiment can be Though the pathogen interaction with a monocul-
conducted rapidly; the secondary cells are ture can give a glimpse of information about
immortal, provided nutrients and proper cultur- pathogens behavior, it is difficult to extrapolate
ing conditions are maintained, and relatively the functional information for an animal model
inexpensive compared to the animal models. since multiple different cells in a tissue are
However, there are several limitations. involved during pathogen interaction in vivo.
Cultured mammalian cells are generally derived Co-culturing of two or more cell types in vitro
from tumor cells; therefore, genetic aberrations can overcome some of the limitations of mono-
have occurred in these cells, and the cells may culture system. For example, alveolar epithelial
not be genetically identical to the parent cells. cells have been co-cultured with macrophages
Continuous culturing of cells produces mutations and dendritic cells to develop a model for alveo-
and genetic rearrangements in the chromosome; lar epithelial barrier system for respiratory tract
therefore, these cells may lose traits of original infective agents. Likewise, enterocyte-like
tissue and may also lose tissue-specific receptors Caco-2 cells have been co-cultured with mucus-
for bacterial adhesins or unmasking low-affinity secreting HT-29 cells to represent mucoid envi-
receptors. Fundamentally, immortalized cells are ronment encountered by enteric pathogens in the
different from that of the normal cells in the body. intestine. Co-cultured cells grown in a three-
Microbes that normally do not interact with cer- dimensional (3D) scaffold in a gel matrix can
tain host cells, in fact, may adhere and invade cul- provide an opportunity to study pathogen interac-
tured cell lines. Sometimes the cell shape and tion in the 3D organotypic model. Though the
distribution of surface antigens may be affected. cultured cell model can provide in-depth knowl-
Cultured cells are not polarized, while the pri- edge of specific molecular and cellular mecha-
mary cells in the body are polarized in the intact nism of a pathogen, it is necessary to use animal
animal host, i.e., different parts of the cells are models to substantiate the findings from the cell
exposed to different environments, such as culture model and to understand the tissue distri-
lumen, adjacent cells, underlying blood vessels, bution, associated pathology, immune response,
and tissues. The polarized cells contain different and clinical signs.
sets of proteins important for their functions.
Physical disruption of polarized cells exposes
some surface proteins, which may not be exposed Growth and Maintenance
previously. Hormones can help secondary trans- of Cultured Cell Lines
formed cells to convert into polarized cells.
Furthermore, the host tissue consists of multiple To grow cultured cell lines in vitro, appropriate
cell types such as epithelial cells, fibroblasts, growth media and environment must be provided.
muscle cells, and neurons, but the cultured cells The basal growth medium is composed of
Growth and Maintenance of Cultured Cell Lines 123
Table 5.2 List of human, animal, and insect cell lines used to study the interaction of foodborne pathogens
Bacteria Cell line Cell type Source Interaction type
Salmonella CHO Epithelial Chinese hamster ovary Elongation, detachment
Vero Epithelial Monkey kidney Lysis, protein synthesis inhibition
HEp-2 Epithelial Human laryngeal Invasion
Henle-407 Epithelial Human jejunal Intracellular growth
J774 Macrophage Mouse Intracellular growth
HeLa Epithelial Human cervix Toxicity, actin polymerization
HT-29 Epithelial Human colon Apoptosis
E. coli Vero Epithelial Monkey kidney Lysis, protein synthesis inhibition
CHO Epithelial Chinese hamster ovary Lysis, toxicity
Henle-407 Epithelial Human jejunal Adhesion
HEp-2 Epithelial Human laryngeal Adhesion, toxicity
MDBK Epithelial Madin–Darby bovine kidney Invasion
HeLa Epithelial Human cervix Toxicity, apoptosis
T84 Epithelial Human colon Apoptosis
Y1 Epithelial Human adrenal gland? Rounding, detachment, cAMP
J774 Macrophage Mouse Apoptosis
Shigella HeLa Epithelial Human cervix Cell death, protein synthesis
inhibition
Vero Epithelial Monkey kidney Lysis, protein synthesis inhibition
3 T3 Fibroblast Mouse Invasion, actin polymerization
U937 Monocyte Human Apoptosis
Mϕ Macrophage Mouse Invasion, apoptosis
Campylobacter HeLa Epithelial Human cervix Distended cells (CDT effect)
Vero Epithelial Monkey kidney Distended cells (CDT effect)
AZ-521 Epithelial Human stomach Vacuolation
Yersinia J774 Macrophage Mouse Exocytosis, apoptosis
HEp-2 Epithelial Human laryngeal Invasion, lysis
Vibrio CHO Epithelial Chinese hamster ovary Elongation, cAMP accumulation
Clostridium Vero Epithelial Monkey kidney Membrane permeability
perfringens Caco-2 Epithelial Human colon Necrosis, apoptosis, pore formation
MDDK Epithelial Madin–Darby dog kidney Pore formation
Listeria Caco-2 Epithelial Human colon Adhesion, invasion, apoptosis
monocytogenes CHO Epithelial Chinese hamster ovary Detachment, lysis
Henle-407 Epithelial Human jejunal Intracellular growth, death
Vero Epithelial Monkey kidney Toxicity, adhesion, invasion
HEp-2 Epithelial Human laryngeal Invasion
J774 Macrophage Mouse Intracellular growth
RAW Macrophage Mouse Intracellular growth
HeLa Epithelial Human cervix Toxicity
HUVEC Endothelial Human umbilical vein Intracellular growth
endothelial cell
Hep-G Epithelial Human liver Intracellular growth, apoptosis
3 T3 Fibroblast Mouse Invasion, plaque formation, lysis
Ped-2E9 B cell Mouse hybridoma Lysis, apoptosis
RI-37 B cell Human–mouse hybridoma Lysis, apoptosis
S2 Epithelial Drosophila Invasion and infection model
Avian flu virus MDCK Epithelial Madin–Darby canine kidney Cytopathic effects, virus
(H5N1) cells multiplication
Hepatitis A Vero Epithelial African green monkey kidney Virus replication and cytopathic
virus effects
Adapted and modified from Bhunia, A.K. and Wampler, J.L. 2005. Foodborne Pathogens: Microbiology and Molecular
Biology, Caister Academic
Table 5.3 Media used for growing common mammalian cell lines
Cell line Source Morphology Medium used
Caco-2 Human Epithelial cell (colorectal Eagle’s Minimum Essential Medium (EMEM or Dulbecco’s
adenocarcinoma) Modified Eagle’s Medium (DMEM) + serum (10%)
HT-29 Human Epithelial (colorectal McCoy’s 5a + serum (10%)
adenocarcinoma)
HEK293 Human Epithelial Eagle’s Minimum Essential Medium (EMEM) + serum (10%)
HUVEC Human Endothelial Hams’s F-12 + serum (10%) + heparin (100 μg/ml)
3 T3 Mouse Fibroblast Dulbecco’s Modified Eagle’s Medium (DMEM) + serum (10%)
CHO Hamster Epithelial Hams’s F-12 + serum (10%)
Jurkat Human Lymphoblast Roswell Park Memorial Institute 1640 (RPMI-1640) + serum
(10%)
g lucose, amino acids, fatty acids and lipids, vita- ence of phenol red indicator. When the medium
mins, trace elements, salts, and phenol red as pH pH drops below 7.4 due to the buildup of acid,
indicator. Several commercial media such as the medium color changes to yellow. Sometimes
Eagle’s Minimum Essential Medium (EMEM), phenol red may interfere with cellular growth;
Dulbecco’s Modified Eagle’s Medium (DMEM), thus, it may be omitted from the medium
Iscove’s Modified Dulbecco’s Medium (IMDM), formulation.
Roswell Park Memorial Institute 1640 (RPMI-
1640), McCoy’s 5a, and Hams’s F-10 and F-12
media are used depending on the cell type Measurement of Virulence
(Table 5.3). The basal growth medium is then
supplemented with serum (1–10%) to provide Animal Model
proteins, growth factors, cytokines, iron chelators
(transferrin, ferritin), and vitamins. Serum source In an animal model, infectivity or lethality of a
is generally from adult bovine or calf. The pathogen or toxin may vary depending on the age
medium is also supplemented with 2 mM gluta- and immunological health status of the animal
mine since this amino acid tends to degrade over and the route of administration of the infective
time. Antibiotic supplement (i.e., gentamicin, agent. Generally, for experimental purposes, dif-
ampicillin, streptomycin) is also used to prevent ferent routes are used to administer pathogens,
microbial contamination, but antibiotic may slow such as oral, intragastric (i.g.), intravenous (i.v.),
down mammalian cell growth. Mammalian cells intraperitoneal (i.p.), intramuscular (i.m.), subcu-
are seeded in appropriate flasks and dishes and taneous (s.c.), and intradermal (i.d.). To assess
placed in a specialized cell culture incubator at pathogenicity of an infective agent, the natural
37 °C under humidified condition, with a con- route of infection is recommended: an oral or
stant supply of CO2 gas (5–7%). During cell intragastric route for foodborne pathogens or
growth, metabolic activity yields acidic by- enteric pathogens, the intranasal route for respi-
product, which is toxic to cells. Therefore, main- ratory tract infective agents, and so on. Generally,
taining the buffering capacity of the growth the virulence measurement is expressed as the
media is critical, which is naturally adjusted by infectious dose (ID) or as the lethal dose (LD).
the gaseous CO2 and the CO3/HCO3 content in The infectious dose 50 (ID50) is defined by the
the media. HEPES, a zwitterion, has buffering number or concentration of a pathogen required
capacity (in the pH range of 7.2–7.4) and has to infect 50% of the animals. The lethal dose 50
been used in the medium in the absence of CO2 (LD50) is defined by the number or concentration
atmosphere. The serum also contributes to the required to kill 50% of the animals. Kaplan–
media buffering. The standard pH of the growth Meier survival curve is generated to determine
medium is 7.4 and appears red due to the pres- the LD50 value (Fig. 5.2). Infectious dose or lethal
Measurement of Virulence 125
microscope, or electron microscope. The typical

cytopathic effect (CPE) consists of cell detach-
ment, flocculation, rounding or elongation, cell
lysis, cytoplasmic granulation, and cell death
(Fig. 5.3). Highest dilutions of a toxin or patho-
gen showing CPE are considered the cytotoxic
titer for that agent. Furthermore, the nature of cell
death (necrosis vs. apoptosis) could be detected
by using a fluorescence microscope after staining
cells with acridine orange, ethidium bromide, or
propidium iodide. These dyes can permeate only
through the damaged cell membrane and bind to
the DNA emitting green or red fluorescence
indicative of cell death. The healthy cells with
Fig. 5.2 Estimation of LD50 values for pathogens extrap- intact cell membrane prevent dye entrance.
olated from a Kaplan–Meier survival curve from a hypo- Scanning electron microscope is often used to
thetical animal study. LD50 value is 5 × 108 cfu
examine the membrane damage, pore formations,
and apoptotic bodies in the eukaryotic cells
dose data provide the crude measurement of (Fig. 5.4).
infection that includes cumulative effects of colo- One of the most commonly used cytotoxicity
nization, invasion, tissue distribution, and symp- assays is Vero cell (monkey kidney epithelial cell
toms; therefore, ID50 cannot be compared with line) assay, which is performed to determine the
the LD50 value. Moreover, these data provide a cytotoxin (also called verotoxin) production from
relative measure of virulence and cannot com- Shigella species, E. coli, and Clostridium diffi-
pare two different diseases such as bacillary dys- cile. The cytotoxic or cytopathic effects (CPE)
entery vs. cholera. are characterized by cell detachment, floccula-
tion, rounding, and cell death (Fig. 5.3).
Cell Culture Model rypan Blue Exclusion Test

T
Trypan blue is a diazo dye, which is routinely
Using cell culture models, pathogen or toxin used in the cell culture laboratories to assess the
interaction with different cell lines can be studied viability of cultured mammalian cells during sub-
(Table 5.2). Cytotoxicity or cytopathogenicity culturing. In this assay, the cell suspension is
assays are used to determine the cytotoxic poten- mixed with an equal volume of trypan blue (0.4%
tial of pathogens or toxins. Similarly, pathogens’ v/v) for a few seconds, and a small volume is
ability to adhere, to invade, and to move from placed on a hemocytometer, and viable or dead
cell-to-cell can be assessed using specific cell cells are counted under a microscope. Viable
lines. Cytotoxicity assay is generally used as a cells with intact membrane exclude stain (hence
confirmatory test for a pathogen, which has called trypan blue exclusion test) and appear as
already been identified or detected by other meth- bright translucent, while the dead or dying cells
ods. This in vitro assay also allows one to study with damaged membrane allow stain permeation
the mechanism of pathogenesis of various micro- and appear blue. Confluent adherent cell mono-
organisms or toxins, which can be assessed by layers growing on a flask or Petri dishes also can
different methods. be stained with the trypan blue to assess the
degree of cell damage induced by toxins or
Microscopy pathogens. The cell monolayers are then exam-
The degree of cell damage can be assessed under ined under a light microscope. Trypan blue stain-
a phase-contrast light microscope, fluorescence ing cannot differentiate cell death by necrosis or
Fig. 5.3 Effect of

Escherichia coli
O157:H7 (EDL933)
toxin on Vero cells after
24 h exposure.
Toxin-exposed cells
(panel B) show
cytopathic effects
characterized by cell
rounding, granulation,
and detachment
Fig. 5.4 Scanning electron microscopic photographs of listeriolysin O (LLO, a hemolysin). Toxin-induced mem-
lymphocyte cell line, Ped-2E9 (A) control, treated with brane pore formation is marked by arrows
(B) Bacillus cereus toxin, and (C) Listeria monocytogenes
apoptosis, but it can interpret the membrane as hemolysins or phospholipase enzymes pro-
integrity of a cell. duced by species of Listeria, Bacillus,
Clostridium, and Staphylococcus, cause mem-
lkaline Phosphatase Assay
A brane pore formation and allow the release of
Alkaline phosphatase enzyme is expressed by all ALP from cells. The ALP enzyme is analyzed by
living organisms; however, its expression and the addition of a substrate, such as p-
function may be tissue-specific. Certain cells, nitrophenylphosphate (PNPP) or methyl umbel-
especially those of lymphocyte origin, express liferyl phosphate (MUP) yielding colored or
endogenous membrane-anchored alkaline phos- fluorescence product for measurement by a spec-
phatase (ALP) enzyme (100–165 kDa), which is trophotometer or a spectrofluorometer. The draw-
released from cells if the cell membrane is back of this assay is that not all cell types may
severely damaged. Membrane-active toxins, such have adequate amounts of this enzyme and thus
Measurement of Virulence 127
thiazolyl-2]-2,5-diphenyltetrazolium bromide), is a
water-soluble compound, which is reduced by met-
abolically active cells. MTT is converted into
water-insoluble purple formazan by the action of
dehydrogenase enzyme that generates NADH and
NADPH. The purple formazan is then quantified
by a spectrophotometer at 570 nm. In the MTT
assay, pathogen/toxin is added for 1–2 h before the
MTT reagent is added. The assay generally takes
24–48 h to complete. The assay has been used to
Fig. 5.5 Lactate dehydrogenase (LDH)-mediated color
change in the reaction mixture
measure cytotoxic effects of bacterial and viral
pathogens.
may not be a good indicator of cell cytotoxicity. The WST-1-based assay is similar to that of
In general, epithelial cells carry very low levels, the MTT assay, except that the WST-1
while the cells of B lymphocyte origin carry high (4-[3-(4-iodophenyl)-2-(4-nitrophenyl)-2H-5-
amounts. Therefore, pathogen-induced damage tetrazolio]-1,3-benzene disulfonate) reagent is
in epithelial cells cannot be measured accurately used and the products are measured at
with the ALP assay. 450/655 nm. The assay takes about 3 h to
complete.
actate Dehydrogenase Assay
L
All living cells including the mammalian cells ell Death Analysis
C
possess lactate dehydrogenase enzyme (LDH). Pathogen interaction with host cells often results
LDH is a low molecular weight enzyme (35 kDa) in cell death, which could be either necrotic,
and may exist in the tetrameric form. The enzyme necroptotic, apoptotic, or pyroptotic (see Chap.
is released from the cells even due to a minor per- 4). Necrosis, a nonspecific or accidental cell
turbation in membrane integrity, thus making the death, usually leads to cell swelling with a loss of
assay highly sensitive. LDH release assay has membrane integrity, lysis, and initiation of a host
been widely used for studying cell cytotoxicity inflammatory response. In contrast, apoptosis is a
induced by varieties of microbial or nonmicro- tightly regulated method for removal of damaged
bial interactions with mammalian cells and or unneeded “self”-cellular debris, especially
immune effector cells (NK cells and cytotoxic T during growth, development, and cell homeosta-
cells). The enzyme activity can be assayed by sis. Apoptosis is characterized by cell shrinkage,
using an appropriate substrate. LDH converts lac- chromatin condensation and margination, DNA
tate to pyruvate and the NAD receives an electron fragmentation, and membrane blebbing
(H+) from NADH, and during this process, yel- (Fig. 5.6). There is no inflammatory response
low tetrazolium is converted to red formazan, during apoptosis. Many cytokines and cellular
which can be measured by a spectrophotometer proteins regulate cells death via two main path-
for a quantitative cytotoxic response (Fig. 5.5). ways (1) by activating tumor necrosis receptor-
Commercial LDH assay kits are available for associated death domain (TRADD) and (2) by
performing cytotoxicity assays. activating CD95 receptor (also known as Fas) or
Fas-associated death domain (FADD). These
TT- and WST-1-Based Cytotoxicity
M receptors bind effector molecules and set an
Assays intercellular apoptotic cascade in motion by
The eukaryotic cell viability or proliferation could recruiting, binding, and activating caspase (Cas
be assayed by a metabolic staining method. 3, Cas 6, Cas 7, Cas 8, Cas 9) enzymes or down-
Pathogens or toxins that interfere with the meta- regulating apoptosis by interacting with inhibitor
bolic activity of cells can be measured by this assay. proteins, such as proteins in the Bcl-2 family (see
The yellow tetrazolium, MTT (3-[4,5-dimethyl Chap. 4).
Fig. 5.6 (A) Different stages of apoptotic cell death in fragmentation; and (e) membrane blebbing of apoptotic
human B lymphoma cell line by Listeria monocytogenes: bodies. (B) DNA fragmentation in hybridoma B cells by
(a) cell shrinkage; (b) zeiosis stage, cell morphology L. monocytogenes (Adapted from Bhunia and Feng 1999.
alters and shrinkage continues; (c) chromatin condensa- J Microbiol Biotechnol 9, 398–403; Menon et al. 2003.
tion/margination, crescent-
shaped nucleus; (d) DNA Comp Immunol Microbiol Infect Dis 26, 157–174)
Apoptosis is regarded as a noninflammatory easurement of Specific Steps

M
response that targets specific cell without full- in Colonization and Invasion
scale tissue damage. However, in some cases,
pathogens such as Shigella and Salmonella Animal Model
induce massive macrophage apoptosis, and this
process is often proinflammatory. Consequently, icrobial Enumeration and Staining
M
speculation surrounds the question of whether To study foodborne pathogen colonization, adhe-
host cells initiate apoptosis in an attempt to sion, invasion, and translocation to the extraintes-
reduce widespread infection or if pathogens tinal sites or to study localized tissue damage,
themselves may trigger apoptosis through activa- pathogens are administered orally or intragastri-
tion of the apoptotic protein cascade as an infec- cally. Sometimes animals are starved up to 12 h
tion strategy without alarming the immune and/or fed with antacids to neutralize gastric pH
system. before administration to allow safe microbial
Apoptosis and necrotic cell deaths are differ- passage to the intestinal tract. To determine
entiated by using a DNA fragmentation assay, pathogen colonization and localized cell damage,
characterized by DNA laddering, by flow cytom- intestinal sections are collected and intestinal
etry analysis of annexin V binding to outer leaflet contents removed, homogenized, and analyzed
of the apoptotic cell membrane, or through a spe- for bacterial counts in the mucus membrane by a
cific cell staining method such as acridine orange, plating method. In some cases, intestinal sections
ethidium bromide, or propidium iodide staining are treated with a broad-spectrum antibiotic
which intercalate host cell DNA (Fig. 5.6). (gentamicin) to kill extracellular bacteria and

Measurement of Specific Steps in Colonization and Invasion 129
analyzed for bacterial invasion into the intestinal body bioluminescence imaging has been used
epithelial cells or submucosal layers. Histological for studying bacterial pathogens, such as
sections of intestine can be stained with hema- Salmonella, Listeria, Escherichia, Bacillus,
toxylin and eosin and examined under a micro- Haemophilus, Staphylococcus, Streptococcus,
scope to assess recruitment of inflammatory cells and Mycobacterium, and parasitic (Entamoeba,
(neutrophils and lymphocytes). Bacteria-specific Toxoplasma, Leishmania, Plasmodium), fungal
immunostaining can be performed to localize (Aspergillus fumigatus and Candida albicans),
bacterial cells in these sections. To determine and viral (Vaccinia) diseases.
bacterial invasion and translocation across the
epithelial barrier to the extraintestinal sites, such
as the liver, spleen, lymph nodes, brain, kidney, Cell Culture Model
and placenta (pregnant model), each organ/tissue
is collected, homogenized, and serially diluted, Adhesion Assay
and bacterial counts are determined by a plating Microbial adhesion is generally performed on
method. confluent cell monolayers formed in the wells of
Intravenous or intraperitoneal route of admin- cell culture plates, small flaskets, chambered
istration of foodborne pathogens has been used to slides, and Petri dishes or on coverslips.
investigate the pathogenic potential, tissue distri- Depending on the cell type, some cells must be
bution, immune response, and clinical signs grown for a specified time to allow for cell dif-
when the gastrointestinal route is bypassed. ferentiation and polarization before the adhesion
In the ligated intestinal loop model, pathogens assay can be performed. Cell monolayers are
or toxin preparations are injected directly into the inoculated with the test organisms at MOI (multi-
loop to determine fluid accumulation or to study plicity of infection) of 0.1, 1, 10, or 100 and incu-
bacterial adhesion and translocation to deeper tis- bated for 30 min to 1 h at 37°C in a cell culture
sues in a controlled environment. Staining of his- incubator. The unbound organisms are removed
tological sections of the loop can reveal the by washing the cell monolayers at least for three
bacterial adhesion and invasion patterns, the times using cell culture media or a buffer. MOI
involvement of the M cells in pathogenesis, and needs to be optimized for each pathogen – gener-
the infiltration of inflammatory cells. ally, a lower MOI of 1 or 10 is recommended.
Bacterial attachment is assessed by performing
hole-Body Bioluminescence Imaging
W specific immunostaining, fluorescence staining,
Whole-body bioluminescence imaging is also a or Giemsa staining. Differential fluorescence
powerful tool for real-time visualization of staining is also used to distinguish intracellular
pathogen localization and tissue distribution bacteria from surface-attached bacteria.
during infection in live animals. In this case, Quantitative bacterial counts are obtained by a
pathogens are engineered to express luciferase plating method. To achieve bacterial counts, cell
enzyme (lux operon from Photorhabdus lumi- monolayers are first disrupted by treating with a
nescens) that produces bioluminescence. After mild detergent solution such as Triton X-100
the animals are infected with the bioluminescent (0.1–1%), and then cell suspensions are serially
microbes, the entire animal body can be imaged diluted and plated on agar plates.
at time intervals to visualize real-time progres-
sion of infection and tissue distribution in live Invasion Assay
animals. This method complements conven- To determine bacterial invasion and intracellular
tional microbial enumeration method from har- localization, polarized and differentiated cell
vested tissues. This method can also reveal monolayers are infected with the test microbes at
unexpected spread of infection in different tis- MOI of 0.1, 1, 10, or 100 for 1–2 h to allow
sues, which may otherwise be missed if the pathogen entry into the cell. Cell monolayers are
tissues were not harvested from that site. Whole- then washed (three times) with cell culture media
or buffer to remove loosely attached or unbound transwell setup and incubated in a humidified
microbes and treated with an antibiotic (gentami- CO2 incubator at 37 °C for 1–2 h. The liquid from
cin at 10–100 μg ml−1) for 1–2 h to kill extracel- the basal compartment is collected and the trans-
lular bacteria. Gentamicin at low concentrations located bacteria are enumerated by plating.
cannot permeate through the cell membrane; Often, fluorescein isothiocyanate (FITC)-
thus, intracellular bacteria are protected. conjugated dextran (4–40 kDa) is used to assess
Subsequently, the cell monolayers are washed as tight junction integrity. FITC-dextran is loaded
before and treated with mild detergent (Triton into the apical well, and after a period of incuba-
X-100) or cold buffer to lyse the cells, serially tion, translocated FITC-dextran level in the liq-
diluted, and plated to determine the intracellular uid in the basal compartment is measured by a
bacterial counts. Immunofluorescence staining of spectrofluorometer. The higher the fluorescence
cells whose membranes are permeabilized before reading, the greater is the epithelial permeability.
staining can reveal intracellular bacterial Epithelial tight junction integrity or disruption
localization. can also be visualized under a confocal micros-
copy following immunostaining with the tight
ell-to-Cell Spread or Plaque Assay
C junction and adherens junction-specific proteins:
This assay is performed to determine the micro- ZO-1, occludin and claudin, and E-cadherin.
bial ability to move from cell to cell. Generally, Enterohemorrhagic E. coli (EHEC), enteropatho-
fibroblast (L2) or epithelial (Caco-2 or HT-29) genic E. coli (EPEC), Helicobacter pylori,
cells are used for this purpose. Differentiated or Campylobacter jejuni, Yersinia pseudotuberculo-
polarized cell monolayers are first inoculated sis, Listeria monocytogenes, and Shigella species
with the test organisms for 1–2 h, washed, and are known to disrupt epithelial barrier for para-
treated with gentamicin (100 μg ml−1) for another cellular translocation.
1.5 h. After washing the monolayers with a buffer
solution, the cell monolayers are overlaid with
tempered agarose (0.7%) containing gentamicin Summary
(10 μg ml−1) and incubated for about 72 h in a cell
culture incubator. The monolayers are stained Much of our knowledge and understanding of the
with 0.1% neutral red and examined for the for- pathogenic mechanism of foodborne pathogens
mation of clear plaques. Clear plaques indicate are gained from using animal and cell culture
the capacity of the pathogen to move from one models. In modern day, these models are indis-
cell to another. This technique has been used to pensable research tools; thus, one must have
assess the infectivity of intracellular pathogens, apprehension for the availability of different
such as Shigella and Listeria species. models, their usage, advantages, and limitations.
In addition, one must choose an appropriate
Paracellular Translocation Assay model for a pathogen in order to learn the micro-
Many pathogens disrupt epithelial barrier to gain organism’s behavior in that environment. Cell
entrance into the subcellular space and for the culture models, especially the secondary cell
systemic spread. Bacterial ability to translocate lines, are the most valuable tools that allow deter-
through the paracellular route (in between cells) mining virulence potential of pathogens.
can be examined in an in vitro transwell setup Cytotoxicity assays are measured by microscopic
(Fig. 5.7). Confluent cell monolayers are first analysis and enzyme release assays (lactate dehy-
established on transwell filter inserts with pore drogenase, alkaline phosphatase, or metabolic
sizes of about 3–4 μm. Often, transepithelial staining assay). Cell culture models also allow
electrical resistance (TEER) of the polarized studying bacteria-induced cell death such as
monolayer is measured using a voltmeter to apoptosis or necrosis. In addition, these models
ensure monolayer integrity before conducting the help us to study the molecular mechanism of
translocation experiment. Bacteria (MOI ~10) to pathogen–host interaction such as microbial
be tested are added to the apical well of the adhesion, invasion, paracellular translocation and
Fig. 5.7 Transwell

setup for measuring
paracellular
transepithelial
translocation of bacteria
cell-to-cell movement, and host cell signaling 2. Andreu, N., Zelmer, A. and Wiles, S. (2011)
Noninvasive biophotonic imaging for studies of infec-
events. Animal models are often used to substan- tious disease. FEMS Microbiol Rev 35, 360–394
tiate the pathogenic mechanism that has been 3. Banerjee, P. and Bhunia, A.K. (2009) Mammalian
established in an in vitro cell culture model. cell-based biosensors for pathogens and toxins.
Whole animals provide a better picture of dis- Trends Biotechnol 27, 179–188
4. Banerjee, P., Franz, B. and Bhunia, A. (2010)
semination of bacterial pathogens in different Mammalian Cell-Based Sensor System. In Whole
organs and tissues, and some models show clini- Cell Sensing Systems I eds. Belkin, S. and Gu, M.B.
cal symptoms similar to humans. Sometimes ani- pp.21–55: Springer
mals are insensitive to human pathogens. In such 5. Bhunia, A.K. and Feng, X. (1999) Examination
of cytopathic effect and apoptosis in Listeria
situations, immunocompromised or immunodefi- monocytogenes- infected hybridoma B-lymphocyte
cient animals are employed to study microbial (Ped-2E9) line in vitro. J Microbiol Biotechnol 9,
pathogenesis. In addition, transgenic mice with 398–403
targeted gene knockout can elucidate the role of a 6. Bhunia, A.K. and Wampler, J.L. (2005) Animal and
cell culture models for foodborne bacterial patho-
specific gene product in microbial pathogenesis. gens. In Foodborne Pathogens: Microbiology and
Pathogen interactions with animals are measured Molecular Biology eds. Fratamico, P., Bhunia, A.K.
by determining the lethal dose (LD50) or infective and Smith, J.L. pp.15–32. Norfolk, UK: Caister
dose 50 (ID50). Tissue distribution of pathogens Academic Press
7. Decker, T. and Lohmann-Matthes, M.-L. (1988) A
can be analyzed by whole-body imaging using quick and simple method for the quantitation of lac-
bioluminescence reporter microbes, conventional tate dehydrogenase release in measurements of cel-
microbiological analysis from harvested tissues, lular cytotoxicity and tumor necrosis factor (TNF)
and microscopic analysis of histological sections. activity. J Immunol Methods 115, 61–69
8. Duell, B.L., Cripps, A.W., Schembri, M.A. and Ulett,
Animal models also provide the nature of the G.C. (2011) Epithelial cell coculture models for study-
immune response and the efficacy of a vaccine ing infectious diseases: Benefits and Limitations.
against a pathogen. Animal use in research is J Biomed Biotechnol 2011, 852419
extremely regulated and alternative to animal 9. Glavis-Bloom, J., Muhammed, M. and Mylonakis, E.
(2012) Of model hosts and man: Using Caenorhabditis
models is highly desirable. Zebrafish, fruit fly, elegans, Drosophila melanogaster and Galleria mel-
and nematodes (C. elegans) are some of the lonella as model hosts for infectious disease research.
uncommon models which are gaining popularity In Recent Advances on Model Hosts eds. Mylonakis,
as a substitute for laboratory animal models. E., Ausubel, F.M., Gilmore, M. and Casadevall, A.
pp.11–17. New York, NY: Springer New York
10. Hoelzer, K., Pouillot, R. and Dennis, S. (2012) Animal
models of listeriosis: a comparative review of the cur-
Further Readings rent state of the art and lessons learned. Vet Res 43, 18
11. Hutchens, M. and Luker, G.D. (2007) Applications of
1. Andersson, C., Gripenland, J. and Johansson, J. bioluminescence imaging to the study of infectious
(2015) Using the chicken embryo to assess virulence diseases. Cell Microbiol 9, 2315–2322
of Listeria monocytogenes and to model other micro- 12. Ito, R., Takahashi, T., Katano, I. and Ito, M. (2012)
bial infections. Nat Protocols 10, 1155–1164 Current advances in humanized mouse models. Cell
Mol Immunol 9, 208–214
13. Martín, R., Bermúdez-Humarán, L.G. and Langella, 16. Ngamwongsatit, P., Banada, P.P., Panbangred, W. and
P. (2016) Gnotobiotic rodents: An in vivo model for Bhunia, A.K. (2008) WST-1-based cell cytotoxicity
the study of microbe–microbe interactions. Front assay as a substitute for MTT-based assay for rapid
Microbiol 7, 409 detection of toxigenic Bacillus species using CHO
14. McCormick, B.A. (2003) The use of transepithelial cell line. J Microbiol Methods 73, 211–215
models to examine host–pathogen interactions. Curr 17. Salyers, A.A. and Whitt, D. (2002) Bacterial patho-
Opin Microbiol 6, 77–81 genesis: A molecular approach. Washington, D.C.:
15. Menon, A., Shroyer, M.L., Wampler, J.L., Chawan, ASM Press
C.B. and Bhunia, A.K. (2003) In vitro study of 18. Trevijano-Contador, N. and Zaragoza, O. (2014)
Listeria monocytogenes infection to murine primary Expanding the use of alternative models to investigate
and human transformed B cells. Comp Immunol novel aspects of immunity to microbial pathogens.
Microbiol Infect Dis 26, 157–174 Virulence 5, 454–456
Foodborne Viral Pathogens
and Infective Protein 6
Introduction including: (1) attachment to host receptor, (2)

penetration of the host cells, (3) uncoating of
Foodborne viral diseases account for about 5.5 RNA/DNA, (4) transcription and/or translation,
million illnesses, 15,284 hospitalizations, and (5) RNA/DNA replication, (6) assembly and
156 deaths each year in the United States of packaging of nucleic acid with viral proteins, and
America. Estimated economic burden for food- (7) release of matured virus particles (see Chap.
borne viruses is about 3 billion dollars. Viruses 2). RNA virus encodes genes for RNA-dependent
are obligate intracellular parasites, requiring a RNA polymerase, a nonstructural enzyme (repli-
live host for replication. They cannot grow out- case), which is needed for RNA replication inside
side their specific host or within foods. Viruses the host, while the DNA-dependent RNA poly-
are generally “host-specific,” i.e., infecting spe- merase (called RNA transcriptase) is required for
cific plants, animal, human, or bacteria and usu- transcription of RNA from DNA and protein syn-
ally do not establish cross-species infections. thesis such as capsid protein for viral packaging.
However, zoonotic viruses are able to infect Viruses do not carry any genes for metabolism
humans, and other viruses can occasionally and hence rely on host cells for propagation.
undergo genetic modifications to adapt them-
selves to different hosts.
The majority of foodborne viruses are consid- Sources and Transmission
ered enteric due to their fecal–oral mode of
transmission. Enteric viruses are generally non- Contaminated foods readily transmit the virus.
enveloped virus and are stable in the environ- Primary contamination occurs before harvesting.
ment. There are four acute gastroenteritis-causing Examples include oyster and clams (which con-
viruses: Calicivirus, Rotavirus, Astrovirus, and centrate virus particles) and vegetables that are
Adenovirus. Norovirus (Calicivirus) causes about irrigated with contaminated or polluted water.
5.4 million illnesses in the USA per annum, while Secondary transmission occurs during process-
Rotavirus, Astrovirus, and Sapovirus cause ing or handling of products where fecally con-
approximately 15,000 cases, and hepatitis A taminated hands are in contact with foods.
virus causes approximately 1500 cases. Secondary transmission can also occur when
Enteric viruses (Table 6.1) are highly infec- vomitus, containing virus particles, is aerosol-
tious. A low dose of virus consisting of as few as ized, contaminating foods and food contact sur-
10–100 particles is sufficient to cause foodborne faces. Uncooked foods that receive no heat
infection. Virus life cycle consists of several steps treatment are common sources of virus infection.

134 6 Foodborne Viral Pathogens and Infective Protein
Table 6.1 Foodborne viruses

Incubation period in
Name Family (Genus) Size (genome) Foodborne days (median)
Poliovirus, Coxsackievirus, Picornaviridae 28 nm (ssRNA) Yes, mainly water, 1–5 (3)
Echovirus, Enterovirus (Enterovirus) present in shellfish
Astrovirus Astroviridae 28 nm (ssRNA) Yes, shellfish 2–3
Hepatitis A virus Picornaviridae 28 nm (ssRNA) Yes 15–50 (28)
(Hepatovirus)
Hepatitis E virus Hepeviridae 34 nm (ssRNA) Mainly water 14–60
(Hepevirus)
Rotavirus Reoviridae 70 nm (dsRNA) Rare often water 1–4 (2)
(Rotavirus)
Sapovirus Caliciviridae 34 nm (ssRNA) Yes (rare), mainly 1–3 (2)
(Sapovirus) shellfish
Adenovirus group F, Adenoviridae 100 nm (dsDNA) Water, shellfish 3–10 (5)
serotypes 40 and 41 (Mastadenovirus)
Norovirus Caliciviridae 34 nm (ssRNA) Yes 1–2 (1)
(Norovirus)
Nipah virus Paramyxoviridae 500 nm (ssRNA) Fruit bat, fruits, 7–14
(Henipavirus) and sap
Ebola virus Filoviridae 1200 nm Yes, Fruit bat and 2–21 (7–10)
(Ebolavirus) (ssRNA) other animals
These foods include salads, bakery products, and 28–45 kb long). Adenovirus is a nonenveloped
raw shellfish. Ice made from virus-contaminated virus. It is a member of Adenoviridae family and
water can also be a source of transmission. genus Mastadenovirus, which includes >20
known viruses (5 human, 3 bovine and 3 porcine,
and 9 other species). There are 51 serotypes of
Virus Classification/Taxonomy human adenovirus. While many adenoviruses
can infect the intestinal tract, serotypes 40 and 41
Enteric viruses are generally nonenveloped cause the majority of human adeno-gastroenteritis
viruses and belong to the family of Adenoviridae, and are shed in feces in large numbers. The incu-
Caliciviridae, Hepeviridae, Picornaviridae, and bation period is about 3–10 days and the illness
Reoviridae. Viruses are classified based on the lasts for about a week. Immunocompetent adults
size, shape, structure, and nucleic acid content are more resistant to these viruses, while children
(Fig. 6.1). Viruses contain either DNA or RNA below 2 years of age are most susceptible. The
with single- or double-stranded nucleic acid mol- clinical symptoms are associated with gastroin-
ecules. Adenovirus has double-stranded DNA, testinal complications involving watery diarrhea.
and RNA viruses generally have single-stranded Waterborne outbreak resulting in conjunctivitis
RNA molecules. Based on the genetic elements, has been reported. Failure in proper chlorination
viruses can be classified into seven types: dsDNA, of water may lead to an outbreak. Adenovirus is
ssDNA, dsRNA, (+) sense ssRNA, (−) sense also frequently found in shellfish.
ssRNA, RNA reverse-transcribing viruses, and
DNA reverse-transcribing viruses.
Astrovirus
Foodborne Viral Pathogens Astroviruses are RNA viruses and are small
(28 nm diameter) with star-like surface projec-
Adenovirus tions. Human astroviruses have eight serotypes,
with serotypes 1 and 2 being predominant in
Adenovirus has a large icosahedral structure children. It causes diarrhea in children and the ill-
containing a double-stranded DNA (genome
ness is generally mild. The incubation period is
Foodborne Viral Pathogens 135
Fig. 6.1 Schematics of

a model virus showing
various structural
components
2–3 days and the disease lasts for about 3–4 days.
A major foodborne outbreak was reported in
Japan in 1994 affecting 1500 school children and
the teachers. Serotype 4 was responsible for that
outbreak. The virus can be cultured using mam-
malian cells.
Rotavirus
Rotavirus looks like a wheel (rota means a

“wheel”) in which capsid proteins are arranged
like spokes of a wheel. The capsid structure con-
sists of an inner core, an intermediate capsid, and Fig. 6.2 Electron microscopic picture of rotaviruses
an outer capsid with short radiating spikes (70 nm particles). Bar = 100 nm (Courtesy of Dr. Erskine
(Fig. 6.2). It is a large dsRNA nonenveloped L. Palmer, CDC, Atlanta, GA)
virus belonging to Reoviridae family. The seg-
mented genome encodes six structural viral pro- Foodborne rotavirus is responsible for an esti-
teins (VP1–VP4, VP6, and VP7) and six mated 15,000 cases per year in the USA world-
nonstructural proteins (NSP1–NSP4, NSP5/ wide; rotavirus causes about 215,000 deaths
NSP6). Among the VP proteins, VP1 is the RNA- annually in children under age 5. The incubation
dependent RNA polymerase, VP2 is core protein, period is about 1–4 days, and the illness is mani-
VP3 is methyltransferase, VP6 is inner capsid, fested by diarrhea, vomiting, and fever, lasting for
and VP4 and VP7 are outer capsids. Among the a week. Severe dehydration and electrolyte imbal-
NSPs, NSP1 is an interferon antagonist, and ance are responsible for fatalities. A cell culture
NSP4 is an enterotoxin. Rotavirus is grouped in model (MA104, monkey kidney epithelial cell
eight (A–H) groups. Groups A, B, C, and H are line) is available to study rotavirus pathogenesis.
known to infect humans. Of which, group A is Rotavirus infection starts with viral attach-
responsible for more than 90% of all infections in ment to matured enterocytes using VP4 to the
humans. host cell glycan (i.e., sialic acid). The virus then
Rotavirus primarily infects children of less than enters the enterocytes at the tip of the small
5 years of age and causes acute gastroenteritis. intestinal villi. Histo-blood group antigens

(HBGAs) may serve as a receptor for some rota- (HBV), C (HCV), and D (HDV) viruses are
viral strains. Virus induces structural changes blood-borne and are responsible for acute and
such as villous atrophy and infiltration of mono- chronic liver diseases. A brief description of
nuclear inflammatory cells in the lamina propria. HBV, HCV, and HDV is included to have some
Rotaviruses are released from infected epithelial understanding of non-foodborne hepatitis
cells without destroying them or causing cell viruses, while HAV and HEV as food−/water-
death. Thus, maldigestion and maladsorption of borne pathogens are discussed in detail below.
nutrients and a consequent inhibition of reab-
sorption of water lead to diarrhea. Nonstructural Hepatitis B Virus
viral protein, NSP4, has been shown to act as an Hepatitis B virus (HBV) is responsible for about
enterotoxin. It promotes chloride secretion and 786,000 deaths per year globally. It is a double-
fluid loss. Chloride secretion response is regu- stranded DNA virus (3.2 kb) and is transmitted
lated by phospholipase C-dependent calcium sig- through contact with infected blood and semen.
naling pathway. NSP4 may disrupt tight junction It causes sexually transmitted disease with a
barrier function and may induce paracellular per- high rate of infection seen in homosexuals or
meability. Viruses are released in the stool in high heterosexuals with multiple sexual partners,
numbers (about 109 particles per gram) and can injection drug users, and healthcare personnel.
contribute to the fecal–oral transmission. Perinatal infection from mother to neonates is
Diagnosis is relatively simple which is accom- common in the high endemic area. It causes
plished by electron microscopy, agglutination chronic infection. Patients suffer from liver cir-
assay or enzyme-linked immunosorbent assay rhosis or hepatic carcinoma. A prophylactic vac-
(ELISA), and reverse transcriptase PCR with cine made from recombinant DNA that expresses
stool specimen. Rotavirus infection can induce a hepatitis B virus surface antigen (HBsAg) is
long-lasting immunity. Two live, attenuated oral highly effective.
vaccines, Rotarix® and RotaTeq® are available
to prevent rotavirus-associated gastroenteritis Hepatitis C Virus
and mortality. Hepatitis C virus (HCV) is a single-stranded
RNA virus with 9.6 kb genome. It exhibits a high
rate of mutation due to the lack of proofreading
Hepatitis Viruses activities of RNA-dependent RNA polymerase. It
belongs to Flaviviridae family. It infects about
Hepatitis viruses are a major public health con- 130–170 million people worldwide, and these
cern worldwide. They are grouped into hepatitis patients are in the high risk of developing hepat-
A, B, C, D, and E (Table 6.2). Hepatitis A virus osteatosis (accumulation of lipids in the liver),
(HAV) and hepatitis E virus (HEV) are known liver fibrosis (scarring), liver cirrhosis, and
food−/waterborne pathogens, while hepatitis B hepatic cancer. Transmission occurs predomi-
Table 6.2 Characteristics of hepatitis viruses

Hepatitis
virus Genome Properties Clinical significance
HAV ssRNA(−); 7.5 kb; Fecal–oral Hepatitis, jaundice
27–32 nm transmission
HBV dsDNA; 3.2 kb Blood-borne Acute and chronic liver disease
HCV ssRNA(+); 9.6 kb Blood-borne Acute and chronic liver disease; hepatosteatosis
(accumulation of lipids in the liver), liver fibrosis (scarring),
liver cirrhosis, and hepatic cancer
HDV ssRNA; 1.7 kb Blood-borne Coinfection with HBV
HEV ssRNA(+); 7.5 kb; Fecal–oral Acute hepatitis; increased mortality during pregnancy
27–34 nm transmission; water
Foodborne Viral Pathogens 137
nantly through contaminated blood due to blood single-stranded RNA virus (7.5 kb) and belongs
transfusion, intravenous drugs, organ transplants, to Picornaviridae family and genus, Hepatovirus.
and vertical transmission from mother to child. HAV has seven genotypes and four of them (IA,
IB, II, III) are associated with human infection
Hepatitis D Virus and three others (IV, V, and VI) are associated
Hepatitis D Virus (HDV) is also known as “delta with nonhuman primates. The viral genome
hepatitis” infection and is often associated with encodes a single open reading frame for a poly-
HBV infection. HDV contains a small RNA protein of 250 kDa. The polyprotein has three
genome (1.7 kb) with single ORF (open reading regions: P1 represents structural protein, i.e., the
frame) and is unable to synthesize its own enve- capsid protein, and P2 and P3 represent nonstruc-
lope protein to form a fully functional virion. tural proteins required for RNA synthesis and
Thus, it is called defective virus particle or satellite virion assembly.
virus and depends on HBV to supply the envelope Pathogenesis
protein or surface antigens (HBsAgs) for packag- The incubation period of the disease is about
ing during coinfection with HBV. HDV infection 15–50 days (median 28 days). From the intestine,
is blood-borne, and infection can spread during HAV reaches to the liver after a systemic circula-
coinfection with HBV. HDV infection is involved tion. It interacts with the host cell receptor pro-
in acute or chronic liver disease and infection is tein called HAVCR1 (hepatitis A virus cell
uncommon in the USA. Globally more than 15–20 receptor 1) before entry into hepatocytes. It repli-
million people are infected by HDV. cates inside hepatocytes, moves to the gall blad-
der and released into bile, and eventually is shed
Hepatitis A Virus in the stool. Infection results in inflammation in
Introduction the liver. Viruses impair liver function and as a
The hepatitis A virus (HAV) was identified in result, bilirubin accumulates in blood and jaun-
1972 when an immunoelectron microscopy was dice develops. Two to three weeks after the
used for diagnosis. The most common infection infection, the immune response to virus develops.
routes are person-to-person (fecal–oral route) via Consequently, activated immune cells, CD8+ T,
household contact, or in homosexual men, intra- and NK cells attack virus-infected hepatocytes to
venous drug users sharing the same needles, and eliminate the virally infected cells. As a result,
exposure to contaminated food and water. Foods hepatocytes are severely damaged manifesting
implicated in outbreaks including clams, mus- characteristic viral pathogenesis.
sels, raw oysters, lettuce, ice slush beverages, fro- The major symptom of hepatitis is jaundice,
zen strawberries, blueberries, raspberries, and characterized by yellow discoloration of the skin
green onions. The largest outbreak occurred in and the white part of the eye. In jaundice patient,
1988 in Shanghai (China) due to consumption of the stool is pale colored and the urine becomes
contaminated clams, and about 300,000 people dark. Anorexia, vomiting, malaise, and fever are
showed the symptom of acute hepatitis with 47 manifested in the hepatitis patients, and virus
deaths. Generally, the HAV infection is asymp- particles are shed in large numbers in the stool
tomatic in children under 6 years of age, while it (about 109 particles per gram). Viruses are also
is symptomatic in older children and adults with shed through saliva. Liver failure may occur in
jaundice occurring in greater than 70% patients. patients with the underlying chronic liver dis-
Annually, approximately 1.4 million people suf- ease. Children may exhibit asymptomatic HAV
fer from HAV infection worldwide. infection and shed viruses longer than the adults
do, while older children and adults show symp-
Biology toms. The long-lasting immunity primarily
HAV particle is 27–32 nm in diameter and has humoral (antibody) response, after primary infec-
icosahedral symmetry. It is a nonenveloped tion, is seen in patients.
Immune Response Hepatitis E Virus

The innate immunity involves the production of
Introduction
interferons (IFNα, IFNβ) and antiviral state of
Originally, identified as an “atypical hepatitis A
viral recognition through pathogen recognition
virus” that caused an outbreak in New Delhi
receptors (PRRs) or pathogen-associated molec-
(India) and infected about 29,000 people in 1955,
ular patterns (PAMPs). The adaptive response
hepatitis E virus (HEV) was originally classified
includes the production of IFNγ and activation of
as hepatitis non-A, non-B virus. Now it has been
CD4+ regulatory T cells (Treg) that express
renamed hepatitis E virus. This virus causes acute
CD25+ and FoxP3 and suppress the immune
hepatitis, annually infecting about 20 million
response to the pathogen. Cytotoxic CD8+ T cells
people worldwide. HEV is a member of family
respond to viral antigens through MHC class I
Hepeviridae, genus Hepevirus, and there are four
pathway. HAV also induces a long-term humoral
genotypes. Genotype 1 and 2 are associated with
antibody response.
person-to-person human infection, and genotype
Prevention and Control 3 and 4 are associated with zoonotic transmission
Detection of HAV is achieved by immunologi- to humans, from pigs and other species.
cal methods such as immunofluorescence assay, HEV is primarily a waterborne virus and
dot blot assay, immunoblotting, and ELISA. In caused several outbreaks in developing countries
the cell culture using African green monkey in Asia, Latin America, and Africa. Swine serves
kidney cell line (Vero) and the fetal rhesus as the most important reservoir for HEV. HEV
monkey k idney cell, virus replication could be infection can also transfer through organ trans-
detected in 2–4 weeks by immunoassays. plants. The high-risk group includes patients suf-
Reverse transcriptase- polymerase chain reac- fering from chronic liver disease and pregnant
tion (RT-PCR) and real-time PCR assays have women and young children. Adults and young
been used to detect the virus. More recently, adults are also susceptible. Though it is a
nucleic acid sequencing of the PCR-amplified waterborne virus, several recent outbreaks were
products has been done to confirm and to deter- associated with consumption of deer meat and
mine the genetic relatedness among the HAV raw or undercooked swine liver in Japan. HEV
isolates. has been routinely isolated from swine from
It is difficult to trace the food source because Canada, South Korea, Japan, Spain, the
of the long incubation period of the disease. Netherlands, New Zealand, and the USA imply-
HAV may survive about a month in the environ- ing potential future outbreak of HEV with the
ment. It is resistant to chlorine and requires consumption of undercooked pork meat.
1 min exposure to 1:100 dilutions of household
Biology
bleach (sodium hypochlorite). Inactivation by
HEV is a nonenveloped icosahedral-shaped
heating requires >85 °C for 1 min. Immunization
spherical particle (27–34 nm) containing a single-
has been effective in reducing the hepatitis
stranded RNA with genome size, 7.2 kb. The
cases, especially in children. The reduction in
genome contains three ORFs: ORF1 encodes
children hepatitis cases possibly affects the
nonstructural proteins, RNA-dependent RNA
hepatitis infection cycle thus probably reduces
polymerase (RdRP), protease, methyltransferase,
the number of adult hepatitis cases in recent
and helicase; ORF2 encodes a structural protein,
years. However, total numbers of sporadic hep-
capsid that is required for viral entry; and ORF3
atitis cases have not been reduced. Vaccination
encodes phosphorylated protein required for viral
of food handlers may reduce HAV cases but
release from the host cells.
may not be cost-effective. Personal hygiene and
hand washing after toilet visit are the most Pathogenesis
effective practice in preventing the transmis- The incubation period of HEV varies from
sion of HAV. 2 weeks to 2 months. Propagation of human HEV
Norovirus 139
is currently challenging but hepatic cell lines, Norovirus (NoV). NoV is highly contagious and
Huh7, HepG2, lung cell line A549, and colon cell the transmission routes are food, water, person,
line Caco-2, have been used as cell culture mod- and environment (Fig. 6.4). About 20 million
els to study pathogenesis. Pathogenesis involves Americans are affected every year resulting in
several steps that require viral interaction with 56,000–71,000 hospitalizations and 570–800
the host cell receptor, heparin sulfate proteogly- deaths. Of the total illnesses, about 5.5 million
cans, and to another uncharacterized receptor illnesses are attributed to food.
molecule. Upon internalization, viral uncoating NoV has been responsible for numerous out-
allows RNA release followed by RNA replica- breaks in various establishments: the cruise ships,
tion, packaging, and release of mature virions restaurants, swimming pool, schools, nursing
from the host cells. homes, and hospitals. Primary transmission to
The disease is dose dependent: higher dose humans can happen through food, and then sec-
shows clinical symptoms while lower dose exhib- ondary transmission occurs from fecal–oral or
its subclinical infection. It is responsible for acute person-to-person and from the environment.
viral hepatitis similar to HAV and manifests mild Secondary transmissions are expedited when
jaundice, anorexia, and hepatomegaly. Some people are in close contact in settings like hospi-
patients suffer from abdominal pain, nausea, tals, cruise ships, hotels, restaurants, nursing
vomiting, and fever. homes, day-care centers, prisons, military
installments, and sports stadiums. Fresh produce
such as lettuce, tomato, melons, green onions,
Norovirus strawberries, raspberries, peppers, fresh-cut
fruits, salads, and food handlers, processors, and
Introduction irrigation water are also involved in the transmis-
Norovirus (formerly Norwalk virus) is the lead- sion. Transmission can occur through filter-feed-
ing cause of gastroenteritis and a major public ing bivalves including muscles, oysters, and
health concern worldwide. The first outbreak clams, which collect viruses in their tissues.
occurred in 1968 in children and the adults in
Norwalk, OH (USA), hence the name Norwalk, Biology
but the virus was not identified until 1972. Dr. Norovirus is also known as a small round struc-
Albert Z. Kapikian (1930–2014) at the National tured virus (SRSV) of 27–38 nm diameters with
Institutes of Health was the first person to iden- icosahedral shape (Fig. 6.3). It is a nonenveloped
tify the virus using an electron microscopy virus carrying a plus-sense single-stranded RNA,
(Fig. 6.3). The Norwalk virus is now called and the 7.4–7.7 kb genome is comprised of three
ORFs. ORF1 encodes a nonstructural polypep-
tide consisting of p48, NTPase, p22, VPg (viral
genome-linked protein), viral protease, and
RNA-dependent RNA polymerase (RdRP).
ORF2 encodes a major capsid protein, VP1, and
ORF3 encodes a minor capsid protein, VP2. VP1
binds to the host cell receptor, histo-blood group
antigens (HBGAs), promotes viral entry, and
determines antigenicity and strain specificity.
VP1 also elicits host protective antibody, cellular
and humoral immunity; while the VP2 helps in
RNA packaging, regulates the synthesis of VP1,
and stabilizes the VP1 structure.
Fig. 6.3 Electron microscopic image of norovirus NoV belongs to the family of Caliciviridae
(Courtesy of Charles D. Humphrey, CDC, Atlanta, GA) that has six genera: Norovirus, Sapovirus,
Fig. 6.4 Transmission

pathway for norovirus
(Schematics based on
Ushijima et al. 2014.
Food Safety, 2, 37–54)
cles ingested. The HBGAs distributed in blood

Lagovirus, Vesivirus, Recovirus, and Becovirus.
cells, vascular endothelial cells, and mucosal
Human infections are generally associated with
gastrointestinal epithelial cells serve as the
viruses from the genus Norovirus. NoV is now
receptor for viral interaction. Norovirus infects
grouped into six genogroups (G1-GVI): GI is
mature enterocytes covering the small intestinal
isolated from humans; GII from both humans and
villi leading to massive cell damage and malab-
swine; GIII from cattle; GIV from human, feline,
sorption. Damaged cells are rapidly replaced by
and canine; GV from murine; and GVI from the
undifferentiated immature enterocytes origi-
canine. Genogroups are again subdivided into
nated from the crypt, which are not susceptible
genotypes. Genogroup GI has >9 while GII has
to the virus infection. These immature cells can-
>22 genotypes. GII.4 is currently the most com-
not function properly; thus, malabsorption con-
mon epidemic strain.
tinues until the cells mature. The virus causes
Pathogenesis gastroenteritis, characterized by explosive pro-
There is no reliable cell culture or small animal jectile vomiting, nausea, cramps, diarrhea, dehy-
model, which can be used to study human noro- dration, anorexia, headache, chills, fever, and
virus pathogenesis or viral growth. However, myalgia. Adults are more susceptible to norovi-
gnotobiotic pigs, gnotobiotic calves, rhesus rus than children are, and the incubation period
macaques, and chimpanzees have shown to is 24–48 h.
respond to human norovirus infection. Feline Symptoms appear within 12–24 h after inges-
calicivirus (FCV) and murine norovirus (MNV) tion and last for 2–3 days. The disease is self-
have been used as surrogates. FCV is the mem- limiting, and the viruses are excreted in the
ber of family Caliciviridae and genus Vesivirus. vomitus and feces of infected persons at the rate
Likewise, MNV is the member of Caliciviridae of 104–105 virus particles per gram of vomitus
family and genus Norovirus. Both possess some and 108–1010 particles per gram of feces.
attributes similar to the human norovirus, and Recovering patients shed virus for an extended
they can be cultured in cell culture models thus, period even up to a month or longer.
have been used widely as NoV surrogates. The Immunocompromised patients, children, and the
infectious dose of NoV is very low: about elderly may shed for a prolonged duration. Adults
10–100 virus particles. Severity and the onset of may encounter recurrent infections despite the
disease may depend on a number of virus parti- presence of norovirus-specific antibody in the
Zoonotic Viral Pathogens 141
serum. Cell-mediated immunity is Th1-merase gene. Electron microscopy is used as a

dependent, and IFNγ is the dominant cytokine. confirmatory test.
Prevention and Control
Effective sanitization and disinfection are needed
to prevent and control the spread of NoV. However, Zoonotic Viral Pathogens
it is resistant to standard sanitizers and disinfec-
tants at the permissible levels during food pro- Avian Flu Virus
cessing or food production. Since it is resistant to
standard chlorination treatment, so a concentra- Introduction
tion >10 mg L−1 is needed to disinfect water. As Avian flu is also known as avian influenza and the
an industry practice for leafy green sanitization, predominant strain is H5N1. Avian influenza
chlorine is used at 0.2 mg L−1, which is not effec- virus infects birds including poultry; however, it
tive against NoV; however, 15–30 s treatment is capable of adapting and causing infection in
with 2–5% trisodium phosphate solution (TPS) humans, especially the poultry handlers, and thus
can effectively reduce viral loads on produce. it is considered a zoonotic pathogen (Fig. 6.5).
Human milk, oligosaccharide, milk glycoprotein, Avian influenza virus has been isolated from wild
and milk glycolipid, contain the same epitope as birds such as geese, ducks, and waterfowls and
HBGA and can block viral binding to the epithe- mammals including pig, horse, dog, and sea
lial cells and provide a strategic approach to pre- mammals. The migratory birds can readily trans-
venting viral interaction with enterocytes. mit the virus to different continents. Even though
Currently several vaccines are under develop- the avian influenza virus has been isolated from
ment based on viral proteins; however, due to the poultry meat, there is no evidence for foodborne
lack of a cell culture model to grow the virus, live transmission. Though human-to-human trans-
attenuated viral vaccine development is not pos- mission has not been confirmed, scientists pre-
sible. Noroviruses are genetically and antigeni- dict that avian influenza can possibly cause a
cally diverse; thus, vaccine efficacy would be pandemic, which is now responsible for the epi-
limited. The virus is routinely detected by ELISA demic in Asia, Africa, and Eastern Europe.
and by RT-PCR assay targeting the RNA poly- According to the World Health Organization
Fig. 6.5 Interspecies

transmission pathways
for avian flu virus (avian
influenza H5N1 virus)
(WHO), between 2003 and 2015, avian influenza with pigs and H3 and N8 in dogs. A majority of
virus strain H5N1 infected 840 and killed 447 avian influenza viruses are H5 and H7 subtypes,
worldwide. and H5, H7, and H9 have caused sporadic infec-
There are three types of influenza viruses: A, tions in humans. There are two pathotypes, highly
B, and C. Influenza virus type A is responsible pathogenic avian influenza (HPAI) and low patho-
for human epidemic every year, and it was genic avian influenza (LPAI). Several different
responsible for past pandemics. In the human his- avian influenza virus types have been isolated
tory, three major pandemics of influenza A virus from different regions or countries: H1N1 (Asia),
have been reported: 1918 Spanish flu (H1N1), H4N6 (Canada), H9N2 (China), and H5N1
1957 Asian flu (H2N2), and 1968 Hong Kong flu (Asia). Avian flu virus is currently named by type/
(H3N2). These three pandemics killed millions place isolated/culture number/year of isolation.
of people. The avian flu virus has an avian origin For example, the strain isolated from Shanghai,
or the virus results from avian-human virus China, is designated as B/Shanghai/361/2002
reassortment. (H5N1).
Biology Pathogenesis
Avian influenza virus belongs to the family Typically, the influenza virus affects the respira-
Orthomyxoviridae and genus Influenzavirus A. tory tract. The HA of influenza virus binds to the
Influenza A virus is an enveloped virus and the epithelial sialic acid-containing receptor before
size is about 80–120 nm in diameter. It is a initiating an infection. In human, this receptor
negative-sense single-stranded RNA virus with consists of sialic acid–galactose, and the cross-
eight different segments and encodes ten proteins link between these two molecules consists of
including surface glycoproteins, hemagglutinin α-2,6 linkage (SA α-2,6), while in birds it is α-2,3
(HA), and neuraminidase (NA) and matrix pro- linkage (SA α-2,3). This difference possibly pre-
teins M2 and M1, nonstructural proteins NS1 and vents the avian influenza virus from readily
NS2, the nucleocapsid, and the three polymerase infecting humans. Moreover, the receptors with
enzymes, PB1 (polymerase basic 1), PB2 (poly- the α-2,3 linkages are distributed in the lower
merase basic 2), and PA (polymerase acidic). part of the respiratory tract of humans thus also
HA and NA are the surface antigens and are a reduces the access of the virus. In addition,
determinant of the pathogenicity, transmission, human isolates of avian H5N1 have shown to dis-
and adaptation of the virus to other species. HA is play antigenic variation to show binding to
the most important determinant and binds to the human cellular receptor containing α-2,6 linkage.
host epithelial cell receptor for viral entrance and The virus infects and multiplies in nasopharyn-
replication, while NA is responsible for the release geal and alveolar epithelial cells. The virus also
of newly formed viruses. Both HA and NA are exhibits tropism for the liver, renal system, and
responsible for antigenic variation resulting in other tissues showing signs of diarrhea, renal
antigenic drift and shift and allow the virus to dysfunction, and lymphopenia.
evade the host immune system. The virus lacks a The symptoms of H5N1 infection in humans
proofreading and correction mechanism during appear 2–8 days after exposure, and clinical signs
viral replication; hence, the error introduced due include flu-like symptom, fever, cough, shortness
to nucleotide substitution, deletion, or point muta- of breath, and pneumonia affecting primarily the
tion results in antigenic variation. There are 16 lower respiratory tract. The disease may progress
subtypes of HA (H1–H16) and 9 subtypes of NA to vomiting, diarrhea, and abdominal pain. The
(N1–N9), and many of these are associated with patient requires mechanical ventilation and dies
various animals including humans, dogs, pigs, within 9 days from the onset of symptoms.
horses, and birds. The subtypes H1, H2, and H3 In birds, the low infective pathotype exhibits mild
and N1 and N2 are associated with human infec- symptoms characterized by ruffled feathers and mild
tion; H1 and H3 and N1 and N2 are associated respiratory symptoms lasting approximately for
Zoonotic Viral Pathogens 143
10 days. The high infective pathotype shows severe The greatest risk of exposure is through handling
respiratory and neurological disorders, organ failure, and slaughter of live infected poultry.
and death within 2–3 days.
Nipah Virus
The avian flu virus can be cultured using Madin–
Darby canine kidney (MDCK) cell line or embry-
Introduction
onated eggs (see Chap. 5). Viral antigens can be
The first Nipah virus (NiV) outbreak was reported
detected from a clinical specimen by enzyme
in Malaysia in 1998–1999, which affected 276
immunoassay or immunofluorescence assay, and
people and 39% fatality, and the infection was
viral RNA can be detected by using a reverse
originally transmitted through exposure to
transcriptase PCR (RT-PCR) assay that targets
infected swine. The NiV was amplified at large
genes for HA and NA synthesis. Antiviral drugs,
numbers in the respiratory tracts and facilitated
amantadine, rimantadine, oseltamivir and zana-
the spread of infection in farm workers. NiV was
mivir, show serotype-specific effectiveness. NA
originally isolated from a patient from Sungai
inhibitors (oseltamivir and zanamivir) are shown
Nipah village in Malaysia, and fruit bat (genus,
to be effective against H5N1 in vitro and in a
Pteropus), also called flying fox, acts as a natural
mouse model. Several vaccines based on killed
reservoir. Interaction of fruit bats with swine and
subunit vaccines are under development, but
humans led to increased numbers of outbreaks in
those must be able to protect against different
Malaysia. Bats transmit the virus through saliva
strains currently causing infections in humans
or urine to the fruits. Swine from a farm located
globally.
near the bat habitat acquires the organism and aid
Waterfowl is the natural reservoir for avian
in the zoonotic transmission of the disease
influenza, and the virus can be transferred to
(Fig. 6.6). Domestic animals foraging may eat
domestic birds by respiratory and fecal–oral
virus-laden partially eaten fruits and may trans-
routes through contaminated water, feed, envi-
mit the disease to humans. NiV is also transmit-
ronment, and feces. Infiltration of wild birds
ted through sap (juice) of date palm tree when
should be prevented from poultry farms or
fruit bats feed on sap at night, and this leads to
premises.
numerous epidemic and sporadic outbreaks in
The avian flu virus has the potential for caus-
Bangladesh and the eastern part of India. Date
ing a pandemic; thus, precaution should be taken
palm tree sap is used for making molasses. The
to prevent the spread. Contact with infected
virus survives well in the sap, and unheated sap
domestic or wild birds should be avoided.
can transmit the virus to humans. Person-to-
Routine surveillance of migratory birds (dead or
person transmission also occurs. Nipah virus out-
alive) or birds in a poultry farm for the presence
break was also reported in Singapore, Cambodia,
of influenza virus should be performed.
and Thailand, and virus has been circulating in
Vaccination of human populations may be needed
the natural reservoir in Southeast Asia, including
to control the spread; however, concerns of anti-
Malaysia, Cambodia, Indonesia, East Timor,
genic variation may challenge its efficacy and
Vietnam, Thailand, Bangladesh, India, and Papua
effectiveness.
New Guinea.
Can the avian flu virus be a food safety con-
cern? It has been demonstrated that conventional Biology
cooking temperature (70 °C or more) can readily NiV is a member of the family of Paramyxoviridae
inactivate the H5N1 virus; however, the virus and genus Henipavirus. NiV virus is about
may not be killed by refrigeration or freezing, 500 nm in diameter and is larger than the typical
and in fact, the virus has been isolated from fro- paramyxoviruses (150–400 nm). The Nipah virus
zen duck meat. If the poultry eggs and meats are size may vary from 180 nm to 1900 nm. It is an
properly cooked, they can eliminate the virus. enveloped negative-sense single-stranded RNA
virus with genome size 18.246 kb. Six structural encapsidation of the viral genome and interacts
proteins are encoded in the genome: two enve- with P protein. The L protein possesses all the
lope glycoproteins F (fusion) and G (receptor enzymatic activities responsible for initiation,
binding), the nucleoprotein N, phosphoprotein P, elongation, and termination of both mRNA tran-
matrix protein M, and the RNA-dependent RNA scription and genome replication. P protein
polymerase L. The G and F glycoproteins are serves as a scaffold between the L and the encap-
required for viral attachment and entry into the sidated genome.
host cells. The G protein binds to the receptor
Pathogenesis
molecule, Ephrin-B2, which is expressed on neu-
The incubation period of NiV infection is
rons, smooth muscles, and endothelial cells. The
1–2 weeks. The virus binds to the cellular receptor
F protein (546 amino acid residues) is a type I
Ephrin B2 present on the neuron and endothelium,
transmembrane protein and facilitates the fusion
enters host cells, replicates, and causes cell dam-
of virus and the host cell membrane during the
age. Systemic vasculitis with extensive thrombosis
infection. Both F and G proteins induce neutral-
is seen in patients since virus attacks endothelium
izing antibodies. The M protein (352 amino acid
in the blood vessels and the CNS. The virus causes
residues) provides rigidity and the structural sta-
high fever, headache, myalgia, dizziness, confu-
bility of the virion through its interactions with
sion and lack of consciousness, and encephalitis.
the F protein, the ribonucleoprotein (RNP) com-
In addition, the virus causes acute respiratory tract
plex, and the inner surface of the virion envelope.
infection, pulmonary edema, coma, and death.
The N protein (532 amino acid residues) helps
Fig. 6.6 Transmission

pathways for Nipah
virus
Prevention and Control of Foodborne Viruses 145
Kidneys are also affected showing signs of glo- viral genome encodes for a nucleoprotein (NP),
merular fibrinoid necrosis. The mortality rate in glycoprotein (GP), RNA-dependent RNA
human is about 75%. polymerase (L), and four structural proteins:

Infected bats do not show clinical signs, but VP24, VP30, VP35, and VP40. In addition, it
serve as the carrier, whereas pigs are highly sus- expresses a truncated soluble form of GP (sGP)
ceptible to NiV showing signs of meningitis and through RNA editing. Five strains are reported:
encephalitis, bronchointerstitial pneumonia, sys- Zaire ebolavirus (EBOV), Sudan ebolavirus
temic vasculitis, and focal necrosis in the spleen (SUDV), Tai Forest ebolavirus (TAFV),
and lymph nodes. Viral antigen is detected in the Bundibugyo ebolavirus (BDBV), and Reston
endothelial and smooth muscle cells of the brain, ebolavirus (RESTV). All are pathogenic to
lungs, and lymphoid system. Virus antigen is humans except RESTV, which is thought to be
present in neurons, glial cells, and epithelial cells pathogenic to nonhuman primates.
of the upper and lower respiratory tracts.
Pathogenesis
Prevention and Control The incubation period of the disease is 2–21 days
Culling of infected swine helped reduce NiV (average 7–10 days). The pathogenesis is not
cases in Malaysia. Heat treatment of sap or avoid- fully understood, but the virus is thought to sup-
ing unprocessed sap consumption will also help press both innate and adaptive (cellular and
prevent viral infection. Serologic testing for anti- humoral) immune responses. The virus replicates
body titer in human sera and reverse transcriptase in monocytes, macrophages, and dendritic cells.
PCR assay have been used for diagnosis and The virus is found inside endothelial cells, fibro-
detection from human urine, cerebrospinal fluid, blasts, hepatocytes, and adrenal cells and dis-
and oral swabs. seminates to the lymph nodes, liver, and spleen.
Massive production of proinflammatory cyto-
kines (IL-1, IL-6, IL-8, IL-15, IL-16) and several
Ebola Virus chemokines lead to shock and multi-organ failure
and death. The symptoms of infection include
Introduction lack of appetite, fever, headache, malaise, joint
In the past several years, the Ebola virus caused and muscle aches, abdominal pain, diarrhea and
major outbreaks in West and Central Africa vomiting, and internal and external bleeding at
with a case fatality rate of 25–90%. The Ebola the later stage of the disease. Reverse transcrip-
virus was first identified in 1976 in the tase PCR is used for diagnosis of the disease.
Democratic Republic of the Congo, and it was Vaccines are being developed for humans using
named after the Ebola River located near the inactivated virus and DNA vaccine using
epicenter of the first outbreak. The fruit bats are replication-defective recombinant adenovirus
considered the reservoir. Animals or humans type 5 expressing glycoprotein (GP) and nucleo-
have acquired the disease by consuming or capsid protein (NP).
coming in contact with the infected bats or ani-
mals (gorillas, chimpanzees, and monkeys).
Thus, there is a strong evidence for the infec-
tion to be of foodborne zoonotic disease, and Prevention and Control
bush meat may be an important link. The virus of Foodborne Viruses
can pass through bodily fluids and spread from
human-to-human. Enteric viruses are shed in large numbers from
the host through feces and vomitus, and they
Biology could be airborne or waterborne. The infectious
The Ebola virus is an enveloped single-stranded dose is very low; thus, effective sanitization and
RNA virus (19 kb) of the family Filoviridae and control measures need to be employed to prevent
genus Ebolavirus. It is a filamentous and pleo- contamination and spread. Person-to-person
morphic virus with about 1200 nm in length. The transmission occurs readily when people are in
close contacts, especially in cruise ships, in res- consumption of infected brain tissues of rela-
taurants, and in hospitals. tives. The disease was characterized by the
Depuration helps remove the virus from shell- degenerative brain with spongy appearance, and
fish; however, proper water temperature should the victims suffered from rapid physical and
be maintained. During depuration, harvested mental abnormalities, culminating in paralysis,
shellfish are kept in clean fresh water for 24–48 h coma, and death. It was called slow virus because
where viruses are escaped into the water. Food the incubation period is about 2–10 years.
preservatives (chlorine compounds, detergents, It became a major concern in the early 1990s
etc.), freeze-drying, ultraviolet light, freezing, when the disease was detected in cattle, and the
and heating at 100 °C can inactivate foodborne wasting of brain tissue resulted in abnormal
viruses. In general, viruses are highly stable, behavior in cattle; hence, it was called “mad cow
because the virus coat proteins most likely pro- disease.” Animals exhibit symptoms of abnormal
vide the protection against processing treatments. gait, hyper-responsiveness to stimuli, tremors,
Viruses are remarkably stable at high tempera- aggressive behavior, nervousness or apprehen-
tures such as 90–100 °C, possibly because the sion, changes in temperament, and even frenzy.
virus particles may remain aggregated or pro- Cattle over 24–30 months of age are susceptible
tected by food particles. UV treatment can inacti- to this infection. The incubation period of classi-
vate the virus. During the farming of fruits and cal BSE is about 2–8 years. Though there is no
salad vegetables, clean virus-free water should be human case directly linking the consumption of
used for irrigation. Food handlers serve as a contaminated beef, finding the organism in the
source; thus, workers’ health and hygienic prac- late 1990s and early 2000 (2003) in Canada and
tices should receive the greatest attention. the USA caused a major beef embargo among
Shellfish are a potential source of norovirus and developed countries with huge economic impacts
hepatitis A virus, and these animals should not be costing both countries over 4–6 billion dollars.
harvested from water that may have been pol- WHO reported that from October 1996 to
luted with sewage. March 2011, 175 cases of vCJD have been
reported in the UK; 25 in France; 5 in Spain; 4 in
Ireland; 3 each in the Netherlands and the USA;
Infective Proteins 2 each in Canada, Italy, and Portugal; and 1 each
in Japan, Saudi Arabia, and Taiwan. BSE is
Bovine Spongiform Encephalopathy endemic in the UK with reported 176 cases of
vCJD as of April 2012. The incubation period for
vCJD in humans is 11–12 years. Before 1980,
Introduction vCJD occurred due to the use of (1) cadaveric
A group of neurodegenerative infective agents human growth hormone, (2) contaminated surgi-
(prions) capable of transmission to various hosts cal instruments, (3) infected dura mater graft, and
is termed transmissible spongiform encephalopa- (4) corneal transplant. In recent years, however,
thies (TSEs). Several TSE agents are described in consumption of contaminated animal products
the literature: bovine spongiform encephalopathy with a brain, lymph nodes, or neurons is thought
(BSE) or “mad cow disease” in cattle, “scrapie” to be responsible for transmission. One suspected
in sheep and goat, chronic wasting disease source of BSE in beef is presumably due to the
(CWD) in cervids, transmissible mink encepha- feeding of beef cattle with contaminated meat
lopathy (TME) in minks, and Creutzfeldt–Jakob and bone meal (MBM) preparation, for fast
disease (CJD), variant CJD (vCJD), Gerstmann– growth and increased body weight gain. MBM is
Straussler syndrome, and “kuru” in humans. In often prepared from sheep offal and/or con-
the early 1950s, in the eastern highlands of Papua demned bovines, which are not fit for human
New Guinea, Kuru was prevalent among the food. In 1997, the US-FDA banned the use of
islanders due to a cannibalistic practice of proteins derived from mammalian tissues in
Infective Proteins 147
f eeding to ruminants in an effort to prevent trans- etc). It can withstand dry heat treatments of
mission of TSE to food animals. Conversely, the 160 °C for 24 h, or at 360 °C for 1 h, and satu-
UK delayed imposing such a ban and about 100 rated steam autoclaving at 121 °C for 1 h. The
persons developed fatal cases of vCJD between prion protein is resistant to chemical treatments
roughly 1996 and 2005. such as 0.5% sodium hypochlorite for 1 h, 3%
hydrogen peroxide for 1 h, and ethanol. However,
Biology
complete inactivation is possible by autoclaving
BSE-causing agent was originally thought to be a
at 132 °C for 1.5 h, and treatment with 1 M
virus, but later in 1982, Dr. Prusiner discovered
sodium hydroxide at 20 °C for 1 h, or sodium
that it is a proteinaceous infectious particle called
hypochlorite (2% chloride) for 1 h at 20 °C.
prion protein (PrP). He received the Nobel Prize
for his work in 1997. Prion protein has aberrant Pathogenesis
protein folding, and its accumulation in nervous Transmission of prion through digestive tract has
tissues leads to neurodegeneration. It is resistant been the subject of much investigation in recent
to most treatments including heat, chemicals, and years. Prion possibly passes through the M cells
proteases. The prion is found primarily in the cen- overlying the Peyer’s patches, and it is then trans-
tral nervous systems (CNS) including the brain ported by the dendritic cells to the central ner-
and neurons and in lymphatic system in the gut. vous system and the brain. In another study, it is
Amino acid sequences of PrP from normal and proposed that the prion bypasses the lymphoid
infected brains are identical but show differences system altogether and is directly transmitted via
in biochemical and biophysical behaviors. the peripheral nervous system to reach the
Monomeric form of the PrP protein contains 253 CNS. PrP accumulates in the neural cells and dis-
amino acids with a molecular mass of 22–36 kDa, rupts normal neurological function, causing vac-
while the abnormal or infective molecule is a uolation (spongy appearance) and cell death.
macromolecular aggregate with a molecular mass In humans, the first signs are psychiatric, such
greater than 400 kDa. The normal cellular version as anxiety, depression, insomnia, withdrawal,
of PrP is called PrPc and is encoded by a single paranoid delusions, head and neck pain, and pro-
chromosomal gene, PRNP located on the chro- gressive dementia. Mean duration of suffering is
mosome 20 in humans. PrPC is sensitive (PrPsen) to about 14 months. The neurologic symptom is
proteases such as proteinase K and trypsin. PrP- accompanied by cerebral ataxia (defective mus-
mRNA is 2.1 kb long and is detected primarily in cular coordination) and dementia. In the terminal
the brain (neurons) and small amounts in the stage, the patient becomes bedbound, akinetic,
lungs, spleen, and heart. The infective form is and mute, a state in which the person is not able
resistant to protease and has a drastically different or will not move or make sounds.
secondary structure and referred to as PrPSc (PrP
from scrapie). The α helical structures are pre-
There is no laboratory test available to use in the
dominant in PrPC, while a misfolding of the prion
live animals or humans for testing of abnormal
protein results in the formation of β-sheet, which
PrPSc. Postmortem analysis of brain tissues shows
is abundant in PrPSc. Normal PrPc has α-helix of
characteristics amyloid plaque, which is a waxy
40% and β-sheet 3% while in the disease-causing
translucent substance, composed of complex pro-
prion (PrPSc) has α-helix 30% and β-sheet 40%.
tein fibers and polysaccharides that are formed in
The prions are highly hydrophobic and form
body tissues in some degenerative diseases, such
aggregates easily. Aggregates are highly resistant
as Alzheimer’s disease, and spongy appearance.
to cellular digestion and accumulate in the lym-
Immunoassays (Western blot or ELISA) are used
phoid and nerve tissues and cause a spongiform
to detect PrPSc antigens in cattle after slaughter.
change in the brain.
In humans, magnetic resonance imaging (MRI)
Prion (PrPSc) is highly resistant to heat, certain
has been used as a tentative diagnosis to detect
chemicals, and proteases (proteinase K, trypsin,
cortical atrophy in the brain, coupled with
clinical signs. There is no treatment available foodborne source, but the virus can spread from
for the infectious prion. human-to-human through bodily fluids.
PrPSc is concentrated in certain tissues in Transmissible spongiform encephalopathy (TSE)
infected animals and referred to as SRM (specific diseases such as bovine spongiform encephalopa-
risk material). These include the brain, spinal thy (BSE) and varient Creutzfeldt-Jakob Disease
cord, skull, vertebral column, eyes, tonsil, and (vCJD) are caused by misfolded neurodegenerative
ileum. BSE can be prevented by several ways: (1) infectious prion proteins (PrPSc), which are highly
routine surveillance for BSE-infected cattle, (2) resistant to heat and protease enzymes and can be
prevent entry of BSE agent in cattle population, transmitted by consuming contaminated meat.
(3) stop feeding the beef cattle with animal pro- Preventing the use of meat–bone meal (MBM) or
teins derived from other animals, and (4) identify specific risk materials (SRM) can prevent the
and condemn the infected cattle before entering spread of prions among the meat-producing ani-
into the human food chain. Currently, the mals and to humans.
European Union and the USA have banned the
feeding of animal proteins from other animals.
BSE suspect carcasses should not be used for Further Readings
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Foodborne Parasites
7
host and are known as heteroxenous. Foodborne

Introduction parasites do not proliferate in food but require
live hosts for their growth. Parasites are detected
Parasites are those organisms that derive nourish- by direct means using microscopy, genetic, or
ment and protection from other living creatures. antibody-based assays.
Some parasites are unicellular organisms, such as Contamination of foods with parasites often
protozoa, or multicellular, such as metazoa. indicates inadequate hygienic practices employed
Metazoa includes helminths, nematodes, trema- during production and processing. Water, fruits
todes, and arthropods (flies, mosquitos, ticks). and vegetables, and raw or undercooked meat or
Some of the parasites can be transmitted by the fish are known to be the major source of parasitic
arthropod vectors or food. Parasites are classified pathogens. The occurrence of parasitic diseases
into ectoparasites that harbor outside the body, is high in tropical countries, but global warming,
such as ticks, lice, and mites; endoparasites that the climactic shift in weather patterns, and glo-
reside inside the host such as Toxoplasma, Taenia, balization of food supply may cause increased
Trichinella; and epiparasites that feed on another foodborne parasitic diseases in countries that
parasite such as endoparasite of a flea living on a may not have experienced in the past.
dog. Foodborne parasites are endoparasites, and
the major groups are protozoa, cestodes (tape-
worms), nematodes (roundworm), and trema- Protozoa
todes (flatworms or flukes) (Table 7.1). Parasites
are significantly larger than the bacteria and vary Giardia duodenalis
in their sizes. The general life cycle consists of a
stage, in which the parasite is as an oocyst (egg) Introduction
and can contaminate food and water (Fig. 7.1). Giardia duodenalis also known as Giardia lam-
The oocysts are morphed into larvae (designated blia or G. intestinalis is responsible for giardia-
trophozoites, bradyzoites, or merozoites) and sis. The disease is characterized by watery
then become adults. Larva stage is most infective. diarrhea referred to as traveler’s diarrhea, which
Most parasites require more than one animal occurs in people during travel to countries where
host, i.e., an intermediate host to complete their sanitary practices are inadequate. Giardia is also
life cycle. Generally, the protozoa complete their sometimes responsible for chronic illness. The
life cycle in a single host and are called homoxe- common source for giardiasis is contaminated
nous, while the metazoa require more than one water and vegetables. Cysts (eggs) are also found

152 7 Foodborne Parasites
in sewage affluent. Wild animals, such as b eavers, and contain two nuclei and four flagella, and they
can carry the protozoa and contaminate water. display tumbling motility (Fig. 7.2). Trophozoites
use surface protein, alpha-giardins to attach to
Pathogenesis the intestinal epithelium for colonization and
Giardia is an extracellular parasite and resides in infection. They multiply rapidly through asexual
the intestinal lumen. Giardia life cycle has two reproduction and cause damage to the mucus
stages: cyst and trophozoite. Cysts are infective membrane and disrupt epithelial tight junction
while trophozoites are not. The infective dose is barrier to increase intestinal permeability in the
10–100 cysts, and the incubation period is upper small intestine. During chronic infection,
1–2 weeks. Once ingested, the cyst (ovoid, giardia downregulates tight junction protein,
9–12 μm long) arrives in the duodenum and is claudin 1, and increases epithelial apoptosis.
dissolved by digestive enzymes, and two tropho- Some trophozoites mature into the cyst, before
zoites (9–21 μm long and 5–15 μm wide) are being released into the feces completing its life
released. The trophozoites are teardrop shaped cycle. Giardiasis is characterized by malabsorp-
tion, severe watery diarrhea, bloating, and flatu-
Table 7.1 Foodborne parasites lence. Treatment with metronidazole or tinidazole
Group Genus/species can eliminate the infection. Giardia is resistant to
Protozoa Giardia lamblia; Entamoeba chlorine treatment that is applied to municipal
histolytica water supplies.
Cyclospora; Cryptosporidium;
Cystoisospora
Toxoplasma gondii; Trypanosoma cruzi
Cestodes Taenia saginata; T. solium Entamoeba histolytica
(tapeworms) Echinococcus granulosus
Nematode Ascaris lumbricoides Introduction
(roundworms) Trichinella spiralis The genus Entamoeba consists of several spe-
Trematodes Clonorchis sinensis cies: Entamoeba histolytica, E. dispar, E. mosh-
(flatworms or Paragonimus spp kovski, E. coli, E. hartmanni, and E. polecki.
flukes) Fasciola hepatica Entamoeba histolytica is pathogenic and the
Fig. 7.1 General life

cycle of parasites
Protozoa 153
Fig. 7.2 (a) Illustration

of Giardia duodenalis
protozoa (Courtesy of
Jennifer Oosthuizen and
James Archer, CDC,
Atlanta, GA), (b)
Giardia duodenalis life
cycle and tissue damage
in the intestine
most invasive member of the genus and is respon- is 1–4 weeks. Ingested organisms reach to the
sible for amebic dysentery. Other species of lower small intestine (terminal ileum) and upper
Entamoeba exist as commensal in the human large intestine, where each cyst gives rise to eight
intestine. Worldwide, E. histolytica causes 50 daughter trophozoites. The trophozoites (10–
million cases and 100,000 deaths, and the most 60 μm in diameter) are motile (Fig. 7.3) and
amebiasis cases are associated with the develop- adhere and invade intestinal epithelial cells,
ing countries including Africa, Central and South majorly in the large intestine. They produce sev-
America, and the Indian subcontinent. Amebic eral virulence factors, which promote adhesion
cases are also predominant in tropical and sub- and tissue damage. The parasite uses Gal/
tropical countries. Humans serve as the major GalNAc-inhibitable lectin to interact with the host
reservoir of the organism and can contaminate glycoprotein for adherence, to block complement
water and foods. Unsanitary living conditions activation, and to promote cytotoxicity of neutro-
and unhygienic food preparation practices are the phils and macrophages. The parasite produces
major risk factors for the spread of amebic infec- proteolytic enzymes (collagenase and neutral pro-
tion. Transmission may occur through contami- teases) and cysteine proteases, which promote
nated water, fresh foods, sewage, insects, and parasite invasion and tissue damage. It produces
fecal–oral route or in homosexuals through oral– amoebapore that forms ion channels in the host
anal contacts. cell membrane causing cytolysis. Trophozoites
can cause localized infection by invading mucosal
Pathogenesis membrane in the intestine or pass through the
Entamoeba histolytica produces cysts (10–15 μm bloodstream to reach to extraintestinal sites such
in diameter), which are infective. The infective as the liver, brain, and lungs. The growth of tro-
dose is above 1000 cysts, but a single cyst has the phozoites results in inflammation and ulceration
potential to cause disease. The incubation period in the mucosal layer. The trophozoites multiply by
Fig. 7.3
Photomicrograph of
Entamoeba histolytica
trophozoite carrying a
number of erythrocytes
(arrow) as inclusion
bodies (Courtesy of Dr.
Mae Melvin; Dr.
Greene, CDC, Atlanta,
GA)
binary fission and produce cysts, and both stages ered a zoonotic pathogen and is one of the leading
are found in the feces. Each trophozoite carries causes of foodborne illnesses and deaths. It
single nucleus and can convert into precyst and causes about 87,000 illnesses, 4428 hospitaliza-
matures into tetranucleated cysts, which are tions, and 327 deaths annually in the USA.
released with feces. Cysts can survive outside for Drinking water contaminated with cat feces is
several weeks to months but are sensitive to tem- thought to be responsible for several outbreaks
perature under −5 °C or over 40 °C. worldwide including a large outbreak in Brazil in
The symptoms of amebiasis include abdomi- 2002. Unwashed hands after contact with pet
nal cramp and diarrhea which is watery, mucoid, cats, vegetables washed in contaminated water,
or bloody resembling bacillary dysentery caused and eating of undercooked pork are considered
by Shigella dysenteriae type 1 and S. flexneri (see potential sources for this parasite.
Chap. 19) and enteroinvasive Escherichia coli
(see Chap. 14). Diarrhea may occur with ten or Pathogenesis
more bowel movements per day, and some Toxoplasma gondii is an obligate intracellular
patients show high fever. The patients show signs parasite and it has very low host specificity. Its
of anorexia and continue to loose body weight. life cycle has two phases: intestinal (enteroepi-
The disease becomes chronic in some patients, thelial) phase, which is seen on the primary host,
characterized by intermittent diarrhea, flatulence, cats, and extraintestinal phase, which is seen in
and ulcerative colitis. The extraintestinal disease the secondary host, i.e., all infected animals
condition may include liver abscess, pulmonary (Fig. 7.4). Cats can be infected by eating infected
disease, peritonitis, pericarditis, and brain prey. Cysts or oocysts in the meat are dissolved in
abscess. Treatment with metronidazole (tinida- the lumen of the digestive tract and the bradyzo-
zole) is effective against amebiasis. ites are released, which penetrate intestinal epi-
thelial cells and undergo asexual multiplication.
Later, sexual multiplication follows. Male and
Toxoplasma gondii female gametes form zygotes, which develop
into oocysts and released in the feces. Oocysts
Introduction mature in the feces and infect warm-blooded ani-
The disease caused by Toxoplasma gondii is mals (intermediate host) including rodents, farm
called toxoplasmosis, which normally is trans- animals, and humans. Oocyst dissolves in the gut
mitted by the domestic cat. T. gondii is consid- and sporozoites are released which then penetrate
Protozoa 155
Fig. 7.4 Toxoplasma gondii transmission pathway
the intestinal epithelial cells, reach to blood cir- s ymptoms appear flu-like, swollen lymph nodes,
culation, and invade muscle tissues. Sporozoites fatigue, joint and muscle pain, rash, and head-
bind to host cell receptor, sialic acid, and exhibit ache, and the disease is usually self-limiting. In
gliding motility that helps parasite invasion into immunocompromised host, the disease could be
enterocytes. Invasion is an active process for the fatal. In pregnant women, there is a significant
parasite, and the host cell does not play an active risk of transmission of the parasite to the fetus,
role in invasion, i.e., invasion does not alter host resulting in spontaneous abortion. Infection
cell actin cytoskeleton or phosphorylation of during the later stage of pregnancy may be less
tyrosine residues. Inside the host cell, Toxoplasma severe and may not affect the fetus.
is trapped in a specially modified vacuole that is An experimental infection induced in mice
primarily derived from the host cell plasma mem- shares morphologic and histologic characteristics
brane. The parasite avoids fusion of vacuole with with human inflammatory bowel disease (IBD),
normal host endocytic or exocytic vesicle and which is characterized by the loss of intestinal
begins replication. Toxoplasma then migrates to epithelial architecture, shortened villi, the influx
the subepithelial region in the basement mem- of inflammatory cells (macrophages, monocytes,
brane and penetrates deep into the submucosa neutrophils, and lymphocytes), and scattered
and disseminates into central nervous system necrotic patches. The parasite is found mostly in
(CNS), retina, and placenta. the muscle and CNS. When the infection is
A human can also acquire by eating raw or unregulated, the inflammatory process results in
undercooked meats, water, or food contaminated early mortality. Female animals are more suscep-
with cat feces. In healthy individuals, the tible than the males, suggesting that gender and
sex hormones play an important role in determin- gliding motility using parasite-driven process.
ing the susceptibility of the small intestine to T. Cryptosporidium induces host cytoskeletal struc-
gondii. ture to create a platform made of host actin fila-
ments composed of α-actinin, ezrin, talin, and
I mmune Response to Toxoplasmosis vinculin causing severe cellular damage resulting
Toxoplasma gondii infection exhibits a signifi- in gastroenteritis.
cant increase in chemokine secretion and macro- The disease is characterized by watery diar-
phage inflammatory proteins and recruitment of rhea or cholera-like illness with abdominal
polymorphonuclear cells (PMNs) such as neutro- cramp, fever, and muscle ache. Anorexia, weight
phils, macrophages, monocytes, dendritic cells loss, dehydration, and abdominal discomforts are
(DCs), and T lymphocytes. Infiltration of neutro- generally associated with this disease. In immu-
phils is essential to remove T. gondii during the nocompromised patients (e.g., the AIDS patients),
first few days of infection. Neutrophil-depleted the organism is highly invasive and can infect
mice exhibit lesions and greater parasite burden. lungs and bile ducts which are often life threaten-
Activated CD4+ T cells produce increased IFN-γ ing. Infected cattle or human shed large numbers
and TNF-α that are responsible for recruitment of of oocysts in the feces, and the stool samples are
inflammatory cells including macrophages for used for diagnosis. Oocysts are resistant to chlo-
clearance of parasite. rine treatment; thus, contaminated water supply
becomes a threat for infection. A large outbreak
of Cryptosporidium occurred among HIV
Cryptosporidium parvum patients in Milwaukee (Wisconsin, USA) in the
mid-1990s due to the consumption of contami-
Introduction nated water from the municipal water supply. It
Cryptosporidium parvum is a coccidian protozoa was determined that the water was contaminated
and it causes cryptosporidiosis. The major source with cattle manure runoff and the parasite sur-
of this pathogen is water including agricultural vived the water treatment system.
runoff and the sewage effluent. Vegetables or
foods exposed to contaminated water serve as the
carrier for this pathogen. The first human case Cyclospora cayetanensis
was reported in 1976. There are several species in
the genus, but Cryptosporidium parvum is the There are 17 species of Cyclospora. Cyclospora
most common. Two types of C. parvum exist, cayetanensis is the newest member of the coccid-
type I and type II. Type I is exclusively from ian family that is responsible for food and water-
humans and type II from cattle or humans borne diseases. Consumption of
exposed to infected cattle. C. parvum completes oocyst-contaminated food or water leads to the
its life cycle in one host. The oocyst is 4–6 μm in onset of cyclosporiasis disease. The first human
diameter and carries four sporozoites inside. case was reported in 1977. Since then, outbreaks
of Cyclospora have been associated with raspber-
Pathogenesis ries, basil, and lettuce. In the late 1990s, contami-
The infective dose is thought to be very low, about nated raspberries from Guatemala caused
10 oocysts, and the incubation period is 2–10 days. widespread outbreaks in 20 states in the USA,
After consumption of contaminated food or water, and again in 2004, Guatemalan snow pea caused
one oocyst releases four sporozoites, which an outbreak in Pennsylvania (USA).
undergo asexual reproduction followed by sexual Infected people shed unsporulated oocyst,
reproduction, and the zygotes are formed. The which is non-infective and immature. The oocysts
sporozoites adhere to the epithelial cells and are 8–10 μm in diameter and oval shaped, and
invade apical surface of the epithelial cells by each differentiates or matures into sporocyst
Protozoa 157
under favorable condition. Each sporocyst carries Trypanosoma cruzi

two infective crescent-shaped sporozoites.
The infective dose is unknown but the incubation Introduction
period is typically 1 week. The disease usually Trypanosoma cruzi causes Chagas disease, also
lasts for 2 weeks. Oocysts excyst in the small known as American trypanosomiasis, and is a
intestine usually in the jejunum and sporozoites serious tropical disease in Latin America.
infect the epithelial cells. The detailed mecha- Trypanosoma cruzi was first identified by Carlos
nism of infection is not clear; however, histopa- Chagas, and he named it Trypanosoma cruzi after
thology shows villous atrophy, crypt hyperplasia his mentor, Oswaldo Cruz. It belongs to
with inflammation in the small intestine. Trypanosomatidae family. T. cruzi infects both
Cyclospora cayetanensis causes self-limiting vertebrates and invertebrates, hence called het-
watery diarrhea, fatigue, nausea, vomiting, myal- eroxenic. This protozoon is transmitted by blood-
gia, anorexia, and weight loss, and sometimes it sucking Triatomine insect, also called kissing bug
may cause explosive diarrhea. Cyclosporiasis that lives in dwellings in rural poor communities.
sometimes may lead to chronic sequelae like Contaminated fruit juice laden with feces of
Guillain–Barré syndrome, reactive arthritis, and Triatominae or crushed bugs during juice prepara-
acalculous cholecystitis. Trimethoprim–sulfa- tion is a major source of T. cruzi foodborne infec-
methoxazole treatment is effective in controlling tion in Brazil and Venezuela. Besides transmission
the infection. of T. cruzi through blood meal feeding during
Triatomine bite to humans, it can also transmit
through blood transfusions, organ transplantation,
Cystoisospora belli and from mother to fetus in the womb. Dogs have
been recognized as an animal reservoir.
Cystoisospora belli, formerly known as Isospora
belli, is a coccidian protozoan parasite and causes Pathogenesis
cystoisosporiasis, which is manifested by diar- Trypanosome generally enters through the skin
rhea. It is distributed in tropical and subtropical after an insect bite. During a blood meal,
countries including Latin America, Caribbean Triatomine releases Trypanosome through feces
countries, Africa, Southeast Asia, and Australia. on the bite wound. However, as a foodborne
It is generally transmitted through vegetables and pathogen, T. cruzi enters gastrointestinal tract
contaminated water. Cystoisospora oocysts (10– through contaminated food and interacts with
19 × 20–30 μm) are much larger than Cyclospora intestinal mucosal epithelial cells. T. cruzi is an
or Cryptosporidium. Oocysts are elliptical, and intracellular pathogen. It uses surface glycopro-
one oocyst forms two sporocysts, and each car- tein, gp82, to interact with host sialic acid and
ries four sporozoites. Like other coccidian proto- other unknown host cell receptors to invade cells.
zoa, the infective sporozoites invade intestinal Parasite interaction initiates signaling cascade
epithelial cells, undergo sexual and asexual and promotes cytoskeletal rearrangement facili-
reproduction, and cause tissue damage. Villous tating parasite invasion. During this process, the
atrophy, hypertrophic crypts, and inflammation host cell adenylate cyclase is activated promoting
and infiltration of epithelial cells with eosino- an enhancement of cAMP that contributes to the
phils are characteristics of cystoisosporiasis Ca2+ release from the endoplasmic reticulum,
infection. Watery diarrhea, abdominal pain, which promotes lysosome recruitment forming
anorexia, and weight loss are symptoms of this the parasitophorous vacuole where parasite rests
infection, and the disease can persist for months and transform into trypomastigote. Ca2+ can also
to years. It could be fatal in immunocompro- facilitate disorganization of the actin cytoskele-
mised hosts, and the AIDS patients are highly ton, aiding parasite invasion by disrupting micro-
susceptible to this infection. filaments. T. cruzi produces a toxin (TcTox),
which forms pores in the vacuole, and the trypo- some parts of Asia), and parasite life cycle is sus-
mastigotes escape into the cytoplasm. tained (Fig. 7.5).
Trypomastigotes transform into amastigotes, The length of an adult tapeworm varies
multiply by binary fission inside the cytoplasm, between 4 m and 12 m. T. solium is much smaller
burst out of the cell, and enter the bloodstream. and thinner than the T. saginata. Suckers in the
Triatomine insect acquires T. cruzi from humans scolex (head) help the worm to remain attached
during feeding on human or animal blood. to the intestinal wall. The segments or proglottids
The symptoms of trypanosomiasis (Chagas could give rise to several new progenies. Gravid
disease) may include fever, fatigue, body aches, proglottids may carry up to 80,000 eggs and each
headache, skin rash, loss of appetite, diarrhea, egg represents a single unit. Before eggs are
and vomiting. The acute Chagas disease is char- released, the scolex evaginates exposing the
acterized by swelling of the eyelids if the bug suckers outside the body, which can attach to the
feces are deposited near the eyes during the bite mucus membrane when ingested.
or accidentally rubbed into the eye. Acute phase
symptoms disappear within a few weeks or Pathogenesis
months. Infection persists if untreated. During Taenia solium causes taeniasis or cysticercosis in
chronic infection, enlarged heart or cardiomyop- humans. Taeniasis is caused by adult worms
athy, altered heart rhythm, and cardiac arrest may while cysticercosis is caused by ingestion of
occur. Intestinal complications include enlarged cysts (eggs). The lethal form of cysticercosis is
esophagus (megaesophagus) or colon (megaco- neurocysticercosis (NCC). Other forms include
lon), and disease becomes fatal, if untreated. ocular (OCC) and subcutaneous cysticercosis
Vector control is the key to controlling Chagas (SCC). For completion of the life cycle, the dogs
disease in the endemic areas. and pigs serve as the intermediate host and
humans the definitive host. In humans, after
ingestion of contaminated meat, adult tapeworm
Cestodes (Tapeworms) matures in the intestine, and eggs are released
from the gravid proglottids. The gravid proglot-
Taenia solium and Taenia saginata tids carrying thousands of eggs can be separated
from the adult parasites and are discharged with
Introduction the feces. Eggs also are released with the feces.
Cestodes are also known as tapeworms. Two spe- Pigs and dogs become infected by eating con-
cies of tapeworms are of major concerns to taminated food and water or direct eating of
humans: Taenia saginata, known as “beef tape,” human feces. A human can also be infected by
associated with beef, lamb, and fish, and Taenia Taenia eggs through food and water. Eggs reach
solium, associated with pork hence “pork tape.” CNS and infect the brain to cause neurocysticer-
T. asiatica also occurs in cattle and pigs and is cosis which results in seizures, epilepsy, menin-
another major pathogen of concern for humans. gitis, hydrocephalus, increased intracranial
Several other Taenia species infect dogs, cats, pressure, and death. Subcutaneous cysticercosis
wolves, and foxes and are of less public health is characterized by the formation of the firm and
concern (Table 7.2). Raw or undercooked pork or mobile nodules in the skin. Muscular cysticerco-
beef are the major sources of transmission to sis is characterized by myositis, muscular pseu-
humans. Asia and perhaps Africa and Latin dohypertrophy, fibrosis, and eosinophilia; and
America are considered endemic zones. The dis- ocular cysticercosis presents visual difficulties,
ease is considered a highly neglected disease, and retinal edema, hemorrhage, and vision loss.
the parasite is maintained in the rural impover- During the intestinal phase of infection (taenia-
ished parts of the communities where humans, sis), the tapeworms cause severe abdominal pain
pigs, and dogs coexist. Pigs and dogs feed on resembling colic-like sharp pain, headache, and
human feces or feces-contaminated foodstuff, diarrhea. Treatment regimen includes praziquan-
and the humans consume both pigs and dogs (in tel and corticosteroid.
Cestodes (Tapeworms) 159
Table 7.2 Taenia species and their host range

Species Intermediate host Definitive host
Taenia solium Pigs, dogs, humans Humans
Taenia saginata Cattle Humans
Taenia asiatica Pigs, cattle Humans
Taenia crassiceps Rodents, humans (rare) Dogs, foxes
Taenia ovis Sheep, humans (rare) Dogs
Taenia hydatigena Pigs, sheep, goats, humans (rare) Dogs
Taenia pisiformis Rabbits, humans (rare) Dogs, foxes
Taenia taeniaeformis Rodents, humans (rare) Cats
Taenia coenurus Sheep, rabbits, humans (rare) Dogs, cats, wolves
Taenia serialis Rabbits, rodents, humans (rare) Dogs, wolves, foxes
Adapted from Ito et al. (2016). Infect. Gen. Evol. 40, 357–367
Fig. 7.5 Life cycle of Taenia species (Schematic based on lifecycle illustration created by CDC, Atlanta, GA)
Echinococcus granulosus Humans are the accidental intermediate host and

acquire parasite eggs through contaminated fruits
Echinococcus is a very small tapeworm (3–6 mm) and vegetables. The disease is called echinococ-
found in carnivores. Dogs and other carnivores cosis or hydatidosis or hydatid disease. Four
are the definitive host, and ungulates (horses, species of Echinococcus are of public health con-
cattle, goats, sheep, pigs, giraffes, camels, deer, cerns: Echinococcus granulosus, Echinococcus
and hippopotamuses) are the intermediate host. multilocularis, Echinococcus oligarthus, and
Echinococcus vogeli. E. granulosus causes cystic first-stage larvae are called L1 and are released
echinococcosis, E. multilocularis causes alveolar from the eggs. The larvae molt into L2 form,
echinococcosis, and E. oligarthus and E. vogeli which is then eaten by crustaceans, where they
cause polycystic echinococcosis. mature into L3 form. Through predation, fish
E. granulosus is the most important species of acquires L3 form, which is infective to fish, and
concern, and the genotype sheep G1 is responsi- migrates from the intestine to muscle tissues.
ble for the majority of echinococcosis infection Once contaminated fish is ingested by humans,
in humans. After ingestion, the larvae reach to the the Anisakis larvae reach the stomach and attach
liver, which is the most common extraintestinal to the mucosa using their projections in the
site, and the parasite forms polyp (cyst). The mouth. They release proteolytic enzymes, which
lungs, spleen, kidney, heart, and CNS are also cause erosion of mucus and submucus layers
affected. The polyp grows very slowly in the leading to the hemorrhagic lesion. The acute gas-
organs at the rate of 1–5 cm per year, eventually tric symptoms appear within 2 h of infection
causing organ dysfunction, and the onset of clini- characterized by severe abdominal pain, vomit-
cal symptoms. A sudden burst of polyp results in ing, diarrhea, and mild fever. Untreated disease
severe hypersensitivity (allergic reaction) may lead to chronic ulcer-like infection. The
response due to the release of polyp content and onset of the intestinal form of anisakiasis appears
fatal anaphylactic shock. Treatment involves sur- within 5–7 days after ingestion of larvae, which
gical removal of the cysts (polyps), chemother- primarily affect terminal ileum causing inflam-
apy with benzimidazoles, and percutaneous mation and granulomatous lesion. The symptoms
aspiration, injection, and re-aspiration (PAIR). include constant or intermittent abdominal pain
and in rare cases, ascites, small bowel obstruc-
tion, ileal stenosis, and pneumoperitoneum.
Nematodes (Roundworm) Anisakis can also migrate to the peritoneal cavity,
liver, pancreas, and ovary.
The major foodborne nematodes include Anisakis The immunological response involves adap-
and Trichinella. In addition, other nematodes that tive immune response leading to both Th1 and
are also involved in foodborne infections are Th2 induction and production of IL-2, IL-4, IL-5,
Ascaris, Gnathostoma, and Angiostrongylus spp. and IFN-γ and proliferation of B lymphocytes for
the production of IgE. The cellular immune
response involves activation of eosinophils and
Anisakis simplex mast cells leading to a typical allergic response.
Anisakis simplex is a nematode responsible for

fish-borne disease called “anisakiasis,” first Trichinella spiralis
reported in the 1960s in the Netherlands. It is also
referred to as “worm-herring disease” resulting Introduction
from the consumption of lightly salted herrings. Trichinella species are transmitted through inges-
Consumption of raw or undercooked fish is tion of muscle tissues containing the encysted
responsible for this disease. The symptoms asso- Trichinella larvae. Trichinella spiralis causes
ciated with anisakiasis are described as gastric, trichinellosis and is mostly associated with the
intestinal, extraintestinal, and allergic. consumption of raw or undercooked pork prod-
Worldwide, about 20,000 people suffer from ani- ucts. Both wild and domestic swine are the major
sakiasis and about 90% of which are from Japan. source for Trichinella; however, other animals
Anisakis life cycle is complex and is main- such as horses, reptiles, birds, and wild carni-
tained in marine mammals and fish. Adult A. sim- vores (fox, hyena, and bear) serve as the reservoir
plex harbors in the stomach of marine mammals for Trichinella spp. Trichinella spiralis is the
and lays eggs, which pass through the feces. The major species associated with swine maintaining
Nematodes (Roundworm) 161
Fig. 7.6 Life cycle of Trichinella spiralis
a bioburden of greater than 8000 larvae per gram undergo molting to develop into sexually matured
of tissue without showing clinical symptoms and adults within 2 days. The male and female copu-
is a major public health concern. Other species of late, and the female releases newborn larvae
Trichinella include T. britovi and T. nelsoni, and within 5–7 days of post-infection. The larvae
both can infect swine in lesser frequency than T. reach to muscle tissue through blood circulation
spiralis. Other Trichinella spp. such as T. nativa, and are encapsulated in the muscle. Some larvae
T. murrelli, T. patagoniensis, etc., rarely infect may be found in the intestinal mucosa (Fig. 7.6).
pigs are of less public health concerns. Calcification of the encapsulated larvae in mus-
cle tissue is seen after 6 months of infection, but
Pathogenesis the larvae can survive in muscle tissues in humans
Trichinella species completes its life cycle in one for up to 40 years. Within months, due to immu-
host (Fig. 7.6). A gravid female worm living in nological action in the intestine, the adult para-
the mucosal layer in the host intestine releases sites are continuously expelled in the feces.
the newborn larvae, which migrate to lymphatics The trichinellosis is manifested in two phases:
and blood and then to muscle tissues forming the intestinal phase and the muscular phase. The
cysts (polyps). Larvae grow in the striated skele- intestinal phase of the disease is characterized by
tal muscle. When the undercooked infected meat gastroenteritis seen 2 days after infection: nau-
is ingested by man, swine, and horse, the disease sea, abdominal pain, diarrhea, and vomiting. The
propagates. Parasite larvae are released from the muscular phase is characterized by muscle pain.
meat tissues in the intestine after proteolytic Trichinellosis is again categorized as acute – or
enzymatic digestion, and the larvae penetrate the chronic – stage disease. Acute stage is character-
epithelial lining of the small intestine. Larvae ized by a headache, fever (lasting for 1–3 weeks),
chills, myalgia, pyrexia, eyelid swelling, occa- Curing and smoking are ineffective in controlling
sional gastrointestinal disorders, myocarditis, Trichinella larvae in meat. For treatment of trich-
thromboembolic disease, and encephalitis. In inellosis in humans, anthelmintic drugs such as
some patients infection may be severe, and albendazole and mebendazole are used.
clinical pathology includes conjunctivitis with
ocular edema and vascular lesion (bleeding) in
the conjunctiva, uvea, and retina. Some patients Ascaris lumbricoids
experience intense pain in the eyeballs due to the
invasion of larvae into the ocular muscle. Patients Introduction
may also suffer from myocarditis including peri- Ascaris lumbricoids, a nematode, is associated
cardial pain and tachycardia. In the chronic stage with food when prepared under poor sanitary
of disease (usually after 3–4 weeks after infec- conditions. The disease is called ascariasis and is
tion), the patients exhibit signs of encephalitis, manifested by enlarged liver and malnutrition. It
neurological disorders, and bronchopneumonia is a soil-transmitted helminth (STH); hence,
due to secondary infection by bacteria. fruits and vegetables from contaminated soil may
Symptoms may include persistent formication carry the worm. The disease affects adults and
(insect walking sensation on skin), numbness, children primarily in economically poor coun-
excessive sweating, impaired muscle strength, tries. Children in tropical and subtropical areas
and conjunctivitis. carry this parasite for a long period. The disease
The immunological response involves inflam- is prevalent in Asia, Africa, and Europe. Although
mation characterized by infiltration of eosino- there have not been any major outbreaks in the
phils, mast cells, monocytes, and lymphocytes. USA, the high occurrence of the eggs in sewage
The immune response is primarily mediated by municipal waters does present a potential high
Th2, and cytokine production leads to prolifera- risk for infection.
tion and activation of eosinophils as the major
effector cells. Eosinophilia (>1000 cells/ml) is Pathogenesis
common in trichinellosis patients; however, IgE Ascaris lumbricoides is mainly a human pathogen
response is inconsistent. while Ascaris suum infects swine. Report of A.
suum infection in humans is also documented.
iagnosis, Prevention and Control
D Both are prevalent in soil, and soil moisture,
The disease can be diagnosed by clinical symp- atmospheric humidity, and warm temperature
toms: fever, muscle ache, gastrointestinal symp- help maintain eggs and larvae and facilitate faster
toms, conjunctivitis, retinal hemorrhage, and development of ova. Fruits and vegetables con-
eosinophilia. Laboratory diagnosis involves taminated with fecal matter or soil containing the
PCR, ELISA, and Western blot (for seroconver- eggs is the source of infection in humans. Larvae
sion). Trichinellosis can be prevented by routine hatched from eggs, invade the intestinal mucosa,
postmortem inspection of meats from pigs, and are transported to lungs via lymphatics and
horses, wild boars, and bear for the presence of blood circulation. The matured larvae invade the
larvae or encapsulated calcified nodules in meat. alveolar wall, ascend the bronchial tree to the
The predilection sites for inspection include the throat, and are swallowed by the host. In the small
diaphragm, tongue, and masseter muscle (facial intestine, the larvae develop into adult worms and
jaw muscle). Microscopic examination of enzyme engage in sexual reproduction. The adult Ascaris
digested muscle tissues, and magnetic stirrer survives in the intestine for 1–2 years. The females
methods have been used for detection of larvae are larger (30 cm × 5 mm) than the males. A
from meats. The cyst could be easily killed by female may lay 200,000 eggs per day, and eggs
cooking meat at 71 °C or 159.8 °F (internal tem- are released through feces. Only fertilized eggs
perature) at least for 1 min, freezing meat at least are infective. The whole cycle of infection requires
at −18 °C for 30 days, and irradiation (0.3 kGy). 2–3 months for completion.
Trematodes (Flatworm or Fluke) 163
Ascariasis affects adults but is more common aquatic foods such as freshwater fish, frogs,
in children from 5–15 years of age. The disease is shellfish, snails, tadpoles, snakes, and water
manifested 4–16 days after infection. plants. The life cycle of trematodes starts with the
Malnourished children are highly susceptible to production of a large number of eggs by adult
ascariasis. Infected children exhibit slower worms following sexual reproduction in the final
growth, poor cognitive development, and slow host (humans or domestic or wild animals). For
weight gain since the parasite feed on nutrients of most foodborne trematodes, eggs are released
the partially digested food in the intestine. through feces or sputum (for Paragonimus spp.).
Patients infected heavily with parasite show Embryonated eggs hatch under appropriate envi-
symptoms of abdominal pain and obstruction of ronmental conditions including temperature,
the intestines accompanied with fever, inflamma- moisture, and oxygen tension. Hatched eggs
tion, and diarrhea. Other complications include release “saclike” larvae called miracidium, which
pancreatitis, acute cholecystitis, and liver abscess. begins swimming, and snails (intermediate host)
The diagnosis involves identification of eggs can ingest the miracidium. Alternatively, mira-
(40–70 μm × 35–50 μm) or the worm from the cidium larvae can penetrate the molluscan tissue
stool sample. In addition, the larvae can be found for life cycle completion. Two representative
in pulmonary secretions (sputum). It is also com- trematodes are described below.
mon for the patients to cough the worm when
they are migrating through the throat.
Clonorchis sinensis
Immune Response
The migration of the larvae to the pulmonary Clonorchis sinensis is also known as “Oriental
tract results in hemorrhagic lesions in the lungs liver fluke,” a major trematode of concern in parts
with eosinophil infiltration and inflammation. of the Asia including China, Taiwan, Japan,
The migration of the larvae to the intestine trig- Korea, and Vietnam. Snails serve as the first
gers an immunological response with mucosal intermediate host, which transfers the flukes to
mast cell recruitment and elevated levels of hista- fish. Freshwater or brackish water fish is the
mine. The immune response consists of Th2- second intermediate host, and human acquires
derived cytokines (IL-4 and IL-13) that stimulate infection upon consumption of raw or under-
intestinal smooth muscle contractibility, decrease cooked fish. Globally about 35 million people are
glucose intake, and enhance fluid accumulation infected with C. sinensis, of which 15 million
leading to the expulsion of the parasite. The lar- infections are reported in China. The parasite
vae, however, can overcome host defense by affects the liver and can obstruct the biliary duct.
upregulating gene expression to enhance motility The symptoms include jaundice, ascites, gastro-
and metabolic activity. intestinal bleeding, pancreatitis, gallstone, and
fatal cholangiocarcinoma.
Trematodes (Flatworm or Fluke)

Paragonimus Species
Foodborne trematodes pose a significant public
health concern and globally about 750 million Paragonimus species is also known as lung fluke
people are at risk. Trematodes comprised of liver (about 10 mm long) and is found in Southeast
flukes (Clonorchis sinensis, Fasciola gigantica, Asia, America, and parts of Africa. P. wester-
Fasciola hepatica, Opisthorchis felineus, and manni is distributed in Asia while P. kellicotti in
Opisthorchis viverrini), lung flukes (Paragonimus North America. Freshwater snail is the first inter-
spp.), and intestinal flukes (Echinostoma spp.). mediate host, and crustaceans (crab and crayfish)
Transmission to humans occurs through con- are the second intermediate host. Humans and
sumption of contaminated raw or undercooked other mammals (cats and dogs) are infected after
consumption of raw, undercooked, or pickled and migrate through several tissues and reside in
crustaceans. Humans release eggs through spu- the veins. Adult worms reside in the mesenteric
tum. Symptoms for paragonimiasis include veins, bladder, and rectal venules depending on
fatigue, malaise, myalgias, fevers, night sweats, the species. Symptoms and pathology include
vomiting, swollen lymph nodes, gastroenteritis, fever, hepatic granulomas, fibrosis, and occa-
hepatosplenomegaly, eosinophilia, and tubercu- sional embolic egg granulomas in the brain or
losis (TB)-like symptoms such as a cough, brown spinal cord, hematuria, scarring, calcification,
tinged sputum, and chest pain. Paragonimiasis and squamous cell carcinoma.
could be easily misdiagnosed. High numbers in Schistosoma spp. also infect ruminants (cattle)
lungs can result in serious complications. Patients causing intestinal, hepatic, and nasal schistoso-
showing symptoms of coughing, fever, hemopty- miasis. The intestinal disease is characterized by
sis (coughing of blood), and eosinophilia should diarrhea, weight loss, anemia, hypoalbuminemia,
be examined for Paragonimus. Avoiding eating hyperglobulinemia, and severe eosinophilia. In
raw crayfish is a good preventive strategy. severe cases, the animal dies within a few months
Praziquantel is the drug of choice for treatment. of infection. In the hepatic form, granulomas
develop in the liver and in the intestine. Lesions
are also found in the lungs, pancreas, and bladder
Schistosoma Species of heavily infected animals. In the nasal form of
the disease, a cauliflower-like granuloma growth
Schistosoma species is a blood fluke and causes is seen on the nasal mucosa. Partial obstruction of
schistosomiasis. It has not been implicated in any the nasal cavity leads to snoring sounds during
foodborne infection. Schistosoma species are breathing and grazing. A blockage also causes
considered zoonotic pathogens. There are several dyspnea. Nasal discharge is hemorrhagic and/or
species distributed around the globe: Schistosoma mucopurulent, and adult flukes are found in the
haematobium, S. mansoni, S. japonicum, S. blood vessels of the nasal mucosa; however, the
mekongi, and S. intercalatum. S. haematobium is pathogenesis is associated with the eggs.
widely distributed over the African continent Rupturing of the abscess releases eggs, which
with smaller foci in the Middle East, Turkey, and eventually leads to extensive fibrosis.
India. S. mansoni is widespread in Africa and
Middle East and the only species seen in the
Western Hemisphere in parts of South America revention and Control of Parasitic
P
and some Caribbean islands. S. japonicum is pre- Diseases
dominant in Asia, mainly in China, the
Philippines, Thailand, and Indonesia. S. mekongi Water treatment, hygienic practices, and proper
is predominantly found in Southeast Asia. S. washing and cooking of foods are necessary to
intercalatum is found in Central and West Africa. prevent parasite infection. Proper disposal of fecal
Schistosoma infects both humans and bovine spe- material is necessary to prevent the spread of par-
cies (water buffalo, cattle, goats). asitic diseases in economically poor countries
Schistosoma life cycle consists of a single since the eggs are present in the stools of an
intermediate host, a freshwater snail. Schistosoma infected person. Arthropod vectors should be con-
larvae enter through the host skin, while all other trolled to prevent parasite transmission among
trematodes enter by ingestion. Schistosoma eggs intermediate and definitive hosts. Food should be
are released with feces or urine and the eggs stored below 10 °C or frozen since parasites are
hatch and release miracidia, which penetrate tis- susceptible to cold. Wash fresh fruits and vegeta-
sues of the snail. In the snail, miracidia undergo bles thoroughly before consumption. Meat should
transformation to form sporocysts and then cer- be cooked thoroughly. For trematodes control in
cariae. Cercariae (infective) are released into the fish, the FDA recommend freezing at −20 °C or
water, swim freely, and penetrate human skin. below for 7 days or at −35 °C or below for 15 h
In the human host, they become schistosomula intended for raw consumption.
Chemotherapy should be used that can affect maintained as oocyst (egg) and can contaminate
cysts, larvae, and adult parasites. The drug should food and water. Oocysts or eggs hatch to produce
kill or paralyze parasites. Many parasites cause larvae and then mature into adults. Larvae stage
chronic diseases so treatments are often for the is the most infective, and some parasites require
long term. Thus, drugs present serious side an intermediate host and a definitive host to com-
effects. Anti-protozoan drugs include tinidazole, plete their life cycles. Immunocompromised peo-
which is effective against trichomoniasis, giardi- ple are highly susceptible to the intracellular
asis, and amebiasis. Antinematode drug is protozoan parasites. The hygienic practices dur-
mebendazole and is effective against pinworms, ing food production and processing are essential
roundworms, and hookworms. Anthelminthic for controlling and preventing parasitic diseases.
such as praziquantel is effective against schisto- Antiparasitic drugs are effective but often present
somiasis, cysticercosis, and paragonimiasis. serious side effects to the patients. Vector control
Anisakis simplex larvae are removed by endos- is also an important step for parasite control.
copy from infected patients, and granulomas are
removed by surgical method.
Common side effects of antiparasitic drugs are Further Readings
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cycle consists of a stage, in which the parasite is
Molds and Mycotoxins
8
crops that are affected include wheat, maize, pea-

Introduction nuts, tree nuts, cottonseed, and coffee. Food and
Agriculture Organization (FAO) of the United
Fungi are also called molds or microfungi. Molds Nations estimates 25% of the world’s crops are
are ubiquitous in nature and cause diseases in affected each year with annual losses account for
humans, animals, and plants. Fungi infection in about 1 billion metric tons of foods. Economic
mammals is referred to as mycosis, which is seen losses are attributed to (1) yield loss due to dis-
in the form of skin infections: ringworm and ath- eases by toxigenic fungi, (2) reduced crop value
lete’s foot. Invasive mycoses are caused by inha- resulting from mycotoxin contamination, (3)
lation of spores, and systemic spread of the losses in animal productivity from mycotoxin-
infection has been seen in healthy as well as in related health problems, and (4) human health
immunocompromised hosts, which can be fatal. costs. Additional costs associated with mycotox-
Mycoses are caused by two categories of patho- ins include the cost of management at all levels:
gens: primary pathogens such as Coccidioides prevention, sampling, mitigation, litigation, and
immitis and Histoplasma capsulatum and the research.
opportunistic pathogens such as Aspergillus Filamentous toxigenic molds produce myco-
fumigatus and Candida albicans. Some filamen- toxins, and both molds and mycotoxins affect
tous molds produce mycotoxin, a secondary food and feed supply chains including crop pro-
metabolite with no apparent function in the nor- ducers, animal producers, grain handlers and dis-
mal metabolism of fungi. It is, however, toxic to tributors, processors, consumers, and society as a
humans, animals, and birds. whole. Twenty-five to 50% of the foodstuffs are
Mold contamination can occur at various presumed contaminated with mycotoxins.
stages of plant-based food production: in the Generally, the cereal foods and nuts are the major
field, during harvest, processing, transportation, sources of mycotoxins. In addition, spices, fruits,
and storage. Mold infection in crops causes and their by-products may be a source. The dis-
reduced yield, and feeding of meat animals with ease caused by mycotoxin is called mycotoxico-
the contaminated feed results in poor animal sis, and it is nontransmissible and does not
health, death, and substantial economic losses. respond to antibiotics, and the associated out-
Molds also cause spoilage of foods. The major break is usually seasonal.

168 8 Molds and Mycotoxins
Biology tion or frameshift mutation due to deletion, addi-

tion, or substitution of nucleotide bases in DNA,
Molds are multicellular microorganisms, and a and can affect liver function (hence hepatotoxic).
typical mold possesses hyphae, conidiophore – Teratogenic action leads to birth defect, and the
consisting of stalk, vesicle, sterigmata, and carcinogenesis causes irreversible defects in cell
conidia (spores) (Fig. 8.1). Often the identifica- physiology resulting in abnormal cell growth and
tion of mold species is done by examining the metastasis.
morphological shape and size of the spores under Three major genera of molds, Aspergillus,
a light microscope. The cell wall consists of a Penicillium, and Fusarium, are of significant
polymer of hexose chitin and N-acetyl glucos- interest in food safety for the production of
amine (NAG). Mold spores or hyphae are aller- mycotoxins (Table 8.1); however, several other
genic to humans, and frequent exposure may mold species are also recognized in recent years
occur in damp buildings with high humidity with for production of a variety of mycotoxins.
poor air circulations.
Toxigenic filamentous molds produce myco-
toxins. There are about 400 different mycotoxins, Aflatoxin
and only a few have been studied in depth. The
same mycotoxin can be produced by different Aflatoxin (Aspergillus flavus toxin) is produced
species of mold, or many species can produce the primarily by Aspergillus flavus and A. parasiticus
same mycotoxin. Mycotoxins are low molecular (Fig. 8.2). Aflatoxins (AF) are difuranocouma-
weight secondary metabolites. The molecular rins and occur in different chemical forms: AFB1,
weight may vary from 50 Da to >500 Da and gen- AFB2, AFG1, AFG2, and AFM1. The B and G
erally non-immunogenic. The structure may vary forms stand for the blue fluorescence and green
from a simple heterocyclic ring to 6–8 irregularly fluorescence, respectively, which are evident
arranged heterocyclic rings. They are highly sta- when the samples containing those mycotoxins
ble, hence difficult to destroy by traditional food are exposed to UV light. Aflatoxins are found in
processing conditions. Under certain wavelength nuts, spices, and figs and are produced during
of UV light, they emit fluorescence thus can be storage under hot and humid conditions. The
used for screening of contaminated food or feed allowable toxin limits are 20 ppb in nuts such as
stuff. The optimal condition for mycotoxin proin Brazil nuts, peanuts, and pistachio. Aflatoxin-
duction is at a water activity (Aw) of 0.85–0.99 contaminated feed causes high mortality in farm
and at a temperature range of 10–30 °C animals. Cows fed with aflatoxin-contaminated
(Table 8.1). feed convert the aflatoxin B1 into hydroxylated
The toxigenic molds are a major problem in form called aflatoxin M1 (milk), and the toxin is
agriculture products such as grains, cereals, nuts, released through milk, and the allowable limit in
and fruits. Mycotoxins (Fig. 8.1) produced in milk is 0.5 ppb. The allowable limit in meats,
these products can cause severe health problems corn, and wheat is also 0.5 ppb. The 50% lethal
in both humans and animals. When mycotoxins dose (LD50) for rat is 1.2 mg/kg. The acute lethal
are consumed at high dosage or over a long dose for an adult human is thought to be
period, they may cause severe disease. 10–20 mg. The primary target organ for aflatoxin
Mycotoxins can cause acute disease manifested is the liver. Mitochondrial cytochrome P450
by kidney or liver failure or chronic diseases such enzyme in the liver converts aflatoxin into reac-
as carcinoma, birth defects, skin irritation, neuro- tive 8,9-epoxide form, which binds to DNA and
toxicity, and death. Three general mechanisms of results in GC to TA transversions, leading to car-
mycotoxin action are described: mutagenic, tera- cinogenesis. Aflatoxin causes gross liver damage,
togenic, and carcinogenic. During the mutagenic resulting in liver cancer (hepatocarcinogen). It
action, the toxin binds to DNA, especially the can also cause colon and lung cancer. The
liver mitochondrial DNA resulting in point muta- International Agency for Research on Cancer
Biology 169
Fig. 8.1 Schematics of (a) Aspergillus species and (b) icroscopic photograph of (c) Aspergillus niger and (d)
m
Penicillium species. Panels (c) and (d) are the light Penicillium citrinum (magnification 400×)
Table 8.1 Optimal condition for mycotoxin production from molds

Mold Mycotoxin Optimum temperature (°C) Optimum water activity (Aw)
Aspergillus flavus Aflatoxin B1, B2, G1, G2 33 0.99
A. parasiticus
Aspergillus ochraceus Ochratoxin 30 0.98
Penicillium verrucosum Ochratoxin 25 0.90–0.98
Aspergillus carbonarius Ochratoxin 15–20 0.85–0.90
Fusarium verticillioides Fumonisin 10–30 0.93
Fusarium verticillioides Deoxynivalenol (DON) 11 0.90
Fusarium graminearum Zearalenone 25–30 0.98
Penicillium expansum Patulin 0–25 0.95–0.99
Adapted and modified from Murphy et al. (2006). J Food Sci 71, R51–R65
Fig. 8.2 Chemical structures of selected mycotoxins
(IARC) has classified aflatoxin B1 as a group I ochratoxin (Fig. 8.2). Ochratoxin is a phenylala-
carcinogen. AFB1 suppresses the immune sys- nyl derivative of a substituted isocoumarin and is
tem, promotes inflammation, and retards the found in a large variety of foods including wheat,
growth of humans and animals. corn, soybeans, oats, barley, coffee beans, meats,
Aspergillus flavus also causes “Aspergillus ear and cheese. Barley is thought to be the predomi-
rot” in corn. Climate change can affect Aspergillus nant source. The toxin is heat stable requiring
flavus growth and aflatoxin B1 production. Hot exposure to 250 °C for several minutes to reduce
and dry climates have the propensity for high risk activity. Ochratoxin is hepatotoxic and nephro-
of aflatoxin production. toxic and a potent teratogen and a carcinogen.
Nephropathy and renal pathology are predomi-
nant consequences of ochratoxin poisoning. The
Ochratoxin toxin is structurally similar to phenylalanine
(Phe), thus inhibits enzymes that use Phe as a
Aspergillus ochraceus and several other species substrate. Specifically, it inhibits phenylalanine–
including Penicillium spp. produce seven struc- tRNA synthetase to synthesize phenylalanine
turally related secondary metabolites called required for protein synthesis. It causes mito-
Biology 171
chondrial damage, oxidative burst, and lipid per- Trichothecenes

oxidation and inhibits cellular function and ATP
production. It also causes apoptosis in some Trichothecenes are a group of structurally related
mammalian cells. The LD50 value in rats is compounds. Over 180 trichothecenes are
20–22 mg/kg–1. The IARC considers ochratoxin reported, and they are produced by a number of
as the category 2B carcinogen. The provisional fungal genera including Fusarium, Trichoderma,
tolerable weekly intake (PTWI) is 120 ng kg−1 Myrothecium, Stachybotrys, Trichothecium, and
body weight. The toxin is analyzed by using the others. The trichothecenes (T) are divided into
high-performance liquid chromatography four groups (A–D). Group A consists of the most
(HPLC) technique and a mass spectrometry. common trichothecenes: 3-acetyl deoxynivalenol
(DON), HT-2, and T-2 toxin, and group B are
represented by DON. Groups C and D trichothe-
Fumonisins cenes are of less importance. T-2 and HT-2 are
produced by Fusarium sporotrichioides, F. langs-
Fumonisins are synthesized by the condensation ethiae, F. acuminatum, and F. poae, while DON
of amino acid alanine into acetate-derived pre- is produced by F. graminearum, F. culmorum,
cursor, and the most abundant form is fumonisin and F. cerealis. These toxins are associated with
B1 (Fig. 8.2). Other forms of the toxins are FB2 several different cereal products, meat, and dairy
and FB3. The FB1 has structural similarity to products. The toxins can be detected by HPLC
sphinganine, the backbone precursor of sphingo- and the thin layer chromatography.
lipids. Fumonisins are produced by Fusarium Trichothecenes inhibit eukaryotic protein syn-
verticillioides, F. proliferatum, and F. nygamai. thesis by binding to the 60S ribosomal subunit
Fusarium verticillioides under ideal conditions and by interacting with the peptidyl transferase
can infect corn causing seedling blight, stalk rot, enzyme thus preventing peptide bond formation.
and ear rot and are present virtually in all matured The T-2 toxin can also inhibit DNA and RNA
corns. Corns, tomatoes, asparagus, and garlic are syntheses and cell death. Toxins cause hemor-
the major source of fumonisin. Fusarium ear rot rhage in the gastrointestinal tract, diarrhea, and
of corn is also caused by Fusarium verticillioi- vomiting. Toxins are cytotoxic, immunotoxic,
des. Fumonisins are highly water-soluble, and and hepatotoxic, and direct contact may cause
they do not have any aromatic structure or unique dermatitis. For trichothecenes (T-2 and HT-2),
chromophore for easy analytical detection; how- the tolerable daily intake (TDI) is 0.1 μg per kg
ever, HPLC with a fluorescence detector has been body weight, and provisional maximum tolerable
used for identification. Fumonisins are highly daily intake (PMTDI) is 0.06 μg per kg body
stable to a variety of heat (150 °C) and chemical weight per day. For DON, both TDI and PMTDI
processing treatments and are not degraded dur- are 1 μg per kg body weight per day, and acute
ing fermentation. reference dose (for 1-day exposure) (ARfD) is
Fumonisin B1 strongly inhibits ceramide 8 μg per kg body weight per day.
(CER) synthase enzyme that catalyzes the acyla-
tion of sphinganine and recycling of sphingosine.
As a result, the intracellular sphinganine and Patulin
other sphingoid levels increase, which are highly
cytotoxic. This cellular imbalance of sphinganine Patulin (Fig. 8.2) is known as toxic lactones pro-
and sphingosine may be responsible for toxicity duced by Penicillium clariform, P. expansum, P.
and possibly carcinogenicity. In animals, fumoni- patulum, Aspergillus spp., and Byssochlamys
sins cause a variety of diseases including leuko- spp. Penicillium expansum is a common contami-
encephalomalacia, pulmonary edema, and nant of damaged fruits. Therefore, fruits (apri-
hydrothorax. The toxins are reported to cause cots, grapes, peaches, pears, and apples) or juices
esophageal cancers in humans. are the major sources. Patulin does not survive
the apple cider making process; hence, it is sensi- Citrinin

tive to some processing treatments. In addition,
patulin is also found in bread and sausage. Patulin Citrinin (Fig. 8.2) is produced by Penicillium
is needed in high dosage to cause diseases such citrinum, Penicillium expansum, and Penicillium
as gastrointestinal ulceration, immunotoxicity, viridicatum including some strains of Penicillium
and neurotoxicity. It has a strong affinity for camemberti, used in cheese production. Citrinin
sulfhydryl group in enzymes thus inhibits enzyme is also produced by several species of Aspergillus
activity. Patulin can affect immune response and including Aspergillus oryzae, used in the produc-
suppress macrophage function. The LD50 value in tion of sake, miso, and soy sauce. The major
rats is 15–25 mg per kg. It is a carcinogenic toxin source of this toxin is rice (also called yellow
and is reported to be responsible for subcutane- rice), bread, ham, wheat, oats, rye, and barley.
ous sarcoma. The provisional maximum tolerable Citrinin is a nephrotoxin and causes nephropathy
daily intake limit is 0.4 mg kg−1 body weight. in animals. The LD50 for citrinin in chicken is
95 mg per kg; in rabbits, 134 mg per kg, and its
significance in human health is unknown.
Zearalenone
Fusarium graminearum and other Fusarium spe- Alternaria Toxin

cies (F. culmorum, F. cerealis, F. equiseti, F. ver-
ticillioides, and F. incarnatum) produce There are more than 70 Alternaria mycotoxins
zearalenone (Fig. 8.2). Zearalenone has two produced by several species of Alternaria:
derivatives: α-zearalenone and β-zearalenone. All Alternaria alternata, A. citri, A. solani, and A.
zearalenones have strong estrogenic properties tenuissima. Alternaria toxins are fetotoxic and
and resemble the 17β-estradiol, the principal hor- have teratogenic and mutagenic effects. Other
mone produced by the human ovary. The estro- known Alternaria mycotoxins are alternariol,
genic potential of α-zearalenol is higher than that altenuene, and tenuazonic acid. A. alternata is
of β-zearalenol due to a greater affinity toward the most important mycotoxin-producing spe-
the estrogen receptor. Zearalenone is classified as cies. This toxin is generally associated with
a nonsteroidal estrogen or a mycoestrogen. Thus, apples, tomatoes, and blueberries.
the toxin designation does not appear to be appro-
priate for zearalenone. Occasional outbreak of
mycotoxin in livestock results in infertility. Emerging Mycotoxins
Zearalenone concentrations of 1.0 ppm can cause
the hyperestrogenic syndrome in pigs, and even There are several other mycotoxins reported in
higher concentrations can cause abortion and recent years, for example, fusaproliferin, enniat-
other fertility-related problems. Reproductive ins, beauvericin, and moniliformin produced by
problems are also reported in cattle and sheep. different species of Fusarium. These mycotoxins
The major concern is that zearalenone can dis- are teratogenic and cytotoxic, and detailed mech-
rupt sex steroid function in humans. It has been anism of action is yet to be determined.
used to treat postmenopausal problems in women
and has been patented as oral contraceptives.
Zearalenone promotes estrus in mice, and LD50 in Ergot Alkaloids
the rat is reported to be 10,000 mg per kg. Corn,
wheat, oats, and barley are known sources for this Claviceps purpurea produces a toxic cocktail of
toxin. The provisional maximum tolerable daily alkaloid, ergot, which is not considered a typical
intake in human is 0.5 μg per kg body weight per mycotoxin. Claviceps purpurea grows on the
day, and the tolerable daily intake is 0.25 μg per heads of grasses such as wheat and ryes, and the
kg body weight. Zearalenone is sensitive to heat ergot is transmitted after ingestion of bread or
exceeding 150 °C. other products prepared with contaminated rye or
Summary 173
wheat. The alkaloids are relatively thermolabile, duced into the food can also be used for detoxifi-
and some may not survive during the bread mak- cation. Other modern food processing
ing process. In addition, other Claviceps species technologies, such as cold plasma and irradia-
including Claviceps paspali (forage grass), tions both ionizing (gamma) and non-ionizing
Claviceps fusiformis, Claviceps gigantea, and (UV, microwave), can inactivate molds and
Sphacelia sorghi (an anamorphic form of reduce mycotoxins.
Claviceps) produce alkaloids called ergometrine, In modern day, HACCP (hazard analysis criti-
ergotamine, and ergotoxine. cal control points) is employed to reduce mold
The disease caused by ergot is called ergot- and mycotoxins in products. Implementation of
ism. It is also known as “holy fire” or “St. HACCP is aided by improved analytical tech-
Anthony’s fire” because of severe burning sensa- niques for sensitive detection of mycotoxins and
tions in the limbs and extremities of the victim. stringent regulatory standards to exclude prod-
Two forms of ergotism are reported: gangrenous ucts for human consumption that contain myco-
and convulsive. In the gangrenous form, the toxin levels over the allowable limits. In addition,
blood supply is affected causing tissue damage. development of transgenic plants that are able to
In the convulsive form, the toxin affects the cen- increase the insect and mold resistance may aid
tral nervous system. With advanced food process- in reduced levels of mycotoxins in products.
ing techniques, the ergotism may have been Except for supportive therapy, there is no
eliminated from humans, but it remains a serious treatment currently available for foodborne
problem in animals including cattle, sheep, pigs, mycotoxin poisoning.
and chicken resulting in gangrene, convulsions,
abortion, hypersensitivity, and ataxia. In cattle,
ergotism spreads around the hooves, and the ani- Summary
mal may lose hooves and is unable to walk and
die by starvation. Molds (fungi) or mycotoxins infect humans, ani-
mals and plants. The common mold genera of
importance in food safety are Aspergillus,
Prevention and Control Penicillium, Fusarium, and Alternaria. Mold
of Mycotoxins causes mycosis, while the mycotoxin causes
mycotoxicosis in humans and animals. Molds
Good agricultural practices (GAP) and good also cause food spoilage, and the major crops that
manufacturing practices (GMP) to control molds are affected include wheat, maize, peanuts and
in preharvest and postharvest crops should be other nut crops, cottonseed, and coffee. The Food
employed. Those include soil testing, crop rota- and Agriculture Organization (FAO) of the WHO
tion, irrigation, antifungal treatments, appropri- estimates that about 25% of the world’s crops are
ate harvesting conditions, drying, and storage. affected by molds. The toxigenic molds produce
Traditionally mold was controlled by adjusting a variety of mycotoxins, and some are studied in
the temperature, pH, and moister levels of the detail. A majority of mycotoxins such as afla-
stored grains, cereals, and fruits. Proper food pro- toxin, fumonisin, trichothecene, deoxynivalenol
cessing approaches such as physical removal of (DON), patulin, ochratoxin, and citrinin are car-
the moldy grains, nuts, and fruits can lower the cinogenic, mutagenic, teratogenic, or cytotoxic
mycotoxin levels. Washing, flotation and density and are acquired by consuming mycotoxin-
segregation, dehulling, milling, steeping, and contaminated cereal foods, nuts, milk, and meat.
extrusion can also reduce mycotoxin content. Some mycotoxins are hepatotoxic and nephro-
High temperature, acids, and alkaline conditions toxic and are life-threatening. Claviceps pur-
in food can also chemically modify the mycotox- purea produces a toxic cocktail of alkaloid, ergot,
ins. Natural enzymes in the food or enzymes which is responsible for ergotism characterized
derived from fermentation or deliberately intro- by gangrene and neurological disorder. A variety
of mycotoxins may be present in a food, but sen- on human and animal health. Food Control 36,
159–165.
sitive analytical tools are needed to monitor their 4. Karlovsky, P., Suman, M., Berthiller, F., De Meester,
presence. Avoiding the use of mold-contaminated J., Eisenbrand, G., Perrin, I., Oswald, I.P., Speijers,
raw products for food/feed production can reduce G., Chiodini, A., Recker, T. and Dussort, P. (2016)
mycotoxicosis in humans and animals. There is Impact of food processing and detoxification treat-
ments on mycotoxin contamination. Mycotox Res,
no treatment currently available for foodborne 1–27.
mycotoxin poisoning. 5. Marin, S., Ramos, A.J., Cano-Sancho, G. and Sanchis,
V. (2013) Mycotoxins: Occurrence, toxicology,
and exposure assessment. Food Chem Toxicol 60,
218–237.
Further Readings 6. Marroquín-Cardona, A.G., Johnson, N.M., Phillips,
T.D. and Hayes, A.W. (2014) Mycotoxins in a chang-
1. Bennett, J.W. and Klich, M. (2003) Mycotoxins. Clin ing global environment – A review. Food Chem
Microbiol Rev 16, 497–516. Toxicol 69, 220–230.
2. Cousin, M.A., Riley, R.T. and Pestka, J.J. (2005) 7. Medina, A., Rodriguez, A. and Magan, N. (2014)
Foodborne mycotoxins:chemistry, biology, ecol- Effect of climate change on Aspergillus flavus and
ogy, and toxicology. In Foodborne Pathogens: aflatoxin B1 production. Front Microbiol 5.
Microbiology and Molecular Biology eds. Fratamico, 8. Murphy, P.A., Hendrich, S., Landgren, C. and Bryant,
P., Bhunia, A.K. and Smith, J.L. pp.163–226. Norfolk: C.M. (2006) Food mycotoxins: An update. J Food Sci
Caister Academic Press. 71, R51–R65.
3. Edite Bezerra da Rocha, M., Freire, F.d.C.O., Erlan 9. Zheng, M.Z., Richard, J.L. and Binder, J. (2006) A
Feitosa Maia, F., Izabel Florindo Guedes, M. and review of rapid methods for the analysis of mycotox-
Rondina, D. (2014) Mycotoxins and their effects ins. Mycopathologia 161, 261–273.
Fish and Shellfish Toxins
9
can cause increased release of nutrients, nitrogen,

Introduction and phosphorous to the coastal and marine waters
that can alter phytoplankton community and
Fish and shellfish harvested from marine, brack- algal bloom. The highest incidence of seafood-
ish (a mixture of fresh and seawater), and fresh- related outbreaks is associated with coastal
water environments may carry marine biotoxins inhabitants. Algal toxins are originated from
and when consumed can cause foodborne intoxi- dinoflagellates, while bacterial toxins are from
cation. The toxins are generally derived from cyanobacteria (blue-green algae). Dinoflagellates
harmful single-cell marine plant (phytoplankton) produce several polyether ladder toxins which
algae, in which fish or shellfish feeds on. The tox- include ciguatoxin (CTX), brevetoxin A (BTX
ins could also be originated from microbial A), brevetoxin B (BTX B), maitotoxin (MTX),
breakdown of fish proteins. Worldwide, about and yessotoxin (YTX).
60–80 different species of toxic microalgae are
reported, of which dinoflagellates represent about
75% of all marine species. Marine toxins can kill iseases Caused by Fish
D
fish, shellfish, or marine animals. Fish and shell- and Shellfish Toxins
fish also accumulate toxins in their tissues upon
feeding of toxic algae, and humans suffer from The majority of fish and shellfish toxins are
fish and shellfish poisoning after consuming raw, derived from single-cell marine algae. There are
cooked, or partially cooked products, because a six classes of algal toxins that are responsible for
majority of toxins are heat stable. The toxins are food poisoning: (i) paralytic shellfish poisoning
responsible for two types of symptoms: diarrhea- (PSP), (ii) diarrhetic shellfish poisoning (DSP),
genic and neurotoxic. Diarrheagenic toxins affect (iii) neurotoxic shellfish poisoning (NSP), (iv)
intestinal epithelial barrier and cause fluid loss, ciguatera fish poisoning (CFP), (v) azaspiracid
while the neurotoxins block Na-channel and shellfish poisoning (AZP), and (vi) amnesic
interfere with nerve impulse. shellfish poisoning (ASP). In addition, the scom-
There is a direct correlation between algae broid toxin is an important class of toxin repre-
bloom and the toxins in seafood, which generally senting the biogenic amines, histamine,
occur in the late spring to summer months. Global cadaverine, putrescine, and saurine, produced by
warming and the climate change can have a sig- bacterial decarboxylation of fish proteins.
nificant impact on marine organisms leading to Pufferfish poisoning is associated with tetrodo-
toxic algal bloom. Sea level rise and flash flood toxin produced by marine bacteria (Table 9.1).

Table 9.1 Fish and shellfish toxins and their regulatory limits
176
Toxin(s) involved Symptom

Type of poisoning [microbial source] FDA regulatory limit Fish and shellfish source Symptoms onset time
Scombroid Histamine USA: 50 ppm Tuna, mahi-mahi, bonito, Severe headache, dizziness, nausea, Minutes to
[multiple bacterial species] Europe: marlin, bluefish, wahoo, vomiting, allergic response (flushed skin, 1h
200 mg/kg in fresh fish mackerel, and salmon urticaria, and wheezing)
and 400 mg/kg in
fishery products
Ciguatera fish Ciguatoxin (CTX) 0.01 ppb (Pacific Coral reef fish: amberjack, Abdominal pain, diarrhea, vomiting, 30 min–4 h
poisoning (CFP) [algae: Gambierdiscus ciguatoxin) snappers, grouper, goat fish, paresthesias (tingling, burning,
toxicus] 0.1 ppb (Caribbean barracuda, sea bass, numbness), cold-to-hot sensory reversal,
ciguatoxin) surgeonfish, ulua, and papio weakness, and myalgias
Paralytic shellfish Saxitoxins 0.8 ppm (80 μg/100 g) Molluscan shellfish: mussels, Vomiting, diarrhea, facial paresthesias, 5–30 min
poisoning (PSP) [algae: Alexandrium clams, and oysters and respiratory paralysis leading to death
acatenella]
Neurotoxic Brevetoxins (BTX) 0.8 ppm Mussels and clams Diarrhea, vomiting, abdominal pain, 30 min–3 h
shellfish [algae: Karenia brevis] myalgias, paresthesias, and ataxia
poisoning (NSP)
Amnesic shellfish Domoic acid (neurotoxin) 20 ppm Mussels, clams, crabs, and Vomiting, diarrhea, headache, myoclonus, 15 min–>
poisoning (ASP) [algae: Pseudo-nitzschia anchovies loss of short-term memory, confusion, 35 h
pungens] disorientation, seizures, coma, and
hemiparesis
Diarrhetic Okadaic acid, 0.16 ppm Mussels, clams, and scallops Diarrhea, nausea, vomiting, and 30 min–3 h
shellfish dinophysistoxins, abdominal pain
poisoning (DSP) pectenotoxins, yessotoxin
[algae: Dinophysis fortii]
Azaspiracids Azaspiracid-1 0.16 ppm Mussels Diarrhea, nausea, vomiting, and 30 min–6 h
shellfish [algae: Azadinium spinosum] abdominal pain
poisoning (AZP)
9
Pufferfish Tetrodotoxin (TTX) – Ocean sunfishes, porcupine Paresthesias, headache, vomiting, 10–45 min
poisoning (PFP) fishes, and fugu (pufferfish) diaphoresis, respiratory paralysis,
circulatory collapse, and death
Cyanotoxin Nodularin and – Crayfish Hepatotoxicity, gastroenteritis
cylindrospermopsin
[cyanobacterial blooms]
Adapted from Kalaitzis et al. (2010). Toxicon 56, 244–258; Tsabouri et al. (2012). Ped. Allergy Immunol. 23, 608–615; FDA (http://www.fda.gov/downloads/Food/
GuidanceRegulation/UCM252395.pdf)
Fish and Shellfish Toxins
Diseases Caused by Fish and Shellfish Toxins 177
Scombroid Toxin originally isolated from a clam, Saxidomus

giganteus. However, the toxin is now found in
Scombroid toxin means “mackerel-like,” i.e., the mackerel, snapper, grouper, sea bass, barracudas,
toxin production is associated with scombroid mollusks (moon snails and whelk), scallops, and
fish (dark meat fish). These fish include tuna, oysters that feed on the toxic dinoflagellates
mackerel, skipjack, bonito, marlin, sardine, yel- (algae). The toxigenic dinoflagellate species are
low tail (amberjack), and dolphin (mahi-mahi) Alexandrium acatenella, A. andersonii, A.
fish. The toxin is also found in bluefish, herring, catenella, A. tamarense, A. fundyense, A. hiranoi,
and anchovies. Most of these fish have a high A. monilatum, A. minutum, A. lusitanicum, A.
level of free L-histidine amino acid in their tis- tamiyavanichii, A. taylori, A. peruvianum,
sues. The scombroid toxin is produced due to the Gymnodinium catenatum, and Pyrodinium baha-
breakdown of L-histidine of flesh by bacterial mense. Many cyanobacterial species also pro-
histidine decarboxylase within 3–4 h. Bacterial duce saxitoxin: Aphanizomenon flosaquae,
decarboxylation of histidine produces small Anabaena circinalis, Lyngbya wollei,
amines such as histamine, saurine, putrescine, Planktothrix, and Cylindrospermopsis racibor-
and cadaverine, which produce typical allergic skii. When dinoflagellates bloom, the water
symptoms for the toxin when consumed. appears red, and it is referred to as “red tide.”
The most important bacterial species associ- Saxitoxin is a heat-stable potent neurotoxin
ated with histamine production in fish is and blocks the production of action potentials in
Morganella morganii. Several other species are neuronal cells. The guanidinium moiety of the
also involved, namely, Klebsiella pneumoniae, saxitoxin binds to a region of the sodium channel
Proteus spp., Enterobacter aerogenes, Hafnia known as “site1” and blocks the opening of the
alvei, and Vibrio alginolyticus, which are present sodium channel. Saxitoxin is responsible for par-
in the gill and the gastrointestinal tract. In fer- alytic and neurotoxic shellfish poisoning. The
mented fish products, Lactobacillus spp. can also symptoms appear within 30 min–2 h after con-
produce histamine. Toxin level increases with sumption and include paresthesia (tingling and
increasing fish decomposition (spoilage). pricking sensation) of the mouth and extremities,
The scombroid toxin is highly heat-stable. burning sensation, drowsiness, incoherent
Cooking, canning, or freezing cannot reduce the speech, numbness, rash, and paralysis. In severe
toxicity of the fish products. Symptom severity cases, saxitoxin poisoning may cause respiratory
depends on the amount of toxin ingested. The paralysis leading to death if respiratory support is
symptoms are generally manifested by an aller- not provided.
gic reaction such as flush, body rash, headache,
shortness of breath, dizziness, and tachycardia,
which appear within hours of consumption of Diarrhetic Shellfish Poisoning
toxin. Dilatation of the peripheral blood vessels
results in hypotension, flushing, and headache, Diarrhetic shellfish poisoning (DSP) is a severe
while the increased capillary permeability causes form of gastrointestinal illness. Two major tox-
urticaria, hemoconcentration, and eyelids edema. ins, okadaic acid (OA) and dinophysistoxins
The gastrointestinal symptoms are nausea, vom- (DTXs) are responsible for diarrhetic shellfish
iting, cramp, and diarrhea. Antihistamine drugs poisoning. These toxins are produced by dinofla-
can be used to treat scombroid fish poisoning. gellates: Dinophysis acuta, D. acuminate, D.
caudata, D. fortii, and other Dinophysis species.
They are also produced by Prorocentrum arenar-
Paralytic Shellfish Poisoning ium, P. lima, P. hoffmannianum, P. maculosum,
and other species. OA also acts as a tumor pro-
Saxitoxin is responsible for causing paralytic moter, induces lipid peroxidation, cytotoxicity,
shellfish poisonings (PSP). The toxin was and apoptosis in cultured mammalian cells. DSP
178 9 Fish and Shellfish Toxins
is associated with the consumption of scallops, harvested from the coastal waters of Florida,
mussels, and clams that feed on the toxic algae. Hawaii, Puerto Rico, and the Virgin Islands dur-
The major symptoms are diarrhea, nausea, vom- ing late spring through summer months.
iting, and abdominal pain, which appear within CTX is a neurotoxin and causes paralysis.
30 min–3 h and may last up to 4 days. The toxins CTX has the similar mode of action as breve-
are heat-stable; thus, cooking cannot inactivate toxin, which selectively targets the common
them. The incidence of the disease can be reduced binding “site 5” on the α-subunit of the neuronal
by removing the digestive organs of the shellfish, sodium channel. The symptoms are tingling sen-
which tend to accumulate the majority of toxins. sation of the lips, tongue, and throat, irregular
heartbeat, and reduced blood pressure. It also
causes a headache, muscle pain, and progressive
Neurotoxic Shellfish Poisoning weakness leading to paralysis. The gastrointesti-
nal symptoms that develop within 2 h are nausea,
Neurotoxic shellfish poisoning (NSP) is caused vomiting, cramps, and diarrhea and last for a
by brevetoxin (brevetoxin A, BTX A; brevetoxin short time. Neurological and cardiovascular
B, BTX B) produced by dinoflagellate Karenia symptoms usually emerge within 6 h.
brevis. Algae growth in water produces red tide,
and the molluscan shellfish feeds on the algae
and accumulates toxin in their muscle. Humans Azaspiracid Shellfish Poisoning
consuming the contaminated shellfish exhibit
neurological symptoms similar to the paralytic Azaspiracid shellfish poisoning (AZP) is associ-
shellfish poisoning, such as tingling and numb- ated with the consumption of molluscan mussels,
ness of the lips, tongue, and throat, muscular which accumulate azaspiracid (AZA) toxin in tis-
aches, and dizziness, but less severe. sues. This toxin is produced by a dinoflagellate,
Gastrointestinal symptoms include diarrhea and Azadinium spinosum. The symptoms that develop
vomiting. The symptoms appear very quickly within minutes to hours after consumption of the
within 30 min–3 h and generally subside in a few contaminated shellfish last for several days.
hours. Karenia brevis bloom is a recurring prob- Symptoms are similar to the diarrhetic shellfish
lem in the Gulf of Mexico. poisoning and are manifested as diarrhea, nausea,
vomiting, and abdominal pain.
Ciguatera Fish Poisoning

Amnesic Shellfish Poisoning
Ciguatera fish poisoning (CFP) is associated with
the consumption of ciguatera toxin (CTX), which Amnesic shellfish poisoning (ASP) results from
is derived from the single-celled marine macroal- consumption of the molluscan shellfish (mussels,
gae, Gambierdiscus toxicus. CTX is a lipid- clams) and crabs due to a neurotoxin, domoic
soluble and heat-stable toxin and is produced and acid, which is produced by an alga, Pseudo-
released into the water by G. toxicus bloom. nitzschia pungens. The neurological and gastro-
Toxins enter the marine food chain and ultimately intestinal symptoms associated with ASP
affect humans. Fish such as snapper, amberjack, consumption include vomiting, diarrhea, head-
grouper, barracuda, and sea bass live in reefs or ache, disorientation, seizures, and hemiparesis
shallow water which is the potential source of (lack of voluntary movement of limbs and fingers
this toxin. CTX is concentrated in herbivorous similar to patients suffering from a stroke), which
fish consuming G. toxicus in the reefs of tropical appear within 24–48 h after consumption. In
and subtropical waters. The carnivore fish eats addition, the patient may suffer from a short-term
herbivore and acquires the toxin. CTX toxin memory loss and confusion, respiratory diffi-
occurrence is seasonal and found in fish culty, and coma.
Summary 179
Pufferfish Poisoning Toxins are heat-stable; therefore, cooking may

not be able to inactivate the toxins. A severe heat-
Pufferfish poisoning (PFP) is associated with ing process such as retorting may be able to
consumption of pufferfish (also known as fugu), reduce, but may not completely eliminate the
ocean sunfish, and porcupine fish. Pufferfish is a toxicity.
culinary delicacy in Japan. The active agent is Good manufacturing practice (GMP) and the
tetrodotoxin (TTX), which is produced by marine hazard analysis critical control points (HACCP)
bacteria, and the toxin may accumulate in the should be applied to reduce the scombroid toxin
skin and viscera (liver and ovary) of the puffer- production on fish. Preventing temperature abuse
fish. Tetrodotoxin is a neurotoxin, which blocks is essential. Low-temperature storage of fish can
sodium channel in the neuron. The toxin is heat- slow down the bacterial growth and the scom-
stable and heating may enhance toxicity. The broid toxin production. Once histidine decarbox-
symptoms appear within 10–45 min of consump- ylase has been formed, it continues to produce
tion and manifest as tingling, burning, numbness, histamine even at the refrigeration temperature.
headache, vomiting, diaphoresis, respiratory In frozen fish, histamine remains stable and can
paralysis, circulatory collapse, and death. The be reactivated after thawing. Storage of fish at
lethal potency of TTX is 5000–6000 MU (mouse −18 °C or below can stop the bacterial growth
unit) mg−1. One MU is defined as the amount of and prevent preformed histidine decarboxylase
toxin needed to kill a 20 g male mouse within from producing histamine. Cooking can inacti-
30 min after intraperitoneal injection. In humans, vate both histidine decarboxylase enzyme and
the minimum lethal dose is about 10,000 MU the microbes, but not histamine. Antihistamine
(~2 mg). Pufferfish poisoning is most prevalent drug can be used to treat scombroid poisoning.
in Asian countries with an estimated two to three For other toxin poisoning cases, supportive ther-
deaths happening annually in Japan. Cases have apy is recommended.
also been reported from China, Taiwan,
Singapore, Bangladesh, and Europe. There is no
antidote for TTX. Summary
Fish- and shellfish-associated toxins are gener-

Prevention and Control ally derived from toxic microalgae or cyanobac-
teria growing in the water from which the fishes
To prevent fish- and shellfish-related food poi- are harvested with the exception of the scombroid
soning, fish and shellfish should be harvested toxin, which is associated with bacterial decar-
only from officially approved bodies of water. boxylation of fish protein. Six types of algal tox-
Growth (bloom) of toxic algae is influenced by ins that are involved in fish and shellfish poisoning
water temperature, salinity, runoff, and the pres- include (i) paralytic shellfish poisoning, (ii) diar-
ence of nutrients in the water. Therefore, harvest- rhetic shellfish poisoning, (iii) neurotoxic shell-
ing of fish should be avoided when there is an fish poisoning, (iv) ciguatera fish poisoning, (v)
evidence of algal bloom or red tide. Satellite is azaspiracid shellfish poisoning, and (vi) amnesic
used to monitor ocean color including the red shellfish poisoning. Scombroid toxin shows aller-
tide. Shellfish growing in the shallow water tend genic response, while the majority of the algal
to accumulate more toxins. Periodic monitoring toxins (Na-channel blocker) affect nerve impulse
of shellfish for toxins can also help make a deci- propagation. Fish and shellfish poisoning are
sion on closing contaminated shellfish beds for manifested by the diarrhetic, neurological, or
harvesting. Furthermore, shellfish should be pur- anaphylactic response and are prevalent in people
chased from a certified or licensed angler or living in the coastal areas because of the increased
fishmonger. consumption of seafood. Global warming and
180 9 Fish and Shellfish Toxins
climate change have been attributed to increased Toxic marine microalgae and shellfish poisoning in
the British isles: history, review of epidemiology, and
fish-related food poisoning due to increased toxic future implications. Environ Health 10, 54.
algal bloom. Toxins are heat-stable; thus, cook- 4. Hungerford, J.M. (2010) Scombroid poisoning: A
ing or heating may not be able to inactivate them. review. Toxicon 56, 231–243.
To prevent food poisoning, fish and shellfish 5. Kalaitzis, J.A., Chau, R., Kohli, G.S., Murray,
S.A. and Neilan, B.A. (2010) Biosynthesis of toxic
should be harvested from clean water or pur- naturally-
occurring seafood contaminants. Toxicon
chased from a certified or licensed source before 56, 244–258.
consumption. 6. Noguchi, T., Onuki, K. and Arakawa, O. (2011)
Tetrodotoxin poisoning due to pufferfish and gastro-
pods, and their intoxication mechanism. ISRN Toxicol
2011, 10.
Further Readings 7. Ray, B. and Bhunia, A. (2014) Opportunistic Bacterial
Pathogens, Molds and Mycotoxins, Viruse, Parasites,
1. Bane, V., Lehane, M., Dikshit, M., Riordan, A. and and Fish and Shellfish Toxins. In Fundamental Food
Furey, A. (2014) Tetrodotoxin: Chemistry, toxic- Microbiology. pp.387–406. CRC Press, Taylor and
ity, source, distribution and detection. Toxins 6, Francis Group.
693–755. 8. Tirado, M.C., Clarke, R., Jaykus, L.A., McQuatters-
2. Furey, A., O'Doherty, S., O'Callaghan, K., Lehane, M. Gollop, A. and Frank, J.M. (2010) Climate change and
and James, K.J. (2010) Azaspiracid poisoning (AZP) food safety: A review. Food Res Int 43, 1745–1765.
toxins in shellfish: Toxicological and health consider- 9. Tsabouri, S., Triga, M., Makris, M., Kalogeromitros,
ations. Toxicon 56, 173–190. D., Church, M.K. and Priftis, K.N. (2012) Fish and
3. Hinder, S.L., Hays, G.C., Brooks, C.J., Davies, A.P., shellfish allergy in children: Review of a persistent
Edwards, M., Walne, A.W. and Gravenor, M.B. (2011) food allergy. Ped Allergy Immunol 23, 608–615.

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Food Science Text Series

More information about this series at http://www.springernature.com/series/5999

ISSN 1572-0330 ISSN 2214-7799 (electronic)

Library of Congress Control Number: 2017959346

© Springer Science+Business Media, LLC, part of Springer Nature 2018

Printed on acid-free paper

The first edition of this graduate-level Foodborne Microbial Pathogens:

West Lafayette, IN, USA Arun K. Bhunia

caused by viruses, parasites, mycotoxins, and seafood toxins have been

Dr. M. Rostagna, Purdue University; Chap. 12, Dr. R. Nannapaneni,

West Lafayette, IN, USA Arun K. Bhunia

1 Introduction to Foodborne Pathogens ���������������������������������������� 1

2 Biology of Microbial Pathogens���������������������������������������������������� 25

Small Intestine ���������������������������������������������������������������������������� 54

The Complement System���������������������������������������������������������������� 75

5 Animal and Cell Culture Models to Study Foodborne

Cryptosporidium parvum ������������������������������������������������������������ 156

10 Staphylococcus aureus�������������������������������������������������������������������� 181

12 Clostridium botulinum, Clostridium perfringens,

T3SS and Delivery of Effector Proteins�������������������������������������� 261

Type III Secretion System���������������������������������������������������������������� 279

Plasmid (pYV)-Linked Virulence Gene Products������������������������ 305

Introduction to Food Microbiology immune system to provide protection against

© Springer Science+Business Media, LLC, part of Springer Nature 2018 1

Bacillus species, and some mold species are

Fig. 1.2 The

 hat Are the Attributes

Fig. 1.3 Gene transfer

Fig. 1.4 Food

Poultry be the source of Pseudomonas, coryneforms, and

litica outbreaks also were associated with pas- Low-Moisture Products

I mproved Detection Methods Federal food safety authorities in the USA

antibiotics also affect food production and supply

Fig. 1.6 Street food

 ontrol of Foodborne Diseases

Introduction environments or physiological conditions or

© Springer Science+Business Media, LLC, part of Springer Nature 2018 25

Fig. 2.1 Universal

Fig. 2.2 A flow diagram showing the taxonomic classification of bacteria

Fig. 2.4 A schematic

The cytoplasmic membrane consists of a lipid

The cell envelope of Gram-negative bacteria is

bacterial pathogenesis by activating an immune Protein Secretion Systems

Table 2.2 Protein secretion systems in bacteria

f­ actors for pathogenic bacteria by promoting bac-

Fimbriae (Pili) and Curli

Gram-negative bacteria carry cilia-like long

shared by Klebsiella pneumoniae and Serratia Endospore Formation

Fig. 2.9 Drawing of an

cell. The cortex located surrounding the cell wall

Fig. 2.11 Schematic

immature cells are unable to perform a normal

Parasites are another type of organisms that

Mycotoxins ­optimal condition for mycotoxin production is at a

The ability of the human body to protect

© Springer Science+Business Media, LLC, part of Springer Nature 2018 43

Skin The epithelial layer environment is moist,

Goblet Cells and Mucus increases progressively distally along the

In the mucus membrane, M (microfold) cells Antimicrobial Peptides

Fig. 3.4 Schematic of

West Lafayette, IN, USA Arun K. Bhunia

West Lafayette, IN, USA Arun K. Bhunia

1 Introduction to Foodborne Pathogens �� 1

2 Biology of Microbial Pathogens�� 25

Small Intestine �� 54

The Complement System�� 75

5 Animal and Cell Culture Models to Study Foodborne

Cryptosporidium parvum �� 156

10 Staphylococcus aureus�� 181

12 Clostridium botulinum, Clostridium perfringens,

T3SS and Delivery of Effector Proteins�� 261

Type III Secretion System�� 279

Plasmid (pYV)-Linked Virulence Gene Products�� 305

Introduction to Food Microbiology immune system to provide protection against

hat Are the Attributes

Poultry be the source of Pseudomonas, coryneforms, and

litica outbreaks also were associated with pas- Low-Moisture Products

I mproved Detection Methods Federal food safety authorities in the USA

ontrol of Foodborne Diseases

Introduction environments or physiological conditions or

bacterial pathogenesis by activating an immune Protein Secretion Systems

f actors for pathogenic bacteria by promoting bac-

Fimbriae (Pili) and Curli

shared by Klebsiella pneumoniae and Serratia Endospore Formation

Mycotoxins optimal condition for mycotoxin production is at a

Skin The epithelial layer environment is moist,

Goblet Cells and Mucus increases progressively distally along the

In the mucus membrane, M (microfold) cells Antimicrobial Peptides

row. The secondary lymphoid organs are lymph Lymph Node

fibroblasts and vascular endothelium to repair Eosinophil

p roinflammatory cytokines (IL-1α, IL-1β, IL-6,

Cytokines ytokines in Innate and Adaptive

interferon and interleukins are predominant. Tumor Necrosis Factor

has a hypervariable region, which is also called Classes of Immunoglobulins

Fig. 3.18 Allergen-

Antigen–Antibody Reaction molecular mass of α-chain in human is 44 kDa

The Classical Pathway forms a donut-shaped hole on the surface of the

inflammation thus facilitating increased a ntigen–antibody complex is rapidly cleared

Innate Immunity MHC–SEB complex interacts with T-cell

vasion of Immune System

Introduction toward the liver, which is rich in iron bound to the

Colonization and Adhesion Factors ili, Fimbriae, and Flagella

Curli Adhesion Proteins

pili (TCP), mannose-sensitive hemagglutinin Biofilm Formation

Ingestion of preformed exotoxins such as staphy- Toxins

coagulation (DIC) allowing neutrophils, lympho- enetic Regulation and Secretion

Introduction cell culture or in an animal model. Therefore,

Trangenic Animal Models Organ Culture

Cultured Cell Lines

Cell Culture Model rypan Blue Exclusion Test

Apoptosis is regarded as a noninflammatory easurement of Specific Steps

Introduction including: (1) attachment to host receptor, (2)