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Manure as a source of zoonotic pathogens
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ZOONOTIC PATHOGENS IN THE FOOD CHAIN
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ZOONOTIC PATHOGENS
IN THE FOOD CHAIN
Edited by
Denis O. Krause
Department of Animal Science
University of Manitoba, Winnipeg, Canada
and
Stephen Hendrick
Department of Large Animal Clinical Sciences
Western College of Veterinary Medicine
University of Saskatchewan, Saskatoon, Canada
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A catalogue record for this book is available from the British Library, London, UK.
Library of Congress Cataloging-in-Publication Data
Krause Denis.
Zoonotic pathogens inthe food chain / Denis Krause, Stephen Hendrick.
p. cm.
ISBN 978-1-84593-681-5 (alk. paper)
1. Foodborne diseases. 2. Zoonoses. I. Hendrick, Stephen. II. Title.
RA601.5.K73 2010
615.9'54--dc22
2010021482
ISBN-13: 978 1 84593 681 5
Commissioning editor: Sarah Hulbert
Production editor: Tracy Head
Typeset by AMA Dataset, Preston, UK.
Printed and bound in the UK by CPI Antony Rowe, Chippenham.
Contents
Contributors
vii
Preface
xi
1. Globalization of the Food Supply and the Spread
of Disease
Susan C. Cork and Sylvia Checkley
1
2. Epidemiology of Pathogens in the Food Supply
Susan C. Cork
21
3. Manure as a Source of Zoonotic Pathogens
Gabriel J. Milinovich and Athol V. Klieve
59
4. Animal Feed as a Source of Zoonotic Pathogens
Richard A. Holley
84
5. Milk and Raw Milk Consumption as a Vector
for Human Disease
Stephen P. Oliver and Shelton E. Murinda
6. The Contribution of Antibiotic Residues and Antibiotic
Resistance Genes from Livestock Operations to Antibiotic
Resistance in the Environment and Food Chain
Pei-Ying Hong, Anthony Yannarell and Roderick I. Mackie
7. On-farm Mitigation of Enteric Pathogens to Prevent
Human Disease
Trevor W. Alexander, Kim Stanford and Tim A. McAllister
99
119
140
v
vi
Contents
8. Organic Agriculture and its Contribution to Zoonotic Pathogens
Bastiaan G. Meerburg and Fred H.M. Borgsteede
167
9. Zoonotic Implications of Avian and Swine Influenza
Juan C. Rodriguez-Lecompte, Sudhanshu Sekhar and Tomy Joseph
182
10. Crohn’s Disease in Humans and Johne’s Disease
in Cattle – Linked Diseases?
Herman W. Barkema, Stephen Hendrick, Jeroen M. De Buck,
Subrata Ghosh, Gilaad G. Kaplan and Kevin P. Rioux
197
11. Transmissible Spongiform Encephalopathies as a Case Study
in Policy Development for Zoonoses
Michael Trevan
214
Index
237
Contributors
Editors
Denis O. Krause, PhD, Professor of Microbiology, Department of Medical
Microbiology and Department of Animal Science, Director of Large
Animal Biosecurity and Gut Microbiome Laboratory, University of
Manitoba, Winnipeg, Canada. E-mail: denis_krause@umanitoba.ca.
Research interests: comparative gut microbiome analysis, bioinformatics,
food safety, chronic microbial human and animal infections.
Stephen Hendrick, DVM, DVSc, Assistant Professor, Western College
of Veterinary Medicine, University of Saskatchewan, Saskatoon,
Saskatchewan, Canada. E-mail: steve.hendrick@usask.ca. Research
interests: epidemiology of infectious and non-infectious diseases of beef
and dairy cattle.
Authors
Trevor W. Alexander, PhD, Scientist, Agriculture and Agri-Food Canada,
Lethbridge, Alberta, Canada. E-mail: Trevor.Alexander@agr.gc.ca.
Research interests: microbiology of the rumen, food safety, feed
fermentation, forage and feed evaluations.
Herman W. Barkema, DVM, PhD, Professor, Epidemiology of Infectious
Diseases, Faculty of Veterinary Medicine and Faculty of Medicine
(joint appointment), Head, Department of Production Animal Health,
Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta,
Canada. E-mail: barkema@ucalgary.ca. Research interests: application of
epidemiology to the prevention and control of infectious diseases, such as
Johne’s disease and mastitis, with animal and public health perspectives;
overall goal is to ensure a safe and economical food supply with a reduced
vii
viii
Contributors
risk of transmission of zoonotic diseases to farm families and the general
population.
Fred H.M. Borgsteede, PhD, Wageningen University and Research Centre,
Central Veterinary Institute, Lelystad, the Netherlands. Current address:
Old-Ruitenburg, 8219 PH Lelystad, the Netherlands. E-mail: Fred.
Borgsteede@wur.nl. Research interests: veterinary parasitology with
expertise in field of nematodes and arthropods; occurrence of parasites
within organic livestock systems.
Sylvia Checkley, DVM, PhD, Assistant Professor, Public Health and Veterinary
Epidemiology, Department of Ecosystem and Public Health, Faculty of
Veterinary Medicine, University of Calgary, Calgary, Alberta, Canada.
Research interests: veterinary epidemiology, public health and
surveillance systems, public veterinary practice.
Susan C. Cork, BPhil (vet), BVSc, PhD, PG Dip. Public Policy, MRCVS, MSB,
CBiol. Head of Department and Professor of Ecosystem and Public Health,
Faculty of Veterinary Medicine, University of Calgary, Calgary, Alberta,
Canada. E-mail: sccork@ucalgary.ca. Research interests: wildlife disease
ecology, animal and public health policy, public veterinary practice and
risk assessment.
Jeroen M. De Buck, MSc, PhD, Assistant Professor, Veterinary Microbiology,
Alberta Ingenuity New Faculty, Faculty of Veterinary Medicine, University
of Calgary, Calgary, Alberta, Canada. E-mail: jeroen.debuck@gmail.com.
Research interests: molecular biology, with interest in the pathogenesis
of bacterial pathogens, focusing on Mycobacterium avium subsp.
paratuberculosis (the causative agent of Johne’s disease in ruminants) and
specializing in the study of bacterial virulence factors, strain differentiation
and the development of novel diagnostics.
Subrata Ghosh, MD (Edin.), FRCP, FRCPE, FRCPC, Head, Division of
Gastroenterology, Professor of Medicine, Teaching, Research and
Wellness Centre, University of Calgary, Calgary, Alberta, Canada. E-mail:
ghosh@ucalgary.ca. Research interests: pathogenesis of inflammatory
bowel diseases and novel therapeutic targets of inflammation.
Richard A. Holley, PhD, Professor, Department of Food Science, University
of Manitoba, Winnipeg, Canada. E-mail: Rick_Holley@umanitoba.ca.
Research interests: microbial ecology of foods (meats and processed meat
products), and bacterial foodborne pathogens in animals and the animal
environment.
Pei-Ying Hong, PhD, Department of Animal Sciences and Institute for
Genomic Biology, University of Illinois, Urbana, Illinois, USA. E-mail:
pyhong@illinois.edu. Research interests: microbial communities present
in ‘impacted’ ecosystems, including air and water bodies affected by
the use of antibiotics; utilization of molecular microbial ecology tools
to investigate the abundance and diversity of antibiotic resistance genes
associated with these microbial communities.
Tomy Joseph, DVM, MSc, PhD, Virologist, Veterinary Diagnostic Services
Laboratory, Livestock Knowledge Centre, Manitoba Agriculture, Food
and Rural Initiatives, Winnipeg, Manitoba, Canada. Research interests:
Contributors
ix
diagnostic virology and the molecular basis of pathogenesis of avian and
swine influenza viruses.
Gilaad G. Kaplan, MD, MPH, FRCPC, Assistant Professor, CIHR New
Investigator and AHFMR Population Health Investigator, Departments of
Medicine and Community Health Sciences, Division of Gastroenterology,
Inflammatory Bowel Disease Clinic, University of Calgary, Calgary,
Alberta, Canada. Research interests: epidemiology of inflammatory bowel
disease; role of the environment and air pollution in human health.
Athol V. Klieve, PhD, Associate Professor, Schools of Animal Studies
and Veterinary Science, University of Queensland, Gatton Campus,
Queensland, and Agri-Science Queensland (DEEDI), Animal Research
Institute, Yeerongpilly, Queensland, Australia. E-mail: a.klieve@uq.edu.
au. Research interests: general – knowledge and manipulation of complex
gut microbial ecosystems; specific – reducing methane emissions from
the rumen ecosystem and unravelling the multiple roles that viruses
(bacteriophages in particular) play within these ecosystems.
Roderick I. Mackie, PhD, Professor, Department of Animal Sciences and
Institute for Genomic Biology, University of Illinois, Urbana, Illinois,
USA. E-mail: r-mackie@illinois.edu. Research interests: microbial ecology,
including intestinal microbial ecology and environmental impacts of
animal production systems, with a focus on the ecology and evolution of
antibiotic resistance genes, and using molecular microbial ecology tools
to answer questions related to the abundance and diversity of populations
and genes.
Tim A. McAllister, PhD, Principal Research Scientist, Agriculture and AgriFood Canada, Lethbridge, Alberta, Canada. E-mail: Tim.McAllister@agr.
gc.ca. Research interests: microbiology of the rumen, food safety, feed
fermentation, forage and feed evaluations.
Bastiaan G. Meerburg, PhD, Wageningen University and Research Centre,
Plant Research International, Wageningen, the Netherlands. E-mail:
Bastiaan.Meerburg@wur.nl. Research interests: expertise in the field of
pests and diseases (particularly rodent pests) and studies concerning
organic agriculture and possible food safety consequences.
Gabriel J. Milinovich, PhD, Room 1.094, Department of Genetics in Ecology,
University of Vienna, Vienna, Austria. E-mail: gabriel.milinovich@univie.
ac.at. Research interests: structure and function of gastrointestinal
microbiomes and how changes in these systems relate to disease in
humans and animals.
Shelton E. Murinda, PhD, Associate Professor, Animal and Veterinary Sciences
Department, Director, Center for Antimicrobial Research and Food
Safety, California State Polytechnic University, Pomona, California, USA.
E-mail: semurinda@csupomona.edu. Research interests: conventional
and molecular detection, and control of foodborne pathogens and
zoonotic disease-causing agents; application of natural antimicrobials in
pathogen reduction.
Stephen P. Oliver, PhD, Professor, Department of Animal Science, The
University of Tennessee, Knoxville, Tennessee, USA. E-mail: soliver@
x
Contributors
utk.edu. Research interests: mammary gland physiology, immunology
and microbiology, with emphasis on development of non-antibiotic
approaches for the prevention and control of mastitis in dairy cows;
development and evaluation of strategies to control/reduce foodborne
pathogens in food-producing animals.
Kevin P. Rioux, MD, PhD, FRCPC, Assistant Professor, Department of Medicine,
Faculty of Medicine, University of Calgary, Calgary, Alberta, Canada.
Research interests: composition and ecology of bacterial communities in
the human intestinal tract and their role in inflammatory bowel disease,
energy balance and nutrition.
Juan C. Rodriguez-Lecompte, DVM, MSc, PhD, Assistant Professor –
Immunology, Director of University of Manitoba Poultry Research Unit,
Department of Animal Science, University of Manitoba, Winnipeg,
Manitoba, Canada. E-mail: JC_Rodriguez-Lecompte@umanitoba.ca.
Research interests: avian immunology, nutritional immunology, epigenetics and embryology.
Sudhanshu Sekhar, PhD candidate, Department of Animal Science, University
of Manitoba, Winnipeg, Canada. Research interests: immunology.
Kim Stanford, PhD, Beef Specialist – Production System, Livestock Research
Branch, Government of Alberta, Lethbridge, Alberta, Canada. E-mail:
kim.stanford@gov.ab.ca. Research interests: environmental microbiology,
rumen microbiology, food safety, beef production systems and composting
systems.
Michael Trevan, PhD, FIBiol, PAg, FRSM, Dean of Faculty of Agricultural and
Food Sciences and Professor of Food Science, University of Manitoba,
Winnipeg, Manitoba, Canada. E-mail: michael_trevan@umanitoba.ca.
Research interests: food biotechnology; the use of science in policy formulation.
Anthony Yannarell, PhD, Assistant Professor, Department of Natural Resources
and Environmental Sciences and Institute for Genomic Biology,
University of Illinois, Urbana, Illinois, USA. E-mail: acyann@illinois.edu.
Research interests: microbial ecology – study of changes in microbial
community structure related to human activities; particular interest in
feedbacks between microbial community dynamics, their activities and
the operation of human-managed systems.
Preface
Historically the transmission of disease from animals has increased as humans
have evolved from a hunter–gatherer existence to the domestication of animals. As animals became domesticated, humans were in closer proximity to
animals themselves, their excreta and the pathogens they carried. These
pathogens could contaminate food and water, and resulted in human sickness.
During the 1950s and 1960s, there was an explosion in agricultural productivity, a dramatic decline in animal and human infectious disease – largely
as a result of the use of antimicrobials, and the emergence of a relatively cheap
food supply. During the 1970s and 1980s there was a perception that the ‘food
production problem’ had been solved, but we have seen an increase of not
only classical food safety issues but also of concern about how livestock
production may affect human health in general.
New problems have arisen in the food supply. Chapter 1 of this book
describes the transformation of the food supply from a local activity to a global
activity, and the problems with food safety that globalization brings. Chapter 2
gives a reasonably comprehensive list of zoonoses that are, and are likely to be
in the future, high on the list of important pathogens. Manure, the subject of
Chapter 3, has always been a concern because it is one of the most important
sources of disease to humans, but a less well-studied source of zoonoses in the
food supply is the actual feed that comes on to or leaves a farm (Chapter 4).
In the past, pasteurization had a critical role in making the milk supply
safe for humans, but more recently there has been a trend towards the use of
raw (non-pasteurized) milk (Chapter 5), partly because of a public concern
with the use of antimicrobials in the food production system and the potential
implications of this for human health (Chapter 6). As a result of these new
challenges in securing the food supply, a number of on-farm mitigation strategies have been developed (Chapter 7). Organic agriculture has become popular partly because of the concerns raised by antibiotics in food production,
and some regard organic production as a mitigation strategy for food safety in
xi
xii
Preface
general (Chapter 8). However, food safety is a dynamic area, and mitigation
strategies will be updated in the light of new threats to human health such as
avian (and swine) influenza (Chapter 9), and Crohn’s disease in humans
(Chapter 10).
The last chapter (Chapter 11) is a very interesting case study that illustrates what happens when a human disease threat posed by animal agriculture
becomes public. This chapter examines the course of events and the policy
development surrounding the outbreak of bovine spongiform encephalopathy (BSE) in the UK, with some comparative data from North America also
included. While BSE is a threat to human health, its risk to the public is still
relatively small compared with other food-safety risks.
Denis Krause
May 2010
1
Globalization of the
Food Supply and
the Spread of Disease
SUSAN C. CORK AND SYLVIA CHECKLEY
Historical Perspectives and Current Trends
In the globalized political economy of the late 20th century, increasing social,
political and economic interdependence occurred as a result of the rapid
movement of people, produce and other commodities across national borders
(Harlan and Jacobs, 2008). A consequence of increased trade, travel and
migration is the growing risk of transmitting biological and other hazards
from country to country on a large scale. With greater connectedness, new
and emerging diseases have the potential to travel very fast and subsequently
trace back, and control is often difficult. Growing international trade in food
commodities and the development of transnational cooperations (TNCs) has
corresponded with an increase in cross-border reports of foodborne illnesses
highlighting the need for international cooperation in animal and human
disease reporting and control initiatives (Kaferstein et al., 1997; King et al.,
2004; Kobrin, 2008).
The exchange of food and animal products across regions, nations and
continents has occurred for centuries. There have been a number of
anthropological studies on the cultural impact of trade in specific products
such as sugar (Mintz, 1985) and spices (Miller, 1995; Mintz and Du Bois,
2002). More recently, there has been a wide range of literature focused on
the development and implementation of a range of regulations and agreements to facilitate the safe exchange of animal- and plant-based primary
produce.
The international circulation of food products as commodities, along
with the transnational expansion of food-based cooperations, has resulted
in the need for global governance of food safety and quality. To a large
extent, this has occurred through the World Trade Organization (WTO)
and the implementation of standards outlined in the Sanitary and
© CAB International 2011. Zoonotic Pathogens in the Food Chain
(eds D.O. Krause and S. Hendrick)
1
2
S.C. Cork and S. Checkley
Phytosanitary (SPS) Agreement1 and the Codex Alimentarius2 (Vallat and
Mallet, 2006).
Disease and the Food Chain
Global outbreaks of foodborne disease can have socio-economic impacts on
consumer food choices and other behaviour (Sockett, 1993; Knowles et al.,
2007). Our understanding of the epidemiology of foodborne diseases has
evolved in recent decades corresponding, in part, to improvements in pathogen detection and reporting systems. In addition, new pathogens have emerged
to correspond with a changing food supply, an increase in the number of
people with heightened susceptibility to foodborne diseases and a greater
diversity of food preparation practices and food preferences. This has posed a
number of challenges for the veterinary profession and public health agencies
(Epp, 2008; BeVier, 2008). Alongside these changes, the global economy has
facilitated the rapid transport of perishable foods, increasing the potential for
new populations to be exposed to foodborne pathogens prevalent in distant
parts of the world.
There have been a number of studies developed to examine the impact of
trade in food beyond national boundaries, including the effect of the promotion of non-traditional imports to support consumer demand and the implications of evolving free-trade agreements (Barrientos, 2000; Bonanno, 2004;
Phillips, 2006). The key drivers for the emergence of globalization are largely
economic, with large cooperations seeking more cost-effective ways in which
to produce primary produce to include in the compilation of processed animal feeds and foods for human consumption. Other factors include the
greater mobility of ethnic groups and the subsequent greater demand for
‘exotic’ foods not typically grown in the country of residence. The current
trend of facilitating free flow of traded commodities between industrialized
and less industrialized nations has led to the availability of a greater variety of
fresh and preserved produce for citizens. The concurrent rise in modern processing methods and the availability of a wide range of distribution options has
also facilitated enhanced potential for international trade in both perishable
and non-perishable commodities. However, this increased trade is not without
risks. The expansion of food-related TNCs has had some negative consequences. Processed products manufactured from raw ingredients imported
from many different sources makes traceability difficult, and comprehensively
tracking contaminated foodstuffs may be impossible – as seen in milk and pet
food products contaminated with melamine (Brown et al., 2007; Ingelfinger,
2008).
1
2
http://www.wto.org/english/tratop_e/sps_e/sps_e.htm (accessed 17 May 2010).
http://www.codexalimentarius.net/web/index_en.jsp (accessed 17 May 2010).
Globalization of Food Supply and Spread of Disease
3
Foodborne Disease Control: a Transnational Challenge
Disease knows no boundaries and borders are porous to disease.
(Kaferstein et al., 1997)
The world has moved from a situation in which the majority of food was produced locally and sold in local markets to a system in which food is transported
great distances and marketed through large chains of supermarkets. Despite
the international agreement, which provides a framework for the import
health standards required for traded animal- and plant-based products, the
expansion of trade in fresh and processed primary produce has resulted in a
number of significant biosecurity breaches in recent years. The most recent
examples are the 2001 foot-and-mouth disease (FMD) outbreak in the UK,
which probably occurred as a result of feeding contaminated animal protein
to livestock, and the occurrence of bovine spongiform encephalopathy (BSE)
in Europe following the export of BSE-containing feed from the UK. BSE then
spread to North America following the export of young animals infected with
BSE that were later rendered and entered the North American animal feed
chain.3
Owing to the growth in international trade, ever-increasing quantities of
genetic material and animal and plant products are regularly transported
across the world. This has contributed to the spread of some diseases, including those caused by foodborne bacteria, viruses and parasitic agents, to humans
(Seimenis, 2008). Although international regulations have been developed by
the OIE (World Organisation for Animal Health; see later section of this
chapter)4 and other organizations through the SPS agreement and the Codex
Alimentarius to reduce the risk of trading contaminated products, the degree
of implementation of relevant regulations, and the extent of inspection and
enforcement, varies from country to country, and sometimes within countries
(Arambulo, 2008; Pires et al., 2009).
Factors Affecting the Changing Pattern of Foodborne Diseases
Increased surveillance and reporting
In industrialized countries, the systems for reporting foodborne disease outbreaks have become more sophisticated over the past few decades. This, along
with better pathogen detection methods and the ability to trace the origin of
infections to specific food products, has resulted in more awareness of food
safety (Cooke, 1990; Hartung, 2008). Mild disease and sporadic cases of foodborne infection probably still go unreported, but better public education and
3
http://www.inspection.gc.ca/english/anima/heasan/disemala/bseesb/ab2003/evale.
shtml (accessed 17 May 2010).
4 http://www.oie.int/eng/OIE/en_about.htm?e1d1 (accessed 17 May 2010).
4
S.C. Cork and S. Checkley
greater media engagement in communicating potential food hazards and
food recalls have contributed to enhanced reporting of many foodborne diseases. Computer-based databases such as FoodNet and PulseNet in the USA,
Enter-Net for Salmonella spp. and Escherichia coli O157:H7 in Europe, and
WHO-Global Salm-Surv are now available to assess the trends and changes,
and help to predict numbers and types of infection using current and historical data. These and other surveillance networks, as well as the emergence of
disease modelling, show potential for the future prediction of and early intervention for better control of foodborne zoonoses (Singer et al., 2007).
Changes in food manufacturing and agricultural processes
Food safety should be examined from the farm setting, where the primary
produce is grown, right through to the processing, handling and preparation
of food by the consumer (Beuchat and Ryu, 1997). Before food reaches the
consumer, contamination can occur at any stage of the food production
chain – from production and processing through to packaging. Modern foodprocessing facilities are typically large and centralized compared with the traditional small family-run units where food was usually sold locally. In many
countries there has been a drive, usually due to economic factors, to consolidate the food-producing sector (Howard, 2009). Technological changes in the
food-manufacturing industry have enabled producers to maximize output,
but although some risks have been minimized with modern processing methods (Galligan and Kanara, 2008), bulk production does also have a downside.
For example, owing to the need to preserve food for wider distribution over
long distances, appropriate conditions during transport and safe handling of
food stuffs have become especially important (Siegford et al., 2008; Burton,
2009). A number of regulations have been enforced by different countries to
ensure that good handling practice is maintained by food producers and
manufacturers, but compliance can vary from country to country and from
company to company. Because of the rise of transnational food corporations
and wide-scale distribution between and within countries, international agreements have been developed to ensure that minimum standards are set and
enforced. The increase in the trade of fresh produce and the minimal treatment processes required for some ‘natural’ and organic-based products has
also led to an increase in some foodborne diseases, especially in cases where
the consumer fails to wash the product well, cross-contaminates foodstuffs
during preparation and/or fails to cook food properly before eating it. Raw
fruits and vegetables have increasingly been identified as a source of foodborne infections (Beuchat, 2002; Noah, 2009). In order to obtain raw fruit
and vegetables out of season, as is now common in many countries, produce is
transported many thousands of miles from growing areas to various suppliers.
As a result of this trend, outbreaks of foodborne diseases can occur in a number of regions at once. These outbreaks often go unrecognized, but a few key
examples involving Listeria monocytogenes are provided later in this chapter.
Other examples involving protozoal pathogens are discussed in Chapter 2.
Globalization of Food Supply and Spread of Disease
5
Changes in consumer habits
With the rise in the number of middle-class families in many parts of the world,
people are eating more meals outside the home and this has, in some cases,
been associated with an increase in some foodborne diseases, especially where
food-handling practices and hygiene in fast-food outlets and restaurants are
not well regulated. The increased consumption of raw, chilled and fresh produce has also resulted in a rise in some foodborne diseases, especially where
consumers are not well informed about food-safety risks associated with
unwashed fresh produce that may have been imported from parts of the world
where unfamiliar diseases are common. For example, many parasitic diseases
have the intermediate stages of their life cycles in or on vegetation. The emergence of aquaculture, especially of shellfish, as a key export industry in Asia
and other parts of the world (Langan, 2008) also raises concerns about importing produce contaminated by organisms that may not be detected by standard
inspection practices. Organic produce and other produce, such as fresh fruit
juices, may also harbour potential pathogens and these have been associated
with a number of cases of foodborne disease in consumers who are often
unaware of the potential risks (Burnett and Beuchat, 2001; Cooper et al.,
2007). The growing popularity of organic food and the associated publichealth implications are considered in a later section of this chapter.
Increased travel and increased cultural diversity in many large towns has
also resulted in the import and consumption of foods not normally consumed
and this can lead to improper food preparation on the part of the person not
familiar with the food-safety risks associated with that food (Ramos et al., 2008).
International vacations have also led to the exposure of travellers to foodborne pathogens to which they have little or no immunity; some of these
organisms may inadvertently be carried back to their home country where sick
or recovered travellers can still spread the organism, often without knowing it
(Johnston et al., 2009).
Increased ‘at risk’ population (immunocompromised, elderly, etc.)
In some countries, a changing demographic profile with an ageing population
has led to an increase in the number of elderly people, many of whom have
pre-existing health problems. These people, especially those in residential
nursing homes or hospitals, are more susceptible to foodborne diseases. Along
with the growth in the elderly population, better medical care has also resulted
in improved life expectancy for vulnerable members of the population, such
as infants, pregnant women and the immunocompromised. Professional
health-care workers need to be well informed about the risks associated with
certain food products and to ensure that appropriate food-preparation practices are followed (Mainzer, 2008). In cases where individuals are likely to be
more susceptible to foodborne diseases, it is recommended that they avoid
higher-risk foodstuffs such as chilled ready-to-eat (RTE) meals, delicatessen
meats and salads, soft unripened cheeses, raw meat, raw eggs and raw fish.
6
S.C. Cork and S. Checkley
Patients with acquired immune deficiency syndrome (AIDS), those undergoing chemotherapy and those on immunosuppressive treatment following
organ transplant are especially susceptible to infectious disease, including
foodborne disease, so those responsible for the selection and preparation of
food for these people must take this into consideration, i.e. ensure that fresh
foods are well washed and/or cooked properly.
Improved detection methods and tracking of pathogens
Enhanced pathogen-detection methods, especially molecular techniques, have
increased our ability to detect and trace pathogens associated with foodborne
diseases. This has led to better reporting and a greater ability to implement
food recalls and to raise the alert once a food source associated with a disease
outbreak has been identified. Biosensors, immunoassays and PCR techniques
are very sensitive and can be used to screen batches of products before sale as
part of food-safety monitoring programmes. As new technologies have become
available, current and emerging diseases have been more readily detected, thus
allowing better reporting and more effective intervention in disease outbreaks.
New molecular tools have also facilitated studies towards a better understanding of disease ecology (Chandra et al., 2008; Galligan and Kanara, 2008).
Emerging pathogens with improved survivability
In the past decade, a number of new foodborne pathogens have been identified. Some of these have been discovered as a result of better detection
methods and others represent the development of more virulent strains of
pathogens such as bacteria, e.g. E. coli O157:H7. These new variants may
have arisen as a result of selection pressure associated with livestock husbandry practices such as large-scale intensive production systems and the
use of antibiotics as growth promoters. However, unequivocal evidence to
support this hypothesis has, thus far, been lacking. Although antibiotic resistance has been reported in a wide range of enteric bacteria, including E. coli,
Salmonella enterica and Campylobacter spp., it is not known to what extent this is
a result of natural selection versus a consequence of antibiotic use in both the
human and animal population, and subsequent environmental contamination.
As discussed in Chapter 2, it is clear that many foodborne infections in
industrialized nations, especially viral infections such as norovirus, reflect
human-to-human transmission, with animals having a minor or insignificant
role. However, other foodborne diseases such as listeriosis, E. coli O157:H7
and many outbreaks of campylobacteriosis have proven links to livestock production and food preparation practices. Owing to the similarity of the clinical
signs associated with a range of food- and waterborne diseases in the human
population, the causative agent in many cases remains unconfirmed. In developing nations, emerging diseases such as Nipah virus have the potential to be
foodborne and have probably infected humans as a result of pressure on
Globalization of Food Supply and Spread of Disease
7
wildlife habitats, with subsequent spillover of the pathogen from its natural
host (e.g. bats) into livestock (e.g. pigs) and the human population (through
the consumption of palm sap contaminated with bat faeces, or contact with
pigs). Other new and emerging diseases, such as severe acute respiratory syndrome (SARS), may be inadvertently spread across the world as a result of the
increasing, and generally illegal, trade in bushmeat and animal-based medicinal compounds, as well as being spread by humans as a result of international
air travel. These isolated reports of emerging zoonotic pathogens that may be
foodborne may reflect rare events, but they may also act as early indicators of
diseases that may be exported around the world where biosecurity measures,
and food safety and inspection standards are not enforced.
Ensuring the Safety of the Food Supply
Surveillance of foodborne disease outbreaks
Surveillance plays an important role in the early detection of foodborne diseases and their control. Early identification of the source of a disease outbreak
is becoming increasingly important as commodities are traded efficiently in
high volumes internationally. Increased mass production means that disease
outbreaks have the potential to affect hundreds of people in multiple countries. Examples of large foodborne disease outbreaks include over 168,000
cases of salmonellosis in the UK in 1985, 224,000 cases of salmonellosis in the
USA in 1993, >310,000 cases of hepatitis A in China in 1988, >3050 cases of
Norwalk-like virus in Australia in 1991 and >6000 cases of E. coli O157 infection in Japan in 1996 (Kaferstein et al., 1997). Good surveillance and reporting
can help to identify the source of a problem early and so prevent spread; for
example, in a 1993 outbreak of listeriosis in France, which was caused by a potted pork product and involved 39 cases, eight miscarriages and one death, the
public-health authorities traced its source within a week, recalled the product
and reduced further cases (Kaferstein et al., 1997). Early detection of disease
outbreaks should also help to minimize the associated costs, both in terms of
human disease and public health dollars.
International reporting on foodborne diseases is becoming more important as it facilitates an early and effective response to emerging foodborne
disease risks and can prevent disease spread (Berlingieri et al., 2007). Epidemiological data from foodborne disease outbreaks can provide public-health
authorities with important information about the types of food implicated in
outbreaks, as well as about the populations at risk, practices that lead to food
contamination, and the growth and survival of foodborne pathogens; they can
also provide an early warning for the development of new and emerging pathogens. In industrialized nations, effective disease reporting has demonstrated the
potential role of food handlers in spreading infectious disease. Disease databases indicate a growing role of bacterial pathogens such as Campylobacter, Salmonella and E. coli O157 versus chemicals and other hazards in food as a primary
cause of serious disease outbreaks resulting in high morbidity and mortality.
8
S.C. Cork and S. Checkley
Surveillance systems are set up to collect, collate, analyse and report surveillance data from animal-health and food systems so that action can be taken
based on the findings (Thacker, 2000; Salman, 2003). Some surveillance systems, such as reportable disease systems, rely on timely and accurate reporting
by health workers and/or producers (in animal health) when a disease or
event occurs. Other surveillance tools mine existing data systems, for example
diagnostic laboratories, for changes in health outcomes (Marvin et al., 2009).
Some surveillance systems use sentinel cases or syndromic trends for early
warning of foreign or emerging disease. In animal-health systems, one objective is to look at animal health/disease or events of public-heath significance
preharvest (Davies et al., 2007),5 potentially allowing more intervention/mitigation options. Other surveillance systems function further along the food
production continuum, i.e. at the abattoir or processing plant. Knowledge of
the epidemiology of the organisms causing foodborne disease is required to
optimize the use of surveillance dollars by targeting the surveillance correctly.
Some food-safety surveillance systems are hazard focused and collate reports
of non-compliance or problems in food-safety audits (Marvin et al., 2009).
As a complement to better reporting of surveillance data, the growing
acceptance of the Hazard Analysis and Critical Control Point (HACCP)6 system has resulted in a reduction in the number of foodborne disease outbreaks
associated with certain manufacturing and preparation processes. Application
of HACCP to food preparation permits the identification of practices that may
be potentially hazardous and allows manufacturers and food safety inspectors
to suggest modifications. In addition, it also allows food technologists and
food inspectors to identify which practices within the manufacturing process
are critical for ensuring the safety of foods, and therefore to determine the
critical control points that require specific monitoring. The first principle of
HACCP is to conduct a hazard analysis; this requires epidemiological data on
specific foodborne diseases and allows an appraisal of the risk of those pathogens being present at the start of, and during, the food processing and preparation process. The production or persistence of toxins, antibiotics or physical
agents in foods is also considered.
Risk assessment and international food standards
Microbiological risk assessment can be used to provide an estimate of the
probability of a specific pathogen or hazard being present in a given commodity as well as the likelihood of disease arising from the presence of a given
amount of a specific pathogen or hazard in a specific population (Fosse et al.,
2008a,b; Hallman, 2008). It can also be used to identify high-risk foods or
5
http://www1.agric.gov.ab.ca/$department/deptdocs.nsf/all/afs10440 (accessed 24
November 2009).
6 http://www.fao.org/DOCREP/005/Y1579E/y1579e03.htm#TopOfPage (accessed 20
May 2010).
Globalization of Food Supply and Spread of Disease
9
processing methods (Swaminathan and Gerner-Smidt, 2007). However,
although risk assessment is a useful tool, the conclusions derived from the riskassessment process must be viewed in context, as the assessment is usually
qualitative. This is because there is often insufficient data available to produce
a quantitative measure of the risks associated with a specific commodity. Essentially, risk assessment is a structured and objective process comprising four
steps: hazard identification, hazard characterization, exposure assessment and
risk characterization. Risk assessment, risk management and risk communication together constitute risk analysis. Risk analysis has a wide range of applications in food safety, ranging from informing national and international
food-safety policies to the implementation of specific sanitation measures to
achieve pathogen reduction at certain points in the farm-to-fork (table) continuum. The process is well described in a number of publications (Murray,
2002; Mumford and Kihm, 2006; Ross, 2008; Wooldridge, 2008).
In brief, risk assessment is defined as a scientifically based process that has
the following steps:
1. Hazard identification (i.e. the identification of biological, chemical and
physical agents present in a particular food or group of foods that can cause
illness).
2. Hazard characterization (i.e. the qualitative or quantitative evaluation of
the nature of the illness associated with biological, chemical and physical
agents that may be present in food).
3. Exposure assessment (i.e. the qualitative or quantitative evaluation of the
likely intake of the hazard).
4. Risk characterization (i.e. the qualitative or quantitative estimation, including uncertainties, of the probability and severity of known impacts in a
given population on the basis of hazard identification, characterization and
exposure assessment).
In many, if not most, cases there is not enough data to undertake quantitative
risk assessments of biological hazards.
In a broader context, risk analysis that includes risk assessment, risk management and risk communication is used by regulatory authorities to prepare
for and manage ‘risk’. In 2003, the US Department of Agriculture (USDA), in
collaboration with the Food Safety Inspection Service of the USDA and the
(US) Centers for Disease Control and Prevention (CDC), released the results
of a risk assessment to predict the risk of listeriosis from different food types
(Swaminathan and Gerner-Smidt, 2007). As a result of regulations implemented over the past decade to prevent Listeria in food commodities for sale
to the public, the incidence of listeriosis in the USA has declined, with reported
cases reduced by 40% between 1996 and 1998.
International agreements on food standards
Increased trade opportunities following the Uruguay Round of multilateral
trade negotiations in 1986–1994, and the increased liberalization of trade,
10
S.C. Cork and S. Checkley
caused concern within nations over the safety of imported food, and highlighted the need to develop transparent regulations to ensure that traded raw
and processed products would be produced to the same standards as products
in the country importing these products. This is relevant to hazards with the
potential to affect human health as well as to chemical toxins, genetically modified agents and invasive microorganisms and pests that might have an effect
on animal and plant health. The WTO emerged as an entity in 1995 (after the
Uruguay Round) along with guidelines for the Application of Sanitary and
Phytosanitary (SPS) measures designed to address concerns about the assessment and control of hazards in imported products. Along with the SPS measures, food safety issues are specifically addressed by the Codex Alimentarius
Commission, which has standards, guidelines and recommendations to ensure
food safety in traded products. The purpose of these standards is to facilitate
trade among WTO members while ensuring that quality and safety standards
are met.
The increased volume of global food trade also highlights the need for
consistent and effective surveillance and reporting to facilitate effective risk
assessment. In this regard, Article 5 of the SPS agreement explicitly requires
WTO members to prepare, or refer to, scientific and consistent risk assessments. In addition, the World Health Organization (WHO) has recommended
that the application of the HACCP system at every stage of the food chain
represents an effective approach for governments to meet the terms outlined
in the agreement.
Over 60% of known human infectious diseases have their source in animals (whether domestic or wild), as do 75% of emerging human diseases and
80% of the pathogens that could potentially be used in bioterrorism. At the
global level, the OIE has modernized its worldwide information system on
animal diseases (including zoonoses) with the creation of World Animal
Health Information Service (WAHIS), a mechanism whereby all countries are
linked online to a central server that collects all the compulsory notifications
sent to the OIE, which covers 100 priority terrestrial and aquatic animal diseases. The WHO has adopted the International Health Regulations, placing
new obligations on its members. The OIE, WHO and Food and Agriculture
Organization (FAO) have created GLEWS, the Global Early Warning System,
a platform shared by the three organizations to improve early warning on animal diseases and zoonoses worldwide.
The World Organisation for Animal Health (OIE)
The need to fight animal diseases at global level led to the creation of the
Office International des Epizooties (OIE) through an international agreement signed on 25 January 1924. In May 2003, the OIE became the World
Organisation for Animal Health, but kept its historical acronym, OIE. The
OIE is the intergovernmental organization responsible for improving animal
health worldwide. It is recognized as a reference organization by the WTO
and, as of April 2009, had a total of 174 Member Countries and Territories.
Globalization of Food Supply and Spread of Disease
11
The OIE maintains permanent relations with 36 other international and
regional organizations and has regional and subregional offices on every continent.
OIE’s claimed mission is to:
●
●
●
●
guarantee the transparency of animal disease status worldwide;
collect, analyse and disseminate veterinary scientific information;
provide expertise and promote international solidarity for the control of
animal disease; and
guarantee the sanitary safety of world trade by developing sanitary rules
for international trade in animals and animal products.
Codex Alimentarius
The Codex Alimentarius Commission was created in 1963 by FAO and WHO
to develop food standards, guidelines and related texts such as codes of
practice under the Joint FAO/WHO Food Standards Programme. The main
purposes of this Programme are protecting health of the consumers, ensuring fair trade practices in the food trade, and promoting the coordination of all
food standards work undertaken by international governmental and nongovernmental organizations (Dawson, 1995; Droppers, 2006; Slorach, 2006).
The WTO agreement on the Application of Sanitary and Phytosanitary
Measures (SPS Agreement) considers that WTO members that apply the
Codex Alimentarius Standards meet their obligations under this agreement.
Scientifically based risk assessment plays an important role in the setting of
Codex standards. Epidemiological data on foodborne diseases is important
for the development of these risk assessments. One example is an assessment
of the risk of contracting listeriosis following the consumption of products
that may contain varying amounts of L. monocytogenes. A joint study conducted
by the USDA and the US Food and Drug Administration (FDA) identified
foods as being of high, medium and low risk with respect to harbouring L.
monocytogenes (Swaminathan and Gerner-Smidt, 2007). High-risk foods include
delicatessen meats, high-fat dairy products, soft unripened cheese and unpasteurized fluid milk. Medium-risk products include pasteurized milk, fresh soft
cheeses, RTE meals, salami, fruit and vegetables. To reduce the risk of products harbouring L. monocytogenes, good food production standards such as
HACCP have also been implemented. The Codex Alimentarius Commission
adopts standards, codes of practices and other related texts that are prepared
by specialized Codex Committees and ad hoc Task Forces.
Organic food production
There remains a fair bit of controversy about the benefits and risks associated
with organic food production, especially in relation to food safety and food
quality. A well-managed organic farm can provide many environmental benefits
12
S.C. Cork and S. Checkley
and has socio-economic value, especially when catering for small-scale niche
markets (Hovi et al., 2003; Lund, 2006; Dangour et al., 2009). Some studies
have demonstrated a lower prevalence of antimicrobial resistance in bacteria
isolated from animals on organic farms compared with those on conventional
farms (Schwaiger et al., 2008) although this is not the case in all studies. Largescale processing of products for international and national distribution has to
comply with the same basic food safety standards as conventional produce.
Production standards, however, are usually set by national and international
certification bodies that outline what husbandry and veterinary intervention
practices are required and/or allowed, as well as the production standards for
growing, storage, processing, packaging and shipping.
In Europe, the organic sector is quite well developed (Vaarst et al., 2005,
2006; Sundrum et al., 2006, 2007; Sundrum, 2008)with an active research programme underway to examine new approaches to raising livestock with minimal use of chemicals such as anthelmintics (Deane et al., 2002; Keatinge, 2005;
Maurer et al., 2007) and antibiotics (Schwaiger et al., 2008). European organic
legislation is regularly revised.7 Guidelines on Organic Standards in the USA
are provided by the USDA, with information on sustainable livestock production systems available through the Alternative Farming Systems Information
Centre,8 and the National Organic Program.9 Owing to the evolving nature of
organic regulations hard-copy literature is frequently out of date, so agencies
tend to rely on the Internet and news bulletins to update producers. In most
countries, organic certification remains the responsibility of government or
specialized agencies.
Disease and the Globalized Food Supply: Case Studies
Globalization has changed the face of disease outbreaks and how we respond
to them. The globalization of legal and illegal trade in animals, animal parts and
food has led to large-scale disease outbreaks that have thwarted historical
approaches to disease control. These may also have significant public-health
effects. The large centralized food-processing facilities that have emerged in
recent years have been associated with a number of significant foodborne disease outbreaks, some of which have had impact on a number of different
countries owing to the widespread trade of processed foods and animal feed.
Foot-and-mouth disease in the UK: a new era in disease control
In 2001, the UK was besieged by an outbreak of foot-and-mouth disease
(FMD), which resulted in extremely high disease control costs, required
extensive efforts for the reopening of trade markets, and had a number of
7
http://ec.europa.eu/agriculture/organic/splash_en (accessed 17 May 2010).
http://www.sare.org/publications/organic/resource.htm (accessed 17 May 2010).
9 http://www.ams.usda.gov/AMSv1.0/nop (accessed 17 May 2010).
8
Globalization of Food Supply and Spread of Disease
13
direct and indirect impacts on human and animal health and welfare. In previous outbreaks involving exotic diseases such as FMD, the implementation of
response policies that advocated containment and cullings had proved effective in the UK. In this case, though, it proved difficult to control the outbreak
owing to the widespread undetected dissemination of FMD virus throughout
the country before the disease was recognized and/or reported (Scudamore
and Harris, 2002). Although the source of the outbreak is not known for sure,
it is likely to have been introduced into the UK via FMD-infected meat or other
animal products. As a result of the outbreak, large numbers of animals were
culled amid significant public outcry.
FMD is considered to be a minor zoonosis because human infection is
rare and usually mild. However, the psychosocial and economic effects of the
outbreak caused significant stress and distress in the agricultural community and
beyond (Mort et al., 2005). Fear of a new disaster and loss of trust in the government authorities figured prominently (Mort et al., 2005). Since 2001, there has
been a considerable amount of research done, both within the UK and at an international level, to address questions raised during and after the UK outbreak. This
has facilitated better preparedness planning for future outbreaks using a range of
different strategies, including the judicious use of vaccination, improved vaccine
options, enhanced reporting and surveillance systems, and changes to the veterinary infrastructure (Cottam et al., 2006; Green et al., 2006; McLaws et al., 2006;
Bessell et al., 2008; Schley et al., 2009; Tildesley et al., 2009).
Listeriosis – control, risk assessment and regulations
Systemic listeriosis is a serious, but usually sporadic, invasive disease that primarily afflicts pregnant women, neonates and immunocompromised individuals.
The causative agent is L. monocytogenes, which is primarily transmitted to humans
through contaminated foods. Outbreaks of listeriosis are usually spread by the
faecal–oral route, resulting in self-limiting gastroenteritis in healthy humans,
and have been reported in North America, Europe and Australasia. Soft cheeses
made from raw milk and RTE chilled delicatessen meats are high-risk foods for
susceptible individuals. The infectious dose of L. monocytogenes is not known
(Swaminathan and Gerner-Smidt, 2007). Efforts by food processors and food
regulatory agencies to control L. monocytogenes in food products have been successful in many countries, but sporadic outbreaks, often with severe consequences, continue to occur.
Although only a small percentage of the listeriosis cases reported are
traced to a common source, public-health officials place a high priority on
investigating the outbreaks for the following reasons:
1. Listeriosis is a serious disease and has a relatively high mortality rate in some
cases, especially in individuals with compromised immune systems (e.g. immunodeficiency disorders, the elderly, neonates and pregnant women).
2. Morbidity and mortality can be reduced by prompt action by public-health
agencies, i.e. trace back and recall implicated food, etc.
14
S.C. Cork and S. Checkley
Table 1.1. Gastrointestinal disease outbreaks caused by listeriosis (1993–2001) (adapted
from Swaminathan and Gerner-Smidt, 2007).
Location
Year
Food involved
Italy (northern)
USA (Illinois)
Italy (northern)
Finland
New Zealand
USA (California)
Sweden
Japan
1993
1994
1997
1998
2000
2001
2001
2001
Rice salad
Chocolate milk
Cold maize and tuna salad
Cold smoked fish
Ready-to-eat (RTE) meats
RTE meat – turkey
Raw milk/cheese
Cheese
Serotype
No. cases reported
1/2b
1/2b
4b
1/2a
1/2
1/2a
1/2a
1/2b
18
44
1566
–
32
16
48
38
3. Outbreak investigation can help to identify key risk factors and prevent
future cases.
During the past decade there have been several significant outbreaks of
listeriosis reported worldwide (see Table 1.1).
The availability and use of molecular typing plays an important role in the
early recognition of listeriosis and in tracing the source of contamination. It is
especially useful to group cases that may occur in geographically distinct
regions, or where cases occur in several states or countries. This is discussed
further in Chapter 2.
Our knowledge of listeriosis has increased significantly over the past
decade. This has been achieved largely by applying risk assessment to food
processing and implementing the required sanitation at key intervention
points. However, L. monocytogenes, cannot be entirely eliminated as it occurs
naturally in the environment. Education of consumers and international
cooperation in ensuring that food standards are met is important to control
both this and other foodborne diseases.
Salmonellae and antimicrobial resistance
Salmonellae have been associated with a number of foodborne diseases linked
to traded animal products. Aside from the immediate food-safety issues, there
is also significant concern about the growing presence of antibiotic resistance
in S. enterica isolates (Oloya et al., 2009). Determination and characterization
of Salmonella spp. isolated from domestic animals and humans in North
Dakota, USA was performed to assess their potential role in transferring antimicrobial resistance (AMR) to humans. The National Antimicrobial Resistance Monitoring Systems panel was used to compare AMR profiles of animal
and human isolates to assess a possible role of domestic animals in the transfer
of AMR to humans. The panel found that Salmonella enterica serovar
Typhimurium was the predominant serotype in both humans (13.4%) and
Globalization of Food Supply and Spread of Disease
15
domestic animals (34.3%), followed by S. enterica serovar Newport in animals
(2.6%) and humans (3.9%). Salmonella arizona (0.7%), S. enterica serovar Give
(0.9%) and S. enterica serovar Muenster (3.5%) were isolated from sick or dead
animals; the AMR levels were generally higher in isolates from animals than
humans. However, the genes involved in AMR in animal isolates may not
reflect those found in human isolates; therefore, there is a need to assess genotype and phenotype before source attribution can be confirmed (Hopkins
et al., 2007). This topic is discussed further with respect to trade in Chapter 2.
Conclusion
Many of the diseases that have grown in importance in human medicine in
recent decades are transmitted via the food and water supply. As the world has
moved from a situation in which the majority of food was produced locally and
sold in local markets to a system in which food is transported great distances
and marketed through large chains of supermarkets, the pathogen profile to
which humans are exposed has expanded. Despite the international SPS
agreement that provides a framework for the import health standards required
for traded animal- and plant-based products (Domenech et al., 2006), the
expansion of trade in fresh, chilled and processed produce has resulted in a
number of significant biosecurity breaches in recent years. Current SPS
requirements emphasize the importance of science-based risk assessment and
hazard control programmes for the continued reduction of pathogens at relevant points of the ‘farm-to-fork’ food production chain. This approach, along
with good consumer education about food preparation and handling practices is likely to be the best approach for reducing risks to human health in the
modern globalized society.
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2
Epidemiology of Pathogens
in the Food Supply
SUSAN C. CORK
Introduction
The World Health Organization (WHO) estimates that 1.8 million people
in the developing world die each year from complications associated with
diarrhoea (WHO, 2010).1 Many of these deaths are caused by pathogens
transmitted to humans in food and water supplies. Common sources of foodborne infections are directly contaminated food products and foods contaminated by environmental sources, including water (Gajadhar et al., 2006).
Although many of these infections are typically caused by pathogens transmitted from human to human, others also occur in animals and potentially
have a zoonotic origin (see Table 2.1 and associated references). To develop
appropriate interventions it is important to understand the ecology of these
diseases and to be able to attribute significant disease outbreaks to specific
sources. This not only requires a sound grasp of epidemiology and the surveillance tools used to map infections, but also a good knowledge of pathogen
biology and the behaviour and susceptibility of the populations at risk (Pires
et al., 2009). As urban areas expand and previously protected habitats are
developed, new and re-emerging diseases, many of which are zoonotic in
origin, continue to be reported. This has brought about a recognition that
human and animal health agencies need to work together to develop robust
and timely disease detection, reporting and prevention policies (King et al.,
2004; Gray and Kayali, 2009).
1
http://www.who.int/water_sanitation_health/publications/facts2004/en/ (accessed 23
February 2010).
© CAB International 2011. Zoonotic Pathogens in the Food Chain
(eds D.O. Krause and S. Hendrick)
21
22
S.C. Cork
Patterns and Trends in Enteric Infections
The burden of foodborne disease, even in industrialized countries, remains
substantial. It is estimated that 76 million cases of foodborne disease occur
each year in the USA, with 325,000 hospitalizations and 5000 deaths linked
to foodborne and waterborne diseases each year (CDC, 2010).2 Among the
known zoonotic foodborne pathogens, a few that are reported (i.e. Escherichia coli O157, Campylobacter spp., Salmonella spp., Listeria monocytogenes) tend
to dominate the food safety literature. This trend may reflect the investment
provided to develop reliable detection methods and surveillance systems for
these pathogens, thereby ensuring timely reporting and intervention. Other
agents that cause enteric disease have probably remained under-detected,
e.g. emerging protozoal diseases such as caused by Cyclospora spp. (Mead
et al., 1999).
In addition to the emergence or recognition of new enteric pathogens,
the globalization of the food supply, along with modern processing methods,
the availability of imported foodstuffs to a wide range of consumers and a
growing preference for fresh and ready-to-eat produce, has resulted in widely
dispersed outbreaks of some foodborne diseases (e.g. listeriosis, salmonellosis, some protozoal infections) (Kaferstein et al., 1997; Kobrin, 2008; Oloya
et al., 2009). Other trends that have been noted include an increase in the
detection and reporting of antimicrobial resistance (Hopkins et al., 2007)
and enhanced identification of pathogens that are highly opportunistic (i.e.
affecting only the most high-risk subpopulations). New pathogens, or new
variants of well-known organisms, can emerge as a public-health problem as
a result of natural selection pressure or changes in consumer preferences
and food production technologies (i.e. increased availability of fresh chilled
food products) that connect a potential pathogen with the food chain (e.g.
L. monocytogenes) (Little et al., 2003; Bhunia, 2008a).
A selection of some pathogens reported to cause enteric disease in humans
is provided in Table 2.1. Although this list is not comprehensive, it provides an
overview of the wide variety of agents that can be associated with outbreaks of
foodborne diseases in humans. Some of these will be considered in more
detail later in this chapter.
The impact of specific pathogens on human health will depend on the
virulence of the pathogen and the susceptibility of the host, as well as on the
level of exposure, presence of co-infections and host immune status. The epidemiological picture depends on a wide range of other factors, including the
predominant method of disease transmission, the requirement, or not, for
disease vectors or intermediate hosts, the role of reservoir hosts, the demographics of the human population that is exposed and whether or not the
pathogen is an opportunistic or an obligate pathogen.
2
http://www.cdc.gov/ncidod/dbmd/diseaseinfo/foodborneinfections_g.htm (accessed
23 February 2010).
23
Epidemiology of Pathogens in Food Supply
Table 2.1. Pathogens transmitted by food and water supplies that may have
an animal source.
Agent
Food/water source most
commonly implicated
Common symptoms in humans
Viruses
Enteroviruses
Contaminated water
Adenovirusesa
Contaminated water
Hepatitis Ab
Contaminated water
and food, including shellfish
Contaminated food,
including pork and venison
Hepatitis E
Astrovirusesc
Contaminated water
and food, including shellfish
Rotaviruses
Contaminated water and food
Norwalk-like viruses
(noroviruses)b
Bacteria
Escherichia coli
(many variants)b
Salmonellaed
Shigellaeb
Vibrio spp.b
Campylobacter
spp.
Listeria
monocytogenes
Contaminated water
and food, including shellfish
Contaminated food, including
meats (e.g. beef) and vegetables
washed in contaminated water
Contaminated food, including
meats (e.g. chicken)
and vegetables washed
in contaminated water
Contaminated food, including
salads; primates and humans
are the key hosts
Contaminated water and food,
including shellfish and salads;
primates and humans are the
key hosts
Contaminated food, including
meats (e.g. chicken)
Contaminated food, including
shellfish, chilled meats and salads
Yersinia
enterocolitica
Yersinia
pseudotuberculosis
Contaminated food, including meats
Mycobacterium
avium
paratuberculosis
Contaminated milk?
Contaminated meat and
vegetation
Gastroenteritis and systemic
signs
Gastroenteritis, may have
respiratory signs
Hepatitis with or without jaundice
Hepatitis, often without jaundice
(Banks et al., 2007;
Chandra et al., 2008;
Khuroo and Khuroo, 2008)
Gastroenteritis with watery
diarrhoea, vomiting and
anorexia
Gastroenteritis with vomiting and
diarrhoea
Gastroenteritis with vomiting,
diarrhoea and abdominal pain
Gastroenteritis, may have
significant systemic signs
(Oporto et al., 2008)
Gastroenteritis, may have
fever and other systemic
signs (Hopkins et al., 2007;
Oloya et al., 2009)
Gastroenteritis, often with
blood in faeces
Gastroenteritis with vomiting
and fever
Gastroenteritis, may have fever
and other systemic signs
Gastroenteritis, may have
other systemic signs
(Walse et al., 2003)
Gastroenteritis, may have other
systemic signs
Gastroenteritis, may mimic
appendicitis and also have
other systemic signs
May be associated with
irritable bowel disease
and Crohn’s disease
(Cirone et al., 2007;
Scanu et al., 2007)
Continued
24
S.C. Cork
Table 2.1. Continued
Agent
Food/water source most
commonly implicated
Mycobacterium bovis
Contaminated milk
Staphylococcus
aureusb
Clostridium
perfringens
Clostridium difficile
Food contaminated with toxin
Insufficiently cooked
meat or reheated food
Contaminated ground beef,
pork or turkey
Bacillus cereus
Reheated food, especially rice
Coxiella burnetii
Contaminated milk products
Brucella spp.
Milk and milk products from
infected ruminants
Leptospira
interogans –
various
serovars
Protozoa
Humans usually infected via
direct contact with urine from
infected animals or via a
contaminated food or water supply
Giardia lamblia
Contaminated water
Sarcocystis spp.
Raw or undercooked beef
Toxoplasma gondii
Raw or undercooked meat
(pork, sheep, goat)
Entamoeba
histolyticab
Contaminated food
Cryptosporidium
parvumb
Contaminated water and raw
fresh produce
Cyclospora
Contaminated water and
fresh produce; human host
Common symptoms in humans
Associated with lymph gland
enlargement and localized or
systemic signs (Thoen and
LoBue, 2007; de Kantor et al.,
2008)
Gastroenteritis, may have other
systemic signs
Gastroenteritis, may have other
systemic signs
Mild gastrointestinal signs
associated with toxin
(Rupnik, 2007)
Gastroenteritis, may have other
systemic signs
Fever, systemic signs such as
nausea, headache, myalgia
Variable, undulant fever,
aches, headache, may
become chronic (Mantur and
Amarnath, 2008)
Variable, fever, jaundice, myalgia,
vomiting, abdominal pain,
diarrhoea (Monahan et al.,
2009)
Gastroenteritis, abdominal gas
and discomfort (Appelbee et al.,
2003; Smith et al., 2007)
Gastroenteritis, may have other
systemic signs (Vercruysse
et al., 1989; Pathmanathan and
Kan, 1992)
Gastroenteritis (Dubey et al.,
2002a; Lindsay et al., 2002;
Bhopale, 2003)
Gastroenteritis, may develop
abscesses and other
complications (Stanley, 2003)
Gastroenteritis, diarrhoea
(Millar et al., 2002; Graczyk
et al., 2003; Smith et al., 2007;
Jagai et al., 2009)
Gastroenteritis
Nematodes
Trichinella spp.
Consumption of raw or undercooked
meat from infected animals
(wild boar, bears, walruses, pigs,
horses, etc.)
Variable, hypersensitivity, can be
fatal (Pozio et al., 1996; Cui and
Wang, 2005; Kaewpitoon et al.,
2008; Gottstein et al., 2009)
Continued
25
Epidemiology of Pathogens in Food Supply
Table 2.1. Continued
Agent
Angiostrongylus spp.
Anisakis spp.
Capillaria spp.
Food/water source most commonly
implicated
Consumption of raw or
undercooked crabs, crayfish,
snails, also contaminated
fresh produce, etc.
Consumption of raw or
undercooked saltwater
fish, squid
Consumption of raw or
undercooked fish from fresh
or brackish water
Common symptoms in humans
Depends on species of parasite;
acute abdominal or cerebral
involvement (Lin et al., 2005)
Epigastric pains, vomiting
Gastrointestinal signs
Trematodes
Fasciola hepatica
Fasciolopsis buski
Clonorchis sinensis
Paragonimus
Consumption of plant material
contaminated with
metacercariae
Consumption of water
chestnuts contaminated
with metacercariae
Consumption of raw
or undercooked fish
Consumption of freshwater
crabs and crayfish
Variable (Hammami et al., 2007)
Consumption of
undercooked beef
Consumption of undercooked pork
Often no signs
Epigastric and hypogastric
pain, gastrointestinal signs
Variable, cholangitis
Chest pain, cough and fever
(Liu et al., 2008)
Cestodes
Taenia saginata
Taenia solium
Echinococcus spp.
Diphyllobothrium spp.
a
Consumption of vegetation
washed with water
contaminated with dog faeces
Consumption of raw
or undercooked freshwater fish
May be no signs or serious
systemic involvement with
CNS signs (Kyvsgaard
et al., 2007)
Often no signs until cysts are
large, serious if cysts rupture
(Jenkins et al., 2005)
Often no signs (Margono et al.,
2007)
Adenoviruses are commonly found in human sewage and have been isolated from both human and animal sources.
Disease outbreaks in humans have been reported after consumption of contaminated shellfish (Myrmel et al., 2004).
Most adenovirus infections in humans probably result from human-to-human transmission although a zoonotic source
cannot be ruled out.
b Although these pathogens are predominantly human pathogens that can be transmitted by food and water sources to
other humans, new data suggest that some variants may have a potential animal source, especially where non-human
primates and other wildlife species are present (Cunningham, 2005; Ekanayake et al., 2006).
c Astroviruses are frequently associated with mild cases of gastroenteritis in children and are occasionally isolated
from adults. Infections in adults are often associated with the consumption of contaminated shellfish (Le Guyader et al.,
2000).
d Salmonellae typically cause mild-to-severe gastroenteritis in humans but are also responsible for human typhoid fever
(Salmonella enterica serovar Typhi) and paratyphoid (S. enterica serovar Paratyphi), which are considered to be an
important cause of human foodborne infections worldwide. On a global scale it is thought that there are over 16 million
cases of typhoid each year, with 1.3 billion cases of enteritis and 3 million deaths attributed to S. enterica. Although
the majority of these cases are likely to have been transmitted from human to human via contaminated water or food,
rodents, other wildlife and domestic animals can also be a source of infection. Specific source attribution of Salmonella
infections in non-industrialized nations is often difficult owing to the lack of molecular tools and surveillance systems
(Gassama-Sow et al., 2006).
26
S.C. Cork
Bacterial Foodborne Diseases
A large number of the bacteria that cause enteric disease in humans and animals, as well as those that comprise the normal gut flora, are members of the
family Enterobacteriaceae. These are commonly transmitted via contaminated
food and water from human sources, or to humans and animals from material
contaminated with faeces from other animals (Bhunia, 2008b). The epidemiology of these foodborne pathogens depends on locality, food preparation
practices, food preferences, hygiene, access to clean water, level of community
education and availability of public health services, as well as on the development
and enforcement of food and water safety regulations.
Enteric bacteria commonly transmitted from human to human through
food and water sources include E. coli, Klebsiella spp., Salmonella spp., Vibrio
spp., Shigella spp. and Yersinia spp. (Cooke, 1990; Bhunia, 2008a). Human
infection with other enteric bacteria such as Campylobacter is most often associated with specific foodstuffs such as chicken meat, but may also be transmitted to humans from wildlife and other domestic animals. Other agents,
such as L. monocytogenes, are ubiquitous in the environment but can be present in animals and can contaminate vegetation and meats as well as shellfish.
Many of the enteric diseases transmitted to humans via shellfish are not typically considered to be zoonotic as the associated pathogens are often common in the human population and are likely to originate from a human
source. However, as we learn more about the complex ecosystems associated
with some aquaculture practices this assumption may need to be revised
(Graczyk and Schwab, 2000).
A wide range of other bacteria can cause disease in humans, many of these,
such as mycobacteria, Brucella spp., Coxiella burnetii and leptospirosis, cause
occasional sporadic disease in developing countries and are now less commonly reported in the northern hemisphere. In many countries this is the
result of targeted disease control and food safety practices, but these organisms
may still pose a risk to travellers visiting regions where the diseases associated
with these organisms remain endemic.
The bacterial diseases outlined in the following sections have been selected
to illustrate the epidemiology of some specific bacterial pathogens in more
detail.
Listeria monocytogenes
Listeria is a Gram-positive facultative pathogen that was first isolated from
rabbits in 1926. It was initially considered to be primarily an animal pathogen causing ‘circling disease’ in ruminants, pigs and dogs and cats (Cossart,
2007). In the 1970s, Listeria was identified as a foodborne pathogen causing
numerous outbreaks of gastroenteritis in North America, with up to 2500
cases recorded a year. In humans, the incubation period in susceptible
adults is 3–70 days, with the median incubation period estimated to be
3 weeks.
Epidemiology of Pathogens in Food Supply
27
Listeriosis can be a serious problem in pregnant women, newborns, the
elderly, and immunocompromised or debilitated hosts. Pregnant women may
experience either a mild, flu-like syndrome with fever, chills, headache, slight
dizziness or gastrointestinal signs. This may be followed in a few days to
weeks by abortion, stillbirth, premature birth or septicaemia in the newborn. Newborns may be infected either in utero or from bacteria found in the
vagina during delivery. Infected infants can develop septicaemia, disseminated
granulomatosis, respiratory disease or meningitis; symptoms may be present
at birth or develop within a few days to several weeks. In elderly, immunocompromised or debilitated persons, L. monocytogenes can cause meningitis, meningoencephalitis or, less frequently, septicaemia (López et al., 2006). The
organism survives well in chilled food products, including salads, shellfish and
soft cheeses kept in the fridge. The disease attracts the attention of both the
public and regulatory authorities because there is often a higher mortality
rate associated with listeriosis than with most other foodborne bacteria
(Rocourt et al., 2003). In the USA, a zero-tolerance policy was introduced to
ensure that L. monocytogenes was not present in ready-to-eat foods. Internationally, and in the USA, there have been a number of widespread multi-state outbreaks reported. Most of these have been followed up by food recalls. Tainted
turkey meat and other delicatessen meats were implicated with one case in
1998–1999 that involved 22 states in the USA, 101 illnesses, 15 deaths and
six miscarriages (Swaminathan and Gerner-Smidt, 2007). In 2000–2001 a
Mexican type soft cheese made from unpasteurized milk was identified as the
source of infection in North Carolina and caused five miscarriages, with 12
other people reported to be sick. In 2008, in Canada, there was a listeriosis
outbreak linked to ready-to-eat meats produced at a Maple Leaf food plant in
Ontario. Although there are thought to be between 100 and 140 cases of
listeriosis reported in Canada each year, there are usually very few deaths
attributed to Listeria. The 2008 outbreak resulted in 20 deaths across five provinces. The authorities worked effectively with industry partners to control the
outbreak, but its extent generated a lot of media interest and caused significant public concern as outlined in the 2008 Public Health Agency of Canada
report on the outbreak.3
In an attempt to assess the risk of Listeria in food, a joint study was conducted by the US Department of Agriculture (USDA) and the US Food and
Drug Administration (FDA) which categorized foods as being high, medium
and low risk with respect to harbouring L. monocytogenes. High-risk foods
included delicatessen meats, high-fat dairy products, soft unripened cheese
and unpasteurized fluid milk. Medium-risk products included pasteurized
milk, fresh soft cheeses, ready-to-eat meals, salami, fruit and vegetables.
Low-risk products included cultured milk products, hard cheeses and frozen
products. To maximize disease prevention, good food production standards
have been implemented by the major food manufacturers to minimize the risk
3 http://www.phac-aspc.gc.ca/fs-sa/listeria/2008-lessons-lecons-eng.php (accessed
24 February 2010).
28
S.C. Cork
of contamination with pathogens such as L. monocytogenes (Ivanek et al., 2004;
Swaminathan and Gerner-Smidt, 2007).
Increased awareness of listeriosis in both the food-processing industries
and among consumers has helped to reduce some of the risks associated
with L. monocytogenes in certain food sources, but there remains a need to be
proactive in this area. Owing to the ubiquitous nature of the organism and its
presence in the environment, it is unlikely that this pathogen will be totally
eliminated, but continued consumer education about appropriate food selection and preparation remains an important mechanism by which listeriosis
and other foodborne pathogens can be controlled.
Escherichia coli
The majority of E. coli isolates from humans and animals are non-pathogenic
and exist harmlessly in the intestinal tract. Because of the wide range of nonpathogenic strains of E. coli that are present in the environment it is important
to be able to distinguish these from potential pathogens. Pathogenic E. coli is
subdivided into different pathotypes which can then be further classified into
virotypes, based on the virulence genes that they possess. A virotype reflects a
particular combination of virulence genes. Important virulence factors
encoded by these genes include fimbrial adhesins, enterotoxins, cytotoxins,
capsule and lipopolysaccharides (LPS). Pathogenic E. coli may also be differentiated by serotype based on antigenic differences in the O antigen of the
LPS, in the flagellar or H antigens, and in the fimbrial or F antigens. The
pathogenic E. coli virotypes are usually referred to as follows: enterotoxigenic
(ETEC), enteropathogenic (EPEC), verocytotoxigenic (VTEC) (this includes
enterohaemorrhagic (EHEC) strains), enteroinvasive (EIEC), enteroaggregative (EAEC) and diffusely adhering (DAEC). Some VTEC strains produce a
Shiga-like toxin that kills vero cells in vitro; hence, the group is also referred to
as STEC (Bell and Kyriakides, 1998).
Pathogenic E. coli can cause a variety of diseases in humans, including gastroenteritis, dysentery, haemolytic uraemic syndrome (HUS), urinary tract infections (UTI), septicaemia, pneumonia and meningitis. Several of the verotoxic
E. coli (VTEC) have been isolated from cases of bloody diarrhoea and haemolytic
uraemia in humans (Karmali et al., 2003), and a number of disease outbreaks
have recently been recorded in developed countries. The VTEC group includes
E. coli O157:H7, which has also been isolated from the faeces of healthy cattle, as
well as from goats, chickens, sheep, pigs, dogs, cats and seagulls. Foodborne disease outbreaks in humans have been associated with the consumption of contaminated ground beef in the USA, Canada and Europe. Outbreaks have also
been traced back to contaminated lettuce and spinach, raw milk, mayonnaise,
apple cider, fresh fruit, salami and sprouts, and the general assumption is that the
E. coli responsible has originated from infected animal faeces. However, although
a number of studies have been conducted (Cooley et al., 2007) conclusive evidence for this has not always been provided, as is illustrated in a number of
examples discussed in detail by Bell and Kyriakides (1998).
Epidemiology of Pathogens in Food Supply
29
Salmonellae
Salmonellae are Gram-negative, non-spore forming bacilli commonly found
in the intestinal tract of birds, reptiles, farm animals, wildlife and humans.
Isolates of Salmonella enterica can be classified in a number of different ways
and are usually represented as S. enterica followed by the specific serovar, i.e. S.
enterica serovar Choleraesuis (Dubansky, 2008). In Europe, foodborne disease
outbreaks associated with Salmonella spp. have often been attributed to the
consumption of poultry and poultry products. Salmonella enterica serovar
Enteritidis is often implicated although this organism does not generally cause
disease in infected poultry. S. Enteritidis has the ability to colonize the ovary
of the laying hen and can be isolated from the contents of freshly laid eggs
(Keller et al., 1995). Eggs stored at room temperature may contain up to 109
colony forming units (cfu), which may reflect the level of faecal contamination after laying (Lublin and Sela, 2008). In Europe, there are currently a
number of regulations in place to control the level of S. Enteritidis in poultry
on the farm, i.e. the use of vaccination as well as strict regulations over the
processing and packaging of eggs and poultry products to reduce contamination (Korsgaard et al., 2009).
Other Salmonellae of interest include Salmonella dublin, which can cause
gastroenteritis in humans although it is more typically associated with disease
in cattle. S. Choleraesuis is associated with disease in pigs, and Salmonella arizonae has been isolated from healthy and sick reptiles. As with other bacterial
pathogens, when attributing sources for foodborne salmonellosis it is necessary to compare isolates using molecular methods to assess whether or not the
isolates from animals or infected humans match with those in the suspect food
source. In the past this was not always possible, and there was reliance on classical classification techniques which may not be sufficiently specific to prove a
direct causal relationship in an outbreak. In many diagnostic facilities, salmonellae are still classified according to the somatic/outer cell wall (O) and flagella (H) antigens and the capsular (Vi) antigenic patterns. Currently, there
are thought to be over 2443 S. enterica serotypes with six subspecies: type I
(enterica), II, IIIa, IIIb, IV and VI (Bhunia, 2008b).
In industrialized countries, human cases of salmonellosis are frequently
traced back to the consumption of contaminated meat, milk, poultry and eggs.
However, dairy products, including cheese and ice cream, as well as fruit and
vegetables contaminated with infected faecal material, or fresh foodstuff that
has been washed in contaminated water, have also been implicated. The widespread distribution of foodstuffs from central facilities and the blending of
products for sale in supermarkets has possibly led to rapid and widespread
distribution of potentially contaminated produce. For example S. enterica serovar Typhimurium DT104, which is an emerging pathogen in the USA, Canada
and Europe, causes high mortality in cattle and can be transmitted to humans
in contaminated beef, especially ground beef. In 2005, there were four cases
of multi-drug resistant S. Typhimurium DT104 isolated in beef imported into
Norway from Poland. This was reported to the Norwegian Institute of Public
Health reference laboratory, where the source was traced back to Polish
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imported beef. The isolates from the disease outbreak had identical multilocus VNTR (variable number of tandem repeats) analysis profiles and
antibiotic-resistance patterns. This highlights the value of molecular techniques for the characterization of isolates (Lindstedt et al., 2003) for trace
back and re-enforces the need to monitor imported and blended products.
Campylobacter spp.
Campylobacter is a member of the Campylobacteriaceae and was first identified
as a cause of abortion in sheep in 1913 (Bhunia, 2008c). Since then it has been
isolated from a range of animals and is considered to be a significant cause of
foodborne disease in humans (Clements, 2009). Examination of reported
cases of foodborne disease in humans in the USA indicates that 14.2% have
been associated with Campylobacter (Vasickova et al., 2005). Between 1998 and
2002, the Centers for Disease Control and Prevention (CDC)4 reported 61 outbreaks of campylobacteriosis in the USA, with 1440 cases of Campylobacterrelated illness in humans. The higher number of cases reported in recent times
may reflect better isolation methods and enhanced reporting as the microaerophilic nature of the organism meant that it was not routinely detected in
samples unless its isolation was specifically requested (Humphrey et al., 2007).
This and other factors, such as improvements in awareness, have influenced
the prevalence of reported cases of campylobacteriosis in Europe and elsewhere (Gillespie et al., 2009).
Animals are considered to be the main reservoir for Campylobacter spp., with
isolates obtained from rabbits, birds, sheep, horses, cows, pigs, poultry and
domestic pets. Isolates have also been cultured from vegetables and shellfish.
Risk assessments have been used to determine the source of infections in humans,
and contaminated chicken meat is considered to be the main cause of foodborne Campylobacter jejuni infection in humans (Calistri and Giovannini, 2008).
C. jejuni and Campylobacter coli can colonize the caecae of poultry, and contamination of meat often occurs during processing. Currently, campylobacteriosis is
more commonly reported in developed rather than developing countries, but
this may reflect the priorities and focus of the surveillance systems in place.
Campylobacteriosis has been widely studied in New Zealand, where the
high prevalence of infection in humans has been associated with consumption
of contaminated poultry meat that has not been properly cooked (EberhartPhillips et al., 1997). The New Zealand Food Standards Agency, along with the
poultry industry, and university and government research facilities, have done
risk assessment and case-controlled studies, and identified key risk mitigation
measures. The outcome of this work has been a significant reduction in the
number of human cases of campylobacteriosis over the last few years (Lake
et al., 2007).
4
http://www.cdc.gov/foodnet/factsandfigures.htm (accessed 20 May 2010).
Epidemiology of Pathogens in Food Supply
31
Yersinia enterocolitica
Yersinia spp. are members of the Enterobacteriaceae. The genus has 11 species
including Yersinia pestis, Yersinia pseudotuberculosis and Yersinia enterocolitica. The
majority of the others are non-pathogenic except in immunocompromised
individuals. All three of these organisms are facultative intracellular pathogens
and possess a range of virulence factors. Y. pseudotuberculosis and Y. enterocolitica
have both been implicated as causes of foodborne disease whereas
Y. pestis is more typically transmitted through direct contact with sick rodents,
or via flea bites as in the historic outbreaks of plague also known as the Black
Death. Both pneumonic (respiratory form) and bubonic forms of plague still
occur, but outbreaks are usually self-limiting or involve only sporadic cases5
(Bhunia, 2008d).
Yersiniae are common in the environment and can be found in the intestinal tract of healthy and sick birds and mammals (Cork et al., 1995). The CDC
indicates that Y. enterocolitica is responsible for up to 87,000 cases of gastroenteritis annually, and 90% of these are thought to be foodborne. Between 1988
and 2002 there were eight outbreaks of yersiniosis reported as attributable to
Y. enterocolitica, involving 87 cases. In humans, Y. pseudotuberculosis is more frequently associated with sporadic cases of mesenteric adenitis, and occasionally
septicaemia (Cork et al., 1998). The latter is more likely with patients being
treated for iron overload and related conditions. Pseudotuberculosis in animals has been reported following consumption of vegetation contaminated by
bird faeces (Cork, 1994). Y. pseudotuberculosis is less often associated with foodborne disease than Y. enterocolitica, although both can be transmitted via contaminated vegetation. Disease transmission for both is often more common in
the winter owing to the psychrophilic tendency of Yersiniae.
Y. enterocolitica is classified into five biogroups: 1 (1A and 1B), 2, 3, 4 and
5. This grouping is based on pathogenicity and ecological and geographical
distribution. There are about 60 serotypes recognized, with several within
each biogroup, e.g. 1A (O:5; O:6,30; O:7,8, etc.). Those predominantly causing disease worldwide are serotypes O:3, O:8, O:9 and O:5,27. Potentially
pathogenic strains of Y. enterocolitica are found in sewage, environmental
sources and a wide range of animal faeces (ruminants, dogs, cats, birds, etc.),
but human disease has most frequently been linked to pigs. Although Y. enterocolitica is a common commensal present in the pig intestinal tract it can cause
disease in pigs, and is emerging as a significant zoonotic pathogen in pigs
(De Boer et al., 2008; Truszczynski, 2009). In one European study, it was found
that pathogenic strains of Y. entercolitica were isolated from 13 (9.3%) of 140 samples of porcine tonsils and from five (3.3%) samples of pig faeces examined
(De Boer et al., 2008). Another source of foodborne yersiniosis has been chocolate milk, and also other dairy products, which have been associated with
cases of enteritis reported in children. The disease is usually self-limiting,
but in a few cases septicaemia and death have been reported. Prevention of
5
http://www.who.int/mediacentre/factsheets/fs267/en/ (accessed 26 February 2010).
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disease requires pasteurization of dairy products and proper cooking of meat,
especially pork. There have been reports of pasteurized products, especially
milk, containing Y. enterocolitica, but although there are some heat-tolerant
strains, most cases have been traced back to contamination of the product at
or after packaging (Walker and Gilmour, 1986; Schiemann, 1987; Ackers et al.,
2000). Ionizing radiation and other food-preservation methods can also be
used to control Yersinia.
Q fever: Coxiella burnetii
Q fever, or ‘query fever’, is a zoonotic disease caused by Coxiella burnetii, a species of bacteria that is distributed globally although it has not been reported
in New Zealand (Frazer and Rooney, 2009). In 1999, Q fever became a notifiable disease in the USA, but reporting is not required in many other countries.
Because the disease is under-reported, it is not possible to reliably assess how
many cases of Q fever have occurred worldwide (Maurin and Raoult, 1999).
Although there are few reliable statistics on the prevalence and incidence of Q
fever worldwide, it is estimated that there are 50–60 cases of Q fever reported
in the USA each year, and that the average annual reported incidence is 0.28
cases per million persons.6 Similar statistics are reported by Karakousis et al.
(2006). Cattle, sheep, and goats are thought to be the primary reservoirs of C.
burnetii, although infection has also been noted in a wide variety of other
animals, including other species of livestock, birds and domesticated pets. C.
burnetii does not usually cause clinical disease in these animals, although abortion in goats and sheep has been linked to infection with the bacterium. The
organisms are excreted in the milk, urine and faeces of infected animals and,
during parturition, infected dams may pass high numbers of organisms in the
amniotic fluids and the placenta. C. burnetii has also been isolated from poultry
eggs, although foodborne transmission via this and other routes is considered
very rare (Maurin and Raoult, 1999). Organisms of C. burnetii are resistant to
heat, drying and many common disinfectants, thus enabling the bacteria to
survive for long periods in the environment.
The most common route of infection in humans is probably via the inhalation of aerosols containing dried placental material, birth fluids and excreta
of infected herd animals. Other potential modes of transmission include ingestion of infected unpasteurized milk and tick bites.7 However, these routes of
infection are uncommon, and human Q fever is primarily an occupational
disease of farmers, abattoir workers, veterinarians and laboratory workers
(Hartzell et al., 2008).
6 http://www.bt.cdc.gov/agent/qfever/clinicians/epidemiology.asp (accessed 3 March
2010).
7 C. burnetii can be maintained in a tick–vertebrate cycle, although the ticks are not
necessary for the organism to persist. Some ticks may act as a vector between wild
and domestic animals.
Epidemiology of Pathogens in Food Supply
33
Although reported cases are uncommon, humans are considered to be
very susceptible to the disease, and very few organisms are required to cause
infection. In the majority of cases, the disease is a non-specific flu-like illness
with a 1–3 week incubation period, often remaining undiagnosed. In a minority of cases there will be a clinical atypical pneumonia or hepatitis. Should the
course of disease become chronic, endocarditis and chronic hepatitis can
develop. Chronic Q fever is often fatal, and may be more likely to develop in
immunocompromised individuals and pregnant women (Maurin and Raoult,
1999).
Brucellosis: Brucella melitensis, etc.
Brucellosis is an infectious disease caused by bacteria in the genus Brucella,
which include Brucella melitensis, Brucella abortus and Brucella suis. These bacteria are primarily passed among animals and cause clinical disease in a range
of vertebrates, including sheep, goats, cattle, deer, elk, pigs, dogs, hares and
several other species, including wild animals. Although brucellosis is not
commonly reported in developed countries (for example, there are fewer
than 100 cases reported a year in the USA, and fewer than 50 in Australia), it
can be common in countries where animal disease control programmes have
not reduced its prevalence in ruminants.
In humans, brucellosis is a multi-systemic disease with a broad spectrum of clinical presentations. Clinical signs may appear insidiously or
abruptly. Typically, brucellosis begins as an acute febrile illness with nonspecific flu-like signs such as fever, headache, malaise, back pain, myalgia
and generalized aches. Drenching sweats may occur, particularly at night.
Splenomegaly, hepatomegaly, coughing and pleuritic chest pain are sometimes seen. Gastrointestinal signs, including anorexia, nausea, vomiting,
diarrhoea and constipation occur frequently in adults, but less often in
children. In many patients, the symptoms last for 2–4 weeks and are followed by spontaneous recovery. Other patients develop an intermittent
fever and other persistent symptoms that typically undulate at 2–14 day
intervals. Most people with this undulant form recover completely in 3–12
months. A few patients become chronically ill. Relapses can occur months
after the initial symptoms, even in successfully treated cases (Corbel, 1997;
Sauret and Vilissova, 2002).
B. melitensis is considered a food safety concern in Mediterranean regions
because it may be present in local cheeses and other dairy products if they
have been made from the milk of infected sheep and goats. B. abortus is more
frequently associated with bovids, and is passed in high numbers in placental
material and fluids when associated with abortion. B. suis is typically found in
pigs as a cause of infertility and abortion. Other Brucella spp. have been found
to cause disease in humans and animals, but are less commonly reported.
Humans typically become infected with Brucella spp. by coming into contact
with animals or animal products that are contaminated with these bacteria
(Ramos et al., 2008; Swai and Schoonman, 2009).
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Although brucellosis can be found worldwide, it is more common in
countries that do not have standardized and effective public health and
domestic animal health programmes. Areas currently listed as high risk are
the Mediterranean Basin (Portugal, Spain, Southern France, Italy, Greece,
Turkey, North Africa), South and Central America, Eastern Europe, Asia,
Africa, the Caribbean and the Middle East. Unpasteurized cheeses, sometimes called ‘village cheeses’, from these areas may represent a particular risk
for tourists, so brucellosis should be considered in the differential diagnoses
of returned travellers presenting with a fever (Johnston et al., 2009).
Viral Foodborne Diseases
Viruses cause a wide range of diseases in humans and animals, and may be
transmitted by a variety of routes. Although viruses are strict intracellular parasites requiring host cells in which to multiply, some can survive for significant
periods of time in the environment, leading to transmission to new susceptible
hosts. Many common human viruses are transmitted via water, through either
the consumption of contaminated drinking water, the consumption of contaminated shellfish or the ingestion of fresh fruit and vegetables washed in
contaminated water (Appleton, 2000). Other viruses, many of which are
thought to be zoonotic, are transmitted via infected or contaminated meat
(e.g. hepatitis E, some influenza A viruses). The latter can occur if the animal
host is slaughtered for human consumption when it is still viraemic, or as a
result of contamination of the carcass after slaughter. Examination of reported
cases of foodborne disease in humans indicates that 66.6% of food-related
diseases in the USA are associated with viruses, compared with 9.7% and
14.2% for Salmonella and Campylobacter, respectively (Vasickova et al., 2005).
The majority of cases are likely to be associated with direct human-to-human
transmission of enteric viral agents (i.e. most noroviruses, enteroviruses, hepatitis A, astroviruses, some adenoviruses and rotaviruses). However, there is a
growing recognition that animals may also serve as the reservoir for some
emerging foodborne viral diseases. For example, some strains of rotavirus,
hantavirus, foot-and-mouth disease apthoviruses (López-Sánchez et al., 2003),
some flaviviruses, Nipah virus (Looi and Chua, 2007) and others have been
detected in food sources, and have the potential to sporadically infect humans
via the oral route.
Up until quite recently it was assumed that most viruses were highly host
specific, but there are numerous examples of new viral pathogens identified in
humans that may have an animal source – for example, the coronavirus
responsible for SARS (severe acute respiratory syndrome), hepatitis E, some
strains of rotavirus A and some strains of norovirus. All of these viruses have
been detected in the faeces of infected animals so it is possible that they may
be transmitted to humans via the food chain (WHO, 2008). RNA viruses such
as SARS coronavirus, hepatitis E, rotavirus A and noroviruses are known to
have a high mutation rate, and many human strains appear to be closely
related to those found in mammals, although they may not be identical. The
Epidemiology of Pathogens in Food Supply
35
epidemiology of many of these agents is not fully understood. There is also
limited information on the environmental persistence of many of these emerging agents, and more work is required to determine whether current watertreatment methods are able to inactivate all of the potential viral pathogens
that might be present (Gannon et al., 2004).
Norovirus
Noroviruses cause the majority of human cases of acute viral gastroenteritis
worldwide (Hutson et al., 2004). During 1983–1987 in the USA, noroviruses
were responsible for one-fifth of the foodborne disease outbreaks (Cliver,
1997a). They were previously called Norwalk-like viruses and are members of
the family Caliciviridae. Noroviruses were first recognized in 1968 as the cause
of an outbreak of acute gastroenteritis in an elementary school in Ohio (Adler
and Zickl, 1969). Many cases are mild, but many people can be involved, for
example, the outbreak of norovirus infection in Queensland, Australia in
August 1996 associated with the consumption of oysters (Stafford et al., 1997).
Although most of the reported cases are considered to reflect human-tohuman transmission via contaminated food and water sources there have been
some recent studies to indicate that some noroviruses have been identified in
animals (Hutson et al., 2004). In New Zealand, Wolf et al. (2009) studied faecal
specimens from sheep and pigs and compared norovirus isolates with those
isolated from humans. Samples from animals on New Zealand farms were
examined using a multiplex real-time RT-PCR (reverse transcriptase PCR) and
norovirus was found in 9% (2/23) of porcine samples and 24% (8/33) of
ovine samples. Of the porcine samples, all were genogroup II, and those from
ovine samples were genogroup III. Whether or not these cases reflect animal
or human-to-animal transmission versus a true animal reservoir for some
strains of norovirus is not yet determined.
Enterovirus
Another common cause of foodborne gastroenteritis in humans includes
viruses of the genus Enterovirus. These are members of the family Picornaviridae and include five major groups: polio virus, group A and B coxsackie
viruses, echoviruses and some newer enteroviruses. These are ubiquitous
enterically transmitted viruses responsible for a wide spectrum of illnesses
in infants and children (Cliver, 2000). The enteroviruses multiply in the
intestinal tract and are fairly resistant to environmental factors so may persist on fomites and are readily transmitted via fruit and vegetables that
might have been contaminated several days earlier (Koopmans and Duizer,
2004; Cook and Rzezutka, 2006). Shellfish can also be a source of enterovirus
(Beuret et al., 2003).
The hepatitis A virus, another picornavirus, is classified as a hepatovirus.
It is the only member of this genus and is the cause of significant morbidity
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in humans, especially in developing countries; it is a pathogen that can be
transmitted by food and water supplies. There are four recognized human
genotypes of this virus, with three genotypes naturally infecting non-human
primates (Cliver, 1997b; Fiore, 2004). The role of non-human primates in
transmission to humans is not clear, although in some parts of the world this
may have contributed to the evolution of the virus. The virus has a marked
tropism for the liver and causes hepatitis with associated fever, jaundice and
malaise. Shellfish and the consumption of food contaminated by food handlers have also been implicated as the cause of several outbreaks in humans
(Coelho et al., 2003).
Rotavirus
Rotaviruses are members of the family Reoviridae and demonstrate a large
degree of genetic variability. Most clinical cases in humans are caused by group
A rotaviruses, but infection with group B and C rotaviruses have also been
reported. Within group A there are 3 subtypes and 11 serotypes. Other groups
(D–G) have also been reported and some have an animal source. Some cases
of rotavirus gastroenteritis in humans have been associated with the consumption of undercooked meat or linked to fresh or cooked food that has been
contaminated with infected material (Richards, 2001; Cook et al., 2004).
Animal rotaviruses have been detected in drinking water, and researchers
have speculated about the role of water in the spread of animal strains to
humans and the potential for the emergence of new reassorted strains. Bovine–
human reassortment strains have been detected in infants in Bangladesh
(Ward et al., 1996) and may possibly have been transmitted from humans to
cows or vice versa via faecal contamination of food and/or water.
Hepatitis E
Hepatitis E virus (HEV) has emerged as a significant cause of clinical hepatitis
in the developing world (Nicand et al., 2009). HEV was initially classified as a
member of the Caliciviridae but has now been reclassified as a hepevirus. It is
generally transmitted by the faecal–oral route, with outbreaks reported in
tropical and subtropical countries (Yazaki et al., 2003; Tamada et al., 2004).
Cases in humans have been associated with the consumption of pork (Banks et al.,
2007; Leblanc et al., 2007; Nicand et al., 2009) and deer meat (Vasickova et al.,
2005). Both foodborne and direct routes of transmission have been identified.
Genetic analysis indicates that hepatitis E strains isolated from humans are
closely related to swine hepatitis E virus. In one study, anti-HEV antibodies
were detected in 20% of people exposed to infected pig herds. In some countries the growing prevalence of hepatitis E in the human population has
prompted the public-health authorities to consider the development of a
vaccine (Khuroo and Khuroo, 2008).
Epidemiology of Pathogens in Food Supply
37
Other viruses
There is a wide range of other viruses that can infect humans, many of which
are zoonotic. Most of these are primarily transmitted by non-enteric routes,
but some have the potential to be transmitted via the faecal–oral route in some
cases, e.g. Lassa fever and lymphocytic choriomeningitis. These are both
arenaviruses with bat and rodent reservoirs. A potential link between human
cases and the consumption of contaminated food has been reported (Acha
and Szyfres, 2003). Hantaviruses, members of the family Bunyaviridae, are
found in the urine and droppings of infected deer mice, and there have been
occasional reports of human infection after eating faecally contaminated
food/water (Acha and Szyfres, 2003; Heyman et al., 2009).
The apthovirus that causes foot-and-mouth disease has occasionally caused
clinical disease in humans. Several human cases have been reported following
the consumption of raw milk and dairy products from infected ruminants
(López-Sánchez et al., 2003). There are many other animal viruses that have
the potential to cause disease in humans, e.g. tick-borne encephalitis (Kohl et al.,
1996; Appleton, 2000), avian influenza viruses, paramyxoviruses (Alexander
and Brown, 2000; Alexander, 2006) although other routes of transmission are
more common.
Prions
Prion diseases such as bovine spongiform encephalitis (BSE) and chronic
wasting disease (CWD) in deer are caused by a transmissible protein and have
generated a lot of interest over the past few decades. This topic is discussed in
detail in Chapter 11.
Parasitic Foodborne Diseases
It has been estimated that humans harbour about 300 species of parasitic
worms and over 70 species of protozoa. Many have coexisted with humans for
thousands of years, as evidenced by archaeological records. Many of these
parasites are transmitted by food and water sources, but many are not (Doyle,
2003). Parasitic infections are often asymptomatic in humans, but some
can cause significant morbidity and can persist, causing chronic ill health
(Anantaphruti, 2001).
The epidemiology of parasitic diseases can be complex, with the environmental route of transmission being very important for many protozoan and helminth parasites. The availability of suitable environmental conditions, such as
suitable temperature, humidity, food and water sources, and favourable soil and
vegetation, are particularly significant. The parasites’ biological potential for producing large numbers of infective stages, as well as their environmental robustness (they can survive in moist microclimates for prolonged periods of time), can
pose a significant challenge for human- and animal-health authorities.
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Increased demands on natural resources, as well as the growing trade in
non-traditional fresh food products, has the potential to increase the likelihood of humans encountering environments and fresh produce contaminated
with the infective stages of a variety of parasites. Robust, efficient detection,
viability assessments and typing methods are required to assess risks and to
enhance our understanding of the epidemiology of these emerging parasitic
diseases (Anantaphruti, 2001).
There is a wide variety of food products that may be contaminated with one
or more species of parasite. The prevalence of specific parasites in food supplies varies between countries and regions. One of the major factors influencing the prevalence of parasitic infections in the human population is the
preference for, and traditional popularity of, consuming raw or inadequately
cooked foods. The parasites that may be acquired by eating these foods include
nematodes, trematodes, cestodes and protozoa (Pozio, 2008). A number of
significant zoonoses are associated with the consumption of muscle tissues
from infected meat (Toxoplasma gondii, Sarcocystis hominis, Sarcocystis suishominis,
Diphyllobothrium latum, Taenia solium, Taenia saginata, Opisthorchis felineus, Anisakis spp.) and contaminated food and water supplies (i.e. Giardia duodenalis,
Cryptosporidium spp., T. gondii, Echinococcus granulosus sensu latu, Echinococcus
multilocularis, T. solium, Taenia multiceps). The effective control and prevention
of the majority of these agents requires a focus on the education of both
producers and consumers.
Although many parasitic diseases have traditionally been considered to be
confined to tropical countries, and therefore of little concern to industrialized
nations, recent outbreaks of parasitic diseases in North America have demonstrated that this is incorrect. One example is the outbreak of gastroenteritis
caused by Cryptosporidium in Milwaukee (USA), which was transmitted through
the public water supply (MacKenzie et al., 1994). Consumers are also more
frequently exposed to parasites that originate in the tropics during international travel to exotic locations, e.g. Angiostrongylus sp. in tourists returning
from Jamaica, and also as a result of the availability of a wide variety of imported
fresh produce in the local marketplace through the globalization of trade, e.g.
Cyclospora sp. on raspberries from Guatemala (Herwaldt, 2000). Selected
protozoan and helminth parasites will be considered in more detail in the
following sections.
Protozoan parasites
Protozoan parasites are frequently found in freshwater sources that have been
contaminated by human or animal faeces. Fruits and vegetables washed with
contaminated water may also be a source of infection. Some protozoa (e.g.
Sarcocystis sp.) can be transmitted directly through the handling or consumption of fresh meat. Most species of protozoan parasite have environmental
phases in their life cycles and form resistant resting stages (cysts or oocysts)
that can withstand drying and disinfectants. This can make them hard to control. Although many protozoal organisms are transmitted from human to
Epidemiology of Pathogens in Food Supply
39
human with little or no animal involvement, many (i.e. some strains of Giardia,
Cryptosporidium and other protozoa) have been found in both humans and
animals, indicating that zoonotic transmission may occur. Clinical disease
associated with protozoal diseases in humans, and in other vertebrates,
depends on a wide range of factors, including level of exposure, virulence of
the parasite, host immunity and the presence of concurrent bacterial, viral
and other protozoal infections. The latter situation is not uncommon, especially in the young, immunocompromised or travellers visiting areas where
they are exposed to organisms against which they have no immunity.
Toxoplasmosis (Toxoplasma gondii)
The life cycle and epidemiology of T. gondii have been well categorized
(Lindsay et al., 2001; Dubey et al., 2002 a,b,c; Kniel et al., 2002; Hill et al., 2007).
However, in some parts of the world there are data to suggest that the transmission pathways can be more complex than previously thought, for example,
the emerging role of many non-felid species as sources of infection for humans
and other mammalian species. As a zoonotic disease, the clinical picture varies
widely with a large proportion of the human population, even in urban populations, exposed to Toxoplasma oocysts, but only a small percentage showing
clinical signs of infection.
Toxoplasma spp. are widespread worldwide. In a survey of Central and
South America, up to 40% of the human population in developed regions,
and 80% in underdeveloped regions, were found to be infected (Hill and
Dubey, 2002). The prevalence is also high in France, where raw meat is regularly consumed, with 84% of pregnant women in some regions demonstrating
antibodies to Toxoplasma spp. compared with 32% in New York City. In adults,
the infection tends to be asymptomatic, but significant prenatal damage can
occur when pregnant women not previously exposed to Toxoplasma are infected
during pregnancy (Ajzenberg et al., 2002). The organism crosses the placenta
and can cause a range of birth defects, from blindness to hydrocephalus.
Toxoplasmosis is responsible for the deaths of 10–30% of AIDS patients in
Europe and the USA, and has been linked to encephalitis in immunocompromised patients (Bowie et al., 1997; Choi et al., 1997). The clinical picture in
these patients is quite varied (Bhopale, 2003). There does not seem to be
much genetic diversity among strains of Toxoplasma, with only three lineages
recognized (Ajzenburg et al., 2002). T. gondii infects all warm-blooded animals, but only domestic cats and other felids commonly act as the definitive
host that provides a source of infection for other animals. Up to 8000 million
infective oocysts can be excreted in the faeces of felids (wild and domestic) to
contaminate pasture, water supplies and vegetation. In animals other than
cats, Toxoplasma cells migrate out of the intestine and encyst in muscle tissue
where they remain for the life of the animal. Cysts in birds and rodents are
consumed by felids, and the life cycle resumes. Sheep, pigs and other livestock, including poultry, may also become infected with cysts and these can be
a source of infection for humans. Consumption of undercooked mutton or
pork has been thought to be the source of a number of human infections
(Lundén and Uggla, 1992; Lundén et al., 2002).
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Giardiasis
Giardia lamblia (intestinalis) is the most commonly reported intestinal parasite
in humans. The CDC estimates that there are over 2 million cases of giardiasis
a year in the USA. Giardia sp. cysts may be excreted for prolonged periods in
the faeces of infected people (Nichols and Smith, 2002) and can occasionally
cause severe damage to the gastrointestinal tract, with chronic malabsorption
and other complications if untreated. Severe cases can occur especially in
immunocompromised individuals. Most cases of foodborne giardiasis occur
following the consumption of fruit and vegetables irrigated with faecally contaminated water. In one study in the USA and Central America, it was found
that of 25 samples of water used to irrigate food crops that were eaten raw,
60% contained Giardia (Thurston-Enriquez et al., 2002). Food handlers are
also thought to be responsible for transmitting the infection in food; for example, in 1996 an outbreak in the USA was traced back to the consumption of
contaminated ice cream (Olsen et al., 2000). Other human cases have been
traced to pre-prepared food such as sandwiches, salads, etc. (Hancock et al.,
1998; Rose and Slifco, 1999; Nichols, 2000). Giardia cysts have been isolated
from surface water in areas remote from towns, and are thought to have
come from the faeces of wild animals, including beavers and coyotes (Thompson
et al., 2009). Backpackers have been found to be infected after drinking
from freshwater streams, and it is thought that wildlife carry the organism and
shed it in their faeces into watercourses. Domestic livestock such as cattle
(Appelbee et al., 2003; Dixon, 2009) also shed this organism, with up to 5800
cysts g–1 detected in the faeces of domestic cattle (Heitman et al., 2002). Some
strains of Giardia sp. found in other species do not often infect humans (Appelbee
et al., 2003; Snel et al., 2009).
Cryptosporidiosis
There is a wide range of species in the genus Cryptosporidium (i.e. Cryptosporidium
bovis, Cryptosporidium felis, Cryptosporidium hominis, Cryptosporidium suis, Cryptosporidium galli), most of which can be readily transmitted between hosts.
Because of this transmission between host species, and the wide range of
potential vertebrate hosts infected, it was suggested that the Cryptosporidium
spp. should be grouped together under the species name Cryptosporidium
parvum (Xiao and Cama, 2006).
Over the past decade there have been several large outbreaks of gastroenteritis in humans associated with C. parvum (Millar et al., 2002). In 1993,
over 400,000 people in Milwaukee were affected, with 69 fatalities involving
immunocompromised individuals (Naumova et al., 2003). The economic cost
was estimated to have been about US$96.2 million (Corso et al., 2003). In most
cases, the organism causes a mild and self-limiting disease, but this depends
on the infective dose, the immunity of the host, and the virulence of the strain
involved (DuPont et al., 1995). Foodborne infections are often associated
with the consumption of raw produce contaminated by infected food handlers
or water. C. parvum has been found in cider, unpasteurized milk and in the
faeces of cattle and other livestock (Deng and Cliver, 1999, 2001; Dixon, 2009).
Water infected with sewage is often thought to be the source of contamination
Epidemiology of Pathogens in Food Supply
41
involving fresh fruits and vegetables and beverages (Friedman et al., 1997).
The oocysts of C. parvum can survive in fresh, brackish and saltwater for a
number of months (Gomez-Couso et al., 2003) and have been isolated from
clams, oysters and other shellfish (Fayer et al., 1998). However, human infection associated with eating shellfish does not seem to be common, possibly
because cooking destroys the parasite, although raw shellfish do pose a potential
risk (Graczyk et al., 2003).
Cyclospora
Cyclospora cayetensis has been the cause of numerous foodborne disease outbreaks in recent decades. In the 1990s, there was a multi-state outbreak of
cyclosporosis in the USA following the consumption of imported raspberries.
Other outbreaks have been traced back to the consumption of salad greens,
basil and a range of berries, all of which had been contaminated with water
containing Cyclospora spp. during irrigation (Rose and Slifco, 1999). Only
sporulated oocysts are infectious to humans, so food handlers probably have
little role in the transmission of this organism. The epidemiology of Cyclospora
is not fully known, but it is endemic in many developing countries and is
spread by the faecal–oral route. At the present time, it is thought that humans
are the main host for C. cayetensis, but evidence for the absence of the organism in animal hosts is lacking. The organism causes diarrhoea and debility in
infants and the immunocompromised (Bern et al., 1999). Complications of
infection include Guillain–Barré syndrome (Richardson et al., 1998) and
reactive arthritis or Reiter’s syndrome (Connor et al., 2001).
Sarcocystis
Human infections may occur as a result of infection with S. hominis and S.
suihominis involving a two-host cycle and consumption of contaminated undercooked meat, especially beef and pork. There have been only a limited
number of human cases of Sarcocystis infection reported, and most of these are
from Asia. In Malaysia, Sarcocystis has been reported to be common in domestic and wild animals, including rats, bandicoots, slow loris, buffaloes and monkeys. The overall seroprevalence in humans tested in the same region was
19.8% (Kan and Pathmanathan, 1991). Although many human cases present
with gastroenteritis, other clinical signs can occur, such as myositis and also
malignancy (Pathmanathan and Kan, 1992). In New Zealand, muscle tissue
from the oesophagus and diaphragm of 500 beef cattle was examined, and it
was found that all the cattle were infected with Sarcocystis; of these, 98% had
Sarcocystis cruzi and 79.8% had Sarcocystis hirsuta/S. hominis (Böttner et al., 1987).
A similar study in Belgium found that 97% cattle were positive for Sarcocystis.
Thick-walled cysts were recovered from 56% of the animals, but these could
not be specifically identified as S. hirsuta or S. hominis on morphological
grounds (Vercruysse et al., 1989). A study in India, which examined muscle
samples from 890 slaughtered pigs, determined a prevalence rate of 67%, with
Sarcocystis meischeriana identified in over 40% of cases and 47% of cases with S.
suihominis (Saleque and Bhatia, 1991). In Canada, it has been found that 53%
of some caribou herds can be infected (Khan and Fong, 1991). Other studies
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have shown similar results in a range of livestock, including poultry. Sarcocystis
is killed by proper cooking of meats and this is especially recommended for
game meats and pork (Cama, 2006).
Entamoeba
Entamoeba histolytica is an important cause of diarrhoea in people in tropical
and subtropical countries (Barwick et al., 2002). Although it is not traditionally
considered to be zoonotic, it is mentioned here because it has been found in
non-human primates and other wildlife species (Stedman et al., 2003; Verweij
et al., 2003), and is a significant human pathogen. Morphologically it resembles Entamoeba dispar, which is non-pathogenic, so it is important to distinguish
between the two in diagnostic samples. In the USA, Entamoeba is not uncommon in immigrants and travellers returning from areas in which it is endemic.
It is also common in people living along the US border with Mexico. It should
be noted that, in humans, E. histolytica is the second leading parasitic cause
of death (after malaria). It has been estimated that 50 million people worldwide are infected, of whom 40,000–100,000 die each year from associated
complications of the infection, such gut invasion and liver infection, as well as
secondary bacterial and other diseases (Stanley, 2003). In most healthy adults,
the infection is often asymptomatic as a degree of resistance develops over
time. The usual source of infection is sewage-contaminated water that has
been used to wash fresh produce. Food handlers can also transmit the disease
(Barwick et al., 1998; Leber, 1999).
Helminths
Nematoda
The nematodes, or roundworms, include a number of important human
pathogens, such as Trichinella, Ascaris, Anisakis, Angiostrongylus and Gnathostoma.
Some of these parasites have complex life cycles involving an intermediate
host, for example Trichinella, Anisakis and Gnathostoma, which exist as cysts
in the muscles of mammals or fish and develop into adults in humans that
consume the infected flesh. Proper cooking of meats and fish should prevent
human infection (Hayunga, 2007). Other nematode parasites, such as Ascaris,
have a more simple life cycle and are typically transmitted from human to
human, so are not considered to be zoonotic.
(TRICHINELLA SPIRALIS)
Trichinellosis, also called trichinosis, is
caused by eating raw or undercooked meat of animals infected with the larvae
of the nematode Trichinella spiralis and related species (Khumjui et al., 2008).
Infection occurs commonly in certain wild carnivorous animals, but may also
occur in domestic pigs. Humans have become infected after the consumption
of walrus, horse, bear, pig, cougar and seal meats.
When a human or animal eats meat that contains infective Trichinella cysts,
the acid in the stomach dissolves the hard covering of the cyst and releases the
larval worms, which pass into the small intestine and, in 1–2 days, become
TRICHINELLOSIS
Epidemiology of Pathogens in Food Supply
43
mature. After mating, adult females lay eggs. Eggs develop into immature
worms, travel through the arteries, and are transported to muscles. Within the
muscles, the worms curl into a ball and encyst (become enclosed in a capsule).
In humans, clinical signs occur 1–2 days after infection and can include nausea, diarrhoea, vomiting, fatigue, fever and abdominal discomfort. Additional
symptoms are varied and can develop within days, weeks or months after infection; these include fever, joint and muscle pain, difficulty in breathing, itchy
skin, etc. If the infection is heavy, patients may experience difficulty coordinating movements, and have heart and breathing problems. In severe cases,
death can occur. For mild-to-moderate infections, most symptoms subside
within a few months, although fatigue, weakness and diarrhoea may last for
months.
In emerging economies, such as parts of Eastern Europe, the prevalence
of trichinellosis in rural communities can be quite high (Takumi et al., 2009).
Cases are usually related to the consumption of T. spiralis in pig meat originating from small backyard farms. In contrast, in most parts of Europe, infections
are limited because control measures have been implemented and so the
pathogen is rarely found in commercial pig units. Trichinellosis can be
avoided by cooking meat thoroughly – at 60°C or higher for at least 1 min to
kill the infective stage of the parasite – or by freezing meat at –15°C or colder
for at least 20 days. Ordinary curing and salting, smoking or microwaving of
pork products will not kill the juvenile worms. Human trichinellosis remains
a major foodborne zoonosis in Eastern Europe and Asia (Barennes et al.,
2008) with a high health, social and economic impact. Infection can also
occur in travellers as a result of the consumption of infected wild boar, bear
and other game that has not been well cooked (Sterling, 2006; Kurdova et al.,
2008).
Cestoda
Meat and fish may contain larval tapeworms that can develop into adults in
the human intestine. Clinically important cestodes pathogenic to humans are
the Taenia species T. solium (pork tapeworm), T. saginata (beef tapeworm),
the Diphyllobothrium species D. latum (fish or broad tapeworm), Hymenolepis
nana (dwarf tapeworm) and the Echinococcus species E. granulosus and E. multilocularis (hydatids) (Murrell et al., 2005).
TAENIASIS (TAENIA SOLIUM, TAENIA SAGINATA)
The Taenia cestodes have a worldwide
distribution, but the incidence of human infection is higher in developing
countries. In North America, the infection rate is thought to be as low as
1:1000, but it can be as high as 10% in some developing countries. Humans
can be a definitive host for both the beef tapeworm (T. saginata and the closely
related Taenia asiatica) and the pork tapeworm (T. solium), with the latter
more commonly reported (Bowman et al., 2006). These tapeworms are parasites
that have coevolved with humans and domestic livestock, and remain common
in many parts of the world.
Adult Taenia tapeworms can be up to 4–6 m long with a long, flat
body comprising several hundred segments (proglottids). There are some
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morphological differences between adult worms, but the eggs of T. solium and
T. saginata are indistinguishable. In most cases, human infection occurs when
a tapeworm larval cyst (cysticercus) is ingested with poorly cooked infected
meat. Once ingested, the larvae escape from the cyst and pass to the small
intestine where they attach to the intestinal mucosa by the scolex suckers. The
proglottids develop as the worm matures in 3–4 months. The adult cestode
may live in the small intestine for as long as 25 years and pass gravid proglottids with the faeces. Eggs extruded from the proglottids contaminate, and persist on, vegetation for several days, so in areas where sanitation is poor and
latrines are scarce, these proglottids can be consumed by cattle or pigs in
which they hatch and form cysticerci.
Light infections remain asymptomatic, but heavier infections may produce clinical signs, such as abdominal discomfort, epigastric pain, vomiting
and diarrhoea. In the case of the pork tapeworm, eggs can also infect humans
and cause cysticercosis (larval cysts in the lungs, liver, eyes and brain) resulting
in blindness and neurological disorders. In some parts of the world, the incidence of cerebral cysticercosis can be as high 1:1000 in the population and
may account for a significant proportion of reported neurological disease in
some countries (e.g. Mexico, Bhutan); cysticercosis ocular involvement occurs
in about 2.5% of patients, and muscular involvement is as high as 10% (India).
Prevention of human infection requires good education along with a thorough inspection of beef and pork (and game such as wild boar), with condemnation of meat containing cysts (Gonzales et al., 2003; Bowman et al., 2006).
Improving sanitation and providing latrines to prevent ruminants and pigs
gaining access to human faecal material is also important. Adequate cooking
or freezing of meat are also effective precautions because cysticerci do not
survive temperatures below –10°C or above 50°C. Prevention of the disease in
humans requires good cooperation with communities and is based on an
understanding of the epidemiology of the parasites (Bowman et al., 2006).
Apart from the human-health risk associated with taeniasis, cysticercosis renders beef unmarketable and is globally responsible for over US$2 billion in
yearly losses (Hoberg, 2002).
DIPHYLLOBOTHRIASIS (FISH TAPEWORM INFECTION)
Diphyllobothriasis in humans is a
benign tapeworm infection of the small intestine caused by eating raw fish, a
quite common practice in many parts of the world (e.g. in the Baltic countries,
Finland and Canada/Alaska). The causative agents are the Diphyllobothrium
species D. latum and D. pacificum. D. latum is common in northern temperate
regions where fish are eaten raw, whereas D. pacificum is common in coastal
South America, especially Peru. The definitive hosts of D. latum include
humans, dogs and cats. The natural reservoirs for D. pacificum are seals. The
two main intermediate hosts include a crustacean and a freshwater fish. Gravid
proglottids pass in the faeces of the definitive host and the eggs hatch in lakes
and waterways where they infect crustaceans. Freshwater fish consume these
and the larvae encyst in the musculature. These intermediate hosts can be
eaten by larger fish which can still transmit the infection. Humans acquire the
parasite by eating raw infected fish, so although the disease is usually
Epidemiology of Pathogens in Food Supply
45
asymptomatic in humans or other definitive hosts (i.e. dogs, cats, foxes, bears,
pigs), there are food-safety implications for the aquaculture industry. The
prevalence of D. latum in the USA is estimated to be less than 0.5%, although
outbreaks have been recently associated with the increased availability of fresh
salmon and sushi (Bowman et al., 2006).
ECHINOCOCCOSIS (HYDATIDS)
Hydatid tapeworms (Echinococcus spp.) are parasites of canids that can infect humans and animals who accidentally ingest
the eggs of the tapeworm after they have been passed out in the faeces of the
host. In humans, the two main cestodes associated with ‘hydatid’ disease are
E. granulosus, which causes ‘cystic’ disease, and E. multilocularis, which causes
‘alveolar’ disease.
E. granulosus has worldwide distribution and is the most common form of
hydatid in humans. There are typically two forms of the disease recognized:
the European form, which is globally distributed in domestic animals; and the
Northern form, which is restricted to the tundra and taiga of North America
and Eurasia. Sylvatic cycles of transmission in wild animals may also result in
the accidental infection of humans, especially when the level of environmental
contamination with parasite eggs is high (Jenkins et al., 2005).
The definitive hosts for E. granulosus are predominantly members of the
family Canidae, which harbour the adult tapeworm in the small intestine.
Most infections in canids result in no clinical signs. Most human infections
with E. granulosus occur in places where hygiene is poor and dogs are used to
herd grazing animals, particularly sheep and yaks, which act as intermediate
hosts (Yang et al., 2009). In sheep and other ruminants, hydatid cysts can cause
considerable condemnation of meat and loss of production. Humans are considered ‘dead end’ hosts for the parasite, with infection frequently the result
of ingesting vegetation contaminated with infected dog faeces. The disease is
common throughout southern South America, the Mediterranean and the
Middle East, Central Asia and East Africa.
Implementation of proper meat-inspection practices and preventing
carnivores from consuming the infective cysts in meat has reduced the incidence of hydatids in many countries. This, along with improving hygiene,
carefully washing vegetables such as salads before eating them and regularly
treating dogs with suitable anthelmintics, has minimized the risk to humans in
many parts of the world, but isolated cases still occur in Eastern Europe, Russia,
Australasia, India and parts of the Mediterranean (Jenkins, 2006; Garippa and
Manfredi, 2009). In North America, endemic foci have been reported from
the western USA, the lower Mississippi Valley, Alaska and northwestern Canada
(Somily et al., 2005).
The life cycle for E. multilocularis involves foxes as a definitive host and
rodents such as voles and meadow mice as intermediate hosts (Vuitton et al.,
2008). Domestic dogs and cats can also become infected with the adult
tapeworm when they eat infected wild rodents. Human disease associated
with E. multilocularis has been reported in parts of central Europe, much of
Siberia, northwestern Canada, and western Alaska (McManus et al., 2003;
Somily et al., 2005).
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Trematoda
Most trematodes, or flukes, have a life cycle that requires one or two intermediate hosts. Juvenile flukes may be present on aquatic vegetables or foods
washed in contaminated water (e.g. Fasciola and Fasciolopsis), while others
encyst in fish (Clonorchis) or crabs and wild boar (Paragonimus). Although
trematodes have not received a lot of attention from food-safety authorities,
foodborne trematodiasis is an emerging public-health problem, particularly
in South-east Asia and the Western Pacific region. It is estimated that millions
of people are currently at risk of infection with fluke parasites such as Clonorchis sinensis, Paragonimus spp., Fasciola spp., and Opisthorchis spp. In Asia, where
aquaculture is of growing importance, rural and some urban residents living
near the freshwater habitats for these parasites have a 2.15-fold higher risk
(95% confidence interval 1.38–3.36) for infections than residents living further from the water. The exponential growth of aquaculture may be the most
important risk factor for the emergence of foodborne trematodiasis (Keiser
and Utzinger, 2005; Robinson and Dalton, 2009).
Human and animal fascioliasis is a matter of growing concern in
Egypt (Soliman, 2008), but although the parasite occurs worldwide clinical
disease in humans is rarely reported in Western countries. However, there has
been a recent increase in human cases reported from Europe, the Americas,
Oceania, Africa and Asia. Human fascioliasis is considered to be an important
emerging disease, with most cases associated with consumption of infective
stages in vegetation such as watercress, containing intermediate hosts such as
snails (Soliman, 2008).
Other foodborne zoonotic trematodes (FZT) pose risks to human health,
many of these are transmitted in raw or inadequately cooked fish raised in fish
farms. A number of studies in South-east Asia have found that FZT are a
significant problem in hatcheries supplying stock to fish farms. Farmed fish
in Asia include perch (Anabas sp.), carp (Cyprinus sp., Ctenopharyngodon sp.),
gouramy (Osphronemus sp., Helostoma sp.), tilapia (Oreochromis sp.) and other
species that comprise an important source of protein for consumers (Thien
et al., 2009). Recent concerns associated with the presence of trematode metacercariae in fish flesh have been responsible for substantial economic losses
in the aquaculture industry owing to restrictions on exports and reduced consumer demand because of food safety concerns. Current food inspection procedures may not detect the presence of all parasites in imported fish products,
so there can be a risk of human infection if the fish or shellfish are consumed
raw or lightly cooked. International travel and the increasing availability of,
and interest in, ethnic foods also contribute to the risk of infection in nonendemic areas (Soliman, 2008).
FASCIOLIASIS
Conclusions
A wide range of zoonotic pathogens are capable of causing significant morbidity and mortality in the human population. Outbreaks of zoonotic enteric
Epidemiology of Pathogens in Food Supply
47
disease in urban populations of the industrialized world have typically been
attributed to bacterial pathogens (e.g. E. coli O157, Salmonella spp., L. monocytogenes, Campylobacter spp.) present in contaminated dairy products, eggs, meat
and processed foods. Other outbreaks have been traced back to the presence
of bacteria (e.g. E. coli) or protozoa (e.g. Cryptosporidium spp.) in contaminated
water supplies used to wash fresh produce. Several recent outbreaks of foodborne disease have been linked to hygiene failures in modern food production
plants, with further risk associated with the ready availability of fresh and chilled
food products, which are often transported large distances both nationally and
internationally. In an attempt to reduce the risk to consumers, regional and
international regulatory authorities have been working together to regulate
the food-production industry at all levels – from the producer through to the
consumer. In most countries, there are international agreements in place to
ensure that biosecurity and food-safety standards are applied to all traded food
commodities. This applies to both exports and food for sale in local markets
and in food outlets. This topic is discussed further in Chapter 1.
In less industrialized countries, and in more rural settings, the range and
extent of diseases that are transmitted from animals to humans via the food
chain is more varied. The water supplies and food-production methods are
often less closely regulated and there is more potential for disease transmission between humans, wildlife and livestock. The range of pathogens to which
humans are exposed is also greater now that people have the opportunity to
travel away from their home environment and have access to a range of food
options that were previously not available (e.g. game meats, fresh raw fish,
farmed shellfish, and novel fruits and vegetables that may not have been well
washed or cooked). The epidemiology of many of the predominantly foodborne and waterborne diseases outlined in this chapter is complex as it is
dependent on the interaction between the pathogen, the environment and
the host, as well as on the interplay between other potential hosts present in
an area. Food preferences, food availability, water quality and food-preparation
methods will also have an effect on the nature and extent of foodborne diseases
present in a particular area.
A greater understanding of the interplay between humans, the food
supply and the environment has started to emerge with the development of
interdisciplinary studies on the epidemiology of zoonotic and emerging diseases. These studies involve microbiologists, molecular scientists, ecologists,
risk-assessment experts, geographers, veterinarians, wildlife ecologists, medical specialists, food technologists and others (Daszak et al., 2000, 2007; Meslin
et al., 2000; Croft et al., 2003; Bengis et al., 2004; Dagendorf, 2004; de La Rocque
et al., 2008; Tee et al., 2008; Gould and Higgs, 2009; Gray et al., 2009). Broadbased surveillance efforts are required to inform veterinary and public health
authorities about current and emerging disease risks associated with the food
and water supply. The successes of the 20th century and the new challenges we
face mean that public-health vigilance, careful investigation of new problems,
responsible attention to food safety from farm to table, and partnerships to
bring about new foodborne disease control measures will be needed for the
foreseeable future.
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3
Manure as a Source of
Zoonotic Pathogens
GABRIEL J. MILINOVICH AND ATHOL V. KLIEVE*
Introduction
In addition to providing nutrients, the gastrointestinal system plays an integral
role in the physiological, immunological and protective functions of the host
(Zoetendal et al., 2004). The microbiome of the gastrointestinal system, and its
extensive and diverse populations of gut microorganisms, is increasingly
recognized to have a major role in numerous aspects of the host’s health,
including stimulation of the immune response, protection from pathogens,
production and metabolism of toxins and gene expression in host epithelial
tissue (Daly and Shirazi-Beechey, 2003). The gastrointestinal microbiome is
metabolically adaptable and rapidly renewable (Zoetendal et al., 2004).
However, it may also harbour a wide variety of pathogenic organisms of both
veterinary and human significance.
A review of the scientific literature in 2001 identified 1415 species of infectious pathogens reported to cause disease in humans; 61% (868 species) of
these pathogens were identified as known zoonoses (Taylor et al., 2001). These
868 species were determined to be derived from 313 different genera and
included, overall, viruses and prions (19%), bacteria or rickettsiae (31%),
fungi (13%), protozoa (5%) and helminths (32%). Further highlighting the
importance of zoonoses in global public health was the finding in the study
of Taylor et al. (2001) that 75% (132) of emerging pathogens are zoonoses.
These finding are supported by a study of emerging infectious diseases from
1940 to 2004, which found 60.3% of emerging infectious diseases to be
caused by zoonotic pathogens ( Jones et al., 2008). Both the study of Jones et
al. (2008) and that of Taylor et al. (2001) identified two groups as the main
source of emerging infectious diseases: viruses and prions (25.4% and 44% for
the respective studies) and bacteria and rickettsiae (54.3% and 30% for the
* Corresponding author.
CAB International 2011. Zoonotic Pathogens in the Food Chain
(eds D.O. Krause and S. Hendrick)
59
60
G.J. Milinovich and A.V. Klieve
respective studies); these discrepancies were attributable to the classification
by Jones et al. (2008) of each drug-resistant microbial strain as a separate pathogen. The Jones et al. (2008) study determined that the majority of emerging
infectious disease originated from wildlife (71.8%), rather than from companion or production animals. This chapter will focus on the most significant
of these zoonotic pathogens that are derived from manure, the modes by
which humans become exposed to these organisms and the significance of
the diseases caused.
Manure, Zoonoses and Modes of Infection
Manure is generally regarded as livestock excreta (urine and faeces) mixed
with bedding materials, such as straw, that is traditionally disseminated into
the environment as a source of fertilizer for crops and pastures. It is important
to recognize that, in addition to excreta and bedding material, manure may
also contain water (drinking and cleaning) and excretions from the nose,
throat, vagina, mammary glands and skin, as well as blood, and typically contains 1010 bacterial cells g–1 (Pell, 1997). In 1997, manure production by
housed livestock in England and Wales was estimated at around 70 million
tonnes, the majority of which was produced by the beef and dairy industries
(77% combined), followed by the pig (15%), poultry (6%) and sheep industries (2%) (Hutchison et al., 2000). In the USA, estimates indicate that in
excess of 1.2 billion tons of cattle manure are produced yearly (Sanchez et al.,
2002a). Traditionally, manure was seen as an agricultural waste product that
necessitated disposal, but this view has changed and manure is now viewed as
an industry by-product with a number of valuable uses both within the agricultural setting and outside these industries (Bicudo and Goyal, 2003). The primary use of manure is as a fertilizer source. Manure is recycled back into
agricultural land and provides an economical means of reintroducing organic
material and nutrients to soil, and maintaining or improving quality and fertility (Hutchison et al., 2000). While the vast majority of manure is utilized as
fertilizer, other common uses for manure include use as a source of solid
fuel for cooking and heating (Venkataraman et al., 2005) and for biogas
production (Amon et al., 2007). The handling and use of manure for these
applications has the potential to expose humans, either directly or indirectly,
to potential zoonoses, and constitutes a significant public health issue.
Infection may occur either via direct exposure to contaminated faecal
matter, such as through the handling or processing of manure, or through a
number of indirect routes, such as contaminated meat, milk, other produce or
water. A study by Adak et al. (2005) reported that foodborne disease resulted
in over 1.7 million infections and 687 deaths in England and Wales between
1996 and 2000, with animal-based food products (including meat, milk/dairy
products and seafood) accounting for 65% and 68% of cases and deaths,
respectively. Meat products can become contaminated with manure and resident pathogenic bacteria during slaughtering, butchering or processing, and
contamination may originate either directly from the gastrointestinal tract
Manure as a Source of Zoonotic Pathogens
61
or from the hide of the animal. The same study of foodborne disease in
England and Wales reported that fruits and vegetables account for only 3%
and 2% of cases and deaths, respectively (Adak et al., 2005) – yet despite this
relatively low incidence of foodborne diseases from fruits and vegetables,
these are becoming increasingly recognized as potential sources of infection,
particularly with the increase in popularity of organic farming practices
(Leifert et al., 2008; Sofos, 2008). Milk contamination may occur through poor
udder hygiene (Ramirez et al., 2004) and, combined with dairy products, was
reported to account for 7% and 5% of cases of foodborne illness and death,
respectively (Adak et al., 2005). The exact proportion of foodborne infections
that can be directly attributed to manure contamination is, though, not well
established (Cliver, 2009).
Water contaminated with pathogenic microorganisms is a major source
of human morbidity. A large number of pathogens transmissible via contaminated water can be found in livestock manure, but as humans and a wide variety of wildlife species can also be a source of these organisms, documented
outbreaks are often unable to be definitively attributed to a source (Bicudo
and Goyal, 2003). Environmental contamination with manure containing
zoonotic agents may, via a number of hydrological pathways, result in contamination of water sources accessed by humans (Williams et al., 2008). Leaching may result in the contamination of groundwater, while runoff has the
potential to contaminate watercourses and stored water, especially during
heavy rainfall events (Williams et al., 2008). Thawing snow may facilitate the
entry of zoonotic microorganisms into watercourses or catchments in areas
with winter snow cover (Unc and Goss, 2006). It should also be noted that
contamination of ground, irrigation or drinking water supplies provides
not just a source of infection for humans, but also a means by which these
organisms can be spread between animals or herds.
The intensification and industrialization of the animal production industries have resulted in an increase in microbial load in the production environment through the increased presence of animal feed, animals and associated
waste products, including manure (Millner, 2009). These factors have necessitated increased handling and management of both animals and animal
wastes, and put persons in direct contact with either animals or manure at
increased risk of contracting infections associated with manure. Bio-aerosols,
defined as a collection of aerosolized biological partials (Cox and Wathes,
1995), may potentially be generated within animal housing facilities from
either solid manure or slurries, but also from feed, litter or the animals themselves (Pillai and Ricke, 2002). As a result, within animal housing facilities,
workers can become exposed to aerosolized pathogenic microorganisms or
endotoxins, leading to increased risk of disease (Clark et al., 1983; Pillai and
Ricke, 2002). Mechanical spreading of manure as a fertilizer also results in the
production of aerosols, which constitute a potential source of infection if
inhaled, or in environmental, water or crop contamination through spray drift
(Hutchison et al., 2000). Trials using rain-gun manure sprayers have shown the
potential of these systems to contaminate the environment over 100 m from
the spray site (Hutchison et al., 2008).
62
G.J. Milinovich and A.V. Klieve
Control measures for preventing zoonotic infections arising from
manure can be implemented at multiple steps during animal and crop production, and have been reviewed in detail with particular reference to the
contamination of vegetable crops (Leifert et al., 2008) and verocytotoxigenic Escherichia coli (VTEC) from cattle (Khanna et al., 2008). Briefly, practices should be implemented that reduce pathogen burden within the host
animal (Doyle and Erickson, 2006). Implementation of this policy often
makes economic sense, as many zoonotic pathogens cause disease in the
host, thus reducing productivity and, as such, profitability (Pell, 1997). Prevention of environmental contamination reduces the risk of herd reinfection and of human disease resulting from pathogen contamination of crops
or water sources. Pathogenic organisms may remain viable in manure for
extended periods (for example, E. coli O157:H7 can survive and remain
infectious in faecal matter for up to 21 months; Zhao et al., 2001). Environmental contamination can be reduced through various manure management systems, including the composting of manure, and various physical,
chemical or biological treatments before application to land; these methods have been reviewed in detail (Bicudo and Goyal, 2003; Hutchison et al.,
2005a,b). Appropriate soil management and irrigation practices can also be
beneficial in reducing the risk of contaminating fruit and vegetable crops
(Holley et al., 2008; Leifert et al., 2008), and the use of filter strips has been
shown to be efficacious in reducing watercourse contamination through
runoff (Larsen et al., 1994). Finally, food processing facilities should
implement appropriate risk-minimization practices to reduce the chance
of contamination of products with manure (Zhao et al., 2001; Stecchini and
Del Torre, 2005; Khanna et al., 2008).
Significant Zoonotic Infections Contractible from Manure Exposure
Pathogen types and numbers in manure differ with animal species, geographical location and the physicochemical composition of the manure (Bicudo
and Goyal, 2003). Bacterial, viral and protozoan zoonotic organisms are all
transmissible to humans through manure, although the majority of pathogens
of concern are bacterial (Cliver, 2009). A study of 38,629,641 cases of foodborne diseases of known aetiology in the USA concluded that six pathogens
alone accounted for approximately 70% of total cases and 95% of the 2718
reported deaths: Salmonella (31%), Listeria (28%), Toxoplasma (21%), Norwalklike viruses (7%), Campylobacter (5%) and E. coli O157:H7 (3%) (Mead et al.,
1999). For all of these organisms, with the exception of Norwalk-like viruses
and Toxoplasma, manure can be either the direct source of infection or the
source of contamination by which these pathogens enter the food chain.
Furthermore, a recent publication evaluated and prioritized a list of 51
foodborne and waterborne zoonoses (Cardoen et al., 2009), which corroborated the results of Mead et al. (1999); Salmonella spp., Campylobacter spp.,
Listeria monocytogenes and VTEC were ranked as the most significant of the
diseases analysed.
Manure as a Source of Zoonotic Pathogens
63
Escherichia coli
E. coli is a normal inhabitant of the gastrointestinal system of warm-blooded
animals. Commensal E. coli colonizes the neonate gastrointestinal system
within hours of birth, and plays an intrinsic role in maintaining normal gut
physiology in the host (Mackie et al., 1999). Gut commensal E. coli is rarely
responsible for disease. However, through the acquisition of virulence factors,
strains of E. coli have developed the capacity to cause either enteric or extraintestinal diseases, and are commonly associated with disease resulting from
the exposure of foodstuffs to manure contamination. The E. coli O157:H7
serotype is associated with over 50% of VTEC infections in the EU (European
Centre for Disease Prevention and Control, 2009); this, combined with the
involvement of the organism in large outbreaks, and the severity of the disease, makes E. coli O157:H7 the serotype of most interest. E. coli O157:H7
infection within the EU was reported to affect 0.6 persons per 100,000 in 2007
(European Food Safety Authority, 2009).
Of most concern, with regard to the context of this document, are the
enterohaemorrhagic E. coli (EHEC), a subgroup of VTEC with the capacity to
produce toxins (vero, Shiga or Shiga-like) similar to Shigella dysenteriae type 1
cytotoxin (O’Brien and Holmes, 1987), but also possessing the locus of enterocyte effacement (LEE) pathogenicity island, which encodes for a type III
secretion system (Kaper et al., 2004). Animals are the primary reservoir for
EHEC (Khanna et al., 2008), with bovine products linked to around 75% of
cases of E. coli O157:H7 outbreaks (Callaway et al., 2009). In cattle, E. coli
O157:H7 colonizes mucosal surfaces of the large intestine, and particularly of
the terminal rectum (Naylor et al., 2003), where the organisms may reside and
continue to be shed for up to 3 months (Sanchez et al., 2002a). Prevalence
rates of E. coli O157:H7 range from 0 to 28% for individual cattle, and up to
75% for herds (Sanchez et al., 2002a). Hide contamination with VTEC is typically higher than faecal carriage, and a single animal has the capacity to contaminate, either directly or indirectly through environmental contamination,
the hides of many animals (Rhoades et al., 2009). E. coli O157:H7 is not invasive, and undercooked meat contaminated with cattle faeces constitutes the
leading source of infections (Sanchez et al., 2002a). Studies have shown that as
much as 19% of retail uncooked beef may be contaminated with E. coli, and
4% with E. coli O157:H7 (Zhao et al., 2001); the initial E. coli O157:H7 cases
were associated with the consumption of undercooked hamburgers (Kaper et
al., 2004). It should be noted that other livestock can also act as reservoirs, and
E. coli O157:H7 has been found in pork (1.5%), lamb (2%) and poultry (1.5%)
meat, as well as in various other foods, including unpasteurized milk (Zhao
et al., 2001). Contamination of food products may occur at one or more of
several steps along the food processing, distribution, retail and preparation
line (Zhao et al., 2001), and estimated infectious doses for EHEC are very low
(ID50 between 100 and 1000 cells) (Teunis et al., 2008). Shedding rates for
E. coli O157:H7 are typically lower than 100 colony forming units (cfu) g–1 faecal matter, though shedding rates of greater than 107 cfu g–1 faeces have been
reported (Rhoades et al., 2009). Water sources, such as lakes or ponds
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G.J. Milinovich and A.V. Klieve
contaminated with infected cattle-manure runoff, and swimming pools, have
been identified as sources of E. coli O157:H7 outbreaks, as has contact with
animals, particularly calves, at petting zoos or farms (Sanchez et al., 2002a; Callaway et al., 2009). E. coli O157:H7 has the capacity to survive in the environment for long periods of time, especially in water, with studies showing E. coli
O157:H7 to survive and remain infectious in water (farm drinking troughs)
for 8 months and in faecal matter for 21 months (Zhao et al., 2001).
Salmonella
Of the zoonotic pathogens associated with manure, Salmonella constitutes the
broadest risk (Cliver, 2009). Salmonella is ubiquitous in nature and has been
isolated from a wide variety of vertebrate hosts (Pell, 1997). Salmonellae are
commonly found in production animals, their environments and the manure
produced by animal industries (Thorns, 2000; Guard-Petter, 2001; Hutchison
et al., 2004; Farzan et al., 2009; Rhoades et al., 2009). Furthermore, Salmonella
was ranked by 35 scientific experts in the fields of animal and public health,
food, and clinical microbiology and epidemiology, as the single most important foodborne zoonotic microorganism (Cardoen et al., 2009). Non-typhoidal
salmonellosis is reported to account for 9.7% and 6.6% of total foodborne
disease in the USA and in England and Wales, respectively (Mead et al., 1999;
Adak et al., 2002). Human disease resulting from Salmonella infection is typically self-limiting, and characterized by diarrhoea, stomach cramps, vomiting
and fever (Rhoades et al., 2009). However, Salmonella is reported to account for
more deaths from foodborne infections in the USA and in England and Wales
than any other organism: 30.6% and 29.2%, respectively (Mead et al., 1999;
Adak et al., 2002).
In livestock, salmonellosis results in illness ranging in severity from moderate to severe, but the animals can also be asymptomatic carriers (Van Kessel
et al., 2007). Faecal contamination of hides is common, and hide carriage rates
of Salmonella by feedlot cattle have been reported to be as high as 71.0% (yearly
mean), compared with 4.3% by faecal carriage for the same animals (BarkocyGallagher et al., 2003). Stored manure is reported to contain as many as 106
Salmonella cells g–1 (Hutchison et al., 2004), and the organisms are more likely
to be detected in stored manure than from faecal samples, highlighting the
potential significance of manure as a source of environmental contamination
(Farzan et al., 2009). Long-term Salmonella contamination of farms has been
described and appears to be widespread (Winfield and Groisman, 2003).
Manure may constitute a source of human salmonellosis via a number of
routes of infection. A wide range of food types, ranging from meat products,
milk, eggs, fruit juice, salad and other fresh produce through to peanut butter,
has been associated with Salmonella outbreaks (Denno et al., 2007). Faecal contamination of meat during processing poses a risk of Salmonella entry into the
food chain, and a number of studies have been conducted to evaluate the
level of Salmonella contamination on abattoir carcasses at different stages of
processing. In these studies, up to 45.2% of pre-evisceration carcasses were
Manure as a Source of Zoonotic Pathogens
65
found to be contaminated (Rhoades et al., 2009). Salmonella contamination
of chicken, turkey, pork and beef meat from retail stores in the Greater
Washington, DC area reported contamination rates ranging from 1.9% to
4.2% (Zhao et al., 2001). Enteric disease has been associated with the consumption of a large range of contaminated fresh produce, and Salmonella
constitutes the most commonly identified enteropathogen in these cases
(Heaton and Jones, 2008). Contamination of these products may occur either
postharvest, through mechanisms such as inadequate hygiene, or preharvest,
through mechanisms discussed above such as direct environmental contamination by animals or the application of manure or contaminated water (which
may have initially been contaminated by manure). Salmonella survivability trials in soils amended with pig manure have indicated that the largest decreases
in viable Salmonella occurred in the initial 2 weeks, and this led the authors to
suggest that a 30-day delay between manure application and land use would
minimize the risk of Salmonella contamination of crops and animals (Holley
et al., 2006); in this study, no Salmonella survived for longer than 180 days,
although the organisms have been shown to survive for periods of up to 300
days (Baloda et al., 2001). Occupational contact with animals and animal
waste is reported to constitute a risk for contracting salmonellosis (Sanchez
et al., 2002b). Flies may also act as vectors of Salmonella (Winfield and Groisman,
2003); muscoid flies on dairy and poultry farms have been demonstrated to
have infection rates of 67% and 13%, respectively and total Salmonella defecation rates in experimentally infected flies have been shown to be as high as 107
(Greenberg and Klowden, 1972).
Listeria
Listeriosis constitutes a serious public-health concern owing to the epidemic
potential and high mortality rate characteristic of this disease. Of the six recognized species of Listeria, two are known to cause disease in humans: L. inanovii
and L. monocytogenes (Snapir et al., 2006); virtually all human cases can, however, be attributed to the latter. While direct animal-to-human transmission is
possible, particularly in persons working with aborted fetuses, ingestion of
contaminated food is the primary source of infection, with 99% of listeriosis
cases attributed to this (Mead et al., 1999). L. monocytogenes has been isolated
from 42 mammalian species and 22 avian species, as well as fish, crustaceans
and insects (Wiedmann et al., 1996), and a vast array of plant and soil environments, groundwater, sewage and silage (Freitag et al., 2009). It is the ubiquitous nature of this organism that facilitates its epidemic and zoonotic potential,
but it is cattle and sheep, through the contamination of meat and milk, which
constitute the greatest zoonotic threat as a source of L. monocytogenes infections
(Czuprynski, 2005). L. monocytogenes is resilient, able to grow under a wide
range of temperatures (3–42°C) and pH ranges (<5.5–9.0) and high salt concentrations (Pell, 1997), and has the capacity to become established in foodprocessing facilities if appropriate controls are not introduced (Freitag et al.,
2009).
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Animal infection most commonly occurs through the ingestion of contaminated feed, and studies showing correlations between feeding poorly prepared silage (pH > 5) and L. monocytogenes have been published (Pell, 1997;
Czuprynski, 2005). Livestock infection most commonly presents as neurological signs; altered behaviour and depression, developing into facial hypalgesia,
paralysis, ataxia and, particularly in sheep, head tilt and permanent circling
movement (Wagner et al., 2005). Asymptomatic L. monocytogenes carriage and
faecal shedding is common (Czuprynski, 2005) and has been well reported for
a wide range of livestock and wildlife (Lyautey et al., 2007); L. monocytogenes
prevalence levels as high as 33% have been reported in healthy cattle (Weber
et al., 1995). Under favourable conditions, L. monocytogenes has been shown to
have the capacity to survive in soil amended with bovine manure for several
weeks ( Jiang et al., 2004), thus posing a significant risk of transmission through
the contamination of fruits or vegetables entering the human food chain
(Czuprynski, 2005).
Campylobacter
The Campylobacter genus has long been recognized as associated with both
animals and human disease. Campylobacter-like organisms were first observed
in the colon of babies that had died of diarrhoeal disease in 1886 (Escherich,
1886), and in 1913 Campylobacter-like organisms were observed to be commonly associated with aborted sheep fetuses (McFadyean and Stockman,
1913). It was, however, not until the development of appropriate isolation and
cultivation techniques in the early 1970s that Campylobacter infection became
recognized as both a common and significant cause of enteric illness in
humans (Dekeyser et al., 1972). Campylobacter is now recognized worldwide as
being both a significant public-health concern and an economic burden, and
it is the leading cause of bacterial foodborne illness in the USA, accounting
for 14.2% of total infections and 5.5% of mortalities (Mead et al., 1999). In
England and Wales, Campylobacter was reported to account for around 27% of
total foodborne infections (359,466 of 1,338,772) in 2000 (Adak et al., 2002),
and these acute infections have been estimated to cost, on average, £1315 per
case (Humphrey et al., 2007).
Most human infections are attributable to three species: Campylobacter
jejuni, Campylobacter coli and, particularly in the developing world, Campylobacter
upsaliensis. Around 90% of confirmed cases of campylobacteriosis are attributed to C. jejuni (Altekruse and Tollefson, 2003). Cardoen et al. (2009) ranked
Campylobacter as the second most significant foodborne zoonotic organism
after Salmonella.
Campylobacter spp. may cause gastrointestinal disease and septic abortions
in animals (Cole et al., 1999), although they are generally considered as
normal gastrointestinal microflora of many mammals and birds (Crushell
et al., 2004). Both C. jejuni and C. coli are commonly found in the gastrointestinal tract of production animals, and colonization with these organisms is
thought to occur largely through contact with a contaminated environment
Manure as a Source of Zoonotic Pathogens
67
(Humphrey et al., 2007). Human campylobacteriosis is most commonly sporadic and not linked with environmental contamination (Crushell et al., 2004).
Infections from faecally contaminated water, unpasteurized milk and raw
vegetables are recorded, though campylobacteriosis is most commonly associated with the consumption of raw or undercooked meat products (Horrocks
et al., 2009), particularly poultry meat (Suzuki and Yamamoto, 2009). Direct
transmission of Campylobacter from animals to humans is also possible, but
infections via this route account for only a minority of infections (Crushell et al.,
2004). Studies of meat and animal contamination by Campylobacter spp. have
demonstrated mean rates of contamination for cattle to be 30.0% and 62.1%
for dairy and beef cattle, respectively, and 31.1% for sheep and 61% for pigs
(Humphrey et al., 2007); chicken, turkey and duck flocks had mean contamination rates of 58.7%, 78.0% and 38.0%, respectively, while 3.2% of raw milk
returned positive results for Campylobacter contamination. Of most concern
are the reported contamination rates for meats at retail: chicken (57.4%),
turkey (47.8%), duck (30.2%), pork (2.0%), beef (2.7%) and lamb (6.0%).
C. jejuni is the most commonly isolated Campylobacter species from cattle and
poultry species, while C. coli is more commonly associated with swine (Cole
et al., 1999; Farzan et al., 2009).
Protozoa
The protozoan species of most concern, with regard to manure exposure, are
Cryptosporidium spp. and Giardia duodenalis (Hunter and Thompson, 2005).
Cryptosporidium spp., along with Giardia spp., constitute the most common
enteric parasites of domestic animals and livestock (Thompson et al., 2008)
and, together, are thought to constitute the most common cause of protozoan
diarrhoea in humans (Caccio et al., 2005). It was not, however, until the 1980s,
with the establishment of the role of this organism in the death of AIDS
patients (Current et al., 1983), that Cryptosporidium became recognized as a
significant zoonotic pathogen (Ramirez et al., 2004). Infections by both organisms
occur via mechanisms commonly associated with manure contamination:
the faecal–oral route, predominantly through the ingestion of contaminated
food or water, via host-to-host contact or, for Cryptosporidium, via aerosol
transmission (Leoni et al., 2006; Xiao and Feng, 2008).
Cattle are the major reservoir for Cryptosporidium parvum (Hunter and
Thompson, 2005), and constitute an important source of zoonotic cryptosporidiosis infections, largely through the contamination of food or water with
manure containing Cryptosporidium oocysts (Xiao and Feng, 2008). Overall,
contaminated water constitutes the leading source of human infection
(Ramirez et al., 2004). Disease is usually self-limiting and does not pose longterm health risks for healthy, mature animals (Pell, 1997); infected, immunocompetent animals typically clear the parasite within 3 weeks of infection
(Chappell et al., 1999). If diarrhoea develops, cattle infected with C. parvum
are reported to excrete between 105 and 107 oocysts ml–1 faeces. C. parvum is
among the most common enteropathogens of calves (Thompson et al., 2008),
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and subclinical infections are not uncommon in older animals (Ridley and
Olsen, 1991). Excreted oocysts are immediately infectious once released and
may remain viable in the environment for months (Thompson et al., 2008);
they are resistant to the majority of common disinfectants (Campbell et al.,
1982). C. parvum infectious doses have been determined in healthy, serologically negative (DuPont et al., 1995) and positive (Chappell et al., 1999) adults
and the 50% infectious dose (ID50) was found to be 132 and 1880 oocysts,
respectively.
Overall, the exact significance of cryptosporidiosis as a zoonotic disease
remains unclear. Reports indicate that C. parvum and Cryptosporidium hominis
account for over 90% of cases of cryptosporidiosis (Xiao and Feng, 2008), and
as many as 98% of sporadic cryptosporidiosis cases (Leoni et al., 2006) in
industrialized countries. Of these, C. parvum is responsible for slightly more
cases in the Czech Republic, England, France, New Zealand, Northern Ireland,
Portugal, Slovenia, Switzerland and Wales, while C. hominis is the dominant
species in Australia, Canada, Japan and the USA (Xiao and Feng, 2008). Interestingly, C. hominis alone accounts for 70–90% of cases of cryptosporidiosis in
developing countries, suggesting that cases of zoonotic origin are of less
importance in these countries (Xiao and Feng, 2008). The proportion of
infections occurring from C. parvum of animal source is unclear, as both
humans and animals may be the source of infection (Xiao and Feng, 2008).
Furthermore, Cryptosporidium is often not considered in the differential diagnosis of gastrointestinal diseases of immunocompetent patients, resulting in
the under-reporting of this disease (Ramirez et al., 2004). Persons exposed to
livestock are, however, reportedly at increased risk of Cryptosporidium infection
(Lengerich et al., 1993).
Like cryptosporidiosis, giardiasis presents as an acute, mild-to-severe
gastrointestinal illness, characterized by transient diarrhoea and associated
gastrointestinal symptoms (Wolfe, 1992). Chronic disease may develop and
can persist over a number of years (Pell, 1997). The Giardia genus contains a
number of different species which are known to affect various species of
domestic animals and wildlife, as well as humans. Of these, only assemblages A
and B of G. duodenalis are pathogens of humans (Hunter and Thompson,
2005). The routes of infection and specific roles of animals in the transmission
remain unclear, although the available evidence suggests that livestock only
constitute a small public-health risk with regards to transmission of Giardia
(Caccio et al., 2005).
It should also be noted that Toxoplasma gondii (which was reported by
Mead et al. (1999) to account for 21% of reported deaths) is a zoonotic
organism, for which cats (Felidae) are the primary host. Human infection by
this organism occurs through either direct or indirect contact with feline
faecal matter contaminated with T. gondii oocysts, and while it is not spread
by exposure to manure, it is a possibility that cats can contract toxoplasmosis from manure (Tenter et al., 2000). Livestock may also constitute a
source of infection for both definitive and intermediate hosts, including
humans, through the ingestion of tissue cysts (Tenter et al., 2000; Tenter,
2009).
Manure as a Source of Zoonotic Pathogens
69
Viruses
A wide variety of viruses are known to affect production animals; however, few
of these have been shown to be transmissible to humans through manure
(Cliver, 2009). The study of food-related illness and death in the USA by
Mead et al. (1999) reported Norwalk-like viruses (of the Norovirus genus) to
account for 66.6% of foodborne illnesses and 6.9% of deaths. Norovirus has
recently been detected in the faeces or gastrointestinal tract of a number of
species of production animals (van Der Poel et al., 2000). Furthermore, norovirus resembling a human strain has been detected in swine faeces and retail
meat (Mattison et al., 2007; Wolf et al., 2009), and these human Norovirus
strains have been demonstrated to cause symptoms under experimental infection (Cheetham et al., 2006). While there is concern of the potential of these
organisms to cause zoonotic disease, zoonotic infection has not been detected,
and the general consensus is that while zoonotic infection may be possible,
outbreaks are more likely to occur from human rather than from animal
sources (Farkas et al., 2005; Koopmans, 2008; Cliver, 2009).
Viral zoonotic organisms of more concern with respect to human infection from contact with manure are hepatitis E virus (HEV), avian influenza A
(H5N1) virus (Cole et al., 1999) and, more recently, swine influenza A (H1N1)
virus (Tomley and Shirley, 2009). Described as ‘the last great plague of man’
(Kaplan and Webster, 1977), influenza virus is unique in its potential to rapidly infect billions of people worldwide (Greger, 2007). Wild waterfowl constitute the natural reservoir of avian influenza A viruses, and these can be
transmitted to domestic terrestrial poultry in which they may mutate from
the low-pathogenicity strain acquired into a highly pathogenic form (WHO,
2007). Infected animals shed large quantities of virus, which can become
incorporated into manure and has the capacity to contaminate water supplies
and the environment via surface runoff, through groundwater or via wind
dispersal (WHO, 2007; Halvorson, 2009). The route of transmission of avian
influenza H5N1 from poultry to humans has yet to be fully elucidated. Current evidence suggests that infection may occur via direct animal contact or
through contact with faecal matter via the respiratory or faecal–oral route
(WHO, 2007). Regarding the H1N1 virus, work still needs to be done to establish disease susceptibility in host species, transmission dynamics and virulence
of this organism. However, it is proposed that natural transmissions between
pigs and humans are a feature of this disease (Tomley and Shirley, 2009), and
the role that manure may have in the spread of the disease remains to be
elucidated.
HEV is a non-enveloped, single-stranded RNA virus commonly associated
with large-scale waterborne acute hepatitis epidemics and sporadic infections,
particularly in developing countries (Khuroo, 2008). The transmission of HEV
to humans can occur via a number of routes, including parenterally in association with blood transfusion, or vertically, although transmission primarily
occurs via the faecal–oral route through contaminated water or the meat of
wild or domestic animals (Goens and Perdue, 2004; Khuroo, 2008). In endemic
countries, HEV is reported to account for in excess of 50% of cases of hepatitis
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(Dalton et al., 2008). While primarily associated with developing countries and
with travel within these, autochthonous HEV infections in developed countries are becoming recognized as a more important source of infection than
previously recognized (Dalton et al., 2008). The main source for sporadic
infection in developed countries remains unclear, though virological evidence
of HEV has been found in pigs and antibodies to HEV have been detected in
other domestic livestock, including cattle, sheep, goats and horses (Dalton et
al., 2008). Studies have shown that 50–90% of pigs are anti-HEV seropositive,
and that infected animals have the capacity to shed high levels of infective
HEV in their faeces for weeks post-infection (Teo, 2006). The exact mechanisms of transmission of autochthonous HEV infections in developed
countries are unknown, but it is probable that zoonotic foodborne transmission via manure, particularly from pigs, is involved (Lewis et al., 2009).
Exposure to a number of viruses associated with cancer development in
food animals has been implicated as a potential mediator of cancer development in humans ( Johnson et al., 1997; Fritschi, 2000; Johnson et al., 2007).
Persons at most risk are those in close contact with animals, such as veterinarians, animal workers and meat workers. Of particular note are viruses associated with poultry: the avian leukosis/sarcoma viruses, reticuloendotheliosis
viruses and Marek’s disease virus (Netto and Johnson, 2003), all of which
may be shed in faeces and therefore incorporated into manure (Weyl and
Dougherty, 1977; Cole et al., 1999). The significance of these viruses in
human cancer development, however, remains to be conclusively established.
Manure as a Source of Antimicrobial-resistant Organisms/
Resistance Genes
In addition to contributing to zoonotic disease directly or through the introduction of pathogens into the environment, manure can also contribute indirectly to zoonotic disease via the use of antimicrobials in animal agriculture.
Varying classes of antimicrobials are used in the livestock industries as either
growth promotants or prophylactics, or are used for the treatment of disease,
either at the herd level or in individual animals (Prescott, 2008; Silbergeld
et al., 2008; Venglovsky et al., 2009). The development of communities of
organisms resistant to these compounds is well documented in production
animals (Aarestrup et al., 2008; Call et al., 2008; Gyles, 2008).
The establishment of antimicrobial-resistant bacterial populations in production animals poses a risk to humans in two key areas: (i) as a source of
antimicrobial-resistant organisms with the potential to infect humans; and (ii)
as a reservoir of antimicrobial resistance genes and their precursors, contained
in both pathogenic and non-pathogenic bacteria, and termed the ‘resistome’
(Wright, 2007). The development of such a resistome facilitates the acquisition of antimicrobial resistance by pathogenic bacteria. Horizontal gene
transfer has been demonstrated to occur in a number of complex media,
including both the gastrointestinal tract and animal faeces (Walsh and Fan-
Manure as a Source of Zoonotic Pathogens
71
ning, 2008) and, as such, manure constitutes a potential source of infection
or environmental contamination by organisms (or genetic elements) that
have acquired antimicrobial resistance genes from these environments. Interestingly, this is not the only mechanism by which manure may be involved in
the development of antimicrobial resistance. Manure may also contain significant quantities of antimicrobial residues, and its application to the environment – largely through the use of manure as fertilizer – poses environmental
problems through both the toxicity of these residues to soil microorganisms
and the potential of this practice to increase antimicrobial resistance in environmental microorganisms (Venglovsky et al., 2009). Furthermore, experiments evaluating the effects of applying manure containing varying levels of
sulfamethazine to maize (Zea mays), lettuce (Lactuca sativa) and potato (Solanum tuberosum) crops showed a positive correlation between antimicrobial
concentrations in the manure and in the plant tissue (Dolliver et al., 2007),
leading the authors to question the human-health implications of this process.
Other Notable Zoonoses Contractible from Manure
Disease associated with organisms contractible from manure occurs, largely,
via the faecal–oral route and manifests as gastrointestinal disease. In excess
of 100 zoonotic pathogens have been described that affect humans through
entry into the food chain (Walton and White, 1981). Despite the large number of zoonotic pathogens that have the potential to cause disease in humans,
the vast majority of disease is attributable to only a few organisms (Mead et al.,
1999). This section will, very briefly, focus on a small number of other zoonotic
pathogens of note, contractible through exposure to manure, which have not
been covered in this chapter so far.
Numerous bacterial species constitute potential zoonoses contractible
through manure exposure. Yersinia enterocolitica, a foodborne pathogen responsible for acute gastroenteritis and mesenteric lymphadenitis, is the third most
reported zoonosis in the EU (Laukkanen et al., 2009). Pigs are considered to
be a major reservoir of this organism and infection is most frequently associated with undercooked pork (Tauxe, 1997; Farzan et al., 2009; Laukkanen et
al., 2009). Helicobacter spp. are associated with human gastric disease and have
been found in sheep (Helicobacter pylori), pigs (Helicobacter suis) and cattle (Candidatus Helicobacter bovis) (Haesebrouck et al., 2009). Clostridium difficile has
recently been demonstrated in cattle, pigs and broiler chickens, and in retail
meat from all three animals (Indra et al., 2009), while Clostridium perfringens
inhabits the gastrointestinal system of numerous animal species, including
production animals (Uzal and Songer, 2008).
Maternal and neonatal tetanus is reportedly responsible for around
180,000 deaths worldwide annually (Roper et al., 2007), predominantly in
developing countries, and the elimination of this disease is an objective of the
World Health Organization (WHO, 2006). The organism responsible for tetanus,
Clostridium tetani, can be found in the faeces of numerous domestic animal
species (Edlich et al., 2003). In some developing countries, ghee (clarified
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butter) is applied to the umbilical wounds of neonates for its perceived
healing and strengthening powers. When the ghee is heated with cow dung
fuel, this practice has been shown to be significantly associated with the
development of neonatal tetanus (Bennett et al., 1999).
Brucella melitensis, a re-emerging zoonotic organism and the leading cause
of brucellosis, a febrile disease, has been reported to be associated with occupational contact with manure, particularly from goats, but also from sheep
and cattle (Corbel, 1997; Wallach et al., 1997). Similarly, contact with manure
has been associated with the development of leptospirosis (Diesch, 1971;
Levett, 2001) and Q fever (Coxiella burnetii) (Salmon et al., 1982; Jorm et al.,
1990; Berri et al., 2003), while Rhodococcus equi has been found in the manure
of a wide variety of herbivores (Prescott, 1991; Votava et al., 1997). Erysipelothrix
rhusiopathiae, although most commonly associated with pigs and turkeys, is
shed in the faeces of a number of species, including cattle, sheep and horses
(Pell, 1997); clinical presentation of E. rhusiopathiae infection is, most commonly, a localized, non-pyogenic cellulitis, typically on the hands; however,
other more serious syndromes may also be associated the infection (Brooke
and Riley, 1999). Streptococcus suis type 2 is a communal bacterium of swine
tonsils and may also be found in the gastrointestinal tract and faeces of
swine (Devriese et al., 1994); meningitis resulting from S. suis infection has
been associated with exposure to pigs (Pell, 1997). Mycobacterium avium subsp.
paratuberculosis (M. paratuberculosis) also warrants mention for its putative,
albeit contentious, role in Crohn’s disease (Mendoza et al., 2009; Pierce, 2009).
M. paratuberculosis is the aetiological agent of Johne’s disease, which constitutes
a major economic and veterinary issue of the agricultural industries, particularly the cattle industry (Harris and Barletta, 2001). Cattle are not the only
animals affected by M. paratuberculosis; Johne’s disease has been reported in
other ruminants as well as in pigs and rabbits (Harris and Barletta, 2001).
Infection typically occurs via the faecal–oral route, either vertically or through
exposure to manure-contaminated environments, and studies have shown that
M. paratuberculosis remains viable in faeces, water and cattle slurry for up to
250 days (Harris and Barletta, 2001; Begg and Whittington, 2008).
Poultry constitute a potential source of a number of other zoonotic microorganisms. In birds, Bacillus anthracis infections are usually asymptomatic.
Chickens appear to be particularly resistant and pose a risk of both infection
and environmental contamination through the shedding of spores in faeces
(Senanayake and Baker, 2007). Cryptococcus neoformans is associated with the
faeces of birds, including chickens, but particularly pigeons (Emmons, 1955;
Levitz, 1991; Bovers et al., 2008). Clinical cases of cryptococcosis are relatively
rare, and studies indicate that most people mount a sufficient immune
response to suppress disease upon exposure to the organism (Levitz, 1991);
studies of pigeon breeders indicated that, while exposure to C. neoformans is
common, there was not an increased incidence of cryptococcosis (Newberry
et al., 1967). Psittacosis is a potentially fatal disease resulting from infection
with Chlamydophila psittaci. Clinical symptoms are highly variable, and infection typically presents as respiratory infection, but can progress to affect other
organs (Beeckman and Vanrompay, 2009). C. psittaci infections are reported
Manure as a Source of Zoonotic Pathogens
73
to occur in over 465 bird species, including chickens, turkeys, ducks and geese
(Kaleta and Taday, 2003); disease is predominantly caused through the inhalation of aerosolized urine, respiratory or eye secretions, or from dried faeces
(Beeckman and Vanrompay, 2009).
Treating Manure to Reduce the Risk
The development of zoonotic disease through exposure, either direct or indirect, to livestock manure constitutes a real and significant risk of public-health
concern. Manure may potentially harbour numerous species of zoonotic
pathogens and, for many zoonotic microorganisms, constitutes a significant
reservoir. The public-health risk posed by manure has increased with both the
intensification of the livestock industries and the use of manure outside these
industries (Martens and Bohm, 2009). This risk has driven the development of
numerous methods to mitigate the potential of manure to harbour pathogenic microorganisms, and these are discussed in great detail elsewhere
(Bicudo and Goyal, 2003; Burton and Turner, 2003). Manure-treatment
regimes can be classified as chemical, physical or biological in process, and are
typically applied as either as a general prophylactic hygiene measure or as a
means of controlling or eradicating a specific organism or group of organisms, such as an organism responsible for a notifiable disease (Martens and
Bohm, 2009). It should be noted that reduction in pathogen numbers is not
necessarily the primary objective in manure management regimes; treatment
is often applied for other purposes, such as ammonia stripping or phosphate
precipitation (Martens and Bohm, 2009). Despite this, these practices may
have a positive effect through reduction of pathogen viability.
A number of compounds have been identified that can be used as a means
of chemical treatment of both solid manure and slurries. These chemicals
include: limewash, caustic soda, formalin, peracetic acid and calcium cyanamide (Burton and Turner, 2003). Chemical treatment of manure is, however,
most commonly employed as a means of control during an epidemic, rather
than as a routine means of controlling manure-associated pathogens. Furthermore, the non-microbial residues left from chemical treatment processes may
constitute a greater environmental risk than the microbial pathogens themselves (Cliver, 2009). Physical manure-treatment regimes, such as thermal
treatment or irradiation, can also be employed as a means of disinfecting
manure. This process, with the exception of the drying of poultry manure and
exposure of spread manure to sunlight, is not commonly used for routine
manure management, although it is, once again, employed by some countries
for the control of certain disease outbreaks, such as foot-and-mouth disease
(Martens and Bohm, 2009). Biological systems employed for the control of
pathogenic microorganisms in manure include both aerobic and anaerobic
biotechnological treatments; the function of these systems is described in
greater detail elsewhere (Bicudo and Goyal, 2003).
Biological systems achieve a reduction in viable pathogen numbers
through a number of factors, including: antibiosis, pH alterations, redox-potential
74
G.J. Milinovich and A.V. Klieve
adjustments, antagonism, nutrient deficiencies and exothermic metabolism, but the most effective factor has been demonstrated to be through
elevation in temperature (Martens and Bohm, 2009). Manure is commonly
treated in either batches or under a semi-batch manner (a cycle consisting
of a short feeding period followed by a long stabilization period) and can
be processed either in the mesophilic or the thermophilic range (Martens
and Bohm, 2009). The composting of manure has also been shown to be
effective in reducing pathogen numbers (Bernal et al., 2009). The simple
act of storage alone has been demonstrated to reduce both viable pathogen
levels in manure and the presence of virulence genes (Duriez et al., 2008).
The act of storing manure before use can, essentially, be classified as a biotechnological treatment. Survival rates, however, vary markedly depending
on the microorganism monitored, the source and physical properties of
the manure and the climatic conditions. Furthermore, storage alone is
unlikely to achieve safe and efficient decontamination of all pathogens
(Bicudo and Goyal, 2003).
Appropriate treatment has the capacity to significantly reduce pathogen levels contained in manure, and, consequently, reduce the risk associated with the use of this material. As such, manure management policies
and legislation have been developed within numerous countries aimed at
limiting the negative impacts of manure usage (Burton, 2009). A number
of factors need to be considered in determining the most appropriate system for controlling potentially pathogenic microorganisms within manure.
Considerations to be taken into account include, but are not limited to:
the physical properties of the manure to be treated (solid manure or
slurry); the pathogens that are potentially associated within the material
to be treated; the eventual application of the treated manure (some processes can adversely alter the physiochemical properties of manure; Martens and Bohm, 2009); the risk posed by these microorganisms through its
intended use of the manure; and the economics of treating the manure.
The best results for ameliorating the risk posed by zoonotic organisms
associated with manure are achieved through the adoption of integrated
systemic approaches: employing management strategies to eliminate these
organisms from their animal reservoirs (Cliver, 2009); selecting and implementing an appropriate manure management and treatment system
(Bicudo and Goyal, 2003; Burton and Turner, 2003); identifying and
employing low-risk manure uses, such as utilizing as much manure as possible in low-risk cropping systems (Burton, 2009); and identifying areas of
particular risk and minimizing these through appropriate management
practices (Zhao et al., 2001; Stecchini and Del Torre, 2005).
Summary
Manure is a combination of livestock excreta (urine and faeces) mixed with
bedding materials, traditionally viewed as an agricultural waste product, and
disposed of through its dissemination into the environment. Large amounts
Manure as a Source of Zoonotic Pathogens
75
of manure are produced, utilized for a number of purposes, and eventually
reintroduced into the environment; annual manure production by housed
livestock alone, in England and Wales, is estimated at 70 million tonnes
(Hutchison et al., 2000), while the US cattle industries are estimated to
produce 1.2 billion tons annually. The view of manure as an agricultural
by-product necessitating disposal is, however, shifting and its value is becoming increasingly recognized, despite the fact that it contains high numbers of
microorganisms, including, potentially, a large variety of organisms capable of
causing disease in humans. These organisms comprise a large number of bacterial species, such as E. coli and Salmonella, protozoa and viruses, which are
responsible, in particular, for many of the reported foodborne disease outbreaks. Furthermore, manure can contribute to zoonotic disease through the
introduction of antimicrobial resistance genes, and even antibiotics themselves, into the environment, where they can be horizontally transferred, or
selected for antimicrobial-resistant pathogens, respectively. While manure is a
valuable agricultural commodity, this must be weighed against the risk of
spreading zoonotic disease.
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4
Animal Feed as a Source
of Zoonotic Pathogens
RICHARD A. HOLLEY
Introduction
Steps to effectively control bacterial pathogens in food responsible for causing illness in humans will be possible once it is understood where vulnerabilities lie in the food production, manufacture, distribution and preparation
continuum. Recent foodborne illness outbreaks worldwide, where produce
was contaminated by Salmonella enterica serovars or verocytotoxigenic Escherichia coli (VTEC) (most often E. coli O157:H7), point to a variety of causes,
largely represented by lapses in maintenance of accepted sanitary practice
(Beuchat, 2006). Yet where there has been adoption of these practices, there
appears to have been little recent progress towards reducing overall frequencies of foodborne illness caused by the major bacterial pathogens (CDC,
2009). This suggests that a fresh approach is needed.
Unlike the rest of Europe (Larsson, 2007) and North America (Sapkota et al.,
2007), the Scandinavian countries of Finland (Maijala et al., 2005) and Norway (Vestby et al., 2009) have, for more than 17 years, adopted a policy of zero
tolerance for all serovars of Salmonella in the food and animal feed supply.
This concentration of effort to control Salmonella has yielded reduced rates of
human gastroenteritis caused by these organisms (Mäkelä, 2007), but perhaps
at the expense of illnesses caused by Campylobacter and VTEC. Salmonella control in feed and food in Denmark (Hald et al., 2006; Hermansson, 2007) and
Sweden (Wierup, 2006) is less aggressive than in Norway and Finland, but is
more stringent than in the remaining EU countries.
The link between the frequency of animal carcass contamination by
Campylobacter, Salmonella or VTEC and human illness caused by the consumption of contaminated food of animal origin is well established (Doyle and
Erickson, 2004; Callaway et al., 2008). Evidence indicating that environmental
contamination by manure and waste products from food production can yield
84
© CAB International 2011. Zoonotic Pathogens in the Food Chain
(eds D.O. Krause and S. Hendrick)
Animal Feed as a Source of Zoonotic Pathogens
85
contaminated produce is also strong (Rogers and Haines, 2005; Beuchat,
2006). Our inability to decontaminate produce has highlighted the importance
of the prevention of preharvest contamination of fruits and vegetables to control their contamination at consumption (Beuchat, 2006; Doane et al., 2007).
Infection of livestock and poultry by Campylobacter, Salmonella and E. coli
O157:H7 is followed by variable periods of pathogen shedding (sometimes at
high numbers), but clinical symptoms are rarely seen in the infected animals.
This is also true of Yersinia in hogs (Nesbakken, 2006), but, in contrast, Listeria
monocytogenes causes mortality in small ruminants (sheep and goats) although
it is carried asymptomatically and is shed by cattle (Jemmi and Stephan, 2006).
Further, while L. monocytogenes is ubiquitous in the animal environment, the
other major foodborne illness agents (Salmonella, VTEC and Yersinia) can be
widely distributed but are not truly ubiquitous as animals are commercially
raised in their absence (Forshell and Wierup, 2006). The situation with Campylobacter is poorly understood. Cattle appear to be a major reservoir for E. coli
O157:H7, hogs are a reservoir for Yersinia enterocolitica (Laukkanen et al.,
2009), and poultry are more commonly contaminated by Salmonella and Campylobacter. Sources of these zoonotic pathogens include other animals, the animal environment, and feed and water (Doyle and Erickson, 2004). Intensified
animal production facilitates horizontal transfer of these organisms among
flocks and herds when individual contaminated animals are present. Animal
passage amplifies the numbers of these zoonotic pathogens and accelerates
their spread on the farm.
Studies in the UK and the USA show that VTEC is present on most cattle
farms, at least occasionally, with frequencies of positive faecal samples ranging
from 0.5% to 36%, with a mean of 15.7% (Collins and Wall, 2004). In the USA,
27–31% of dairy, 25–48% of swine herds and 19% of poultry flocks were found
to have animals shedding Salmonella (Callaway et al., 2008), and the latter summary included the observation that 60% of swine farms in Alberta, Canada,
were Salmonella positive. Rogers and Haines (2005) noted that, in the USA,
18% of whole chickens and 45% of turkeys were Salmonella positive, but 95%
of chickens and 2% of turkeys were Campylobacter positive. In the EU, Salmonella contamination of broiler flocks ranged from 0.2% to 66%, and 0–55% of
swine herds and 0–7% of cattle herds were Salmonella positive. The largest
variation in Salmonella frequencies occurred at the country level, with values
being higher in southern Europe (Mäkelä, 2007).
Scandinavian countries reported values of animal and food contamination
by Salmonella of <1% (Maijala et al., 2005; Wierup, 2006). The rare occurrence
of salmonellosis in Finland and Norway (80% of cases are travel related,
according to Lunestad et al., 2007), and the lower frequency in Sweden than
in most of the other countries for which data are available (Table 4.1), suggest
that a solution to the problem of salmonellosis in the rest of the EU and North
America might be evident by examining country-specific policies regarding
animal husbandry. Data from Denmark are less likely to provide a solution
because salmonellosis frequency is higher than in the USA or Canada.
Several years ago, upper limits or National Health Objectives were set in the
USA for illnesses caused by Salmonella, VTEC, Listeria and Campylobacter.
86
R.A. Holley
Table 4.1. Frequencies of human illnesses per 100,000 persons caused by zoonotic bacteria.
Country/region
Escherichia coli
Listeria
Salmonella Campylobacter (verocytotoxigenic) monocytogenes
Yersinia
USAa
16.2
12.7
1.1
<0.29
0.36
EUb
35.7
46.7
1.5
<1.29
2.06
Denmarkc
26.7
–f
–f
–f
–f
Swedend
5.6
–f
–f
–f
–f
Canadae
18.7
29.7
3.1
–f
1.86
a CDC
(2009) (data from 2008). Salmonella frequency in the state of Georgia was 30.2 per 100,000
persons.
b Mäkelä (2007) European Food Safety Agency (data from 2006, applies to 27 EU countries).
c Wegener (2009) Danish Zoonosis Centre (data from 2007), DTU National Food Institute.
d Forshell (2006).
e PHAC (2007) Public Health Agency of Canada (data from 2006).
f Data not available.
Targets were to be achieved by 2010, but in 2009 salmonellosis in the USA was
2.4 times higher than the objective (CDC, 2009).
In the following discussion, the available data will be evaluated to determine
the importance of feed contamination as a vector for zoonotic pathogen contamination of livestock and to examine the link between this contamination
and foodborne illness in humans caused by the pathogens.
Feed as a Potential Disease Vector
The animal feed industry is international, and ingredients are frequently
accessed from developing countries in South America, South Asia and Africa.
The industry plays a vitally important role by formulating livestock and poultry diets to satisfy species-specific nutritional needs. In addition to forages,
grain, seed meals (canola or rapeseed, cotton, soybean, groundnut, safflower),
fats, oils, minerals and vitamins, feed will contain by-products of the agri-food
industry and waste streams from the poultry, livestock (rendered, inedible
protein, carcasses) and marine food (fishmeal) industries (Sapkota et al.,
2007). Although this recycling of nutrients contributes significantly to the
sustainability of agriculture, it may also serve to concentrate, amplify and recycle
zoonotic pathogens as well as redistribute heavy metals and other toxic
substances.
Realization of the risk to human health from an unprotected animal feed
supply has come from the UK and North American experience with mad cow
disease (bovine spongiform encephalopathy, BSE), and dioxin contamination
of poultry feed in Belgium in 1999 (Sapkota et al., 2007) and of hog (pig/
swine) feed in Ireland in 2008 (Wall, 2009).
The animal feed industry since the early 1970s has become large and complex to serve the needs of an integrated and intense animal production industry
Animal Feed as a Source of Zoonotic Pathogens
87
in developed countries. The feed industry in the USA produced 120 million
tons of primary feed in 2004 (Sapkota et al., 2007) and in the EU feed production was estimated to be 420 million tonnes yearly (EFSA, 2008). To address
issues associated with contamination by biological and chemical agents, the
EU officially recognized that animal feed was part of the human food chain by
passing the Feed Hygiene Regulation (EC183/2005). This regulation does not
specify zero tolerance for specific zoonotic bacterial pathogens in animal feed
but does require that they be ‘controlled’ (EC, 2005). In practice, in the EU,
Salmonella control in the feed industry ranges from the very strict control in
place in most of the Scandinavian countries to essentially no strategy at all in
others (Larsson, 2007).
Zoonotic Pathogens in Feed
Work conducted since the early 1990s to evaluate the extent of Salmonella
contamination of animal feed in North America has yielded widely variable
results. Feed ingredient and mixed (compound) feed contained Salmonella in
25–50% of samples (Whyte et al., 2003; Maciorowski et al., 2006, 2007; Sapkota
et al., 2007). Rates of <8% were not uncommon (Jones and Richardson, 2004;
Rodriguez et al., 2006), but these usually climbed once there was uncontrolled
access to the feed (Kidd et al., 2002). Pelleted feed was usually less frequently
contaminated (4–9%) (Jones and Richardson 2004; Bucher et al., 2007). Similarly, in the EU, results from Salmonella analyses of feed varied widely among
countries: from 0.3% in Norwegian fish feed (Lunestad et al., 2007) to <8% in
feed imported to Sweden (Lofstrom et al., 2004), and 3.4% in pig feed before
final heat treatment in Switzerland (Sauli et al., 2005). In the EU in general,
Salmonella was present in compound feed at rates of 0–9.5% and in feed ingredients at rates of <2.5% (Mäkelä, 2007). Younus et al. (2000) reported that
21% of poultry feed samples analysed in Pakistan were Salmonella positive. In
most EU countries, poultry feed is given a lethal heat treatment, often after
the addition of an organic acid (1–2% formic acid) to kill Salmonella (75°C for
>30 s). This is a mandatory requirement in Sweden (Häggblom, 2006) and
Ireland, but 60 s is used in Ireland (Whyte et al., 2003). Factors affecting the
lethality of these treatments and combinations of formic and propionic acid
have been discussed (Maciorowski et al., 2006).
Less work has been done to examine animal feed for contamination by
E. coli O157:H7, and work cited here is from the USA. Hancock et al. (2001)
and Davis et al. (2003) found E. coli O157:H7 present in <0.8% of feed samples.
However, Doane et al. (2007) found the organism present in >17% of fresh
poultry and swine feed in a five-farm survey conducted over a 2-year period.
Levels of feed contamination by both Salmonella and E. coli O157:H7 are
significant when it is considered that cattle consume about 35 kg feed day–1.
Numbers of Salmonella present in feed have been reported to range from 1 cell g–1
to 3 log cfu (colony forming units) g–1 (Lofstrum et al., 2004; Franco, 2005).
The frequencies of feed contamination reported here do not include data
where animals had contact with the feed and have not considered the
88
R.A. Holley
potential growth of both of these organisms in moistened feed. Campylobacter
does not appear to survive long enough in animal feed to be detected (Whyte
et al., 2003; Hansson et al., 2007; Rasschaert et al., 2007). When tests have been
done, Y. enterocolitica has not been found in animal (hog) feed (Gurtler et al.,
2005). In contrast, poor-quality or improperly fermented silage is a vehicle for
the transmission of L. monocytogenes to cattle (Nightingale et al., 2004).
Risk of Feed Ingredient Contamination
Salmonella is regarded in many countries as the most important bacterial
pathogen in feed because it is a major cause of human illnesses worldwide
(Forshell and Wierup, 2006). In the opinion of many, Salmonella-contaminated
feed is of concern because its consumption increases periods of asymptomatic
animal shedding, increases carcass contamination and influences the occurrence of human illnesses (Davies, 2006; Sapkota et al., 2007). Of significant
importance in some countries is the presence of VTEC O157:H7 in feed
(Doyle and Erickson 2004; Doane et al., 2007) because of its potential, as
with Salmonella, to contaminate produce that is not usually cooked before
consumption (Rogers and Haines 2005; Beuchat, 2006).
Although many ingredients may find their way into feed (Sapkota et al.,
2007) the risk that some may be contaminated by Salmonella is greater than
other risks, and this is likely to be true for E.coli O157:H7 as well, although
little information on the latter organism is currently available. Maciorowski
et al. (2007) and Hancock et al. (2001) provided evidence that E. coli O157:H7
is transmitted to cattle by contaminated feed. Animal feed ingredients considered by regulators in most countries to be at high risk of contamination by
Salmonella are rendered animal protein meals and products from the vegetable
oil seed-crushing industry, including meals from soybean seeds, rapeseed
(canola), palm kernels, sunflower seeds, cotton-seed and safflower seeds
(Salomonsson et al., 2005). In addition, fishmeal and eggshells also represent considerable risk of Salmonella contamination when used as feed ingredients (Larsson, 2007). Most often, cereals and cereal products are
considered low-risk materials, but some suppliers (poor performers) can
change the level of risk associated with these ingredients (Hermansson,
2007). In reiterating the high-risk ingredients for Salmonella contamination of feed noted above, EFSA (2008) noted that non-processed soybeans
were often found to be Salmonella positive.
With the withdrawal of bonemeal as a feed ingredient in 1991 – during the
BSE crisis – oilseed meals increasingly have become a popular protein and
energy substitute (Morita et al., 2006). Their more widespread use as feed
ingredients will have a greater influence on the final contamination of
compound feeds by Salmonella.
Salmonella spp. are uniquely adapted to survive in the dry environments
found in oilseed, fishmeal, animal-rendering plants and feed compounding
mills, as well as in poultry-rearing environments, and can survive in niches there
for years (Pedersen et al., 2008). Thermal treatments used in animal-protein
Animal Feed as a Source of Zoonotic Pathogens
89
rendering and oilseed extraction are sufficient to eliminate the organism; however, final meals become contaminated after the heating step. Salmonella can
survive on oily equipment surfaces and floors, be aerosolized in dust in the plant
and recontaminate the meal at cooling. Salmonella spp. are able to grow in
regions of the meal where condensation from the cooling of equipment has
contacted the meal (Morita et al., 2006).
Both E. coli O157:H7 (Davis et al., 2003) and Salmonella have been
frequently found in compounding feed mills (Whyte et al., 2003; Maciorowski
et al., 2006; Lunestad et al., 2007). Although a persistent strain of S. enterica
serovar Senftenberg (S. Senftenberg) was not found to have enhanced resistance
to desiccation (Pedersen et al., 2008), strains of Salmonella more capable of
forming biofilms in feed mills were most likely to contaminate the plant
and feed (Vestby et al., 2009).
As with oilseed crushing mills, in compound feed mills at the pellet forming
operation, condensation at the pellet cooler and contaminated dust at the cooler
air intake have been implicated as sites where Salmonella contamination of feed
occurs (Jones and Richardson, 2004; Maciorowski et al., 2006). Even though thermal treatment of pelleted feed reduced Salmonella contamination, Lo Fo Wong
et al. (2004) found during hog feeding studies that hogs fed pelleted feed were
more likely to be Salmonella positive than those fed mash feed and whey. Particle
size may have been a factor that influenced Salmonella survival in the animals.
None the less, the recently revised and released code of practice for voluntary
control of Salmonella in animal feeds by the UK (DEFRA, 2009) should prove
to be of value in setting standards for minimizing Salmonella contamination
during feed-mill operation.
Animal Feed-derived Human Health Effects
Although it is clear that animals are infected by Salmonella or E. coli O157:H7
in contaminated feed, it is less clearly established whether these infectious
agents in feed are responsible for clinical illness in humans. Proof for such a
relationship is only possible from a continuous line of evidence from surveillance of veterinary and human health programmes that monitor agents in
feed, health effects in animals (detection of infection), presence of the contaminant in animal- (or plant-) based foods, and illnesses in humans. Systems
capable of monitoring sequential movement of a pathogen through this chain
of locations do not exist worldwide. Various reports from many countries have
only characterized either the first or last two steps of the chain of transmission
(Crump et al., 2002; Sapkota et al., 2007).
Perhaps the most complete line of evidence for concluding that feed can
be the source of a zoonotic pathogen causing human foodborne illness comes
from the report of an outbreak of illness in the USA caused by S. enterica serovar Agona (S. Agona) contamination of imported Peruvian fishmeal formulated into poultry feed (Clark et al., 1973). Chickens contaminated by the feed
were consumed and caused illness, initially in Arkansas, but later in several
other US states. In parallel, the Peruvian fish meal was shipped to the UK,
90
R.A. Holley
Israel and the Netherlands, and similarly caused foodborne illness there. This
introduction of S. Agona was visible because at that time (1968) this serovar
was unusual in humans. During the next few years, S. Agona became established and caused over 1 million human cases of salmonellosis in the USA
alone (Crump et al., 2002), and in 2005 it was one of the top ten serovars of
Salmonella isolated from most livestock and poultry species in the USA (Foley
and Lynne, 2008). Currently, the two serovars causing most cases of salmonellosis in the USA are S. enterica serovar Typhimurium (S. Typhimurium)
and S. enterica serovar Enteritidis (S. Enteritidis). Serovars of human-health
importance change over time. In the 1970s, S. Agona was important; in the
1980s S. Enteritidis was most important; and in the 1990s S. Typhimurium DT
104 dominated in the USA (Maciorowski et al., 2007). Franco (2003) questioned the reliability of evidence in the Crump et al. (2002) report linking feed
contamination to human illness, and noted that the majority of S. enterica
serovars isolated from feed were rarely found in humans. Other studies support this observation (Franco, 2005; Häggblom, 2006). Crump et al. (2003)
pointed out that although Salmonella serovars differ in virulence, data indicate
that all serovars have the potential to cause human illness. Hald et al. (2006)
essentially agreed, and the Danish government regulatory authority (Plant
Directorate) used their data in adopting the position that any Salmonella
serovar was dangerous in feed.
Salmonella serovar distribution
Variable results regarding the similarity of Salmonella serovars in feed and
human disease are found in the literature. Although Younus et al. (2009)
found S. Enteritidis or S. Typhimurium in 10–21% of feed in Pakistan, these
serovars were rarely isolated from feed in Sweden (Häggblom, 2006) or
Norway (Lunestad et al., 2007). Of 194 S. enterica isolates serotyped following
their isolation from rendered animal protein meal, Franco (2005) found that,
when combined, 7.5% were S. Typhimurium, S. Enteritidis, S. Agona and S.
enterica serovar Infantis (S. Infantis). Maciorowski et al. (2006) noted earlier
studies in which isolates of S. enterica serovar Hadar (S. Hadar), S. enterica
serovar Heidelberg (S. Heidelberg), S. enterica serovar Virchow (S. Virchow)
and S. Agona from human illness and present in chickens and cattle were
traced back to contaminated bone and fish meal. Lunestad et al. (2007) found
that the most common serovars in Norwegian fish meal were S. Senftenberg
and S. enterica serovar Montevideo (S. Montevideo), which had established
themselves as house strains in feed factories. They also noted from epidemiological studies that common Salmonella serovars in feed ingredients, fish feed
and feed-factory environments accounted for about 2% of the Salmonella
isolates from domestically acquired human salmonellosis cases in Norway.
Similarly, Hald et al. (2006) estimated that 1.7–2.1% of human salmonellosis
cases in Denmark were the result of contaminated feed. Angulo (2004) in the
USA estimated that 10% of foodborne salmonellosis cases were caused by
contaminated feed.
Animal Feed as a Source of Zoonotic Pathogens
91
Underestimating Salmonella serovar importance
An approach to understanding the frequent disparity in serovar identity
between isolates from feed and those from animals (Hald et al., 2006) was
offered by Davies (2006). It was suggested that where differences have been
observed, a source other than feed was likely to be predominant. However,
this conclusion was not considered to have ruled out the possibility that feed
may have been the original source of infection at some earlier point in time.
Complicating understanding of this issue is the different ability of Salmonella
serovars to be dominant in the inhospitable feed environment or the more
hospitable animal intestinal environment. Further, in animals the dominant
serovars change as shedding occurs. Infection may be briefly transient or
sustained for months in animal reservoirs, and require intervention. In addition, the detection of one serovar may obscure the simultaneous presence of
another. A concern here is that an ‘exotic’ or rare strain in imported feed may
become established in new regions (Clark et al., 1973). While there appears to
be doubt about the public-health significance of feed-related Salmonella
serovars, there is little doubt that feed (especially imported products) represents significant risk as a vehicle for the establishment of future epidemic
strains in the food chain (Davies, 2006).
Weight of evidence
At an expert meeting in Rome convened by FAO/WHO (2008), it was concluded that there was insufficient scientific evidence to define the importance
of feed in disease transmission and food safety. There appeared to be little correlation between contaminated feed and the infection of livestock by the same
strain of Salmonella implicated in the contamination of meat, milk or eggs
produced from these animals. Clearly there is a need for further study of the
transmission of zoonotic pathogens, particularly Salmonella, along the feed–
animal–food chain to humans. But even more basic studies are needed to better characterize the dynamics of Salmonella serovar dominance in the chain.
Another neglected area is the contribution that on-farm feed formulation and
mixing of feed may make to the overall burden of animal contamination.
Feed Decontamination
Some study has been directed toward feed decontamination, but there is need
for further work in this area (EFSA, 2008). In the Nordic countries – Sweden,
Norway, Finland and Iceland – all feed must be analysed and found Salmonella
negative before it is delivered to farms (Herland, 2006). Feed for poultry farms
in most EU countries is heat treated at ≥75°C for 30 s to 2 min (Hermansson,
2007; Larsson, 2007; Hendricksen et al., 2008). When found Salmonella positive,
feed is treated first with an organic acid (formic acid) and then heated at
pelleting (Häggblom, 2006). However, Aspar (2007) offered an industry (EU
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R.A. Holley
Grain and Oilseed Trader’s Association) view that when large volumes of bulk
feed are imported, heating becomes expensive and it is difficult to apply
organic acid treatments. Although Hazard Analysis Critical Control Point
(HACCP) systems are strongly recommended in the EU countries, it is not a
requirement on farm at mixing. It was suggested that Salmonella should be a
point of attention (POA) rather than a critical control point (CCP) in feed,
and that the presence of Salmonella serovars other than those mentioned in EU
Regulation 1003/2005 should not trigger the requirement for heat treatment
or a report to the EFSA rapid alert system for food and feed (RASFF). Clearly,
the feed industry is unconvinced that Salmonella in feed represents an important risk to human health, even in Europe. It is also significant that Danish
Salmonella control over increasingly larger quantities of imported feed is not as
stringent as it was when smaller quantities were imported (Hald et al., 2006).
Use of Antibiotics in Feed
The challenge of proving that the use of antibiotics as growth promoters in
feed yields antibiotic-resistant pathogens that cause human foodborne illness
is as complex as proving that zoonotic pathogens in animal feed cause foodborne illness in humans. None the less, the EU has again taken the lead and
has banned antibiotic use at sub-therapeutic levels in animal feed. Presently, a
restricted list is permitted for the latter use in North America, which includes
some antibiotics used in human clinical medicine (Sapkota et al., 2007). Concern has been repeatedly expressed that use of sub-therapeutic levels of antibiotics in feed to promote livestock growth elicits the development of antibiotic
resistance (sometimes multiple resistances) in bacterial populations (Kidd
et al., 2002; Velge et al., 2005; Forshell and Wierup 2006). Examples of resistance development in response to antibiotic use, and the reverse where reductions in isolations of antibiotic-resistant bacteria occur following the removal
of antibiotics from the animal diet have been reported (Sapkota et al., 2007).
Antibiotic use in animals can have undesirable effects, sometimes triggering
the spread of Salmonella through a group of closely confined animals (Forshell
and Wierup, 2006). In a recent study in the USA, multi-drug resistance was
found in 60% of Salmonella isolates from poultry raised conventionally (fed
feed-grade antimicrobials), but these were found in only 11% of isolates from
poultry raised on pasture without access to antibiotics (Siemon et al., 2007). In
animals raised intensively, roughly 10–30% of Salmonella isolates were antibiotic resistant, but this rose to 60–90% when antibiotics were used (Forshell
and Wierup, 2006). Continued use of antibiotics in North American animal
feeds is motivated by the economic advantages associated with greater animal
production efficiency. In addition, there is the suspicion in North America
that withdrawal of antibiotic use as growth promoters will result in a greater
total use of antibiotics for animal therapy in response to the development of
more frequent clinical conditions in livestock and poultry requiring intervention. However, it is likely in the longer term that antibiotics will not be
permitted for use as growth promoters in North America.
Animal Feed as a Source of Zoonotic Pathogens
93
Conclusion
Evidence linking the use of feed contaminated by Salmonella or E. coli O157:H7
to animal infection is strong, and if the expectation is that by reducing the
shedding of these zoonotic pathogens we can reduce human illnesses (Doyle
and Erickson, 2004), then continuing to use feed contaminated by these organisms on farms is not completely logical. If evidence directly linking animal-feed
contamination by these organisms to human illness were stronger, motivation
might exist in developed countries to halt this practice. Our current inability
to control fresh produce contamination by Salmonella and VTEC should serve
as a warning sign that a new approach is needed to reduce our vulnerability to
foodborne illness. That approach must include the prevention of feed contamination by these organisms. Adoption of zero tolerance in feed, at the
beginning of the food continuum, will afford on-farm HACCP plans a critical
control point, and thereby make them useful.
In animal environments where Salmonella contamination is almost ubiquitous, and cycles of animal reinfection are continuous, the use of Salmonellacontaminated feed probably makes little contribution to the level or frequency
of animal contamination (Davies, 2006). However, use of contaminated feed
ensures maintenance of the positive Salmonella status of livestock and poultry
(Sapkota et al., 2007), and lengthens the period and extent of animal shedding. Contamination of poultry and hogs by E. coli O157:H7 from contaminated feed is a recent development; previously, this organism was limited to
cattle (Doane et al., 2007). On farms where Salmonella in the environment and
animals is controlled, the use of Salmonella-contaminated feed can almost
instantly change the Salmonella status of the animals.
As it is accepted that the frequencies of Salmonella and E. coli O157:H7
shedding by animals are directly related to foodborne illness (the link is
strongest with Salmonella and poultry), effort to reduce zoonotic pathogen
shedding in animals should be productive in reducing foodborne illness.
Recent progress has been achieved in the EU (EFSA, 2008) but the pattern of
foodborne illness in the USA during the last 3 years has not responded to the
preharvest interventions adopted there (CDC, 2009). The issue of zoonotic
pathogen contamination of feed has largely been ignored in North America,
but not in Europe. But even in Europe, FAO/WHO (2008) felt there was a
need for more scientific evidence to directly link feed contamination to
human foodborne illness. This is likely to be partly the result of the Finnish
and Norwegian experience, where Salmonella serovars in feed are substantially
different from those causing illnesses in humans (Maijala et al., 2005;
Lunestad et al., 2007). However, the infrequent occurrence of salmonellosis
in humans in those countries makes it difficult to draw such conclusions
legitimately.
Reluctance to tackle the issue of feed contamination by zoonotic pathogens is understandable given the international character, size and complexity
of the feed industry. The existence of reservoirs of Salmonella and E. coli
O157:H7 on the farm serving to reinfect animals also fosters acceptance of the
present situation. As neither Salmonella nor VTEC is yet ubiquitous on farms,
94
R.A. Holley
initiatives to halt the initial introduction of zoonotic pathogens to the farm in
the short term will, with other measures such as HACCP, have an immense
payback in reducing foodborne illness in humans in the longer term.
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5
Milk and Raw Milk
Consumption as a Vector
for Human Disease
STEPHEN P. OLIVER AND SHELTON E. MURINDA
Introduction
In many parts of the world, especially in underdeveloped and developing
countries, the sale of raw milk is commonplace and a large segment of society
consumes raw milk and/or products made from raw milk. An increasing
number of people are consuming raw milk in developed countries as well,
even if the sale of raw milk is discouraged or prohibited by law. Raw milk is
milk from cows, sheep, goats and other animals that has not been pasteurized.
Those that advocate consumption of raw milk cite enhanced nutritional qualities, better taste, demand for natural, unprocessed foods and freedom of
choice as reasons for increased interest in raw milk consumption. On the one
hand, there is a perception by some that raw milk consumption confers health
benefits. On the other hand, raw milk has long been recognized as an important source of pathogens that can cause disease in humans. Consequently,
many public health and regulatory agencies in various countries throughout
the world recommend that milk be pasteurized and oppose the consumption
of raw milk because of the potential risks of foodborne pathogen contamination. Pasteurization is a process in which raw milk is heated for a short time
to destroy pathogens that may be present. Raw milk advocates claim that pasteurization of milk results in several undesirable effects, which, for the most
part, have not been substantiated. The controversy surrounding the consumption of raw or pasteurized milk has been around for decades. However,
the increased demand for raw milk has intensified the raw milk debate – and
so the debate continues! This chapter re-examines some of these issues,
and discusses the potential threats that raw milk consumption poses to
consumers.
© CAB International 2011. Zoonotic Pathogens in the Food Chain
(eds D.O. Krause and S. Hendrick)
99
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S.P. Oliver and S.E. Murinda
Raw Milk Consumption
The sale of raw, unpasteurized milk is allowed in many parts of the world,
including underdeveloped, developing and developed countries, while in
other countries, such as Canada, the sale of raw milk is prohibited. Over 75%
of milk marketed in many developing countries is sold raw through informal
channels (Staal and Kaguongo, 2003). For example, in East Africa, most milk
is produced by smallholders, and the bulk of the milk (86% in Kenya and 92%
in Uganda) is traded through informal channels as unpasteurized milk or
milk products (Grace et al., 2008). These informal milk markets thrive because
they provide social and economic benefits to smallholder producers, small
market agents and consumers in terms of higher farm-gate prices, and they
also create employment and competitive consumer prices (Kang’ethe et al.,
2007). In the USA, it is a violation of federal law to sell raw milk packaged
for consumer use across state lines, although intra-state sale of raw milk is
legal in many states. According to a recent survey by the US National Association of State Departments of Agriculture (NASDA, 2008a,b), 29 states allow
the sale of raw milk by some means. In places where the purchase of raw milk
is prohibited and/or illegal, cow-share or leasing programmes, and the sale of
raw milk as pet food, have been used as means for consumers to obtain raw
milk.
Estimates of raw milk consumption in the USA are difficult to obtain. The
consumption of raw milk has always been common among farm families and
farm employees, ranging from 35% to 60% (Rohrbach et al., 1992; Jayarao
and Henning, 2001; Jayarao et al., 2006), probably because it is a traditional
practice and it is less expensive to take milk from the bulk tank than to buy
pasteurized retail milk (Hegarty et al., 2002). Consumption of raw milk by the
urban community is more difficult to estimate. Headrick et al. (1998), in a
study on the epidemiology of raw milk-associated foodborne disease outbreaks
in the USA from 1973 to 1992, indicated that raw milk accounted for <1% of
the total milk sold in states that permit the sale of raw milk. Headrick et al.
(1997) conducted another study to determine the prevalence of raw milk
consumption in California which, at the time of the study, was the largest producer of certified raw milk in the USA. Among 3999 survey respondents, 3.2%
reported drinking raw milk in the previous year. The demographic and behavioural characteristics of raw milk consumers were as follows: raw milk drinkers
were more likely than non-drinkers to be younger than age 40, male, Hispanic
and to have less than a high school education (Headrick et al., 1997). A more
recent estimate reported that 3.5% of people who participated in a survey
conducted in 2002 consumed unpasteurized milk within a 7-day period before
the survey was taken (CDC, 2004). If the results of this survey and the report
by Headrick et al. (1997) are representative of the US population, this would
imply that over 10.5 million people are consuming raw milk regularly, perhaps
daily. This estimate may be too low based on information from The Weston A.
Price Foundation (2007), a non-profit education foundation that promotes
the consumption of clean raw milk from healthy grass-fed cows, which indicated that the demand for raw milk is growing rapidly – by some estimates at
Milk as a Human Disease Vector
101
40% per year. The concept of ‘produce, sell and buy local’ and the demand
for natural and unprocessed foods are growing consumer trends that are likely
to have resulted in an increased interest in raw milk consumption.
Proposed Benefits of Raw Milk Consumption
A variety of reasons appear to influence the consumption of raw milk by consumers. These include: the perception of a better quality product; enhanced
nutritional qualities; health benefits; superior taste; a demand for natural,
unprocessed food; consumers who are interested in sustainable agriculture
and support producers who use methods that are environmentally friendly;
and freedom of choice. An important tenet of raw milk advocates is that milk
to be pasteurized is inferior in quality to raw milk that is to be consumed
directly. The number of somatic cells in milk, referred to as the somatic cell
count or SCC, is used throughout the world as an indicator of milk quality.
Poor-quality milk has a high number of somatic cells, while excellent-quality
milk has a very low number of somatic cells. The current regulatory limit for
somatic cells in milk in the USA, as defined in the Pasteurized Milk Ordinance, is 750,000 ml–1 (US FDA, 2007). There is continuing pressure in the
USA from a variety of groups and organizations to reduce the regulatory limit
for somatic cells in milk from the current 750,000 ml–1 to 400,000 ml–1 or less
in order to be more competitive with the EU and other countries that have a
lower SCC limit. A recent report published by the US Department of Agriculture (USDA) Animal Improvement Program Laboratory (Norman et al., 2009)
summarized SCC data from all herds in the USA enrolled in the Dairy Herd
Improvement (DHI) testing programme for 2008. The good news is that the
national SCC average for 2008 was 262,000 cells ml–1 of milk, which is 14,000
cells ml–1, or 3%, lower than it was in 2007 (Miller et al., 2008). The bad news,
however, was that 3.4% of herds in the USA had SCCs > 750,000 ml–1, and
22.4% of the national dairy herd had SCCs > 400,000 ml–1. State-average SCCs
were often lower than the national average in the north-east and the far west,
and higher in the south-east, mid-Atlantic and central states, a finding that is
consistent with previous reports. What is not known from this report is which
herds are producing milk that is pasteurized and which are producing milk
that is consumed directly without pasteurization – but to assume that milk to
be consumed directly without pasteurization is a better quality product than
milk to be pasteurized is clearly without merit. Regardless of whether milk is
pasteurized, or consumed directly without pasteurization, some herds produce maximum quantities of very high-quality milk, other herds produce average quantities of average-quality milk, while some herds produce poor-quality
milk.
Raw milk advocates feel that the pasteurization of milk is associated with
lactose intolerance, increased allergic reactions and reduced nutritional value
of milk, and causes pathogens to multiply, destroys antibodies and other
protective bioactive factors found in milk, destroys milk proteins and polypeptides, kills beneficial bacteria, and is associated with the development of
102
S.P. Oliver and S.E. Murinda
arthritis and autism. Most of these claims are anecdotal and/or based on testimonials with little to no science-based data to support the contentions. The
reader is referred to the Marler Blog (http://www.marlerblog.com) for an
overview of the pros (Marler, 2008a) and cons (Marler, 2008b) of consuming
raw milk, and other interesting reviews on raw milk.
Influence of pasteurization on nutritional qualities of milk
Pasteurization is the most effective known method of enhancing the microbiological safety of milk and milk products. Pasteurization is a process that kills
harmful bacteria by heating milk to a specified temperature for a set period
of time. Pasteurization protocols approved and commonly used in the USA
are summarized in Table 5.1. Similar protocols and/or their equivalents are
employed internationally.
Over 25 years ago, Potter et al. (1984) stated that ‘Meaningful differences
in nutritional value between pasteurized and unpasteurized milk have not
been demonstrated, and other purported benefits of raw milk consumption
have not been substantiated’. From a nutritional perspective, the major components of milk, including lactose, casein and most milk proteins, are not
affected significantly following pasteurization (Potter et al. 1984; LeJeune and
Rajala-Schultz, 2009). Heating milk can result in degradation of lactose to
lactulose and epilactose (Lopez-Fandino and Olano, 1999; Teuri et al., 1999),
and large amounts of indigestible carbohydrates such as lactulose can cause
digestive disturbances in individuals who have difficulties digesting lactose.
However, pasteurization generally does not cause detectable levels of lactulose
in pasteurized milk. In addition, pasteurization will kill lactase-producing
bacteria that might be beneficial to those with lactose intolerance (LopezFandino and Olano, 1999; Teuri et al., 1999). Whey proteins such as lactoferrin and immunoglobulins retain their biological activity except following
ultrahigh temperature pasteurization (Paulsson et al., 1993; Li-Chan et al.,
Table 5.1. US FDA’s times and temperatures
for pasteurization of fluid milk.a
Temperature
Time
63°C (145°F)
72°C (161°F)
89°C (191°F)
90°C (194°F)
94°C (201°F)
96°C (204°F)
100°C (212°F)
30 min
15.0 s
1.0 s
0.5 s
0.1 s
0.05 s
0.01 s
a Compiled from US FDA (Food and Drug Administration)
Center for Food Safety and Applied Nutrition (2007).
Milk as a Human Disease Vector
103
1995). Some bovine enzymes in milk are reduced by pasteurization, although
most are not used by humans to aid in digestion. Other enzymes found in low
concentrations in bovine milk, such as lactoperoxidase (Marks et al., 2001),
lysozyme (Fox and Kelly, 2006) and xanthine oxidase (Walstra et al., 1999) are
still active following pasteurization. Pasteurization has little effect on vitamins
A, D, E, and K, but does result in slight reductions in vitamin C (Fox and Kelly,
2006).
Perceived health benefits of raw milk
Raw milk advocates claim that raw milk has medicinal qualities, including
those of allowing lactose-intolerant individuals to consume such milk without
digestive problems, reducing allergies and asthma, and reducing digestive
problems caused by Crohn’s disease. More recently, there have been testimonials that raw milk was associated with the improvement of autistic children. Raw
milk advocates feel that pasteurization of milk is associated with lactose intolerance, increases allergic reactions, destroys antibodies and other protective bioactive factors found in milk associated with disease prevention/resistance, and
is associated with the development of arthritis and autism. Few studies have
been done in the USA or in other countries to prove or disprove the health
benefits associated with the consumption of raw milk, but many of the health
benefit claims are anecdotal or based on testimonials. Additional studies are
certainly needed to determine whether raw milk is indeed associated with
health benefits, and the specific factor(s) in raw milk that are protective.
Some published studies, primarily from the EU, have indicated that children
from farming environments had fewer allergic conditions (asthma, hay fever
and eczema, among others), and that the consumption of raw milk was one of
the protective factors associated with these reduced allergies (Kilpelainen
et al., 2000; Riedler et al., 2000; Wickens et al., 2002; Waser et al., 2007). Other
factors, including barn exposure and contact with animals, were also associated with reduced allergies. Because of the potential health hazards from
foodborne pathogens in raw milk, the authors indicated that raw milk could
not be recommended to prevent allergies.
Nutritional significance of bovine milk and milk products
According to the (US) National Research Council (1995), good nutrition
starts with a balanced diet that provides the necessary levels of essential nutrients and adequate energy. The USDA recommends daily consumption of two
to three servings of dairy products, thus the nutritional significance of these
products cannot be overstated (US Department of Health and Human Services and USDA, 2005). Milk and milk products, primarily from cattle, water
buffaloes, goats, sheep and other species, are important components of the
human diet (LeJeune and Rajala-Schultz, 2009). Inclusion of dairy products
in the diet aids in the prevention of diseases such as obesity, hypertension and
104
S.P. Oliver and S.E. Murinda
diabetes, and dairy products are also a source of calcium, which is important
for growing bones and the prevention of osteoporosis. Furthermore, dairy
products are an important dietary source of protein, vitamins and other minerals. The consumption of milk products has been associated with overall diet
quality and adequacy of intake of many nutrients, including calcium, potassium, magnesium, zinc, iron, riboflavin, vitamin A, folate, vitamin D and protein (McCarron and Heaney, 2004; US Department of Health and Human
Services and USDA, 2005; Huth et al., 2006).
Prevalence of Foodborne Pathogens in Raw Milk and Milk
Products
Prevalence data for raw milk-borne pathogens were obtained from peerreviewed literature published internationally from 1994 to 2009, although
most of the reports were from 2000 to 2009. Prevalence rates for the common
zoonotic pathogens that were isolated worldwide from raw milk and products
derived from raw milk are summarized in Tables 5.2–5.8. The majority of the
reports are from the USA, which has a highly developed capacity for research
and surveillance, and where data are easily available via international publications and the Internet. From the literature that was reviewed, the most commonly researched and/or reported bacterial pathogens from raw milk and
raw milk products were Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp., Staphylococcus aureus and non-O157:H7 Shiga toxin-producing
E. coli, in order of decreasing number of prevalence data reports. Oliver et al.
(2009a) recently reviewed the literature on food-safety hazards associated with
the consumption of raw milk in the USA, focusing primarily on the prevalence
of foodborne pathogens in raw milk and raw milk-borne disease outbreaks.
Other recent reviews on this topic focused on foodborne pathogens in the
farm environment (Oliver et al., 2005) and developments in and the future
outlook for preharvest food safety (Oliver et al., 2009b).
Isolation rates for L. monocytogenes in raw milk (Table 5.2) ranged from
2.7% to 7.0% in North America (Jayarao and Henning, 2001; Muraoka et al.,
2003; Van Kessel et al., 2004; Goff and Griffiths, 2006; Jayarao et al., 2006;
D’Amico et al., 2008) and were higher (12.6%) in in-line milk filters (Hassan
et al., 2000). In Asia (Table 5.3), reported isolation rates of L. monocytogenes
were 0% in China (Chao et al., 2007) and 1.9% in Malaysia (Chye et al., 2004).
In South America (Table 5.4), prevalence rates for L. monocytogenes were 0% in
two studies in Brazil (Nero et al., 2008; Moraes et al., 2009). L. monocytogenes
isolation rates were not available from other continents. Overall isolation rates
for L. monocytogenes reported internationally in raw milk worldwide were 0–7%.
In North America, Salmonella isolation rates (Table 5.2) ranged from 0%
to 11.8% in normal bulk tank milk (Jayarao and Henning, 2001; Murinda
et al., 2002b; Warnick et al., 2003; Van Kessel et al., 2004; Karns et al., 2005; Goff
and Griffiths, 2006; Jayarao et al., 2006; D’Amico et al., 2008; Van Kessel et al.,
2008), and from 1.5% to 66% in in-line milk filters (Hassan et al., 2000; Warnick
et al., 2003; Van Kessel et al., 2008), whereas isolation rates were 15% in
105
Milk as a Human Disease Vector
Table 5.2. Prevalence rates for isolation of bacterial pathogens from bulk tank milk
in the USA and Canada.
Product
Pathogen
Country
Prevalence
rates (%) Reference
Raw milk
Raw milk
Raw milk
Raw milk
Raw milk
Raw milk
Filters
Raw milk
Raw milk
Raw milk
Raw milk
Raw milk
Raw milk
Raw milk
Colostrum
Milk and filters
Milk and filters
Filter
Raw milk
Raw milk
Raw milk
Raw milk
Raw milk
Raw milk
Raw milk
Raw milk
Raw milk
Raw milk
Raw milk
Raw milk
Raw milk
Raw milk
Listeria monocytogenes
L. monocytogenes
L. monocytogenes
L. monocytogenes
L. monocytogenes
L. monocytogenes
L. monocytogenes
Salmonella spp.
Salmonella spp.
Salmonella spp.
Salmonella spp.
Salmonella spp.
Salmonella spp.
Salmonella spp.
Salmonella spp.
Salmonella spp.
Salmonella spp.
Salmonella spp.
Campylobacter jejuni
C. jejuni
Campylobacter spp.
Yersinia enterocolitica
Y. enterocolitica
O157:H7 STECc
O157:H7 STEC
O157:H7 STEC
O157:H7 STEC
Non-O157:H7 STECd
Non-O157:H7 STEC
Non-O157:H7 STEC
Non-O157:H7 STEC
Non-O157:H7 STEC
Canada
USA
USA
USA
USA
USA
USA
Canada
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
Canada
USA
USA
USA
USA
USA
USA
USA
USA
USA
USA
Canada
12.73
12.83
14.63
15.63
4.9–7.0
14.83
12.63
10.17
16.03
12.24
16.13
12.63
12.6/11.8a
10.87
15.87
1.1/12.6
11/666
11.5bb
19.23
12.03
10.47
16.13
11.23
10.23
10.87
10.75
10.87
12.43
13.83
13.96
13.53
10.87
Goff and Griffiths (2006)
Jayarao et al. (2006)
Jayarao and Henning (2001)
Van Kessel et al. (2004)
Muraoka et al. (2003)
D’Amico et al. (2008)
Hassan et al. (2000)
Goff and Griffiths (2006)
Jayarao et al. (2006)
Murinda et al. (2002b)
Jayarao and Henning (2001)
Van Kessel et al. (2004)
Karns et al. (2005)
D’Amico et al. (2008)
Houser et al. (2008)
Warnick et al. (2003)
Van Kessel et al. (2008)
Hassan et al. (2000)
Jayarao and Henning (2001)
Jayarao et al. (2006)
Goff and Griffiths (2006)
Jayarao and Henning (2001)
Jayarao et al. (2006)
Karns et al. (2007)
Jayarao and Henning (2001)
Murinda et al. (2002a)
D’Amico et al. (2008)
Jayarao et al. (2006)
Jayarao and Henning (2001)
Karns et al. (2007)
Cobbold et al. (2008)
Goff and Griffiths (2006)
a Conventional
versus real-time PCR method.
of six isolates was Salmonella enterica serotype Typhimurium DT104.
c O157:H7 STEC, Shiga toxin-producing Escherichia coli O157:H7.
d Non-O157:H7 STEC, Shiga toxin-producing E. coli (non-O157:H7).
b One
colostrum samples (Houser et al., 2008). This implies that feeding colostrum
to calves and the consumption of colostrum by humans could be a potential
health hazard to both calves and humans. A strain of Salmonella enterica serovar
Typhimurium DT104 was isolated from one in six Salmonella-positive milk filter samples (Hassan et al., 2000). In general, isolation rates of pathogens were
higher in in-line milk filter samples than in bulk tank milk. The prevalence
106
S.P. Oliver and S.E. Murinda
Table 5.3. Prevalence rates for isolation of bacterial pathogens from raw milk and milk
products in Asia and the Middle East.
Product
Pathogen
Country
Milk and milk
productsa
Raw milk
Raw milk
Raw milk
Raw milk
Raw milk
Raw milk
Raw milk
Raw milk
O157:H7 STECb
India
O157:H7 STEC
O157:H7 STEC
Listeria monocytogenes
L. monocytogenes
Salmonella
Salmonella
Vibrio parahemolyticus
Mycobacterium avium
subsp. paratuberculosis
M. avium subsp.
paratuberculosis
Malaysia
China
Malaysia
China
Malaysia
China
China
Iran
Raw milk
a It
Iran
Prevalence
rate (%)
1.8
33.5
0
1.9
0
1.4
4
0
110
(herd)
8.6–23
(sample)
Reference
Sehgal et al. (2008)
Chye et al. (2004)
Chao et al. (2007)
Chye et al. (2004)
Chao et al. (2007)
Chye et al. (2004)
Chao et al. (2007)
Chao et al. (2007)
Haghkhah et al.
(2008)
Haghkhah et al.
(2008)
was assumed they were both unpasteurized.
STEC, Shiga toxin-producing Escherichia coli O157:H7.
b O157:H7
Table 5.4. Prevalence rates for isolation of bacterial pathogens from milk and dairy products
in South America and the Caribbean.
Prevalence
rates (%)
Product
Pathogen
Country
Raw milk
Raw milk soft
cheeses
Raw milk
Raw milk soft
cheeses
Raw milk
Salmonella
Salmonella
Brazil
Brazil
0
0
Listeria monocytogenes
L. monocytogenes
Brazil
Brazil
0
0
O157:H7 STECa
Trinidad
a O157:H7
23.6
Reference
Nero et al. (2008)
Moraes et al.
(2009)
Nero et al. (2008)
Moraes et al.
(2009)
Adesiyun (1994)
STEC, Shiga toxin-producing Escherichia coli O157:H7.
rates of Salmonella spp. in Asia (Table 5.3) were 1.4–4% (Chye et al., 2004;
Chao et al., 2007). However, in two studies conducted in Brazil (Table 5.4), the
pathogen was not isolated (Nero et al., 2008; Moraes et al., 2009).
Isolation rates for Campylobacter jejuni were 2.0% (Jayarao et al., 2006) and
9.2% (Jayarao and Henning, 2001) in the USA, whereas, Goff and Griffiths
(2006) reported prevalence rates of 0.47% in Canada for Campylobacter
spp. (Table 5.2).
The prevalence of Shiga toxin-producing E. coli (STEC) in bulk tank milk
was investigated in the majority of studies. Worldwide prevalence rates for the
107
Milk as a Human Disease Vector
Table 5.5. Prevalence rates for isolation of bacterial pathogens from raw milk and milk
products in Europe.
Prevalence
rates (%) Reference
Product
Pathogen
Country
Raw milka
Bulk tank milka
Fresh cheese
curdsa,d
Cheesea,d
Raw milk
O157:H7 STECb
Non-O157:H7 STECc
Non-O157:H7 STEC
Spain
Spain
Spain
0.3
10.8
3.9
Spain
UK
5.
1.6
Raw milk
Non-O157:H7 STEC
Mycobacterium
paratuberculosis
M. paratuberculosis
Switzerland
19.7
Raw milk
Tick-borne encephalitis
Czech
Republic
11.7
Rey et al. (2006)
Rey et al. (2006)
Rey et al. (2006)
Rey et al. (2006)
Goff and Griffiths
(2006)
Corti and Stephan
(2002)
Kríz et al. (2009)
a Milk
and milk products from goats and sheep.
O157:H7 STEC, Shiga toxin-producing Escherichia coli O157:H7.
c Non-O157:H7 STEC, Shiga toxin-producing E.coli (non-O157:H7); the non-O157:H7 STEC serotypes
isolated from these products included E. coli O27:H18, O45:H38, O76:H19, O91:H28, ONT:H7, ONT:H9
and ONT:H21.
d Made from raw milk.
b
Table 5.6. Prevalence rates for isolation of bacterial pathogens from raw milk and milk
products in Africa.
Product
Pathogen
Country
Raw milk
Raw milk
Raw milk
Raw milk
Jbenb
Raw milk
O157:H7 STECa
O157:H7 STEC
O157:H7 STEC
O157:H7 STEC
O157:H7 STEC
Brucella abortus
Kenya
Uganda
Egypt
Morocco
Morocco
Kenya
Prevalence
rates (%) Reference
0.7–0.8
11.2
16.2
14.3
40.2
0.0–10c
Kang’ethe et al. (2007)
Nasinyama and Randolf (2005)
Abdul-Raouf et al. (1996)
Benkerroum et al. (2004)
Benkerroum et al. (2004)
Arimi et al. (2005)
a O157:H7
STEC, Shiga toxin-producing Escherichia coli O157:H7.
fresh Moroccan cheese.
c Prevalence rates increased with increasing intensity of farming operation.
b Traditional
isolation of O157:H7 STEC in raw milk ranged from 0% to 33.5% (Tables
5.2–5.6). Low prevalence rates of 0–0.75% (Table 5.2) were established in the
USA (Murinda et al., 2000a; Jayarao and Henning, 2001; Karns et al., 2007;
D’Amico et al., 2008). The pathogen was found at the high prevalence rates of
33.5% (Table 5.3) and 23.6% (Table 5.4) in raw milk in Malaysia (Chye et al.,
2004) and Trinidad (Adesiyun, 1994), respectively. In general, most researchers have indicated low prevalence rates for non-O157:H7 STEC in bulk tank
108
S.P. Oliver and S.E. Murinda
Table 5.7. Prevalence rates for isolation of mastitis pathogens in raw milk worldwide.
Pathogen
Staphylococcus aureus
S. aureus
S. aureus
S. aureus
S. aureus
S. aureus
S. aureus
S. aureus
S. aureus
Coagulase-negative
Staphylococcus spp.
Coagulase-negative
Staphylococcus spp.
Streptococci
Streptococcus agalactiae
S. agalactiae
Mycoplasma
Country
Prevalence rates (%)
USA
USA
USA
USA
Kenya
Malaysia
Trinidad
China
Brazil
USA
42a
Reference
Houser et al. (2008)
Jayarao et al. (2004)
Khaitsa et al. (2000)
D’Amico et al. (2008)
Ombui et al. (1992)
Chye et al. (2004)
Adesiyun (1994)
Chao et al. (2007)
Moraes et al. (2009)
Gillespie et al. (2009)
31
37b
27.4
61
60
94.3
34
30.9
11.4c
USA
>74a
Houser et al. (2008)
USA
USA
USA
USA
>71a
10
0
0
Houser et al. (2008)
Jayarao et al. (2004)
Khaitsa et al. (2000)
Khaitsa et al. (2000)
a Colostrum.
b Herd
prevalence.
prevalence.
c Quarter
Table 5.8. Prevalence rates for isolation of bacterial pathogens in raw non-bovine milk
and milk products worldwide.
Product
Pathogen
Country
Prevalence
rates (%) Reference
Water buffalo milk O157:H7 STECa
Water buffalo milk Non-tuberculosis
mycobacteria
Ewe milk
O157:H7 STEC
Ewe milk
O157:H7 STEC
Goat milk
O157:H7 STEC
Listeria
Goat milk
monocytogenes
Goat milk
Tick-borne
encephalitis
Goat cheese
O157:H7 STEC
Turkey
Brazil
1.4
21.7
Seker et al. (2008)
Jordao et al. (2009)
Spain
Greece
USA
USA
3.5
1
0.75
17
Czech Republic
56.3
Caro et al. (2006)
Dontorou et al. (2003)
D’Amico et al. (2008)
Abou-Eleinin et al.
(2000)
Kríz et al. (2009)
Morocco
0
Camel milk
O157:H7 STEC
Morocco
0
Ewe milk cheese
Tick-borne
encephalitis
Czech Republic
a O157:H7
STEC, Shiga toxin-producing Escherichia coli O157:H7.
33
Benkerroum et al.
(2004)
Benkerroum et al.
(2004)
Kríz et al. (2009)
Milk as a Human Disease Vector
109
milk in North America compared with O157:H7 STEC. Non-O157:H7 STEC
has also been isolated (Rey et al., 2006) in raw milk products such as fresh
cheese curds (3.9%) and cheese (5%) in Spain (Table 5.5).
Prevalence rates of some less frequently targeted zoonotic pathogens from
raw milk, such as Yersinia, Vibrio, Brucella and Mycobacterium spp., have also
been reported; rates reported for Yersinia enterocolitica in the USA (Table 5.2)
were 1.2% (Jayarao et al., 2006) and 6.1% (Jayarao and Henning, 2001). In
Kenya (Table 5.6), Brucella abortus was isolated in 0–10% of raw milk samples
that were tested; the prevalence rates increased with degree of intensity of
dairying operations (Arimi et al., 2005). In studies conducted in China (Table
5.3), Vibrio parahemolyticus was not isolated from any of the samples that were
evaluated for the pathogen (Chao et al., 2007). Mycobacterium avium subsp.
paratuberculosis (MAP) was isolated in 1.6% (Goff and Griffiths, 2006) and
8.6–23% (Haghkhah et al., 2008) of raw milk samples in the UK (Table 5.5) and
Iran (Table 5.3), respectively; additionally, herd prevalences of 11% were established in Iran (Table 5.3) (Haghkhah et al., 2008). Corti and Stephan (2002)
reported that 19.7% of bulk tank milk samples in Switzerland were positive for
MAP (Table 5.5). Bovine tuberculosis due to Mycobacterium bovis is a major
cause of human gastrointestinal tuberculosis in developing countries where
raw milk is consumed; Bonsu et al. (2000) reported prevalences for M. bovis of
13.8% in cattle, and as high as 50% in some kraals (i.e. cattle pens) in Ghana.
Interestingly, studies conducted in the Czech Republic (Table 5.5) indicated
that 11% of raw bovine milk samples tested positive for tick-borne encephalitis,
a viral disease that is normally transmitted via tick bites (Kríz et al., 2009).
Prevalence of mastitis pathogens in raw bovine milk
The percentage of bulk tank milk samples that were positive for S. aureus, the
major pathogen associated with contagious mastitis worldwide, ranged from
27.4% to 94.3% (Table 5.7). In the USA, colostrum had higher S. aureus prevalence rates (42%) than raw milk (Jayarao et al., 2004; D’Amico et al., 2008;
Houser et al., 2008). In other countries, prevalence rates ranged from 30.9%
to 94.3%. These rates were higher in countries with hotter climates, namely,
Kenya, Malaysia and Trinidad (Table 5.7). Contamination of raw milk and
colostrum with coagulase-negative Staphylococcus spp. (CNS) (Houser et al.,
2008; Gillespie et al., 2009) and Streptococcus species (Houser et al., 2008),
including Streptococcus agalactiae (Jayarao et al., 2004), has also been reported.
In the USA, Jayarao et al. (2004) reported an increase in the frequency of isolation of Staphylococcus aureus and Streptococcus agalactiae, which were significantly
associated with increased bulk tank milk somatic cell counts. Staphylococcus
aureus isolated from the milk of cows with mastitis has been demonstrated to
harbour enterotoxin genes at high frequencies (Srinivasan et al., 2005). This is
a concern of epidemiological significance to raw milk consumers because
S. aureus is a common zoonotic foodborne pathogen. The CNS isolated by
Gillespie et al. (2009) from milk samples were further characterized by Sawant
et al. (2009), who demonstrated that some of the Staphylococcus epidermidis
110
S.P. Oliver and S.E. Murinda
samples isolated from milk were antibiotic resistant; these could pose a publichealth hazard to those who consume raw milk. Khaitsa et al. (2000) did not
isolate Mycoplasma or Streptococcus agalactiae from the raw milk samples they
evaluated.
Bovine colostrum is used traditionally for feeding dairy calves and providing
passive immunity to calves that are born hypogammaglobulonaemic. More
recently, colostrum has gained popularity as a human food because it has been
advocated as an excellent source of bioactive proteins, which have been
claimed to inhibit viral and bacterial pathogens, improve gastrointestinal
health and enhance body condition (Houser et al., 2008). In a study that was
conducted to determine bacteriological quality and the occurrence of Staphylococcus aureus, Streptococcus agalactiae, CNS, other streptococci and other
parameters, bovine colostrum was associated with high contamination rates
with milk-borne pathogens, with Staphylococcus aureus (42%), CNS (>74%) and
Streptococcus spp. (>71%) predominating (Houser et al., 2008). Thus, consumption
of unpasteurized colostrum could pose a potential health risk to those consuming this product. Results from these studies also emphasize that pathogens are
frequently found in colostrum even though it contains high concentrations of
bioactive compounds and antibacterial factors such as antibodies and lactoferrin
(Oliver and Sordillo, 1989).
Contamination of non-bovine milk samples by foodborne pathogens
Although worldwide milk and milk products come primarily from dairy cattle,
in some countries water buffaloes, goats, sheep and other species are important milk-producing animals. With regard to carriage of zoonotic foodborne
pathogens, these alternative sources of raw milk for consumers are not any
safer than bovine milk (Table 5.8). In the USA, Abou-Eleinin et al. (2000) isolated L. monocytogenes from 17% of raw goat bulk milk samples that were tested.
D’Amico et al. (2008) isolated E. coli O157:H7 from one goat milk sample
(prevalence rate, 0.75%; n = 49), whereas milk samples from cows (n = 62) and
sheep (n = 22) were found to be negative for the pathogen.
A total of 1% of raw ewe’s milk samples that were tested in Greece were
positive for E. coli O157:H7 (Dontorou et al., 2003), whereas in Turkey 1.4%
of raw milk samples from water buffaloes tested positive for this pathogen
(Seker et al., 2008). Escherichia coli O157:H7 was also isolated from 3.5% of
ewe’s milk samples in Spain (Caro et al., 2006). Benkerroum et al. (2004) did
not isolate this pathogen from the goat’s cheese and camel milk they analysed.
Unpasteurized goat milk (56.3%) and sheep milk cheeses (33%) made from
unpasteurized milk were associated with tick-borne encephalitis (a disease
that is commonly associated with tick-bite transmission) in the Czech Republic
(Kríz et al., 2009); correspondingly lower prevalence rates, i.e. 11% of dairy
cow milk samples, were associated with this disease. Raw milk from water buffalo is popularly used for the manufacture of mozzarella cheese in Brazil; five
of 23 (21.7%) water buffalo milk samples that were examined tested positive
for non-tuberculosis mycobacteria (NTM), which are considered to be emerging
Milk as a Human Disease Vector
111
milk-borne pathogens in Brazil (Jordao et al., 2009). The isolation of opportunistic NTM pathogens, such as Mycobacterium kansaii, Mycobacterium simiae and
Mycobacterium lentiflavum, represents a risk to consumers of mozzarella cheese
made from raw buffalo milk.
Potential Threats that Raw Milk Consumption Poses to
Consumers
Milk can harbour a variety of microorganisms and can be an important source
of foodborne pathogens. The presence of foodborne pathogens in milk can
be due to direct contact with contaminated sources in the dairy farm environment and to excretion from the udder of an infected animal. Raw milk and
raw milk products have long been regarded as an important source of zoonotic
bacterial pathogens that cause disease in humans. The symptoms caused by
the foodborne pathogens found in raw milk range from nausea, vomiting,
chills and diarrhoea to, in some cases, death (Table 5.9). For example, L. monocytogenes is associated with flu-like symptoms, diarrhoea and meningitis, and
can also cause abortions. O157:H7 STEC is associated with several different
conditions including haemorrhagic colitis, haemolytic uraemic syndrome
(HUS), thrombotic thrombocytopenic purpura (i.e. HUS + fever), kidney
failure, fever and death.
People continue to consume raw milk even though numerous epidemiological studies have shown clearly that it can be contaminated by a variety of
pathogens, some of which are associated with human illness and disease.
Table 5.9. Diseases caused by zoonotic pathogens commonly isolated from milk and milk
products.a
Zoonotic pathogen
Disease symptoms
Salmonella spp.
Nausea, vomiting, abdominal pain, headache, chills,
diarrhoea, fever
Listeria monocytogenes
Yersinia enterocolitica
Flu-like illness, diarrhoea, meningitis, abortions
Diarrhoea, fever, vomiting, abdominal pain
Vibrio parahemolyticus
Acute gastroenteritis, nausea, vomiting, abdominal cramps,
fever, chills, diarrhoea
Nausea, vomiting, diarrhoea
Haemorrhagic colitis, haemolytic uraemic syndrome (HUS),
thrombotic thrombocytopenic purpura (HUS + fever), kidney
failure, death, fever
Profuse diarrhoea, sometimes bloody diarrhoea, stomach
cramps, nausea, dizziness, fever
Staphylococcus aureus
Shiga toxin-producing
Escherichia coli O157:H7
Campylobacter jejuni
Viruses
a Compiled
Gastroenteritis, fever, vomiting, diarrhoea
from Mortimore and Wallace (1994).
112
S.P. Oliver and S.E. Murinda
Several documented milk-borne disease outbreaks occurred from 2000 to
2008 and were traced back to the consumption of raw unpasteurized milk
(Oliver et al., 2009a). Numerous people were diagnosed with infections, some
were hospitalized, and a few died. In the majority of these outbreaks, the
organism associated with the milk-borne outbreak was isolated from the implicated product(s) or from subsequent products made at the suspected dairy or
source. In contrast, fewer milk-borne disease outbreaks were associated with
consumption of pasteurized milk during this same time period. For a comprehensive review of the hazards associated with the consumption of raw milk see
Oliver et al. (2009a).
Summary
Safety and quality of dairy products start at the farm and continue throughout
the processing continuum. One thing is certain – it is impossible to transform
a low-quality raw milk product into a high-quality finished dairy product! To
meet increased raw milk quality standards, dairy producers must adopt production practices that reduce mastitis and reduce the bacterial contamination
of bulk tank milk. Use of effective management strategies to minimize the
contamination of raw milk and proven mastitis control strategies will help
dairy producers achieve these important goals. However, it is important to
recognize that use of these methods will not eliminate the risk of pathogen
contamination of raw milk and the potential for milk-borne diseases.
An increasing number of people are consuming raw unpasteurized milk.
Enhanced nutritional qualities, superior taste, health benefits, demand for
natural, unprocessed foods and freedom of choice, among others, have all
been advocated as reasons for increased interest in raw milk consumption,
although science-based data to substantiate these claims are limited or lacking. Although milk and milk products are important components of a healthy
diet, if consumed unpasteurized, they can present a health hazard resulting
from possible contamination with pathogenic bacteria. People continue to
consume raw milk even though numerous studies have shown clearly that it
can be contaminated by a variety of pathogens, some of which are associated
with human illness and disease.
Where raw milk is offered for sale, strategies to reduce the risks associated
with raw milk and products made from raw milk are needed. The development of uniform regulations, including microbial standards for raw milk to be
sold for human consumption, the labelling of raw milk, improving sanitation
during milking, and enhancing and targeting educational efforts directed
towards producers and consumers are potential approaches to this issue.
Development of on-farm preharvest and postharvest control measures to
effectively reduce bacterial contamination is critical to the control of pathogens in raw milk. The International Dairy Foods Association and the US
National Milk Producers Federation have called for legislation requiring all
facilities producing raw or unpasteurized milk products for direct human consumption to register with the US Food and Drug Administration (FDA) and
Milk as a Human Disease Vector
113
adhere to food-safety requirements that are followed by all other facilities
producing milk products.
Liability is another very important aspect that should be considered by
both producers and consumers of raw milk. Dairy producers supplying raw
milk must be well informed of the risks and liabilities associated with the milk
they sell to consumers. In addition, consumers purchasing raw milk need to
recognize the inherent risks of the product and that they may not have protection if they become ill from consuming the contaminated product.
Advocates of raw milk are long on the proposed benefits of consuming
raw milk but short on data to support most of these claims. Given the current
body of knowledge on this topic, it is clear that the disadvantages of raw milk
consumption far outweigh the proposed benefits. Even in studies that have
shown a benefit of raw milk consumption for allergic conditions, authors of
many of these studies do not recommend this practice because of the potential health hazards from contamination by foodborne pathogens. Additional
science-based studies are needed to evaluate raw milk quality, determine the
potential benefits of raw milk consumption and delineate the factors in milk
that are beneficial. However, until those studies are done, one sure way to prevent raw milk-associated foodborne illness is for consumers to refrain from
drinking raw milk and from consuming dairy products manufactured using
raw milk.
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6
The Contribution of Antibiotic
Residues and Antibiotic
Resistance Genes from
Livestock Operations
to Antibiotic Resistance
in the Environment
and Food Chain
PEI-YING HONG, ANTHONY YANNARELL AND RODERICK I.
MACKIE*
Introduction
Antibiotic-resistant bacteria and their antibiotic-resistance genes (ARGs) have
become emerging contaminants of concern in the 21st century. One of the
main reasons that this issue is gaining public interest is the adverse health
impacts that are associated with these contaminants. According to the World
Health Organization (WHO), deaths arising from acute respiratory infections,
diarrhoeal diseases, measles, AIDS, malaria and tuberculosis account for more
than 85% of the mortality from infection worldwide. Further compounding
the problem is that the pathogens causing these diseases are gaining resistance to first-line drugs for treatment of disease, and, in some instances, resistance to second- and third-line drug agents. An additional complication is that
immunocompromised groups are at a heightened risk of developing adverse
health implications from these infections (WHO, 2001).
Many factors can possibly account for the emergence of antibiotic resistance.
The rampant use of antibiotics in clinical settings, coupled with a large pool of
* Corresponding author.
CAB International 2011. Zoonotic Pathogens in the Food Chain
(eds D.O. Krause and S. Hendrick)
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immunocompromised patients, has resulted in selective pressure that favours
the rapid emergence of antibiotic-resistant bacterial strains (McDonald et al.,
1997). For example, one of the most notorious antibiotic-resistant bacterial
strains to have emerged from clinical settings includes methicillin-resistant
Staphylococcus aureus (MRSA). In the USA in 2005, MRSA has been associated
to 18,650 deaths (Klevens et al., 2007), and MRSA-associated infections have
also been widely reported globally (Diekema et al., 2001). To curb the dissemination of antibiotic-resistant bacteria from clinical settings, initiatives that
promote the prudent use of antibiotics and consumer education are currently
in place to reduce unnecessary antibiotic prescriptions (CDC, 2009).
Despite these efforts, it is widely recognized that the emergence of antibiotic
resistance still remains as a serious threat, in part because of the use of antibiotics
in livestock-production operations. Antibiotics are routinely used in livestock
production for therapeutic treatment of disease, at sub-therapeutic concentrations to prevent disease (prophylaxis) and for growth promotion. The classes of
antibiotics so used include tetracyclines, macrolides, lincosamides, polypeptides,
streptogramins, cephalosporins, penicillins, sulfonamides, aminoglycosides and
fluoroquinolones (Schmidt, 2002; Chee-Sanford et al., 2009), all of which include
drug members that were originally intended for disease treatment in humans
but are also being used for livestock-production purposes.
Because the use of antibiotics in livestock production may lead to increased
emergence and spread of antibiotic-resistant bacteria, it is a cause for concern
and requires deeper understanding in order to control and prevent the dissemination of resistance. This chapter aims to provide a summary of the global
use of antibiotics in the livestock industry, and of the possible dissemination
routes of antibiotic resistance from livestock-production operations into the
environment. Scientific evidence will be provided to support claims that antibiotic use in livestock production has adversely affected the environment.
Lastly, we provide an outlook on the potential health impact and the current
knowledge gaps that need to be addressed to tackle this global problem.
Global Use of Antibiotics in the Livestock Industry
The use of antibiotics in livestock production seems to be a ubiquitous practice.
Swine, poultry and cattle production, as well as aquaculture, have reported
the use of antibiotics for the treatment (i.e. therapeutic use) of and prevention (i.e. prophylactic use) of animal diseases, and for growth promotion. An
estimate of the quantity of antibiotics used in animal husbandry is difficult to
obtain and is often not reliable. For example, the Animal Health Institute
(AHI) and Union of Concerned Scientists (UCS) have reported two different
estimates of antibiotic usage in animal production in the USA. AHI reported
that approximately 9.3 million kg of antibiotics were sold for animal use in
1999, while UCS reported that more than 11.2 million kg of antibiotics were
used for non-therapeutic purposes alone (Chee-Sanford et al., 2009). In the
USA, no regulation is currently enacted to ban the use of antibiotics for livestock production that were originally intended for use in human medicine.
Antibiotic Residues and Resistance Genes in Livestock
121
Livestock production companies can, however, voluntarily withdraw the
use of antibiotics in their production systems, or consumers can purchase
meat produced without the use of antibiotics (Schmidt, 2002), usually at
increased cost.
In the EU, antibiotics that are used in human medicine have been progressively phased out from being added to animal feed as growth promoters. The
animal growth promoter avoparcin, a glycopeptide antibiotic related to vancomycin, was one of the first such antibiotics to be banned in 1997, followed by
tylosin, spiramycin, bacitracin and virginiamycin in 1999. Subsequently, a ban
on monensin sodium, salinomycin sodium, avilamycin and flavomycin for
growth promotion entered into effect from 2006 (Casewell et al., 2003; Europa,
2005). However, it is noted that antibiotics are still allowed and critical for
therapeutic and prophylactic purposes. Also, a ban on the use of antibiotics as
growth promoters has not necessarily equated to an end of the resistance
problem. Casewell et al. (2003) argued that banned antibiotics that were previously used as growth promoters also showed prophylactic effects. A ban on
their use has led to an increase in the number of diseased animals and an
associated increase in the usage of therapeutic antibiotics in animal feed.
This may explain why the amount of antibiotics used in the EU, although
lower than in the USA, has remained high even after the ban. For example,
the European Federation of Animal Health reported in 1998 that approximately 5 million kg of antibiotics were used for veterinary applications and
for growth promotion, a mere twofold lower consumption than in the USA
(Barton, 2000).
In China, regulatory and surveillance frameworks to encourage the
responsible use of antibiotics in animal husbandry are generally not in place.
Public awareness of the potential health impact remains low, and data on the
quantity of antibiotics given to livestock are not easily available to the public or
government agencies. WHO estimates that quinolone consumption in animals approximates 0.5 million kg year–1 in China (WHO, 1998). In a report
published by WHO, it was noted that in practice other antibiotics such as tetracyclines and the mycelial by-products from the production of antibiotics are
also added into animal feeds (Jin, 1997). As developing countries typically rely
on the agricultural industry to contribute to their gross domestic product,
concentrated animal farming operations (CAFOs) are becoming the norm,
and the use of antibiotics in the feed formulations of these operations is likely
to increase even further in the near future (Sarmah et al., 2006).
In addition to swine, poultry and beef livestock production, aquaculture is
a rapidly growing industry in both developing and developed countries, and it
is estimated that more than twofold growth in industrial aquaculture was achieved
worldwide over the past 15 years (Naylor et al., 2000; Cabello, 2006). Antibiotics
such as oxytetracycline, erythromycin and sulfonamides are typically administered in the feed or medication that is added to ponds and holding tanks in
aquaculture (Sorum, 2006). In many instances, fish do not effectively metabolize antibiotics and will excrete largely unused and untransformed antibiotic
residues back into the environment. Coupled with the good mobility and,
therefore, the better dissemination capabilities of water, it is anticipated that
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the use of antibiotics in aquaculture will have an environmental and health
impact that is equal to or more adverse than that of swine, poultry and beef
livestock production (Cabello, 2006).
Despite the lack of statistically reliable data on the global usage of antibiotics
in livestock production, it is clear that the use of antibiotics that were originally
intended for use in human medicine is now routine in livestock production
(Chee-Sanford et al., 2009). As such, animal production and animal feed have
often been implicated in the spread of antibiotic resistance, with adverse
impacts on human medicine.
Sources of Antibiotic Residues and Antibiotic Resistance Genes
and their Dissemination Routes
With the increase in the global demand for meat production, the livestock
industry responded by implementing the widespread use of CAFOs to meet
production demands. Livestock housed in CAFOs are concentrated in a single location at high densities, typically ranging from a few hundred to a few
thousand animals at one location. To achieve rapid weight gains and to keep
animals healthy under such conditions, most CAFOs resort to administering
antibiotic growth promoters to the animals through their feeding (Aarestrup
and Jenser, 2007). The US Food and Drug Administration (FDA) defines a
subtherapeutic concentration of antibiotics as an amount of <0.2 g kg–1 of
feed. In practice, the actual amount of antibiotics added to the feed varies
according to the class and treatment effectiveness of the drug. On a daily
basis, some amount of these antibiotics may be atmospherically dispersed
into the environment through the ventilation fans equipped in a CAFO and
through the dispersion of dust generated during feed mixing and the filling
of feed troughs.
Given the dense population of livestock within a single confined area, a
large quantity of manure is also produced. It is estimated that each pig typically
produces approximately 1500 kg of manure during a 5–6 month production
cycle, and that each gram of animal manure in turn contains approximately
1011–1015 bacteria (Dowd et al., 2008). Besides the high bacterial load emitted
on a daily basis, animal faeces and urine also contain antibiotic residues and
their breakdown by-products. For example, approximately 50–90% of the
erythromycin parent compound is excreted via faeces, and approximately
10% is discharged in the urine. The remaining percentage is metabolized by
demethylation in the body to generate erythromycin-H2O, and is discharged
from the body as waste products (Schlüsener et al., 2006; Yang et al., 2006).
Owing to the large amount of waste products generated on a daily basis, routine cleaning of the housing areas is necessary to maintain a basic level of
hygiene within the confined area. Typical management practices include
scraping and flushing waste products in the housing areas to a trench. As illustrated in Fig. 6.1, accidental spillage and atmospheric dispersion of antibiotics
into the environment can happen at this point by releases via construction
flaws (e.g. broken liners or cracks in trenches).
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Antibiotic Residues and Resistance Genes in Livestock
Concentrated animal farming operation
Waste lagoon
Atmospheric dispersion of
antibiotic contaminants
Leaching through flawed liner
Accidental spillage while
transporting manure to
lagoon
Daily cleaning and accidental release
through engineering flaws
Land application
Affected ecosystems
Soil
Ground water
Surface water
Vertical seepage and surface
runoff
Fig. 6.1. Possible dissemination routes for antibiotic contaminants in concentrated livestock
production farms.
In most CAFOs, the waste products are next transported to an aerated
lagoon for removal of biochemical and chemical oxygen demand, as well as
volatilization of the organic nitrogen content. Solid retention time (SRT) in a
manure lagoon is typically 5–30 days depending on the extent of treatment
desired (Burton and Turner, 2003). However, this treatment process is not sufficient to remove all antibiotics present in the manure. For instance, given a
10-day SRT, Kim et al. (2005) reported that the removal of tetracycline in an
activated sludge process was approximately 85%, and was primarily achieved
by physical sorption of antibiotics to the sludge flocs. Therefore, the removal
efficiency of an antibiotic is highly dependent on the sorption capabilities
exhibited by that particular antibiotic. Although lagoon treatment achieves a
certain percentage removal of antibiotics from the manure, a major limitation
of such lagoons is that they are susceptible to deterioration with time. Leachate
contaminated with antibiotics of low sorption capabilities can diffuse
through flawed liner or sealing systems and contaminate groundwater
sources (Fig. 6.1). Furthermore, manure lagoons require periodic removal of
the accumulated solids that are contaminated with antibiotics of high sorption capabilities. Thus antibiotic contaminants are merely transported from
the manure lagoon to another location during the desludging process.
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Subsequently, the treated manure, which may still contain antibiotic contaminants, is disposed of by land application (Fig. 6.1), which is a common
practice in most CAFOs, as faecal material contains a high content of nitrogen
and/or phosphorus, and it can be used to enrich soil for agriculture (CheeSanford et al., 2009). Land application is also one of the most economical ways
to dispose of waste products. However, this practice can facilitate the dissemination of antibiotic contaminants into the soil and water environment. For
instance, antibiotics that were previously adsorbed on to the manure particulates may leach out to contaminate groundwater and nearby surface waters
during seasons of heavy rainfall, and under extreme irrigation conditions
(Blackwell et al., 2009).
Transfer of Antibiotic-resistance Genes at the Microbial Cell
Level
Upon entry into the environment, different antibiotics have different halflives and fates, and these may include physical sorption on to solid particulates,
chemical degradation and biodegradation (Chee-Sanford et al., 2009). Some
antibiotic contaminants can persist for up to 100 days in the manure (Boxall
et al., 2006; Chee-Sanford et al., 2009), and coupled with regular dosing and
application of the contaminated manure on to fields, the build-up of antibiotic
residues will favour the selection of antibiotic-resistant bacteria in the environment. Together with the antibiotic-resistant bacteria that originate from the
manure, these bacteria can promiscuously disseminate a vast amount of ARGs
via mobile genetic elements.
Plasmids, transposons and integron gene cassettes are three different
types of mobile genetic elements that can disseminate ARGs among microorganisms (Fig. 6.2). A plasmid is defined as a collection of functional genetic
modules that are organized into a stable, self-replicating entity, and usually
confers non-core genetic functions (e.g. antibiotic resistance) on to the microbial recipient (Frost et al., 2005). The first resistance plasmid (R plasmid) was
discovered in Japan in strains of enteric bacteria, and was found to carry more
than one ARG. For example, plasmid R100 carries genes encoding resistance
to sulfonamides, streptomycin, spectinomycin, chloramphenicol and tetracycline. Furthermore, it can be transferred promiscuously between various
enteric bacteria, therefore making it a serious threat to human health (Mathur
and Singh, 2005; Madigan, 2009). Transposons are genetic elements that can
relocate or transpose themselves to different positions within the chromosomal DNA or plasmid. A key distinctive feature of transposons is that they
carry transposase, which is an enzyme necessary for transposons to move
around the chromosomal DNA (Madigan, 2009). An integron gene cassette is
a combination of an integron, which is a chromosomal insertion site for genes,
and one or more modular gene cassettes that encode the functional genes. An
integron gene cassette has several distinctive traits: it comprises the integrase
gene (int), an adjacent recombination site (attI), and one or more gene cassettes
that can be expressed based on the promoter in the integron (PANT) (Hall and
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Antibiotic Residues and Resistance Genes in Livestock
Donor bacterial cell
Recipient bacterial cell
Bacteriophage
Chromosome
Transduction
Chromosome
Chromosome
Plasmids
Transposons
Plasmids
Pilus
Integron gene
cassette
Transposon
Transposon
Conjugation
Integron gene
cassette
Naked DNA
Transformation
Fig. 6.2. Mobile genetic elements and different mechanisms for horizontal gene transfer
(courtesy Frost et al., 2005).
Collis, 1995). Unlike transposons, the insertion of integrons is highly site
specific. A survey of over 600 full or partially sequenced bacterial genomes
revealed that 9% of the sequenced genomes contained integrons that can
be categorized into classes 1, 2 or 3. Among these three classes, class 1 integrons
have been frequently identified as the central player in the worldwide dissemination of ARGs (Boucher et al., 2007; Gillings et al., 2008).
A recent PCR-based survey of 17 European habitats, including farm soils,
waste water, and cattle, chicken, and pig manure, was able to detect the presence
of markers for several mobile genetic elements, including broad-host-range
plasmids, conjugative transposons and integron gene cassettes (Smalla et al.,
2000). These mobile genetic elements encode genes that confer resistance
to the antibiotics gentamycin (Heuer et al., 2002), sulfadiazine (Heuer and
Smalla, 2007), and amoxicillin (Binh et al., 2007). These surveys provided an
outlook on the ubiquity, persistence and stability of a vast pool of mobile
genetic elements in antibiotic-affected hot spots or sites where gene transfer
and recombination are higher than in non-affected locations.
Under favourable conditions, the pool of mobile genetic elements can be
transferred promiscuously among different bacteria by three mechanisms:
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P.-Y. Hong et al.
(i) conjugation, a process of genetic transfer that involves cell-to-cell contact;
(ii) transformation, a process by which free DNA is incorporated into a competent cell and brings about genetic change in the recipient; and (iii) transduction, a process by which DNA is transferred by bacteriophage (Fig. 6.2).
Conjugation was originally thought to play a more important role in disseminating ARGs across microorganisms of different genera and species (Courvalin,
1994) because the ubiquity of biofilm formation or high cell densities and close
cell contact found in gut ecosystems would favour the dissemination of ARGs
via conjugation (Molin and Tolker-Nielsen, 2003). However, with the advent of
molecular tools, it is now recognized that various ecosystems, such as activated
sludge and marine waters, harbour a large diversity and abundance of bacteriophages along with the bacterial population. The high numbers of bacteriophages suggest that transduction may also be a major force for horizontal gene
transfer in ecosystems such as those in close proximity to aquaculture farms
(Cabello, 2006). Likewise, the stability of naked DNA in the soil environment
suggests the possibility for bacteria to acquire genetic elements through transformation. Therefore, although the relative contribution of each mechanism
remains unknown, it is likely that all three may play significant roles in
disseminating ARGs among different microorganisms.
The Spread of Antibiotic Resistance
In this section, we provide evidence to show that the use of antibiotics in animal
husbandry can contribute to the spread of ARGs and antibiotic-resistant bacteria
to the environment by presenting two case studies. The first involves data that we
have gathered over a 3-year monitoring study conducted in three Illinois swineproduction facilities. This case study documents the presence of ARGs in waste
lagoons and nearby groundwater-monitoring wells and soil ecosystems. The
second case study, which is based on several reports from published literature,
documents the presence of ARGs and antibiotic-resistant bacteria in food
products sold to consumers. This provides indirect evidence that antibiotic use
in livestock production can possibly affect human health.
Case study 1: swine production farms
Two swine production farms in Illinois (designated as Sites A and C) were
monitored extensively over a 3-year period. As illustrated in Fig. 6.3a and b, each
site was fitted out with a network of groundwater sampling wells (designated
A1–A16 and C1–C7, respectively) for the monitoring of antibiotic contaminants
and ARGs. These wells were distributed around the open waste-treatment
lagoons, allowing monitoring of groundwater both ‘upstream’ and ‘downstream’
of the waste lagoons. The ‘upstream’ wells served as on-site controls, allowing us
to distinguish ARGs originating in waste lagoons from those derived from other
sources in the landscape (e.g. the indigenous environmental microbiota). Additionally, we surveyed agricultural soils at a third site (Site E, Fig. 6.3c), where
manure from a deep pit treatment system was applied as fertilizer.
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Antibiotic Residues and Resistance Genes in Livestock
(a)
0m
A7 (7.2)
Stream
A4 (3.8)
Upper sand layer
A3 (7.8)
3m
A11 (4.3)
Direction
of
flow
A5
A12 (4.1)(7.8)
A6
A14 (4.6)
(2.8)
A15
(7.5)
Stream
A16
(8.1)
A13
(3.6)
Waste
lagoon
6m
Confinement
buildings
9m
A2
(3.8)
Concrete
settling basin
N
A10 (4.0)
0 40 80 m
Fine-grained
glacial deposits
(silt loam)
Lower sand layer
A1
(7.1)
A8 (5.2)
A9
(3.9)
Topsoil
Gray silt
12 m
Glacial deposits
(silt loam)
15 m
(b)
Confinement
buildings
Wind-blown silt
Silty loam
3m
Thin, gravelly
loam layer
C1 (6.6)
Stream
Direction
of
flow
0m
6m
C5
(4.5)
C7
(13.5)
C4
(4.9)
C2
(4.1)
Waste
lagoon
9m
C3
(7.3)
N
Fine-grained
glacial deposits
(silt loam)
12 m
C6
(6.4)
Stream
0
50
100 m
15 m
Fig. 6.3. Site maps and corresponding stratigraphic columns indicating the characteristics of
sand layers at different vertical depths at three experimental sites. (a) Site A: wells A1 to A16
are groundwater monitoring wells; A7 is an upstream background monitoring well. (b) Site C:
wells C1 to C7 are groundwater monitoring wells; C1 is an upstream background monitoring
well.
Continued
128
(c)
P.-Y. Hong et al.
Crop field
(manure applied)
Stream
E4
(7.1)
E12 (5.8)
E8
(6.2)
0m
Topsoil
(clayey silt)
E10 (6.0)
3m
Silt loam
E7 (6.2)
E6 (5.6)
E3
(6.8)
E2
(6.8)
E11 (5.5)
E9 (5.9)
Waste
pit
6m
Sandstone
E5 (8.3)
Crop field
(manure applied)
Stream
Tree-lined
fence row
Direction
of
flow
9m
Facility well Facility well
(36.6)
(30.5)
E1(5.3)
Road
N
0
15 m
Fig. 6.3. Continued. (c) Site E: wells E1 to E12 are groundwater monitoring wells; there are
two wells providing water to the facility (facility wells); well E1 is an upstream background
monitoring well. Agricultural wells were surveyed at Site E.
Preliminary surveys of the study sites suggested that genes conferring
resistance to tetracycline and tylosin (a macrolide similar to erythromycin)
were of particular interest. Tetracycline and tylosin are both commonly used
for prophylaxis and growth promotion at Sites C and E. Site A had used both
antibiotics before our study but had reduced its antibiotic usage considerably
in the later stages by maintaining a high-health status herd. Erythromycin,
which is strictly reserved for human use, was not used at these sites. However,
erythromycin and tylosin are both macrolide antibiotics, and erm genes have
been shown to confer resistance to both these antibiotics.
Detection and quantification of ARGs were first achieved by designing
primer sets that targeted four major groups of antibiotic resistance genes,
namely: (i) four classes of genes (tet(M), tet(O), tet(Q), tet(W)) that confer resistance to tetracycline by means of ribosomal protection proteins; (ii) three
classes of genes (tet(C), tet(H), tet(Z)) that confer resistance to tetracycline by
means of efflux pump proteins; (iii) two classes of genes (tlr(B), tlr(D)) that
confer resistance to tylosin; and (iv) six classes of methylase genes (erm(A),
erm(B), erm(C), erm(F), erm(G), erm(Q)) that confer resistance to erythromycin
(Aminov et al., 2001, 2002; Mackie et al., 2006; Koike et al., 2007, 2010).
Our findings showed that manure-treatment lagoons and storage pits at
these sites always contained every tet gene for which we surveyed (Koike et al.,
2007), and, likewise, five out of six erm genes found at these sites were detected
in nearly every lagoon sample (Koike et al., 2010). A subset of groundwater
Antibiotic Residues and Resistance Genes in Livestock
129
wells contained both tet and erm genes with much higher frequencies than
other wells, and the detection frequencies of most tet and erm genes for these
wells were close to 100%. The wells concerned were all located in close proximity
to the source lagoon, and most of them were situated in a relatively porous
aquifer that bisected the lagoon.
Cloning and sequencing of the tet(W) genes revealed phylogenetic patterns that correlated well with gene detection frequency patterns of affected
versus non-affected wells. There was a distinct sub-cluster consisting of tet(W)
genes that were found only in ‘upstream’ background control wells, while
tet(W) genes from ‘downstream’ affected wells were often of similar or identical
phylogeny to those from lagoons or swine manure (Koike et al., 2007). Thus, at
least a portion of the tet(W) gene pool of the native groundwater microbiota
was readily distinguishable (99.8% gene similarity) from the pool of genes associated with swine-production activities and affected wells. In addition, the phylogenetic distribution of tet(W) sequences from lagoons was broader than that
of sequences recovered from background wells, which suggests that the tet(W)
gene pool of swine waste was more diverse than that of the native groundwater
microbiota. The detection of erm genes at these sites suggested selection due to
tylosin usage, as no erythromycin was used at all three farms.
Besides detecting ARGs in the groundwater wells, our surveys at Site E
further showed that soils were also affected by the addition of manure. Before
manure injection, we did not detect the presence of tet(C), tet(H) and tet(Z) in
our grab samples. Approximately 3 days after manure injection, it was possible
to detect these three tetracycline-resistant genes, along with tet(M), tet(O),
tet(Q) and tet(W) in most of the soil samples surveyed. Over time, the detection
frequency of tet(C), tet(H), tet(M) and tet(Z) returned to near-zero, while
others, such as tet(O), tet(Q) and tet(W) persisted for up to 7 months
(A. Yannarell, unpublished data).
Interestingly, the temporal and spatial patterns of ARGs in soils and water
did not seem to depend on direct selection pressure resulting from antibiotic
persistence in the environment, as levels of antibiotic residues were generally
found to be low or below the detection limit in soil and water samples. These
results support previous observations that the problem of antibiotic-resistant
bacteria is not necessarily linked to the persistence of antibiotic residues in the
environment (McEwen, 2006). Instead, the spatial and temporal patterns of
antibiotic resistance genes at these three sites suggest that exposure to hog
waste is the most important factor.
Case study 2: contaminated food produce
While the spread of ARGs to soil and water represents an environmental concern, the presence of ARGs in food produce provides a direct pathway for these
contaminants to enter into the human food chain, and thus these contaminants may impose adverse health impacts when consumed. To evaluate this concern, Kumar et al. (2005) first demonstrated that antibiotics are bioaccumulated
in crops that were grown on land to which manure had been applied.
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Garofalo et al. (2007) went on to collect faecal samples from chickens and
pigs, as well as specimens of raw chicken and pork meat from production factories that were located in central Italy. Samples were subsequently extracted
for bacterial DNA, and a PCR-based approach was utilized to detect 11 types of
genes encoding resistance against tetracycline, erythromycin, vancomycin,
aminoglycosidase, methicillin and β-lactams. Genes conferring resistance
against tetracycline and erythromycin, namely tet(K) and erm(B), were prevalent in the meat samples, and Garofalo et al. (2007) concluded that contamination is likely to be due to the breeding process rather than to processing
techniques in the production farms.
Similarly, Donabedian et al. (2003) isolated a total of 360 enterococci from
food purchased in grocery stores, and from faeces of healthy chickens, turkeys,
cattle and pigs reared on farms. The isolates were evaluated for genes that
confer resistance against gentamicin, which is an antibiotic that is commonly
used in swine farming and widely used in chicken and turkey rearing in the
USA. Donabedian et al. (2003) found that when gentamicin resistance genes
are highly prevalent in the faeces of food-producing animals, there was an
equally high prevalence of those resistance genes in the food specimens.
These findings reiterated those reported by Garofalo et al. (2007), and provided evidence of the spread of gentamicin-resistant enterococci from livestock production to food.
Independent studies have also detected the presence of ARGs in fermented dairy products and probiotic food. For example, Huys et al. (2004)
isolated 187 enterococci from European cheeses and found that 24% of these
isolates exhibited phenotypic resistance to tetracycline. Furthermore, 4% of
the isolates exhibited multiple resistance to tetracycline, erythromycin and
chloramphenicol. It was further demonstrated that some of the isolates were
able to transfer ARGs to recipient strains through conjugative transposons
(Huys et al., 2004). In a separate study, Hummel et al. (2007) examined the
antibiotic resistance of 45 strains of lactic acid bacteria. Using a PCR-based
approach, genes that confer resistance to β-lactams, chloramphenicol, tetracycline and erythromycin were found in approximately 7% of the isolates. However, in some of the strains that possessed the chloramphenicol-resistant genes,
the genes were found to be silent after performing a reverse transcription PCR
and phenotypic trait testing. The Hummel et al. (2007) study demonstrated
that genotypic detection of ARGs alone is not sufficient to distinguish between
silent and active resistance genes, and that phenotypic testing of ARGs should
also be performed. However, it should be noted that phenotypic testing typically relies on the isolation of bacteria, and that currently, cultured bacteria
are estimated to constitute only less than 0.001–15% of the total cell counts in
environmental samples (Amann et al., 1995; Tamaki et al., 2005). Therefore,
phenotypic testing often underestimates the total antibiotic resistance gene
pool compared with the genotypic approach (D’Costa et al., 2006; Sommer
et al., 2009). It is important, therefore, to use genotypic and phenotypic testing
as complementary approaches to examine both the total antibiotic resistance
gene pool and the expressed ARGs.
Antibiotic Residues and Resistance Genes in Livestock
131
The Spread of Antibiotic Resistance and Why Do We Care?
The detection of ARGs in soil and water ecosystems, as well as in food produce,
suggests that antibiotic-resistant bacteria can be transmitted to humans
through various routes, such as skin contact and oral ingestion. To evaluate
how the use of antibiotics in livestock production has affected the human population, we typically rely on epidemiology studies to compare the percentage
occurrence of antibiotic-resistant bacterial infections in the human population before and after an antibiotic ban was imposed. An example of such a
study is a comparative evaluation of the occurrence of vancomycin-resistant
enterococci (VRE) in Europe before and after the related antibiotic avoparcin
was banned as an animal growth promoter in 1997.
During the late 1990s, after approximately 20 years of approval of the use
of avoparcin as an animal growth promoter in Europe, the community prevalence of VRE was estimated at 2–12%. Interestingly, the infected population
included people with no prior history of hospitalization, and suggested that
some percentage of the infections within the community reservoir was due to
the use of avoparcin in livestock production (McDonald et al., 1997; Smith
et al., 2005). Since avoparcin was banned in 1997, there has been a marked
reduction in the prevalence of VRE within the European Community (EC)
(Klare et al., 1999). This observation further reiterates the possible impact on
the community due to the use of antibiotics in livestock production.
Besides potential adverse impacts on human health, the dissemination of
antibiotic contaminants from animal husbandry can also create a microbial
‘perfect storm’ in which unpredictable and unforeseen factors converge and
result in the emergence of novel microbial threats. For example, a 2003 report
from the Institute of Medicine of the National Academies (of the USA) identified 13 broad categories of socio-economic, ecological, environmental and
biological factors that play a role in the emergence of novel microbial threats
(Smolinski et al., 2003). Among these are factors relating to: (i) land use and
agricultural practices, particularly the promiscuous use of antibiotics in food
production; (ii) changes in human susceptibility to microbial risk, including
the acquisition of antibiotic resistance by pathogens; and (iii) microbial adaptation, which is enhanced when microorganisms are introduced to novel environments, interact with each other in species-rich environments, and
participate in horizontal gene exchange.
As Smolinski et al. (2003) point out, the rampant use of antibiotics in livestock production can create situations where the combined gene pools of
animal-associated enteric bacteria, antibiotic-resistant bacteria and environmental microbiota are conducive to the generation of novel and possibly
undesirable and even dangerous microbial threats. For example, environmental bacteria may acquire ‘enteric’ genes that enable them to spread to human
or animal hosts. Conversely, enteric pathogens may acquire genes that enable
them to persist in new soil or water environments, which then serve as reservoirs for the spread of these microorganisms. In addition, enteric bacteria may
acquire from environmental microorganisms traits that serve one purpose in
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P.-Y. Hong et al.
soil or water but that also contribute to virulence in a mammalian host (Casadevall,
2006). For example, capsules that help organisms to survive predation in the
soil may help pathogens to evade mammalian immune systems (Steenbergen et al.,
2001), and molecules that serve nutritional functions in the environment (e.g.
proteases or phospholipase) can cause damage to host cells in a mammalian
context (Casadevall and Pirofski, 2003; Casadevall, 2006).
The confluence of several of these factors can generate favourable conditions in which novel microbial threats emerge with elevated frequency and
impose a compounded health risk (Fig. 6.4). These threats include new
human and animal pathogens, zoonotic pathogens that can pass between
human and animal hosts, and previously seen pathogens that have acquired
new traits, such as antibiotic resistance, that increase their potential to cause
harm. Novel zoonotic pathogens can also emerge from genetic rearrangements that allow otherwise harmless organisms to acquire new traits associated with human or animal virulence, including the ability to: (i) infect and
survive in new hosts; (ii) persist in environments that increase their contact
with human or animal hosts; (iii) evade host immune response; (iv) resist
medical/veterinary treatment (e.g. antibiotics); or (v) damage host cells.
In early 2009, we witnessed the emergence of a novel influenza A (H1N1)
viral strain of swine origin (S-OIV). This new strain is a reassortment of two
Soil/water
environment
nm
viro
En
Microbial threats
ns
ABR
Composite
gene pool
Microbial threats
ge
tho
en
tal
/pa
ics
mic
ter
rob
En
iota
Animal-production
environment
HGT
Novel genetic
combinations
Fig. 6.4. Conceptual model for a microbial ‘perfect storm’. Enteric microorganisms,
antibiotic-resistant bacteria (ABR), and environmental microbiota can interact in soil and water
systems affected by animal-production activities. Their combined gene pools drive microbial
adaptation via horizontal gene transfer (HGT). Emergent microbial threats can pose a risk to
human and animal health, or they may persist in the environment, thus increasing the risk of
exposure and driving further adaptation.
Antibiotic Residues and Resistance Genes in Livestock
133
previously circulating strains: a ‘triple-reassortment’ swine influenza that has
been circulating in North America since 1998, and an H1N1 strain that
has been circulating for decades in swine populations in Europe and Asia
(Smith et al., 2009a). The lack of systematic surveillance of influenza in swine
has favoured the mixing of genetic elements, and ultimately led to the emergence of viruses with pandemic potential (Smith et al., 2009b). Summing up,
we need to be cautious and consider the possibility that new antibiotic-resistant
microbial threats can emerge as a consequence of the unchecked use of antibiotics in livestock production.
Knowledge Gaps
Much of what is known about resistance mechanisms and horizontal gene
transfer have come from the study of clinical isolates. In contrast, the total
ARG pool in the agriculture-affected environment remains elusive (D’Costa
et al., 2006). Current genotypic and phenotypic approaches for examining
ARGs rely on prior knowledge and detect only those genes that were already
known. Such approaches do not provide new insights into unknown antibiotic
resistance mechanisms (Sundsfjord et al., 2004).
Recent advances in genome sequencing technologies have provided a
cost-effective way to determine the ARG pool. For example, through the study
of various opportunistic pathogens and clinical isolates, it was revealed that
the genes encoding efflux proteins are commonly found in most sequenced
bacterial genomes (Wright, 2007). The vast collection of efflux pumps presumably provides robustness and flexibility to the microorganism so that it can
survive in diverse environments (Stover et al., 2000; Poole and Srikumar, 2001;
Piddock, 2006a). Opportunistic pathogens of environmental origins can also
utilize such traits to promote pathogenicity (Piddock, 2006b). In the near
future, more sequencing effort should be undertaken to provide information
on pan-microbial genomes and possibly discover new ARGs and antibioticresistant mechanisms (Wright, 2007).
Besides the need to elucidate the ARG pool, there is also a need to increase
our understanding of the potential for gene exchange among environmental,
animal-associated and antibiotic-resistant microorganisms in agricultureaffected environments (Smalla and Sobecky, 2002; van Elsas and Bailey, 2002).
Because horizontal gene transfer can effectively expand the gene pool available to a potential pathogen, it is important to understand what happens when
human activities bring together microorganisms with a variety of threat-related
genes, such as virulence and ARGs. However, this remains a daunting task to
perform, primarily because of the complexities involved in an environmental
setting (Nielsen and Townsend, 2004). For example, soil typically contains
approximately 107 bacterial cells g–1 (Gans et al., 2005), and acquiring virulence genes together with ARGs in an endemic soil microorganism may be a
rare event that occurs at low transformation rates. Therefore, conventional
PCR-based and cultivation methods would not be able to detect the occurrence of this rare event easily unless an extensive sampling and surveillance
effort had been carried out (Nielsen and Townsend, 2004).
134
P.-Y. Hong et al.
Concluding Remarks
In their natural habitat, some microorganisms produce antibiotics that are
selective against their enemies. In response to antibiotics, the targeted microorganisms gradually evolve antibiotic-resistance mechanisms and genes in a
bid to outcompete. When human beings eventually discovered these amazing
resources, we ingeniously harnessed them and hailed new milestones in
human medicine. However, what was once useful to bring humans back to
good health has now become a potential menace as a result of inappropriate
use in clinical settings and livestock production. Such practices have indirectly
caused human pathogens to evolve and rapidly acquire multiple drug resistance that renders our existing antibiotics ineffective.
As of now, many published studies have demonstrated the presence of
ARGs in the environment and in food produce, but they have not provided
extensive epidemiological evidence to conclusively prove the link between
ARGs released by animal husbandry and the emergence of new diseases. However, one should always handle environmental concerns and human-health
related issues with prudence. It has been suggested that if we react fast enough
to remediate the current situation, we could perhaps delay the adverse effects
brought about by rapid dissemination of antibiotic contaminants (Johnsen
et al., 2009). As the race to develop new antibiotics is speeding up, more
research and surveillance studies need to be performed to ensure responsible
use of antibiotics in both clinical settings and livestock production.
Finally, the American Academy for Microbiology has recently published a
report entitled Antibiotic Resistance: An Ecological Perspective on an Old Problem
(AAM, 2009). The scope of the problem is much larger than that which has
been considered in this chapter. However, several of the conclusions of the
report are pertinent and worth reiterating. The first is that antibiotic resistance is a pandemic that compromises treatment of all infectious diseases, and
that is at present uncontrollable. The reasons controlling the establishment
and spread of antibiotic resistance are complex, mostly multifactorial and
largely unknown. The second conclusion is that responsibility is due partly to
medical, veterinary and industrial practice, but also to economics and politics,
as well as to antibiotics themselves. Thus, the use of growth-promoting antibiotics in livestock production is only a part of problem and should not be a
scapegoat. Third and last, ARGs are not new entities; they should be considered as fundamental components of microbial diversity and life, as well as
components that represent evolutionary phenomena.
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7
On-farm Mitigation of Enteric
Pathogens to Prevent Human
Disease
TREVOR W. ALEXANDER, KIM STANFORD AND TIM A. MCALLISTER*
Introduction
The importance of controlling foodborne pathogenic bacteria postharvest,
starting with abattoir processing, is well recognized. Indeed, a recent review of
the beef production chain showed significant reduction in the prevalence of
Escherichia coli O157, Salmonella enterica and Listeria monocytogenes throughout
abattoir processing (Rhoades et al., 2009). However, despite efforts to mitigate
pathogens, contaminated meat products still enter the food chain and pathogenic bacteria are frequently detected in food products. For example, Salmonella spp. (Delhalle et al., 2009), E. coli O157:H7 (Naugle et al., 2005) and
Campylobacter spp. (Moran et al., 2009) have all been isolated from retail
meats.
It is evident that pathogenic contaminants from animals can enter the
food chain during processing (Arthur et al., 2007). Using pulsed-field gel electrophoresis (PFGE), E. coli O157:H7 on carcasses (Barkocy-Gallagher et al.,
2001) and antimicrobial-resistant E. coli in ground beef (Alexander et al., 2010)
have been source tracked to cattle at slaughter. Additionally, Salmonella spp.
(Vieira-Pinto et al., 2006) and Yersinia enterocolitica (Laukkanen et al., 2009)
isolated from pig carcasses had similar PFGE genotypes to isolates originating
from digesta and tonsil samples. While both the animal and bacterial species
influence dissemination, pathogen load also affects the likelihood of carcass
contamination (Brichta-Harhay et al., 2008), with higher carriage levels
making it more difficult to avoid the adulteration of meat products.
The prevalence of foodborne pathogens in cattle (Gansheroff and
O’Brien, 2000), pigs (Letellier et al., 2009) and poultry (Arsenault et al.,
2007) preharvest has been shown to be positively correlated with carcass
* Corresponding author.
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(eds D.O. Krause and S. Hendrick)
On-farm Mitigation of Pathogens to Prevent Disease
141
contamination. Reducing colonization and the shedding of pathogens can,
therefore, play an integral role to mitigating foodborne disease postharvest.
Additionally, contamination of produce with enteric pathogens, such as
occurred during the 2006 E. coli O157:H7 outbreak in the USA (Jay et al.,
2007), has prompted greater assessment of the role of animal waste as a
vehicle for transmission. To address these issues, an increasing number of
studies are investigating on-farm mitigation strategies. Preharvest control
points are being analysed that include altering microbial populations of the
digestive tract or direct targeting of the pathogen at the animal level. Equally
as important are strategies to manage waste in order to prevent environmental contamination and the transfer of pathogens among animals. This
chapter will review the on-farm methods for controlling enteric pathogenic
bacteria. As an example, a schematic of how these control points can be used
to mitigate the spread of E. coli O157:H7 in a feedlot production system is
illustrated in Fig. 7.1.
Reducing Pathogens in Animal Feed and Water
Feeds from poultry, swine and cattle production systems have been identified
as vectors of foodborne pathogens to livestock (Doyle and Erickson, 2006).
For example, isolates of S. enterica serovar Typhimurium (S. Typhimurium) or
E. coli O157:H7 that were cultured from stored feedstuffs at feedlots were
genetically related to isolates collected from faeces of cattle at the same feedlot (Davis et al., 2003). For poultry and swine, feed is often pelleted or extruded,
and exposed to temperatures as high as 90°C (Doyle and Erickson, 2006).
Exposure to heat can denature enzymes that confer anti-nutritional properties, and pelleted feed flows more easily in handling systems; during processing, exposure to heat has also been recognized as an efficient method of
mitigating feed-borne pathogens. Mashed poultry feed has been reported to
be more frequently contaminated than pelleted feed (21% versus 1.4%; Veldman et al., 1995). Himathongkham et al. (1996) showed that heat treatment of
poultry feeds for 90 s at 93°C will cause a 10,000-fold reduction in contaminating Salmonella. The addition of propionic acid to the feed can increase the
sensitivity of pathogens such as Salmonella to heat. For example, heating feed
for 80 s at 71°C in the presence of 0.2% propionic acid resulted in a 4 log
(unit) reduction in Salmonella (Matlho et al., 1997). However, the effectiveness
of heating is dependent on the bacterial species as heating feed at 71°C for
120 s only reduced E. coli O157:H7 in the feed by 2.2 log units (Hutchison
et al., 2007). Additionally, the reduction of pathogen loads in feeds by heat
treatment does not preclude the possibility of subsequent contamination of
the feed during downstream handling, either at the feed mill or on the farm.
Furthermore, many livestock operations do not heat treat feed before providing it to livestock (Davies et al., 2004). Also, rodents, birds and wildlife can
contaminate stored feed with pathogens (Davies et al., 2004), necessitating
the need for proper hygiene practices and storage of feed throughout the
production continuum.
142
Feed
a
Colonized
animal
• Manure
• Faeces
b
c
Water
b
d
Environment
(via manure)
• Water
• Soil
Food crops
• Manure
• Faeces
Abattoir
• Hide
• Digesta
e
Beef
Potential human infection
Agricultural
dissemination
Reinfection cycle
Food processing
dissemination
T.W. Alexander et al.
Fig. 7.1. Dissemination of Escherichia coli O157:H7 within a beef production system and to food and environmental sources. Letters a–d within the arrows indicate possible on-farm mitigation control points for the bacterium: a, feed hygiene; b,
feedlot hygiene, waste management; c, water treatment; d, use of antimicrobials, bacteriophages, probiotics, vaccination,
immunotherapy. Letter e, postharvest mitigation.
On-farm Mitigation of Pathogens to Prevent Disease
143
Water can also serve as a vector of pathogens to livestock. For example,
several studies have shown that water troughs in feedlots can harbour large
numbers of E. coli O157:H7. In one study, 12% of water troughs at feedlots
tested positive for E. coli O157:H7 (Van Donkersgoed et al., 2001), and it
appears that long-term survival of the bacterium in this environment (up to
245 days) is possible (LeJeune et al., 2001). LeJeune et al. (2004) observed no
differences in the prevalence of E. coli O157:H7 in feedlot water troughs that
contained chlorinated or non-chlorinated water. Similarly, Zhao et al. (2006)
reported that chlorine had little effect on E. coli O157:H7 in water that contained rumen contents. Contaminating faeces are often observed in feedlot
water troughs, and the resulting elevated organic matter can negate the efficacy of chlorine as an antimicrobial treatment for water (Doyle and Erickson,
2006). In the study by Zhao et al. (2006), four treatments containing a combination of chemicals that included lactic acid, acidic calcium sulfate, caprylic
acid, sodium benzoate or chlorine dioxide reduced numbers of enterohemorrhagic E. coli by up to 5 log units, even in the presence of rumen or faecal
contents. Unfortunately, these treatments reduced water consumption in a
manner that could adversely affect the health of cattle; the authors suggested
that periodic treatment of the water may overcome this problem, but such a
strategy lacks practicality at the commercial level. Other methods shown to
reduce E. coli O157:H7 in cattle drinking water include the use of electrolysed
oxidizing water (Stevenson et al., 2004), and the addition of sodium caprylate
(Amalaradjou et al., 2006), or the plant essential oil trans-cinnamaldehyde
(Charles et al., 2008). However, many of these strategies are expensive, and
their effects on water consumption have not yet been examined.
Antimicrobial Feed Additives
Several types of antimicrobial feed additives have been investigated for targeting specific or general groups of pathogenic bacteria. The application of neomycin sulfate has been reported to reduce E. coli O157:H7 to undetectable
levels in naturally colonized cattle (Elder et al., 2002) and to lower its prevalence in feedlot cattle (Woerner et al., 2006). While these preliminary results
are impressive, adoption of the mass medication of cattle with an antimicrobial of the same class of antibiotics as those commonly used in human medicine is unlikely. This concern arises over promotion of the emergence of
antimicrobial-resistant bacteria in livestock, which can also enter the food
chain through the contamination of meat in the abattoir (Alexander et al.,
2010). Most research on novel antimicrobials has therefore focused on agents
unrelated to traditional antibiotics.
Some chemicals have been used to target specific metabolic pathways of
pathogens. Salmonella and E. coli encode nitrate reductase, which reduces
nitrate to nitrite under anaerobic conditions (Oliver et al., 2009). The enzyme
does not differentiate between nitrate and chlorate, and when the latter is
present, it is reduced to bactericidal chlorite. Evaluation of sodium chlorate
has shown its potential to reduce Salmonella and E. coli in vivo. Sodium chlorate
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T.W. Alexander et al.
supplementation resulted in a 2 and 3 log reduction of E. coli O157:H7 in the
rumen and faeces, respectively, of cattle challenged with this pathogen (Callaway et al., 2002), with similar results in sheep (Callaway et al., 2003). This
approach has also been shown to be effective against Salmonella. In broilers
challenged with S. Typhimurium, birds administered a chlorate product had
reduced prevalence (2% versus 36.7%) and numbers of this bacterium in
their caeca (0.96 versus 2.52 log colony forming units (cfu) g–1) than birds not
fed the product (Byrd et al., 2003). Reduced concentrations of S. Typhimurium
in the crops of turkeys have also been reported in birds administered chlorate
(Moore et al., 2006).
Campylobacter utilizes amino acids as a principal energy substrate and, as a
result, inhibiting amino acid catabolism can potentially reduce their competitiveness in the gut (Horrocks et al., 2009). For example, the inclusion of the
deaminase inhibitors diphenyliodonium chloride and thymol in mixed faecal
cultures from pigs reduced the number of Campylobacter by more than 3 log
units (Anderson et al., 2009). Caprylic acid, a medium-chain fatty acid with
eight carbons, has also been shown to mitigate Campylobacter (Solis de los
Santos et al., 2008); in chickens challenged with Campylobacter, doses of
0.35–0.87% caprylic acid in dietary dry matter decreased caecal concentrations of this bacterium by 2.0–5.0 log units. Caprylic acid has also been shown
to reduce the numbers of S. enterica serovar Enteritidis (S. Enteridis) colonizing intestinal tissues in experimentally inoculated chickens (Johny et al., 2009).
Similarly, the short-chain fatty acid butyric acid has been reported to reduce
colonization and shedding of Salmonella in chickens (Van Immerseel et al.,
2005). The exact mechanisms by which these acids reduce colonization remain
unknown.
The bacteriostatic and bactericidal activities of plant extracts termed
essential oils have been well described (Ojha and Kostrzynska, 2007) and,
more recently, their antimicrobial effects on pathogenic organisms in livestock have been examined. Tasco-14™ is a proprietary product (from Acadian Seaplants Ltd, Dartmouth, Nova Scotia) obtained from the brown
seaweed Ascophyllum nodosum, which grows along the North Atlantic coastline.
This plant product enhances immune function, improves carcass characteristics
and extends the retail display shelf life of beef (Allen et al., 2001; Montgomery
et al., 2001; Braden et al., 2007). The product has also been investigated for
reducing the shedding of E. coli O157:H7. The inclusion of Tasco-14™ at a
level of 2% in the diet of cattle for a period of 2 weeks before slaughter
reduced the prevalence of E. coli O157:H7 in faecal samples (11%) and hide
swabs (36%) compared with the levels 1 day before being fed the additive
(Braden et al., 2004). Bach et al. (2008) tested the same product on feedlot
cattle experimentally inoculated with E. coli O157:H7. In that study, steers
were fed Tasco-14™ in the diet at a level of 1% for 14 days, 2% for 7 days, or
2% for 14 days after inoculation; the dietary treatments commenced 7 days
after inoculation. Throughout the sampling period, detection and concentration of the pathogen in faecal samples was less frequent when the additive
was fed at 1% for 14 days or 2% for 7 days, compared with samples from animals fed Tasco-14™ at 2% for 14 days after inoculation and control animals
On-farm Mitigation of Pathogens to Prevent Disease
145
receiving no additive. Wang et al. (2009) reported that phlorotannins
extracted from A. nodosum were both bactericidal and bacteriostatic to E. coli
O157:H7. Other studies have indicated that plant tannins (Min et al., 2007)
and essential oils (Callaway et al., 2008b) exhibit bactericidal activity against
E. coli O157:H7, although the extent of this antimicrobial activity varies among
plant sources (Min et al., 2007).
Probiotics
Probiotics are any of a number of live microorganisms, including yeast, Lactobacillus or other bacterial strains, extracts and enzyme preparations (Elam et al.,
2003) that are known to be safe and produce beneficial results when fed individually or as mixtures to livestock. Probiotics have been used in the cattle
industry for over 20 years, primarily to improve growth performance, milk
production or feed conversion efficiency (LeJeune and Wetzel 2007). Recently,
a new generation of probiotics has started to be developed which, along
with growth performance benefits, also exhibits activity against pathogens
in livestock (Callaway et al., 2009).
In the poultry industry, a direct-fed microbial (DFM) containing a
mixture of Lactobacillus acidophilus, Lactobacillus casei, Enterococcus faecium and
Bifidobacterium bifidium (Talebi et al., 2008) and marketed under the trade
name of PrimaLac has been well characterized. This DFM has been shown to
improve poultry gut health and bird performance after oral challenge with S.
Typhimurium, S. enterica serovar Heidelberg (S. Heidelberg) and S. enterica
serovar Kentucky (S. Kentucky) (Rahimi et al., 2009). PrimaLac has also been
shown to reduce the prevalence of Campylobacter jejuni in broiler chicks
(Willis and Reid, 2008) and reduce shedding of sporulated ooycsts of Eimeria
acervulina, thereby acting as an alternative to the anti-protozoal products
that are presently used to control coccidiosis (Dalloul et al., 2003).
In the swine industry, a DFM containing Bacillus subtilis served as an
alternative to the use of sub-therapeutic antibiotics as it reduced scours in
piglets that were challenged with E. coli K88 (Bhandari et al., 2008). A DFM
containing Bifidobacterium lactis Bb12 and Lactobacillus rhamnosus LGG
reduced adhesion of Salmonella, Clostridium and E. coli to swine intestinal tissues in vitro (Collado et al., 2007), and similar results were observed in vivo
(Konstantinov et al., 2008). These researchers demonstrated that a DFM containing Lactobacillus sobrius DSM 16698 was able to reduce the colonization
of enterotoxigenic E. coli in piglets immediately after weaning. Inclusion of
E. faecium in sow diets has also been shown to reduce the rate of shedding of
Chlamydiaceae and the transmission of this pathogen to piglets (Pollman
et al., 2005).
In contrast to poultry and swine studies, DFMs for cattle have focused
primarily on the control of pathogenic strains of E. coli (Elam et al., 2003; Zhao
et al., 2003; Callaway et al., 2004; LeJeune and Wetzel, 2007). The most extensively studied DFM for cattle contains L. acidophilus strain NP51 and this has
been found to reduce shedding of E. coli O157:H7 by 48–80% when fed at
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109 cfu (Brashears et al., 2003; Younts-Dahl et al., 2004, 2005; Stephens et al.,
2007a,b). Tabe et al. (2008) found that L. acidophilus strain BT1386 in the diet
reduced faecal shedding of E. coli O157 in feedlot steers, but had no impact on
the shedding of Salmonella. Reduced faecal shedding of Salmonella was
observed when a combination of 109 cfu L. acidophilus NP51 and 109 cfu Propionibacterium freudenreichii NP24 was fed to steers (Stephens et al., 2007b). The
efficacy of DFMs may also depend on the dosage, a relationship that has been
shown to be apply for L. acidophilus NP51 when it is administered to cattle to
reduce the shedding of E. coli O157 (Younts-Dahl et al., 2005).
Although the ability of DFMs to control pathogens in various livestock
species has been reported, the mechanisms to which this response is attributed are not well characterized (Callaway et al., 2008c). The efficacy of a DFM
may be compromised if the pathogen(s) fully colonizes the gastrointestinal
tract (GIT) before introduction of the DFM. Once pathogens form biofilms,
changes in gene expression can stabilize the population, making it resistant to
a wide variety of antimicrobial agents (Ito et al., 2009). This may explain why
positive responses to DFMs are generally more often observed in younger as
opposed to older animals. For example, administration of a DFM containing
mixtures of lactobacilli to preterm piglets precluded the colonization of the
intestinal tract by Clostridium perfringens (Siggers et al., 2008).
Prebiotics
Prebiotics have been defined as non-digestible dietary ingredients that
stimulate growth or activity of native microbial populations in the digestive
tract, ultimately benefiting the health of the host (Collins and Gibson,
1999). Examples of prebiotics include fructo-oligosaccharides (FOS; e.g.
oligofructose), galacto-oligosaccharides, inulin and lactulose (Collins and
Gibson, 1999). Prebiotics have been used to enhance health in humans,
but their potential to provide a competitive advantage to select resident
bacteria has led to their possible use for preharvest mitigation of pathogens
(Callaway et al., 2008c).
Different types of oligosaccharides have been tested in poultry for their
ability to improve animal health and growth performance. Meta-analyses
have shown that mannan-oligosaccharides (MOS) improve body weight and
reduce mortalities in poultry (Hooge, 2004; Rosen, 2007), but the mechanisms responsible for these effects are not clear. Despite reported differences
in the capacity of prebiotics to alter bacterial populations in poultry, most
reports have consistently documented a decline in intestinal colonization
by Salmonella when the products are administered. For example, the addition of lactose to the diets of broiler chickens inhibited colonization (Corrier et al., 1990) and numerically decreased Salmonella in caecal contents
compared with animals fed no lactose (Ziprin et al., 1990). The response
was attributed to the increased production of bacteriostatic volatile fatty
acid concentrations in the caeca, and the associated lower pH as a result of
the inclusion of lactose in the diet. Furthermore, the impact of MOS (Spring
On-farm Mitigation of Pathogens to Prevent Disease
147
et al., 2000) or FOS (Fukata et al., 1999) may be specific to different species of
microbial pathogens as they have been shown to reduce numbers of Salmonella, but have no effect on the numbers of potentially beneficial Bifidobacterium and Lactobacillus spp in caecal contents. Conversely, in some instances,
MOS may even promote an increase in the numbers of Bifidobacterium and
Lactobacillus within the intestinal tract of poultry (Fernandez et al., 2002).
At times, numbers of Bifidobacterium in the caecal contents of broilers fed
MOS have increased, while populations of E. coli and Campylobacter have
decreased (Baurhoo et al., 2009). Inclusion of MOS in diet has also been
shown to alter intestinal morphology, including increasing villus height and
the number of goblet cells associated with each villus. Goblet cells secrete
mucins which may further inhibit the establishment of pathogens within the
caeca of poultry.
Several types of prebiotics have been shown to increase the concentrations
of Lactobacillus reuteri and Lactobacillus amylovorus as well as the overall genetic
diversity of bacteria in colonic samples from pigs (Konstantinov et al., 2004). A
chito-oligosaccharide also increased Labctobacillus counts in the faeces of weaning pigs and favourably altered intestinal morphology (Liu et al., 2008); additionally, in this study E. coli counts and the incidence of diarrhoea decreased
in animals fed the prebiotic. Similarly, FOS improved intestinal function and
protected pigs against S. Typhimurium, reducing the incidence of diarrhoea
(Correa-Matos et al., 2003). Prebiotics have also been shown to reduce the
adhesion of pathogens that utilize cell-surface oligosaccharide-binding proteins as adhesion receptors through competitive binding (Rhoades et al.,
2006). A galacto-oligosaccharide (GOS) mixture increased Bifidobacterium
concentrations in the caecal contents of pigs and also inhibited attachment of
enteropathogenic E. coli and S. Typhimurium to HT29 cells in vitro (Tzortzis et
al., 2005). The feeding of a synbiotic (probiotic plus prebiotic) containing
Lactobacillus plantarum and FOS has also been reported to reduce adhesion of
E. coli O8:K88 to the intestinal mucosa of the jejunum and colon in pigs (Nemcová et al., 2007).
Few studies have investigated prebiotic use in ruminants, mainly because
of their prohibitive costs as additives and the complex microbial ecosystem of
the rumen (Callaway et al., 2008c). Rumen microbes produce an array of polysaccharidases with the capacity to degrade many of prebiotics, limiting their
ability to exert effects within the lower digestive tract of ruminants. Purified
GOS has been shown to inhibit the adherence of enteropathogenic E. coli to
HEp-2 and Caco-2 cell lines in vitro (Shoaf et al., 2006), but expression of this
activity in the lower intestinal tract of cattle seems unlikely. When sorbitol was
added to rumen cultures, inoculated E. coli O157:H7 was only displaced after
72 h (de Vaux et al., 2002), leaving ample time for this pathogen to pass to the
lower tract with the fluid fraction of digesta. However, the effects of prebiotics
on E. coli O157:H7 in vivo have not been examined. As with probiotics, prebiotics may have a greater efficacy in pre-ruminant calves. Recently, lactulose fed
in combination with E. faecium was shown to improve the immune status of
pre-ruminant calves, but the implications of this response for colonization by
pathogens was not explored (Fleige et al., 2009).
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Immunization
Vaccines have been effectively used to manage disease in livestock. Typically,
vaccine development has been targeted towards pathogens that adversely
affect animal health and production (Oliver et al., 2009). Efforts to elicit
immune responses to foodborne pathogens as a means of potentially reducing
infection in humans have recently been undertaken. In some instances (e.g.
Salmonella), the organism can be pathogenic to both the livestock animal and
humans, raising the possibility that a vaccine could have benefits for livestock
production as well as the general public. In contrast, the foodborne pathogens may be asymptomatic in the animal (e.g. Campylobacter, E. coli O157:H7).
Under these circumstances, the economic incentive for the producer to utilize
the vaccine is not immediately apparent, a factor that may pose a barrier to
vaccine development (LeJeune and Wetzel, 2007).
Infection of poultry with Salmonella induces a serological immune response
(Skov et al., 2000) which can reduce the duration of infection as well as reinfection (Gast, 2007). Vaccination against Salmonella produces similar responses and
may also afford long-term protection (Mastroeni et al., 2001). While live Salmonella vaccines have been shown to enhance cell-mediated immunity in comparison to killed vaccines (Babu et al., 2003), multiple commercial vaccines of both
types are utilized to some extent in the poultry industry. Broiler breeder hens
challenged with Salmonella and vaccinated with a commercial bacterin or live
vaccine exhibited reduced excretion of the pathogen and a reduction in the
extent to which it colonized the spleen, liver and caecum (Penha Filho et al.,
2009). Vaccination with a live Salmonella vaccine at 1 day, 6 weeks and 16 weeks
of age has also been shown to reduce the incidence of oviduct and internal egg
contamination in layer hens intravenously challenged with S. Enteritidis (Gantois et al., 2006). Recently, an analysis of layer-hen flocks across the EU found
that vaccination of hens against Salmonella reduced the prevalence of Salmonella,
with the exception of S. Typhimurium (EFSA, 2007). While current vaccines do
not eliminate Salmonella from poultry, their use may reduce the transmission of
this pathogen, as fewer human-associated infections of S. Enteritidis were
reported in both the UK (Cogan and Humphrey, 2003) and Belgium (Collard
et al., 2008) with the introduction of layer-flock vaccination programmes.
Campylobacter is prevalent in poultry production systems and colonizes the
caeca, large intestine and cloaca. Although colonized birds display no signs of
pathology, the bacterium can trigger a systemic and mucosal immune response
(de Zoete et al., 2007). After oral infection of chickens, serum and mucosal
Campylobacter-specific antibodies increase (Cawthraw et al., 1994), and this
increase has been shown to coincide with a reduction in colonization (Lin,
2009). Additionally, high titres of maternal anti-Campylobacter antibodies are
present in chicks for up to 7 days after hatching, at which point they decline
(Sahin et al., 2001). The decrease in titre also coincides with the point at which
Campylobacter is more commonly isolated from the digestive tract. Combined,
these data suggest that there may be some potential to vaccinate poultry
against Campylobacter, although efforts to develop vaccines against Campylobacter have largely been unsuccessful. This lack of success may be attributable
On-farm Mitigation of Pathogens to Prevent Disease
149
to the genomic and phenotypic instability and diversity of Campylobacter, making it difficult to use attenuated variants of the bacterium or to identify antigen targets with sufficiently broad specificity (de Zoete et al., 2007). In one
study, oral vaccination of chickens with an avirulent Salmonella strain expressing the C. jejuni CjaA antigen resulted in a sixfold reduction of colonization
upon challenge with a wild type C. jejuni strain (Wyszyńska et al., 2004). The
study did not include a control group of birds receiving Salmonella that did not
express CjaA, and only a few birds were included in the experiment. Consequently, it was not possible to determine whether the immune response
resulted from Salmonella or from the presence of the CjaA antigen. However, a
recent study in which chickens were also vaccinated with attenuated Salmonella
expressing CjaA confirmed the induction of CjaA-specific serum antibodies
(Buckley et al., 2010). In this latter study, only a 1.4 log reduction of Campylobacter per gram of caecal contents was observed.
As for poultry, commercial vaccines against Salmonella are available for use
in swine. Only a few clinical trials have been conducted, but a recent review
has shown that vaccination of swine is associated with reduced Salmonella
prevalence just before and at harvest (Denagamage et al., 2007). Roesler et al.
(2004) vaccinated 4-week-old piglets with a live S. Typhimurium vaccine and
then subjected them to a challenge 3 weeks later. Of the vaccinated pigs, 90%
did not show any clinical signs of salmonellosis, whereas all control animals
showed moderate-to-severe clinical symptoms. Additionally, colonization of
internal organs was reduced in the vaccinated group compared with the
control (42.5% versus 87.5%). Vaccination of sows also appears to confer
protection against S. Typhimurium in piglets. From birth to 142 days old,
S. Typhimurium was not detected in faecal samples of piglets originating from
vaccinated sows, whereas a prevalence rate of 47.7% occurred in piglets from
unvaccinated sows (Roesler et al., 2006).
Vaccine development against E. coli O157:H7 is complicated because the
bacterium acts as a commensal in cattle and sheep (LeJeune and Wetzel,
2007). Only recently have vaccines targeting E. coli O157:H7 been tested in
cattle. Most vaccines against E. coli O157:H7 have attempted to capitalize on
proteins excreted by the type III-secretory system as antigens. First-generation
vaccines grew E. coli O157:H7 under conditions that promoted the excretion
of these proteins, whereas second-generation recombinant vaccines will be
likely to focus on specific proteins within this system. Vaccination with a firstgeneration vaccine against E. coli O157:H7 was shown to reduce both the level
and duration of shedding of this bacterium in challenged cattle (Potter et al.,
2004). However, use of this vaccine in a commercial feedlot found no difference in prevalence of E. coli O157:H7 between pens of vaccinated and nonvaccinated cattle, a result that was attributed to the adjuvant used in the
formulation or the need for more than two immunizations (van Donkersgoed
et al., 2005). Subsequent studies using an altered formula of the vaccine and
three vaccinations found that vaccinated cattle were 98% less likely to be colonized by E. coli O157:H7 at the terminal rectum mucosa (Peterson et al., 2007).
Under commercial conditions, vaccination three times over the feeding
period is impractical, and as a result two-dose vaccine strategies have generally
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been used. In a large-scale trial using 20,556 cattle, administering a two-dose
vaccine to cattle resulted in a 12% reduction in the likelihood of pens of cattle
shedding E. coli O157:H7 (Smith et al., 2008). From that same study, a subset
of animals was tested for colonization of the terminal rectum (Smith et al.,
2009a). Compared with unvaccinated cattle (17.0%), there was a lower incidence of colonization in vaccinated cattle (2.9%). This vaccine has also been
shown to reduce faecal shedding and hide contamination, but these effects
were negated when unvaccinated cattle were mixed with those that were vaccinated, demonstrating the likely need to vaccinate all cattle in order for vaccination to be an effective mitigation strategy (Smith et al., 2009b). Vaccination
of feedlot cattle against E. coli O157:H7 with a vaccine based on a siderophore
receptor and porin proteins reduced the prevalence and level of shedding of
E. coli O157:H7 in feedlot cattle (Thomson et al., 2009). An interesting followup to this study involved vaccination of cattle that had previously been identified to be shedding E. coli O157:H7 (Fox et al., 2009). Vaccination of these
animals reduced the prevalence of E. coli O157:H7 (17.7%) compared with
unvaccinated controls (33.7%) and lowered the number of individuals that
were shedding high levels of this pathogen.
Passive immunization using egg-yolk antibodies has also been investigated
to control pathogens in swine and ruminants. Antibodies produced by hens
immunized with specific antigens or whole cells are transferred to the egg
yolk, and these antibodies can be administered to the animal without activating the mammalian complement (Cook et al., 2005). In swine, the use of eggyolk antibodies has been tested for the control of E. coli K88+, but with mixed
results. One study showed that challenged piglets treated with egg yolk containing anti-E. coli K88+ antibodies were protected, whereas a control group
experienced a 62% mortality rate (Marquardt et al., 1999). In contrast, Chernysheva et al. (2003) reported no differences in rates of diarrhoea or mortality
between pigs treated with anti-E. coli K88+ egg yolk and control animals. In
calves, a high dose of Salmonella-specific egg-yolk antibodies resulted in protection from a challenge strain, whereas calves in low-dose or control groups
experienced high mortality rates (Yokoyama et al., 1998). No studies with
Salmonella have been conducted in a commercial setting. Anti-E. coli O157:H7
chicken egg-yolk antibodies have been tested in sheep challenged with E. coli
O157:H7 (Cook et al., 2005). Throughout 85 days post-challenge, the number
of E. coli O157:H7 shed and the duration of shedding was shorter for lambs
given a high and medium dose of egg yolk antibodies, compared with control
animals. A subsequent in vitro study by Cook et al. (2007) showed that polyclonal antibodies targeting adherence-associated factors were effective in preventing adhesion and colonization by E. coli O157:H7 to cultured HeLa cells.
Bacteriophages
Bacteriophages are highly specific viruses that target bacteria. They
naturally inhabit a diversity of ecosystems, including the mammalian GIT
(Dabrowska et al., 2005), and significantly influence microbial communities
On-farm Mitigation of Pathogens to Prevent Disease
151
and function (Rohwer and Thurber, 2009). Their use to mitigate bacterial
pathogens is not novel, but there has been renewed interest in bacteriophages
because of the development of antimicrobial resistance in bacteria. As a preharvest strategy to reduce enteric pathogens, bacteriophages are attractive
because they are highly specific, non-toxic, self-amplifying, and can overcome
multiple-drug resistant bacteria (Ojha and Kostrzynska, 2007). Additionally,
there is evidence that bacteriophages may be transferred between animals,
which may result in spread of a protective agent among herd cohorts (Rozema
et al., 2009).
The administration of two broad-range phages to broiler chickens challenged with S. Enteritidis or S. Typhimurium resulted in 4.2 and 2.19 log
reductions, respectively, of these bacteria in caecal contents within 24 h of
treatment (Atterbury et al., 2007). However, it was also observed in this study
that some of the Salmonella isolates collected were phage-resistant. Similarly,
Sklar and Joerger (2001) isolated phage-resistant S. Enteritidis from chickens
that were challenged with the bacterium and subjected to phage therapy; they
noted a reduction of 0.3–1.3 log reduction in the population of S. Enteritidis
in caecal contents, but the results were not consistent across five experiments.
Two other studies reported that phage therapy was successful in short- but not
long-term elimination of Salmonella from the intestinal tracts of chickens.
Administering two phages separately, or in combination, reduced the numbers of S. Enteritidis after 24 h, but after 48 h populations were similar to those
in the control treatment (Andreatti Filho et al., 2007). Likewise, counts of
S. Enteritidis in digestive contents were reduced 12 h after inoculation with
phages, but this effect was less apparent after 24 and 48 h (Berchieri et al.,
1991). In the latter study, bacteriophage treatment was most effective when
numbers of Salmonella exceeded 106 cfu ml–1.
An epidemiological study in the UK found that the number of C. jejuni
present in the caeca of broiler chickens was noticeably lower if Campylobacterspecific bacteriophages were also detected within intestinal contents (Atterbury
et al., 2005). Subsequent experiments have shown that bacteriophage therapy
can reduce Campylobacter in poultry. A thorough study by Loc Carrillo et al.
(2005) reported a 0.5–5 log reduction in caecal counts of C. jejuni when
challenged birds were administered Campylobacter-specifc bacteriophage. The
results were dependent on several factors, including host specificity, dosage
and the time that samples were collected after phage administration. Wagenaar
et al. (2005) reported therapeutic and preventive applications of bacteriophages in broilers. The time of administration of bacteriophage relative to the
time of infection may be a particular important factor in determining the efficacy of bacteriophage therapy. Broiler chickens administered bacteriophage
before challenge with C. jejuni exhibited delayed colonization, whereas those
that received bacteriophage post-challenge showed a threefold decline in
C. jejuni caecal contents (Wagenaar et al., 2005), although these effects were
short term, with populations of C. jejuni returning to near pretreatment levels
over time.
Less work with bacteriophages has been undertaken in swine. Wall et al.
(2010) showed that administering an anti-Salmonella phage cocktail to pigs
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T.W. Alexander et al.
inoculated with S. Typhimurium reduced colonization by 99%, and gave a
2–3 log reduction of the pathogen population. Under commercial conditions,
pigs inoculated with S. Typhimurium were co-mingled with cohorts treated
with either a phage cocktail or a placebo. Pigs that received the phage cocktail
had lower numbers of S. Typhimurium in their caecal contents.
A recent study of feedlot cattle in a commercial setting reported that a
higher prevalence of E. coli O157:H7-specific bacteriophage in faecal pats or
water trough samples was associated with reduced prevalence of E. coli O157:H7
in rectal faecal samples (Niu et al., 2009a). This suggests that the use of bacteriophages may have applications in mitigating infection by this bacterium,
although the complexity of the ruminant digestive tract may pose challenges.
Bach et al. (2003) showed that administration of phages could accelerate the
elimination of E. coli O157:H7 from an artificial rumen system, but in sheep
challenged with E. coli O157:H7, inoculation with bacteriophages had no
effect. Similarly, phages that displayed in vitro activity against E. coli O157:H7,
and eliminated E. coli O157:H7 in infected mice, did not elicit the same
response in challenged cattle (Sheng et al., 2006); however, in this study, concentrations of E. coli O157:H7 shed in faeces were reduced for up to 10 days by
bacteriophage therapy. Similarly, a short-term reduction in E. coli O157:H7 in
challenged sheep showed that bacteriophage-treated animals had reduced
levels of the pathogen in intestinal contents and faeces 24 h (Callaway et al.,
2008a) and 2 days (Raya et al., 2006) after treatment.
Mitigating bacterial pathogens with bacteriophages has had variable
results, but this can be expected given their diversity. Additionally, the highly
specific nature of phages will limit the susceptibility of bacteria, which has
been shown to change according to bacterial genotype and geographical origin of the bacterial isolates (Niu et al., 2009b). A bacteriophage cocktail would,
therefore, be the most efficacious method of using phages to mitigate pathogens on the farm. Also, the use of phages may be most beneficial directly
before animals are sent to slaughter, given the potential for the development
of bacterial resistance and the transient protection against pathogens observed
in some studies.
Waste Management
Proper management of animal waste is important in preventing the spread of
pathogens from livestock to the greater environment. For example, in the
poultry industry, litter can harbour pathogens, and its prolonged retention in
barns can promote the spread of bacteria (Vicente et al., 2007). Additionally,
compromised vaccine performance has been related to poor hygiene in laying
houses (Gast, 2007). Throughout feedlots, rapid spread of E. coli can occur
within and between animal pens (Stevenson et al., 2003). E. coli O157:H7 can
be present in environmental samples even when cattle do not harbour this
pathogen (Davis et al., 2005), and faeces on pen floors have been implicated
as a significant source of infection (Bach et al., 2005). Removal of waste from
animal housing is therefore necessary to reduce the degree of horizontal
153
On-farm Mitigation of Pathogens to Prevent Disease
10
MAC
9
A44
AS700
Control
8
7
6
0
10
MAC+AMP
7
Log10 cfu g–1 DM
6
5
4
3
2
1
0
10
MAC+TET
9
8
7
6
5
4
3
2
1
0
0
15 30 45 60 75 90 105 120 135 150 165 180
Day
Fig. 7.2. Mean counts (n = 3, plus SE) of total, ampicillin-resistant and tetracycline-resistant
Escherichia coli isolated from faecal deposits using MacConkey agar (MAC) or MAC amended
with ampicillin (MAC+AMP, 32 μg ml–1) or tetracycline (MAC+TET, 16 μg ml–1), respectively.
The treatments were as follows: control, no antimicrobial agents added to the diets of
steers from which faecal deposits originated; A44, chlortetracycline (44 ppm); and AS700,
chlortetracycline and sulfamethazine (each at 44 ppm). Faecal deposits were left under
ambient field conditions for 175 days. DM, dry matter; cfu, colony forming units. (From
Alexander et al., 2009.)
154
T.W. Alexander et al.
transmission among individuals. Bacteria from faecal material can also migrate
to surface water, thereby potentially contaminating water sources used by
humans (Schuster et al., 2005). Containment of waste within and outside
housing structures is critical to preventing environmental contamination.
Typically, livestock waste is applied to land as a fertilizer. Enteric pathogens survive well in manure and can therefore also be transmitted to fields
and waterways. Antimicrobial-resistant E. coli has been shown to grow in cattle
faeces and survive in this environment for more than 175 days (Alexander
et al., 2009; Fig. 7.2). Long-term survival of Salmonella in faeces (Sinton et al.,
2007) and soil (Holley et al., 2006) has also been reported. Therefore,
E. coli O157:H7 (log10 cfu g–1 wet wt)
(a)
10
P80
P160
Control
8
6
4
2
0
Total coliforms (log10 cfu g–1 wet wt)
(b)
6
P80
P160
5
4
3
2
1
0
0 7 14
28
56
84
Days of composting
112
147
Fig. 7.3. Survival of Escherichia coli O157:H7 and total coliforms in compost during 147
days of composting (mean ± SE, n = 4). (a) Effect of heat on E. coli O157:H7 inactivation.
Autoclaved manure was inoculated with E. coli O157:H7, sealed in polypropylene vials, and
embedded at depths of 80 and 160 cm (P80 and P160) in compost. Control samples were
retained on the laboratory bench (20°C). (b) Enumeration of total coliforms in compost at P80
and P160. (From Xu et al., 2009.)
On-farm Mitigation of Pathogens to Prevent Disease
155
treatment of waste before its application to land is recognized as an important
step in reducing the transmission of pathogens to other hosts and/or environments (Leifert et al., 2008).
Composting has been established as an effective technology to reduce
pathogens (Wilkinson, 2007). Within a compost pile of spent broiler litter,
reductions in E. coli, faecal coliforms and enterococci ranged from 5.96 to 8.18
log units over a 110-day period (Mohee et al., 2008). Shepherd et al. (2010)
measured the prevalence of E. coli and Salmonella in compost piles on five poultry farms that employ a variety of composting practices. E. coli was detected in
63% of samples collected from the pile surface and in 9.8% of samples collected from the inside of the pile during the primary composting phase. Prevalence at these locations declined to 16.7% and 0%, respectively, during the
second composting phase. With further turning of the pile, E. coli was no
longer detected, suggesting that that heat-sensitive pathogens can be virtually
eliminated from composted poultry waste. Similar to these results, E. coli
O157:H7 was found to survive for up to 4 months on the surfaces of compost
piles composed of dairy cattle manure (Shepherd et al., 2007). In this study,
most locations within the compost pile reached temperatures of 50°C,
although temperatures were not uniform throughout the compost pile. At
locations where temperatures clearly reached 50°C or higher, such as the centre and bottom of the pile, E. coli O157:H7 was not detected after 14 days. A
biosecure static composting system containing bovine mortalities and manure
also showed temperature stratification (Xu et al., 2009). At depths of 80 cm,
temperatures of 55–65°C were maintained for more than 30 days, whereas at a
depth of 160 cm, temperatures failed to exceed 55°C. Regardless of location,
E. coli O157:H7 was rendered undetectable after 7 days, suggesting that factors
other than heat alone contribute to the ability of the composting process to
reduce the viability of pathogens (Fig. 7.3). DNA isolated from Campylobacter
was amplifiable for 84 days at 80 cm and 160 days at 160 cm but, overall, there
was a greater than 6 log reduction of this bacterium. The above studies indicate that, when executed properly, thermophilic composting can substantially
reduce the likelihood of viable pathogens entering the environment when
compost is applied to land as a fertilizer.
Conclusion
Despite postharvest efforts to control enteric pathogens entering the food
chain, contamination of food can occur. Reduction of both the prevalence
and concentration of pathogens in livestock would ease the burden on existing control measures within abattoirs. Preharvest mitigation strategies have
therefore been investigated and implemented to control on-farm pathogens.
Additionally, methods to mitigate the environmental spread of pathogens,
such as composting animal waste, are important. While none of the methods
results in complete eradication of pathogens, reducing pathogen prevalence
by a single method or a combination of methods reduces the risk of food and
environmental dissemination.
156
T.W. Alexander et al.
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8
Organic Agriculture and its
Contribution to Zoonotic
Pathogens
BASTIAAN G. MEERBURG AND FRED H.M. BORGSTEEDE
Introduction
Organic agriculture is defined as a form of agriculture that relies on crop rotation,
green manure, compost, biological pest control and mechanical cultivation to
maintain soil productivity and control pests, excluding or strictly limiting the
use of synthetic fertilizers and synthetic pesticides, plant growth regulators,
livestock feed additives and genetically modified organisms (GMOs).
The organic movement started in the 1930s as a criticism of mainstream
conventional farming with its increasing industrialization and use of pesticides and chemical fertilizers. A British biologist, Sir Albert Howard, travelled
to India in order to teach the Indians about conventional agriculture.
Instead, he was amazed by traditional Indian farming practices and was specifically interested in the connection between a healthy soil and the healthy
population, livestock and crops. From then on, he started to promote Indian
farming practices. In Howard’s tradition, biodynamic agriculture was then
founded by the Austrian philosopher Rudolf Steiner, who emphasized the
interrelationships of animals, plants and soil, and saw farms as unified
organisms. Steiner’s ideas contributed significantly to the development of
modern organic farming.
Organic agriculture has many advantages above conventional agriculture,
at least at a local scale. In the case of organic animal production, animals have
a lower stocking density, obligatory straw bedding and outdoor access, and
are fed with organic feed and/or roughage. The weaning periods of pigs are
longer, while tail, teeth and beak clipping is prohibited. In poultry production,
broiler breeds that grow more slowly are used in order to lower the number of
broilers that cannot keep up and therefore die.
According to consumer perception, organically raised animals are thus
reared under higher welfare conditions. Moreover, the products from these
CAB International 2011. Zoonotic Pathogens in the Food Chain
(eds D.O. Krause and S. Hendrick)
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animals are thought to contain fewer residues (pesticides and veterinary
drugs) than products from conventional animal production systems, as the
use of chemical pesticides or artificial fertilizers is not permitted. Although
consumers and producers sometimes claim that organic produce is healthier
food than conventional produce (Edwards, 2005; Vaarst et al., 2005), the current
scientific evidence does not support this contention (Trewavas, 2004).
Nowadays, the intentions of organic livestock production have been formulated by the International Federation of Organic Agriculture Movements
(IFOAM). However, each region implements its own version of these intentions
and rules may differ significantly. In the EU, there are strict regulations on the
use of antibiotics (longer waiting times after medical treatment before products
are delivered to the market) and GMOs in feed and application of growth promoters are not allowed. The EU has regulated organic animal husbandry via
EU regulation 2092/91 in the year 2000 (Council of the European Union,
2007). In the USA, the application of antibiotics is forbidden in organic livestock production by the USDA Organic Foods Production Act (OFPA) (Jacob
et al., 2008). If an animal receives medical antibiotic treatment, that animal
loses its organic status and should be sold as a regular conventional product.
In organic plant production, use of pesticides is prohibited. Moreover,
seedlings should come from organic sources, and the use of artificial fertilizer
is not allowed. Farmers should use manure from organic farms instead or use
‘green manure’ (nitrogen-fixing plants) in order to keep their land fertile. As
a result of the reduction of inputs, food production in organic systems is less
efficient than in conventional farming systems, and some therefore question
the sustainability of organic production at a global scale. Moreover, the consumption of organic products is in many countries still marginal: only in
Switzerland and Austria does the consumption of organic products exceed 5%.
The Importance of Safe Food
Despite their relative low market share, it is important that products of
organic origin fulfil the highest standards in the field of food safety (Colles et al.,
2008). Generally, consumers believe that organically grown produce will pose
fewer risks than conventionally grown produce (Williams and Hammitt, 2001).
In a specific consumer study, over 90% of the respondents estimated lower
pesticide-related mortality risks associated with the consumption and production of organically grown produce compared with conventionally grown
produce, while about 45% estimated lower natural toxin and microbial pathogen risks (Williams and Hammitt, 2001). However, this perception is not based
on evidence. Contrary to the popular perception that chemical residues form
the major source of food contamination, most foodborne disease outbreaks
have demonstrated that microbial hazards are much more important for
food safety (Cliver, 1999). Risks due to pesticide residues and food additives
are relatively minor compared with both the acute and chronic effects caused
by microbiological and other naturally occurring toxicants (Cliver, 1999;
Magkos et al., 2003).
Organic Agriculture’s Contribution to Zoonotic Pathogens
169
Organic production systems might experience problems concerning
food safety resulting from: (i) their relatively open character; and (ii) the fact
that organic systems are primarily based on biological cycles.
Concerning the relatively open character of organic production systems,
this is of particular importance in organic animal husbandry. Animals have
the opportunity to go outdoors and may more easily come into contact with
pathogens or vectors that might spread pathogens. Wild fauna (e.g. rodents,
flies, etc.) are often mentioned as a potential source of pathogens for
organic livestock (Meerburg et al., 2006, 2007; Meerburg and Kijlstra, 2007;
Kijlstra et al., 2008), but the role of domestic animals such as cats should also
not be forgotten (Kijlstra et al., 2004a). This is specifically important as these
animals are often used to counter rodent presence, as farmers perceive this
method as more ‘natural’ than the application of rodenticides. Although it is
doubtful whether pathogen transmission is always problematic for the animals themselves (as it is often assumed that animals have a better immune
response towards prevailing pathogens under organic conditions than under
conventional conditions (Kijlstra and Eijck, 2006)), pathogen transmission
might eventually result in the contamination of products in the food chain.
With regard to the principle of biological cycles, which includes the use
of organic manure within the farm, the risk of recirculation of infectious
pathogens emerges (Tauxe et al., 1997). Manure from farm animals that is
used as fertilizer for the crops that are used as animal feed, or are for human
consumption, may contain enteric pathogenic microorganisms, and its use as
fertilizer for organic crops can lead to pathogen entry into the food chain
(Pell, 1997). Plants can become infected via the roots or by water splashing on
to the leaf surfaces (Rembiałkowska, 2007). In pot experiments in which contaminated manure was mixed with soil, it was demonstrated that Salmonella
enterica serovar Typhimurium (S. Typhimurium) declined steadily but could
still be traced after 56 days, while the survival of Escherichia coli O157:H7 varied
from 2 to 56 days (Franz et al., 2005). No pathogens were detected in the edible parts of lettuce grown in the pots 2 weeks after mixing with manure, and
only one plant showed the presence of any pathogen (E. coli O157:H7 on root
samples). Even though the composting and drying of manure on the field may
decrease the number of viable pathogens (Pell, 1997; Semenov et al., 2009),
some authors suggest that traditional composting practices are not sufficient
to render animal manure safe for use on vegetables with the advent of pathogens such as E. coli O157:H7 (Tauxe et al., 1997). Moreover, drying manure on
the field will have negative environmental consequences from the formation
of NH3, N2O and other greenhouse gases (Huijsmans et al., 2008).
Although manure is used as a source of crop fertilizer in both organic
and conventional agriculture, the importance of manure as an alternative
source of plant nutrients is greater in organic production systems. Conventional farmers have a variety of synthetic fertilizers at their disposal, while
organic farmers are not allowed to use these. Therefore, the relative risks for
food contamination will most likely be higher in organic production systems
(Albihn, 2001). However, the risk may be determined by the type of plant to
which the manure is applied and the pathogen species. In a recent study
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B.G. Meerburg and F.H.M. Borgsteede
in which crisphead lettuce was grown in ground that was contaminated with
E. coli O157:H7, these bacteria persisted in the ground for a substantial time, but
the lettuce itself did not contain them in its edible parts (Johannessen et al.,
2005). In contrast, the same pathogen is able to persist on organic onions and
carrots for several months (Islam et al., 2005). Moreover, when another pathogen, S. Typhimurium, was added to the ground, it was detected for up to
63 days on lettuce and 231 days on parsley (Islam et al., 2004a), while it was
found for 84 days on radishes and 203 days on carrots (Islam et al., 2004b).
Risks of Organic Plant Products
Several food incidents have been reported as resulting from the consumption
of organic plant products. In 1992, a 2-year-old child died in the USA after
consuming manured vegetables that were inadequately washed. The cause of
infection was identified as E. coli O157:H7 (Cieslak et al., 1993). In 1995, a summer outbreak of gastroenteritis followed by haemolytic uraemic syndrome
(HUS) and thrombotic thrombocytopenic purpura and death was reported in
a nursery school (Tschäpe et al., 1995). The butter that was used to prepare
sandwiches contained organic parsley and this was thought to be the vehicle of
infection. Clonally identical verocytotoxigenic Citrobacter freundii were found
as the causative agents of HUS and gastroenteritis, and were also detected on
the parsley (Tschäpe et al., 1995).
In the USA, both organic and conventional lucerne sprouts were tested
for the presence of Salmonella spp. (Doyle, 2000); Salmonella was detected in
7.7% (3 of 39 samples) of organic sprouts, but not in 39 samples of conventional sprouts. During the same trial, lettuce was tested for E. coli. The bacterium was found in 16.7% (8 of 48 samples) of organic spring mix (lettuce) at
an average count of 106 cfu E. coli g–1, whereas it was detected in 8.3% (4 of 48
samples) of conventional spring mix at an average count of 104 cfu E. coli g–1
(Doyle, 2000). In a study from Minnesota (Mukherjee et al., 2004), microbiological analyses of both organically and conventionally produced fresh
fruits and vegetables (tomatoes, leafy greens, lettuce, green peppers, cabbage,
cucumbers, broccoli, strawberries, apples) were conducted to determine the
coliform count and the prevalence of E. coli, Salmonella and E. coli O157:H7. A
total of 476 and 129 produce samples were collected from 32 organic (of
which eight were certified) and eight conventional farms, respectively. The
proportion of E. coli-positive samples in conventional and organic produce
were 1.6% and 9.7%, respectively. However, the E. coli prevalence in certified
organic produce was 4.3%, which is not statistically different from other samples (Mukherjee et al., 2004). Organic lettuce had the largest prevalence of
E. coli (22.4%) compared with other produce types. Organic samples from
farms that used manure or compost aged less than 12 months had a prevalence of E. coli 19 times greater than that of farms that used older materials.
Serotype O157:H7 was not detected in any produce samples, but Salmonella
was isolated from one organic lettuce and one organic green pepper (Mukherjee et al., 2004).
Organic Agriculture’s Contribution to Zoonotic Pathogens
171
In 2001, a microbiological study was performed on uncooked ready-toeat organic vegetables in the UK to determine the presence of a number of
pathogens (Listeria monocytogenes, Salmonella, Campylobacter and E. coli O157).
In this study (Sagoo et al., 2001) it was found that the majority of the samples
(3185 of 3200; 99.5%) were of satisfactory/acceptable quality, while only 15
(0.5%) were of unsatisfactory quality. The latter was primarily the result of
levels of E. coli and Listeria spp. (other than L. monocytogenes) in excess of 102
cfu g–1. The authors indicated that, overall, agricultural, hygiene, harvesting
and production practices for organic vegetables were good (Sagoo et al.,
2001). In Northern Ireland, commercially available organic vegetables
(n = 86) were examined for the presence of Salmonella, Campylobacter, E. coli
O157, L. monocytogenes and Aeromonas spp. (McMahon and Wilson, 2001);
Aeromonas spp. were isolated from 34% of the total number of organic vegetables examined, while no other enteric pathogens were found. In a recent
study in the UK on edible dried seeds from the retail stores, no difference
was found between seeds labelled as organic and those that were conventional as regards Salmonella contamination, but a significantly higher proportion of organically produced seed samples had unsatisfactory levels of E. coli
(≥102 cfu g–1) (2.4%) compared to those that were not (1.2%) (Willis et al.,
2009).
In Norway, a study was performed that aimed to investigate bacteriological quality in organically grown leaf lettuce (Loncarevic et al., 2005). In total,
179 organic samples were collected from 12 producers. E. coli was isolated
from 16 of the lettuce samples, but in 12 of these contamination was sufficiently low (<100 cfu g–1) that they would be considered to be of acceptable
bacteriological quality. E. coli O157 and Salmonella were not detected in any of
the samples. L. monocytogenes serogroups 1 and 4 were isolated from two
samples (Loncarevic et al., 2005).
In a recent study in the Netherlands, organic lettuce was compared with
conventional lettuce (Hoogenboom et al., 2008). The organic lettuce was sampled at three distribution centres for supermarkets. Conventional lettuce was
obtained from local stores. None of the samples in this study tested positive
for Salmonella or E. coli O157. The authors concluded that introduction
through the use of animal manure should, therefore, not be considered as a
common problem, but also that the presence of pathogenic microorganisms
cannot be excluded in a small product fraction. To minimize potential contamination risks, several countries (including Canada and the USA) do not
allow the use of non-composted manure (Hoogenboom et al., 2008).
So, although bacterial contaminations are found and the amount of contamination is sometimes higher in organic produce, this is not always the case.
Moreover, the impact of consumption of organic plant products on human
health remains unknown. The incidents that were linked to contamination of
food with E. coli (O157:H7) have stimulated the debate on whether the use of
animal manures in certified organic food production systems might confer any
extra health risk for consumers (Bourn and Prescott, 2002). According to recent
literature however, farmers can take measures to reduce the probability of
infection of their products, e.g. by choosing a clean water source and minimizing
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B.G. Meerburg and F.H.M. Borgsteede
the chances of faecal material splashing on to the crop (Leifert et al., 2008).
Such a risk approach might contribute to a reduction of pathogen presence on
organic plant products, thus decreasing the risks for consumers.
Risks of Organic Products of Animal Origin
In veterinary medicine, the prevention of the transfer of infectious diseases,
such as bacterial diseases (e.g. those caused by L. monocytogenes, E. coli O157:H7,
Salmonella spp. and Mycobacterium paratuberculosis) or parasitic diseases (e.g.
those caused by Toxoplasma gondii and Ascaris suum) is based on the breaking of
cycles. In organic livestock systems, organisms causing disease are essentially
the same as those in conventional production systems. However, breaking of
their life cycles could be more difficult owing to less hygienic circumstances
(the provision of straw and roughage in some production systems) and outdoor access for the animals (Höglund et al., 2001; Hovi et al., 2003). This can
lead to more infections with enteric pathogens of zoonotic potential compared
with conventional systems (Honikel, 1998; Thamsborg et al., 1999; Hermansen,
2003; Eijck and Borgsteede, 2005). There are some helminth and protozoan
infections with zoonotic potential that can enter humans via food or the environment. Examples of foodborne zoonoses are T. gondii, Taenia saginata (cattle),
Taenia solium (pig), Trichinella spp. (wild boar, domestic pigs, horses), Sarcocystis
bovihominis (cattle) and Sarcocystis suihominis (pigs). Of these, T. gondii is by far
the most important and frequent. However, in tropical regions, T. solium can be
a dangerous parasite, not as an adult worm, but as the larval stage, which can
cause neurocysticercosis. Among the zoonotic pathogens that can enter
humans via the environment, there are the helminths Fasciola hepatica and A.
suum, and the protozoans Giardia duodenalis and Cryptosporidium parvum. Infections with these helminths are rare, while the incidence of infections with the
protozoans is rather high, but the origin of the infection is not always clear.
Thus, it is unlikely that organic farming contributes much to infections with
these pathogens. However, a recent human hookworm infection (probably
Ancylostoma duodenale) in Japan was associated with consumption of imported
organic produce (Kajiya et al., 2006), indicating that individual cases do occur.
Bacteria can also cause trouble. In Denmark, an outbreak of Shiga toxinproducing E. coli O26:H11 infection in 20 patients (median age 2 years) was
recently described (Ethelberg et al., 2009); the source of infection was identified as an organic fermented beef sausage that was sold in a supermarket.
In this section, we will discuss the three most relevant pathogens among
the bacteria, protozoa and helminths – T. gondii, Salmonella and Campylobacter
– and see whether differences can be found in their occurrence between
regular and organic produce.
Toxoplasma gondii
Toxoplasmosis, caused by T. gondii, is the most prevalent parasitic zoonotic
disease throughout the world (Tenter et al., 2000). It is an important cause of
Organic Agriculture’s Contribution to Zoonotic Pathogens
173
abortion in humans and livestock (sheep) and was recently shown to be the
third most frequent cause of death following foodborne illnesses (Mead et al.,
1999). Recently, the estimated disease burden of congenital toxoplasmosis in
the Netherlands was estimated to be similar to that for salmonellosis in terms
of disability adjusted life years per year (Havelaar et al., 2007).
In humans the parasite is known to cause encephalitis, mental retardation
and blindness. Treatment is sometimes difficult, especially the treatment of
ocular toxoplasmosis (Stanford et al., 2003). So at present, the prevention of
infection by T. gondii is the best strategy. Cats are definitive hosts for the
parasite and can excrete millions of eggs – once over a short period in their
lifetime – that are spread throughout the environment (Kijlstra et al., 2008).
Favourable climatic conditions may contribute to the survival of the pathogen
(Meerburg and Kijlstra, 2009). When the excreted eggs or infected vectors
(e.g. rodents) are consumed by farm animals, tissue cysts may develop in the
meat of the livestock. If improperly cooked (at a temperature < 67°C) humans
may acquire infection.
In the Netherlands, during 1995–1996, a population-based seroprevalence
study was conducted in pregnant women; the results were compared with
those from a study conducted during 1987–1988 in order to estimate the
change in seroprevalence (Kortbeek et al., 2004). In total, 7521 sera were
tested and the national seroprevalence was 40.5%. The seroprevalence among
women aged 15–49 years was 10% lower in the study of 1995–1996 compared
with that of 1987–1988 (45.8%) (Kortbeek et al., 2004), but the steepest rise in
seroprevalence still occurred among the subjects aged 25–44 years. One of
the reasons for this could be the consumption of undercooked pork meat,
which is considered a major risk factor for contracting toxoplasmosis in humans.
In a previous study, it was found that conventionally (indoors) raised pigs are
free from T. gondii infection, while animal-friendly raised pigs (with outdoor
access) were contaminated in 2.9% of the cases (Kijlstra et al., 2004b). However,
T. gondii infection can also be contracted by consumption of the meat of many
other animals, including poultry (Kijlstra and Jongert, 2008). Therefore, it is
important that all meat is frozen or properly cooked before consumption.
Salmonella
In a recent study (Hoogenboom et al., 2008), the presence of Salmonella in pigs
at regular and organic pig farms was compared. No large differences were
observed, but the authors got the impression that Salmonella incidence at
farms that had transformed from regular farming to organic farming some
years earlier was lower. In Denmark, Zheng et al. (2007) found a lower Salmonella prevalence in pigs with outdoor access (both in conventional and organic
systems) compared with pigs that were reared indoors. Because these researchers did not find a difference in the numbers of antibodies between the groups,
they came to the conclusion that pigs with outdoor access must have a better
defence mechanism against Salmonella. However, in earlier Danish and Dutch
studies, Salmonella seroprevalence has been shown to be higher in free-range
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B.G. Meerburg and F.H.M. Borgsteede
finishing pigs than in those produced in conventional intensive systems (Wingstrand et al., 1999; Van der Wolf, 2000). These contradicting results are a
complicating factor. Another problem is the absence of clinical signs of salmonellosis in the majority of pigs, which may result in the entrance of undetected
carriers into the food production chain.
In a Belgian study in which the health status of organic broiler chickens
and the contamination rates with Salmonella and Campylobacter were compared
at slaughter, no difference could be found in prevalence of Salmonella between
organic and conventional broilers (Van Overbeke et al., 2006). In the USA
(Bailey and Cosby, 2005), it was found that Salmonella was more prevalent
in free-range (31%) and all-natural (25%) chickens than in chickens from
the US commercial poultry industry surveyed for the USDA Food Safety
Inspection Service reports in 2000–2003 (9.1–12.8% prevalence); it should be
noted, though, that ten of the 22 lots of free-range chickens and all of the
natural chickens under study had no detectable Salmonella (Bailey and Cosby,
2005).
In an older prospective case-control study of sporadic Salmonella enterica
servovar Enteridis infection in Denmark (1997–1999), the consumption of
eggs was identified as the key source of infection, but these authors were
unable to demonstrate an association with the consumption of organic eggs
(Molbak and Neimann, 2002).
Campylobacter
Campylobacter in broilers has proved to be higher in organic systems than in
conventional flocks (Heuer et al., 2001; Rodenburg et al., 2004; Van Overbeke
et al., 2006). Heuer et al. (2001) isolated Campylobacter spp. from 100% of
organic broiler flocks, 49.2% of extensive indoor broiler flocks and 36.7%
of conventional broiler flocks. Schwaiger et al. (2008) also found a higher
prevalence in organic flocks, but claim that this is the result of differences in
the testing procedures. Most likely, organic broilers become infected with
Campylobacter between weeks 7 and 10 (Van Overbeke et al., 2006).
In a Danish study in pigs, it was found that the increased exposure of
outdoor pigs to Campylobacter jejuni from the environment may cause a shift
from a normal dominance of Campylobacter coli to more C. jejuni, which may
imply a concern of reduced food safety (Jensen et al., 2006). Apparently, in
the case of Campylobacter, the contact of farm animals with outdoor wildlife,
such as birds and rodents, is vital (Jensen et al., 2006; Meerburg et al., 2006).
Conclusions
Food safety hazards are currently an inherent but probably major risk in
organic production systems. It is difficult to drive out this risk, as the
whole farming system is generally more open, which might facilitate the
introduction or preservation of hazards. Moreover, organic production is
Organic Agriculture’s Contribution to Zoonotic Pathogens
175
not a guarantee that the environmental performance of the farming system
will improve (Cederberg and Mattsson, 2000; Rigby and Cáceres, 2001; Trewavas,
2001; De Boer, 2003), although sometimes it will (Reganold et al., 2001; Kumm,
2002; Pacini et al., 2003).
Nevertheless, there are also advantages to organic production systems. In
organic animal husbandry, the use of antibiotics is not allowed. Consequently,
at organic pig and broiler farms a lower resistance of a number of selected
microorganisms to antibiotics has been demonstrated (Hoogenboom et al.,
2008). Furthermore, organic farmers often claim that animal welfare in
organic animal husbandry is generally better, as farm animals are allowed to
lead a more natural life (Lund, 2006), although it is rather questionable what
effects raising the housing standards will have on the total well-being of the
animals (Sundrum, 2001). Other authors even claim, on the basis of practical
experience, that organic livestock production is not a guarantee of good
animal health and welfare (Hovi et al., 2003; von Borell and Sørensen, 2004).
Some studies have compared nutrient compositions of organically and
conventionally produced crops and animal products (meat, milk and dairy
products). Very few compositional differences have been reported (Brandt
and Mølgaard, 2001; Caris-Veyrat et al., 2004), although there are reasonably
consistent findings for higher nitrate and lower vitamin C contents of conventionally produced vegetables, particularly leafy vegetables (Williams, 2002).
Additionally, in a recent study where the organic food consumption by infants
was associated with developing atopic manifestations in the first 2 years of life,
it was found that the consumption of organic dairy products led to a lower
incidence of eczema (Kummeling et al., 2008). More of these studies need to
be performed in order to substantiate these results.
When considering the above, one might speak about a true dilemma.
The consumer has to decide which factor(s) he or she thinks is most important. Consumers who choose organic foodstuffs should be aware that there is
a potential risk of food contamination – potential, as there are currently gaps
in scientific knowledge that make it difficult to weigh the exact risks of organic
production. The attribution of human illness has been recently recognized as
an important tool to better inform food safety decisions. Analysis of outbreak
data sets is necessary for that purpose, but so far only a limited number of
such studies has been performed. Often, it proves difficult to directly link the
agent and food vehicle. In a recent Canadian study for example, foodborne
outbreak data sets covering 30 years were investigated to estimate food attribution in cases of gastrointestinal illness (Ravel et al., 2009); although overall
6908 foodborne outbreaks were described, the agent and food vehicle were
only identified in 2107 of these cases, and in most of these studies organic
production was not incorporated as a specific factor.
To consumers, it should be made clear that ‘organic’ products are not
by definition ‘safe’. Currently, many consumers perceive organic food as
healthier than conventional food products (Schifferstein and Oude Ophuis,
1998; Trewavas, 2001; Zanoli and Naspetti, 2002; Magnusson et al., 2003). In
a study in the USA, over 90% of survey respondents perceived a reduction
in pesticide residue risk associated with substituting organically grown
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B.G. Meerburg and F.H.M. Borgsteede
produce for conventionally grown produce, and nearly 50% perceived a
reduction in risk due to natural toxins and microbial pathogens (Williams
and Hammitt, 2001). In reality, organic products can be expected to contain
fewer agrochemical residues and lower levels of nitrate than conventional
products, but might contain more food hazards of other origin, such as
microbial pathogens and mycotoxins (Lu et al., 2006; Magkos et al., 2006).
Consequently, there is friction between the consumer perception of the
food safety of organic produce and the true safety risks. Thus, more attention
should be paid to consumer education about possible food safety hazards and
the communication of risk in order to maintain public trust (Kijlstra et al., 2009).
Beside this, producers should adopt proper agricultural practices in order to
limit the risks associated with organic production as much as possible (e.g. by
usage of parasite-safe pastures), and processors of organic products should do
their utmost to prevent food contaminations, e.g. by decontamination.
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9
Zoonotic Implications of Avian
and Swine Influenza
JUAN C. RODRIGUEZ-LECOMPTE, SUDHANSHU SEKHAR AND
TOMY JOSEPH
Avian Influenza
Avian influenza (AI) is an infectious viral disease of birds caused by type A
viruses of the family Orthomyxoviridae (Swayne and Halvorson, 2003).
Although the first official description of AI was documented in Italy in 1878
(Lupiani and Reddy, 2009), it is believed to have existed since ancient times
(Hirsch, 1883). In the 20th century, there have been three influenza pandemics, which were caused by H1N1, H2N2 and H3N2 subtypes of the influenza
virus in the years of 1918, 1957 and 1968, respectively (Morens and Fauci,
2007). Of these, the H1N1 pandemic of 1918, the so-called ‘Spanish Influenza’, was the deadliest, and resulted in more than 50 million deaths worldwide (Erkoreka, 2009). The emergence of the H2N2 and H3N2 subtypes
caused relatively mild pandemics (Scholtissek et al., 1978). Furthermore, AI
causes worldwide morbidity and mortality in domestic and wild birds. The
emergence of new highly pathogenic avian influenza (HPAI) strains of type A
viruses has led to devastating consequences for the poultry industry, resulting
in the culling of hundreds of millions of birds (Swayne and Suarez, 2000).
The greatest threat posed by the AI virus lies in its zoonotic potential and
ability to infect and cause significant morbidity and mortality in humans (Yen
and Webster, 2009). The frequent outbreaks of AI in poultry and the transmission of AI viruses to humans reflect the potential for pandemic spread of these
viruses. In 1997, an AI outbreak in Hong Kong attracted attention worldwide
when a highly pathogenic strain of AI virus, H5N1, crossed the species barrier
and infected humans, resulting in six fatalities out of 18 infected humans (Li et al.,
2004). The culling of all poultry in Hong Kong contained further viral dissemination, but the H5N1 viruses continued to circulate among wild aquatic
birds in coastal regions of China (Chen et al., 2004). Since 1997, more than 500
human cases of AI infections have been reported, with a case fatality rate of
about 60%. Interestingly, HPAI viruses are exhibiting an unprecedented
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(eds D.O. Krause and S. Hendrick)
Zoonotic Implications of Avian and Swine Influenza
183
geographical distribution and increased host range (Alexander, 2007), which
substantiates the belief that an influenza pandemic is imminent. AI has, therefore, become one of the greatest concerns for public health in recent times.
Avian influenza viruses
The influenza viruses have been found to infect a variety of hosts, ranging
from birds to mammals, but only viruses of the influenza A genus are known
to infect birds. The virus has a negative-sense single-stranded RNA genome.
The genome has eight segments with a total length of about 14 kb. Influenza
A viruses may be classified into subtypes on the basis of the antigenic properties of their surface glycoproteins – haemagglutinin (HA) and neuraminidase
(NA). Sixteen subtypes of HA (H1–H16) and nine subtypes of NA (N1–N9)
have been reported with almost all subtype combinations (Palese and Shaw,
2006). HA helps in virus attachment to the eukaryotic cell receptors and the
fusion of viral and cellular membranes, while NA cleaves the sialic acid receptors from the host cell membrane, thereby facilitating the release of the virus
from the host cell. Furthermore, the genes encoding the internal virus proteins are highly conserved between influenza A viruses. The matrix 1 (M1)
protein is found between the core and the membrane, and plays an important
role in the assembly of the nucleocapsids (the genome plus its protein coat, or
capsid) and membrane-bound proteins. The matrix 2 (M2) protein, an integral membrane protein, operates as a hydrogen ion channel regulating the
pH environment of the virus. The multi-segmented viral genome is coated
with the nucleocapsid protein (NP). The NP and the RNA polymerase proteins PB1 and PB2 (polymerase basic proteins 1 and 2) and PA (polymerase
acidic protein) are potential targets of the cell-mediated immune system
(Wright, 2007).
Pathogenesis of avian influenza
On the basis of clinical manifestations in poultry, the influenza viruses can be
divided into two groups: highly pathogenic avian influenza (HPAI) and low
pathogenic avian influenza (LPAI). HPAI viruses cause severe disease in poultry, leading to almost 100% mortality. In contrast to this, the LPAI viruses
cause a much milder form of the disease, causing less morbidity and almost no
mortality in infected birds (Webster et al., 1992). The HPAI and LPAI viruses
in domestic poultry are said to be transmitted from wild birds. Moreover, the
LPAI H5 and H7 viruses circulating into poultry from wild birds may mutate
and transform into HPAI viruses. The LPAI and HPAI viruses show structural
differences at the so-called cleavage site of the precursor of the viral HA, which
must be cleaved into HA1 and HA2 for the virus to become infectious. For all
influenza A viruses, the HA glycoprotein is produced as a precursor, HA0,
which requires post-translational cleavage by host proteases before it is functional and virus particles become infectious (Rott, 1992). The LPAI viruses
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J.C. Rodriguez-Lecompte et al.
have only one or a few basic amino acids at this site, and cleavage occurs
exclusively by trypsin-like host proteases. Virus replication is, therefore,
restricted to sites in the host where such enzymes are found, i.e. the epithelia
of the respiratory and intestinal tracts. HPAI viruses, in contrast, possess
multiple basic amino acids at their HA cleavage sites and can be cleaved by a
broad range of cellular proteases (Chen et al., 1998; Pantin-Lockwood and
Swayne, 2009). The transition from an LPAI phenotype to the HPAI phenotype is achieved by the introduction of basic amino acids into the HA cleavage
site, which facilitates systemic virus replication, causing an acute generalized
disease in poultry in which mortality may be as high as 100% (Webster and
Rott, 1987). However, studies of AI viruses in chickens and ducks show that the
NA, NS (non-structural), PA, PB1, PB2 and NP proteins can also play an
important role in pathogenesis of the disease (Pantin-Lockwood and Swayne,
2009).
Emergence of new avian influenza virus strains
The influenza viruses continuously modify their HA and NA antigenic profiles via antigenic drift and antigenic shift. Antigenic drift is caused by point
mutations leading to minor antigenic changes in the HA or NA proteins; this
results from a lack of proofreading ability by the RNA polymerases during
viral replication (Holland et al., 1982). In contrast, antigenic shift refers to
major antigenic changes resulting in new HA or NA proteins; because the
proteins are distinct from the previously circulating strains, populations will
have no immunity to the new subtype. Antigenic shift may occur as a result of
genetic reassortment, which occurs when a host cell is infected by two influenza A viruses at the same time. The segmented nature of the genome facilitates this reassortment by allowing the mixing of genome segments from
distinct AI viruses inside the infected cell (Hayashida et al., 1985) (Fig. 9.1).
Pandemic influenza occurs when a new virus to which the human population has no or little immunity emerges (Wright, 2007). A pandemic AI virus
possesses certain characteristics, e.g. entry and replication in the human
body, causing the disease, being shed in the excreta and effectively spreading
among humans (Brankston, 2007).
Avian influenza ecology and outbreaks in poultry
Aquatic birds are the natural reservoir of influenza A viruses (Webster et al.,
1992). AI viruses have a broad range of avian hosts, e.g. ducks, geese, swans,
gulls, terns, waders, etc. However, turkeys and domestic poultry are most
susceptible to AI viruses (Alexander, 2000; Olsen et al., 2006). The AI viruses
replicate in the respiratory and intestinal tracts of aquatic birds, which show
no clinical symptoms of the disease (Kida et al., 1980). The mechanisms by
which influenza viruses pass from one bird to another and bring about
infection are poorly understood. In the past, some attempts were made to
185
Zoonotic Implications of Avian and Swine Influenza
Different AI virus strains
Host cell
Reassortment of genome
segments
New virus strains
Fig. 9.1. Schematic of the process of genetic shift during avian influenza virus replication.
assess the transmissibility of LPAI and HPAI viruses in domestic poultry
experimentally. The results suggested that bird-to-bird transmission is
extremely complex and depends on strain of virus, the species of bird and
environmental factors. The warm winter in South-east Asia attracts migratory birds from northern climates that spend the winter in this region. The
high density of human populations and prevalence of backyard poultry and
pigs provide the opportunity for close interactions between these reservoirs
of influenza virus. Pigs possess the receptors for AI viruses (sialic acids with
a 2,3-galactose linkage) as well for as human influenza viruses (sialic acids
with a 2,6-galactose linkage), and have been considered as ‘mixing vessels’
for generating reassortant viruses (Scholtissek, 1995). Furthermore, the
live poultry market system provides optimal conditions for influenza virus
evolution, with transmission between avian species and possible infection of
humans (Peiris et al., 2007). In both natural and experimental infections,
virulent viruses have tended to show much poorer transmission from
infected to susceptible chickens and turkeys than viruses of low pathogenicity. The ability of the virus to spread easily should be related to the
amount of virus shed through the respiratory or intestinal route. The highly
pathogenic viruses cause extremely rapid deaths in these birds and it is
possible that relatively little virus is excreted during the course of such
infections. Nevertheless, this contaminates lake or pond water to the extent
that virus may be isolated from lake water where large numbers of waterfowl
are found.
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J.C. Rodriguez-Lecompte et al.
The prognosis of influenza infection for wild birds is different from
that for domestic birds (Alexander, 2000). In wild birds, the infections do
not usually show clinical symptoms, and most influenza viruses isolated from
wild birds are low pathogenic (LP) for poultry. This supports the hypothesis
that HP (highly pathogenic) H5 or H7 viruses emerged after the introduction of an LPAI virus from wild ducks to poultry. Interestingly, viruses that
are HP for poultry usually replicate poorly or to a limited degree in wild
birds. However, the 1961 H5N3 HPAI outbreak in terns in South Africa and
the HP H5N1 virus have caused significant mortality and morbidity among
wild birds. The H5N1 virus has caused disease and death among migratory
geese populations in western China. The increased virulence of some of the
recent H5N1 isolates for some duck species has also been confirmed in
experimental studies (Sturm-Ramirez et al., 2005). Also, some recent H5N1
subtypes may still cause subclinical disease in aquatic wild birds. The target
tissue for LPAI viruses is mainly the intestine, where they replicate and are
subsequently shed in faeces. The AI H5N1 strain prefers the respiratory tissues of wild birds (Brown et al., 2006). These HPAI viruses include the H5 or
H7 subtypes of the AI viruses, but all H5 or H7 subtypes are not highly virulent. The influenza A viruses of subtype H5 and H7 may become highly
pathogenic after introduction into poultry, and can cause outbreaks of HPAI
(Table 9.1). Taking account of the severity of the disease, HPAI is classified
as a list A disease by the World Organisation for Animal Health (OIE, originally Office Internationale des Epizooties) (Alexander, 2000). The OIE
defines highly pathogenic notifiable AI as any AI virus with an intravenous
pathogenicity index in 6-week-old chickens greater than 1.2 or that causes at
least 75% mortality in 4- to 8-week-old chickens infected intravenously
(Swayne and Halvorson, 2003).
Avian influenza zoonoses
A few subtypes of AI viruses are a public health threat as they have the potential to cause serious illness and death in humans, but only six strains – H5N1,
H7N2, H7N3, H7N7, H9N2 and H10N7 – have been found to infect humans
(Table 9.2). Of these, H5N1 is highly virulent and considered to have the
potential to cause the next pandemic in human populations.
The sudden outbreak of Hong Kong HPAI H5N1 virus in 1997 caused a
devastating effect on the poultry industry, leading to the culling of millions
of poultry (Li et al., 2004). The virus strain from which the H5N1 emerged is
still circulating in aquatic birds in south China, without showing any clinical
symptoms in these birds. The H5N1 infection underwent further expansion
in many South-east Asian countries, including South Korea, Vietnam, Japan,
Cambodia, Indonesia, Thailand, China and Laos. The viruses were contracted by the wild bird population from domestic poultry, resulting in huge
mortality of thousands of migratory birds at the Qinghai Lake Nature Reserve
in China (Olsen et al., 2006). Since the 1997 outbreak, the H5N1 viruses are
showing an increased geographical expansion, and have reached Europe
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Zoonotic Implications of Avian and Swine Influenza
Table 9.1. Highly pathogenic avian influenza (HPAI) outbreaks in poultry since 1959
(adapted from Alexander, 2007).
Approximate numbers of poultry
involved
HPAI virus
Subtype
1 A/chicken/Scotland/59
2 A/turkey/England/63
3 A/turkey/Ontario/7732/66
4 A/chicken/Victoria/76
5 A/chicken/Germany/79
6 A/turkey/England/199/79
7 A/chicken/Pennsylvania/1370/83
8 A/turkey/Ireland/1378/83
9 A/chicken/Victoria/85
10 A/turkey/England/50–92/91
11 A/chicken/Victoria/1/92
12 A/chicken/Queensland/667-6/94
13 A/chicken/Mexico/8623-607/94
14 A/chicken/Pakistan/447/94
15 A/chicken/NSW/97
16 A/chicken/Hong Kong/97b
17 A/chicken/Italy/330/97
18 A/turkey/Italy/99
19 A/chicken/Chile/2002
20 A/chicken/Netherlands/2003
21 A/chicken/Eurasia and Africa/
2003–2006
22 A/chicken/Texas/2004
23 A/chicken/British Columbia/2004
24 A/ostrich/South Africa/2004
25 A/chicken/North Korea/05
26 A/turkey/England/07
27 A/chicken/Canada/2007
28 A/chicken/England/2008
29 A/chicken/Spain/2009
H5N1
H7N3
H5N9
H7N7
H7N7
H7N7
H5N2
H5N8
H7N7
H5N1
H7N3
H7N3
H5N2
H7N3
H7N4
H5N1
H5N2
H7N1
H7N3
H7N7
H5N1
1 small farm
29,000
8,000
58,000
1 chicken farm, 1 goose farm
9,000
17,000,000
307,000, mostly ducksa
240,000
8,000
18,000
22,000
Unknown (? millions)
>6,000,000
160,000
3,000,000
8,000
14,000,000
700,000
>25,000,000
Unknown (100s of millions)
H5N2
H7N3
H5N2
H7N7
H5N1
H7N3
H7N7
H7N7
6,600
16,000,000
30,000
219,000
160,000
540
15,000
30,000
a A very closely related virus was isolated from ducks in the area, and the epidemiological data
suggested that the virus had been maintained in ducks as a subclinical infection before transmission
to turkeys.
and Africa. HPAI H5N1 virus is notorious for its zoonotic potential, and
causes high mortality and morbidity in humans. The recent spread of HPAI
H5N1 across Asia, Europe and Africa raises the concern of a possible new
pandemic, in the case that the virus attains the ability to become transmissible from person to person. The evolution of H5N1 into a pandemic threat
could occur through a single reassortment of its segmented genome or
through the slower process of genetic drift (Fauci, 2006). Furthermore, the
cases of human infection with H5N1 viruses in 1997 in Hong Kong
188
J.C. Rodriguez-Lecompte et al.
Table 9.2.
2009).
Cases of human infection with avian influenza viruses from 1997 (source: WHO,
Virus
strains
Year
Country
Clinical symptoms
H5N1
1997
Hong Kong
2003–2009
1999
1999
2003
2007
2003
Azerbaijan, Bangladesh,
Cambodia, China,
Djibouti, Egypt,
Indonesia, Iraq, Laos,
Myanmar, Nigeria,
Pakistan, Thailand,
Turkey, Vietnam
Hong Kong
China
Hong Kong
Hong Kong
Netherlands
Conjunctivitis, pneumonia,
influenza-like symptoms
Pneumonia, influenza-like
symptoms
2004
2006
2002
2003
Canada
UK
USA
USA
2007
2004
UK
Egypt
H9N2
H7N7
H7N3
H7N2
H10N7
Influenza-like symptoms
Conjunctivitis,
influenza-like
symptoms, pneumonia
Conjunctivitis
Influenza-like symptoms
Upper and lower
respiratory infection
Conjunctivitis
Cough and fever
Number
of cases
(deaths)
18 (6)
444 (262)
2
5
1
1
89 (1)
2
1
11
1
1
2
established the fact that AI viruses could be transmitted directly from
poultry to humans (Claas et al., 1998). However, the transmission of the
H5N1 viruses from birds to humans has been uncommon compared with
the transmission of the virus among birds.
The clinical symptoms of H5N1 infection in humans vary from severe
pneumonia to mild upper respiratory tract infection without pneumonia. Gastrointestinal symptoms are also reported in patients suffering from certain
clades of H5N1 viruses (Abdel-Ghafar et al., 2008). Although the mechanism
behind avian-to-human infection is poorly understood, close contact with
infected birds and the consumption of undercooked poultry meat are important causes of human infection (Beigel et al., 2005; Abdel-Ghafar et al., 2008).
Also, the limited human-to-human infections that have occurred have
been observed in family members taking care of H5N1-infected patients
(Ungchusak et al., 2005). In addition, the intrauterine transmission of H5N1
from mother to fetus has been reported (Gu et al., 2007).
In February 2003, there was an HPAI H7N7 outbreak in poultry in the
Netherlands. The outbreak spread further to the neighbouring countries of
Zoonotic Implications of Avian and Swine Influenza
189
Belgium and Germany. To contain the outbreak, more than 30 million poultry
were culled. This outbreak of H7N7 infected 89 humans with a single mortality. Infected people did not show the typical influenza-like clinical symptoms,
but rather conjunctivitis (Koopmans et al., 2004). In 2004, an HPAI H7N3
outbreak was reported in Canada (British Columbia), leading to the culling
of more than 17 million domestic poultry (Berhane and Hooper-McGrevy,
2009). Two humans were infected and also presented with conjunctivitis
(Hirst et al., 2004).
Swine Influenza
Swine influenza is an economically important respiratory disease of pigs
throughout the world. It was first recognized as a disease of pigs in the Midwestern USA in 1918, coinciding with the ‘Spanish Influenza’ pandemic in
the human population (Koen, 1919; Webster, 2002). Swine influenza is manifested as an acute respiratory disease characterized by fever, lethargy,
decreased food intake, respiratory distress, coughing, sneezing, rhinitis,
nasal discharge and conjunctivitis (Alexander and Brown, 2000; Richt et al.,
2003). Swine influenza also contributes to the porcine respiratory disease
complex in combination with the porcine respiratory and reproductive syndrome virus, Mycoplasma hyopneumoniae and other bacterial pathogens.
Human infections with swine influenza viruses are well documented, and
humans occupationally exposed to pigs are at increased risk of infection
with swine influenza viruses and developing influenza-like illness (Olsen et
al., 2002; Gray et al., 2007; Myers et al., 2007). Thus, swine influenza also
poses a threat to public health.
Swine influenza viruses
Influenza viruses were first isolated from pigs in 1930 (Shope, 1931) and were
the initial examples of the classical H1N1 lineage of swine influenza A viruses.
Since then, H1N1 viruses have circulated in swine populations in North
America, South America, Europe and Asia. However, the predominant virus in
Europe is an avian H1N1 virus introduced by wild ducks into the swine population in 1979 (Van Reeth, 2007). Although there are genetic, antigenic and
pathogenic similarities between early swine and the 1918 human H1N1
viruses, it is not clear whether a progenitor virus was transmitted from pigs to
humans or from humans to pigs (Memoli et al., 2009). For nearly 70 years,
classical H1N1 remained the predominant swine influenza virus subtype in
North America without much genetic and antigenic change (Sheerar et al.,
1989; Luoh et al., 1992; Noble et al., 1993).
In 1998, a new ‘triple reassortant’ H3N2 swine influenza virus emerged
in the USA which caused severe influenza-like illness in pigs. This triple
reassortant H3N2 swine influenza virus contained HA, NA and PB1 genes
of human influenza virus origin, NP, M and NS genes of classical swine
190
J.C. Rodriguez-Lecompte et al.
H1N1 virus origin, and PB2 and PA genes of North American avian virus
origin. Swine influenza viruses antigenically and genetically related to the
triple reassortant H3N2 viruses were subsequently established in the US
swine population (Webby et al., 2000). Further evolution of H3N2 viruses
through genetic mutation and reassortment with the classical H1N1
viruses led to the emergence of a number of reassortant swine influenza
viruses, including new H3N2 viruses (Webby et al., 2000, 2004; Richt et al.,
2003), H1N2 viruses (Choi et al., 2002; Karasin et al., 2002) reassortant
H1N1 viruses (Webby et al., 2004) and H3N1 viruses (Lekcharoensuk et al.,
2006; Ma et al., 2006). Currently, the H3N2, reassortant H1N1 and H1N2
viruses co-circulate in swine populations in most regions of the USA and
Canada. Although the origin and time of introduction are different, reassortant H3N2 and H1N2 viruses are circulating in Europe as well (Van
Reeth, 2007). Emergence of reassortant swine influenza viruses with mixtures of genes from human, swine and avian viruses support the notion
that pigs may serve as ‘mixing vessels’ for genetic reassortment between
human and avian viruses. Thus, pigs may play an important role in the
generation of influenza viruses with pandemic potential.
In addition to the H1N1 and H3N2 reassortant viruses, a number of
novel swine influenza virus subtypes were isolated from pigs in different
parts of the world. An H1N7 virus containing gene segments from human
and equine influenza viruses was isolated from a pig farm in the UK in 1992
(Brown et al., 1994). An avian influenza H4N6 virus of North American
lineage was isolated from pigs with pneumonia on a commercial swine
farm in Canada in October 1999. This avian virus was probably introduced
by waterfowl from a nearby lake (Karasin et al., 2000). In 2003, transmission of human H1N2 to pigs was reported in Ontario. This H1N2 virus was
genetically and antigenically distinct from the classical swine H1 virus
(Karasin et al., 2006). Researchers in the USA identified a new H2N3 subtype of swine influenza virus from two groups of infected pigs at separate
production facilities in 2006. The new H2N3 swine influenza virus
belonged to the group of H2 influenza viruses that caused the influenza
pandemic in 1957 and continued to circulate among the human population until 1968. The affected swine production facilities apparently used
water from ponds frequented by waterfowl, which are natural hosts for all
subtypes of influenza A viruses. Use of this pond water might have introduced avian influenza viruses into the swine population, although the
exact source of this virus remains unclear. Laboratory studies also revealed
that the new H2N3 virus has undergone adaptations that enabled it to
efficiently infect and replicate in mammals, including pigs, mice and ferrets (Ma et al., 2007). Many of the newly emerged viruses are not able to
establish themselves in the swine population. However, the frequent transmission of human and avian viruses to the swine population increases the
possibility of the emergence of new influenza virus subtypes by reassortment which could have an impact on public health. Introduction of new
viruses and constant changes in the genetic make-up of circulating swine
influenza viruses also pose a challenge to disease control programmes.
Zoonotic Implications of Avian and Swine Influenza
191
Swine influenza zoonoses
Human infections with swine influenza viruses have been reported on several
occasions in the USA, Canada, Europe and Asia. In a recent study (Myers et al.,
2007), 50 cases of zoonotic swine influenza infections were identified and
described. This number may not reflect the actual number of cases of human
infection with swine influenza viruses, as there are no unique clinical features
to discriminate between infections caused by swine and human influenza
viruses. Most of the cases resulted from direct exposure to swine or human-tohuman transmission within a family cluster. Swine influenza H1N1 virus subtype caused more infections in humans than the H3N2 virus subtype (Myers
et al., 2007). A notable case is the swine influenza virus infection in humans
caused by an H1N1 virus at Fort Dix, New Jersey, in 1976. This outbreak
resulted in one death, and respiratory disease in 12 soldiers, along with serological evidence of infection in more than 200 soldiers (Gaydos et al., 1977).
No evidence of exposure to swine was found in these cases and the virus
had disappeared from the human population within a short period of time
(Gaydos et al., 1977).
On 21 April 2009, the Centers for Disease Control and Prevention (CDC)
in the USA announced the identification of a new strain of H1N1 influenza
virus, the pandemic H1N1 2009 virus (pH1N1) in humans. This virus consists
of genomic RNA segments PB2 and PA of North American avian virus origin,
the PB1 gene of human H3N2 virus origin, HA, NP and NS genes of classical
swine H1N1 virus origin, and NA and M genes of Eurasian avian-like swine
origin. The pH1N1 virus is believed to have originated from the reassortment of recent North American H1N1 and H3N2 triple reassortant swine
viruses with Eurasian avian-like swine viruses (Brockwell-Staats et al., 2009).
Although the exact nature of origin of this new virus is not known, it spread
in humans so quickly that by 11 June 2009 WHO declared a phase six pandemic (Brockwell-Staats et al., 2009; Neumann et al., 2009). The virus was
also isolated from a swine herd in Canada on 2 May 2009, and pH1N1 has
now become an emerging swine influenza virus in several countries. Experimental and field infections of pH1N1 virus in swine appear to be similar in
nature to the circulating swine influenza virus infections (Lange et al., 2009). The
risk of transmission of this virus from pigs to humans is yet to be determined,
and it is still uncertain whether the pH1N1 virus will become established in
swine populations in any part of the world.
Recent studies have shown that persons who work with swine, including
farmers, meat-processing workers, veterinarians and their spouses are at
increased risk of zoonotic influenza virus infections (Olsen et al., 2002; Gray
et al., 2007; Myers et al., 2007). These zoonotic infections could increase the
possibility of the mixing of swine and human influenza viruses in swine workers, leading to the generation of new influenza viruses with pandemic potential. People who are exposed to swine could accelerate the transmission of
new pandemic influenza viruses to their communities if such viruses become
enzootic in the swine population (Saenz et al., 2006). However, the frequency
of transmission of swine influenza virus to occupationally exposed people
192
J.C. Rodriguez-Lecompte et al.
is low or negligible compared with the number of people exposed to pigs
(Van Reeth, 2007).
Conclusion
Since the first documented case of bird-to-human infection with the HPAI
H5N1 virus in 1997, there have been several reports of human infections with
a variety of HPAI viral strains (Table 9.2), although all these viruses were devoid
of the ability to spread from human to human, which is a prerequisite for them
to become established in the human population. The question still remains as
to whether AI viruses will acquire the capacity for consistent human-to-human
transmission. HPAI is constantly evolving over the course of time by antigenic
drift and shift. If the AI virus attains the capability of human-to-human transmission, a global influenza pandemic will be inevitable. It is also accepted that
AI viruses can have a major role in the initiation of an influenza pandemic in
the human population (Capua and Alexander, 2004). Furthermore, cases of
human infections with swine influenza have been reported, especially among
people occupationally exposed to pigs, which have been claimed to work as a
‘mixing vessel’ for the genetic reassortment of human and avian influenza
viruses (Scholtissek, 1995). However, the significance of zoonotic swine influenza infection is still not very clear owing to the lack of consistent screening
and reporting programmes. Nevertheless, the zoonotic potential of the avian
and swine influenza viruses raises serious public health concerns as the cause
of a future pandemic in the human population, which underscores the need
for a comprehensive influenza pandemic preparedness strategy.
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Topics in Microbiology and Immunology 333, 3–24.
10
Crohn’s Disease in Humans
and Johne’s Disease in
Cattle – Linked Diseases?
HERMAN W. BARKEMA, STEPHEN HENDRICK, JEROEN M. DE
BUCK, SUBRATA GHOSH, GILAAD G. KAPLAN AND KEVIN P. RIOUX
Introduction
With advancing world trade, and as new agricultural regions develop, guaranteed quality and cost-effective production are important to maintain both
local demand and the export position of the dairy and beef sector in Canada.
The consumer’s perception of the cattle sector is important, and is affected
by both the quality of the end product and, increasingly, the way this product
is produced. The concerns of the consumer go beyond the actual risk of
consuming this product; the perception of risk is also important and can
undoubtedly affect the consumer’s behaviour. Animal health is a cornerstone
to providing the consumer with the desired assurances on food safety. One
example of perceived consumer risk is Mycobacterium avium subsp. paratuberculosis (MAP), which is known to cause Johne’s disease (JD) in cattle and may
confer a risk of developing Crohn’s disease (CD) in humans. The cause of CD
has been linked to infection ever since it has been described, and of the
putative infectious agents, MAP has been argued to fulfil Koch’s postulates
(Hansen et al., 2010). Further evidence linking CD to MAP, as well as disproving the link, continue to appear, which may be partly attributed to the
heterogenous phenotype of CD.
Johne’s Disease
Johne’s disease in cattle is a chronic granulomatous inflammation of the gut
and other organs caused by infection with MAP. MAP is a major health problem in ruminants, resulting in intermittent diarrhoea, loss of body condition
and lower productivity. In the terminal phase, which most cows will not reach,
animals die with a very poor body condition. Infected cattle shed MAP in
CAB International 2011. Zoonotic Pathogens in the Food Chain
(eds D.O. Krause and S. Hendrick)
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manure and milk in increasing quantities as the disease progresses. The disease is widespread in cattle populations in almost all countries with a cattle
industry, and causes great economic losses because of lower productivity, but
even more by loss of future income due to early culling (McKenna et al., 2006).
In Canada, the economic damage caused by JD is unknown; however, the
percentage of cattle infected is similar to that in the USA, where the disease
is estimated to cost US$100 per animal on an infected farm, or US$250 million a year to the cattle industry (Ott et al., 1999). JD is likely to spread further
if control measures are not implemented, because as herds increase in size
farmers are more likely to purchase animals, often from herds with unknown
JD status.
Testing for MAP infection in cattle is difficult as many infected animals
do not have detectable organisms in their faeces or antibodies in their blood
until shortly before clinical disease develops. In Canada, using antibody
tests such as enzyme-linked immunosorbent assay (ELISA), 1.3% (Prince
Edward Island) to 9.1% (Alberta) of the dairy cows were found to be
infected, while 10% (Ontario) to 60% (Alberta) of the herds had at least
one infected cow (VanLeeuwen et al., 2001; Scott et al., 2006). Furthermore,
a recent slaughterhouse study performed in Atlantic Canada using bacteriological culture of lymph nodes and intestines revealed that 16% of dairy
cattle were infected (McKenna et al., 2004). When using only an ELISA as
the method of testing, 10–25% of infected cattle are identified – meaning
that the true percentage of infected animals and herds is highly underestimated. Most infected animals do not manifest symptoms of the disease, and
thus act as silent carriers that can infect other cattle. Many MAP-infected
herds will therefore never show any symptoms of the disease as these
animals are often removed for reasons other than clinical JD, i.e. low milk
production, mastitis, etc.
Beef cattle in North America are more extensively managed than dairy
cattle, resulting in lower ELISA prevalence estimates at the cow and herd
levels: 1% and 7%, respectively (Dargatz et al., 2001; Waldner et al., 2002).
However, when MAP is present on a beef farm, the within-herd prevalence
can become quite high. As opposed to dairy production, beef cows and calves
are commingled from birth until weaning, thus allowing ample opportunity
for transmission.
Other ruminants, such as sheep and goats, also become infected with
MAP and may present with JD. The hallmark sign of JD in small ruminants is
weight loss, as signs of malabsorptive diarrhoea are often absent. Sheep are
predominantly infected with a different strain from that of cattle and goats,
although sheep can still be infected with cattle strains, and transmission
between these species is possible (Motiwala et al., 2006). Small ruminant production is much more common in Australia, New Zealand and the UK. Given
the relatively low economic value of the individual animal, JD prevention
has focused more on the flock and, in particular, on the use of vaccination.
As new infections are established very early in life, vaccination is quite
limited in its ability to directly prevent infection, but its ability to reduce
faecal shedding minimizes environmental contamination and thus helps
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to control JD. Vaccination is not used widely for cattle in North America
because of its impact on future JD serological testing and tuberculosis
surveillance.
Complicating the epidemiology of JD are the wild ungulates (deer, elk,
moose, etc.) that can serve as sylvatic reservoirs. The role that these species
play as a nidus of infection for livestock has not been well established, but they
are more likely to be innocent victims of infected cattle, sheep and goats. MAP
survives well in the environment, even in the presence of light (sun and UV),
heat, cold and desiccation. Recently, concerns are also being raised as to MAP
as a waterborne pathogen.
Inflammatory Bowel Disease
The inflammatory bowel diseases (IBDs) of humans consist of CD and ulcerative colitis (UC), and are chronic, relapsing inflammatory conditions of the
gastrointestinal tract. Despite their significant impact on patients and society,
there is no cure and their cause remains unknown. The onset of IBD is greatest in early adulthood, with peak incidence among people aged 18–35 years,
and it affects quality of life, employment and psychosocial functioning. About
10–15% of IBD occurs in childhood or adolescence, when the disease may be
particularly aggressive. For unknown reasons, Canada has among the highest
incidence rates of IBD in the world (Bernstein et al., 2006). IBD is most predominant among industrialized nations, which points, at least in part, to
environmental and microbial influences. The disease has shown a prominent
recent increase in incidence in the Far East, Middle East and Eastern Europe,
suggesting environmental influence.
The prevailing theory describing the aetiology of IBD is that the inflammation results from dysregulation of the gut’s immune system in genetically
predisposed individuals who are exposed to unknown environmental triggers
(Podolsky, 2002). Recent advances have identified a multitude of IBD-specific
genes and environmental risk factors. However, none uniformly explain the
processes that drive the pathogenesis of IBD and none can be shown to be a
contributing factor in more than a minority of cases. Overall, many of these
associated genetic variants impair the clearance of intracellular bacteria.
The difficulty in understanding the aetiology of IBD is in part because of the
complex interactions between genes, intestinal microbes and the environment. While the pathogenesis of this chronic disorder is poorly understood,
the burden to the patient in quality of life, morbidity and hospitalizations, as
well as to the health-care system in direct and indirect costs, is staggering (Yu
et al., 2008).
Crohn’s Disease
As already stated, CD and UC are the two most important manifestations of
IBD in humans. Although they are seen as different diseases, they overlap,
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and both diseases can be classified into several phenotypes that may have
different pathogenetic pathways. CD is a chronic relapsing inflammatory
disease that may affect virtually any part of the intestinal tract. Symptoms
are chronic urgent diarrhoea, abdominal pain, nausea, vomiting, fever,
intestinal bleeding, malnutrition and fatigue. A cure does not exist for CD;
consequently, most patients are on chronic immune-suppressing medications and often require at least one IBD-related intestinal operation. Currently, 50,000–100,000 people have been estimated to be living with CD in
Canada, and over a half a million people are believed to have the disease
in the USA. The percentage of people affected with CD is increasing in
industrialized parts of the world (Loftus, 2004). The pathogenesis of CD is
unclear, and there are different theories on the development of the disease.
The predominant theory of pathogenesis is that interactions between intestinal microflora and exposure to environmental factors (e.g. smoking and
diet) in genetically susceptible individuals results in dysregulated inflammation and chronic gastrointestinal injury. Secondarily, investigators have
proposed that chronic inflammation is provoked following a gastrointestinal infection (e.g. with MAP or strains of Escherichia coli). A characteristic
histology is non-caseating granuloma, which may be present in up to 50%
of patients. In the small intestine, the disease often starts as aphthous
ulcers overlying Peyer’s patches, which provide a portal to bacterial entry
and sampling.
Genetic susceptibility
Genetic susceptibility to IBD is well recognized (Cho and Weaver, 2007; Franke
et al., 2008). Approximately 20% of IBD patients report a family history of
IBD and twin studies have demonstrated higher concordance rates among
monozygotic twins (Halfvarson et al., 2003).
The first susceptibility gene identified for CD was the NOD2 gene. Caucasians who are heterozygote carriers of a NOD2 gene variant are three times
more likely to be diagnosed with CD, while those who are homozygous carriers
have 23 times increased risk (Cuthbert et al., 2002). NOD2 has also been shown
to predispose to CD occurring in the terminal ileum and manifesting with
fibrostenotic disease. The prevalence of NOD2 variants is different across
populations (Economou et al., 2004). For example, Japanese people have not
been shown to carry mutations of the NOD2 gene (Yamazaki et al., 2002).
Recently, a meta-analysis of genome-wide association studies confirmed 11 CD
genetic loci and identified 21 new genetic loci (Mathew, 2008). The genes
identified to date have provided insight into the pathogenesis of CD by implicating defects in innate and adaptive immunity, epithelial barrier function
and mucosal defence (Yamamoto-Furusho, 2007; Hussey et al., 2008; Mathew,
2008). The specific linkage of three genes to CD is of particular interest when
considering the role of bacteria in CD pathogenesis, as these genes are involved
in host sensing (NOD2) and the elimination through autophagy (ATG16L1
and IRGM) of intracellular bacteria.
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Likewise, genetic susceptibility is important in UC. The major histocompatability complex region on chromosome 6p (IBD3) has been shown
to be associated with UC (van Heel et al., 2004). Additionally, the IL1RA,
MDR1 and PTPRS genes have been associated with an increased risk of UC
(Yamamoto-Furusho, 2007). Finally, family clusters of IBD have reported
mixed inheritance patterns. These findings suggest that genetic overlap
may lead to CD in one family member and UC in another (Cho and Weaver,
2007). Alternatively, environmental influences may shift phenotypes in
genetically predisposed family members. Thus, the prevailing view is that
IBD is a spectrum of clinical disorders with the eventual phenotype determined by a combination of both genetic and environmental factors. However, gene–environment interactions in IBD have not been well studied
and so the causal relationships between IBD genes and environmental risk
factors are not known. Specifically, several of the genetic defects identified
in CD may increase susceptibility to MAP. NOD2/CARD15 has been shown
to play a role in the susceptibility of cattle to MAP (Pinedo et al., 2009), and
NOD2 also plays a critical role in the host type I interferon response to
Mycobacterium tuberculosis (Pandey et al., 2009). A genome-wide association
study of leprosy showed that variants of the NOD2 signalling pathway are
associated with susceptibility to Mycobacterium leprae (Zhang et al., 2009),
raising the intriguing possibility of a common genetic fingerprint in a
known mycobacterial disease and CD (Schurr and Gros, 2009). MAP is recognized by NOD2 and TLR2/TLR4 receptors (Ferwerda et al., 2007). The
autophagy gene variants ATG16L1 and IRGM impair the ability to clear
intracellular Mycobacteria, which provides further links between the
known genetic associations of CD and possible MAP infection (Glasser and
Darfeuille-Michaud, 2008).
Environment
While IBD genes have provided insight into disease pathogenesis, genetics
has not completely explained the aetiology of IBD. The majority of patients
with IBD have neither a family history nor a known genetic defect (Loftus,
2004). Moreover, IBD has emerged predominantly in industrialized nations
in the last century; as developing nations have more recently become industrialized, the incidence of IBD in these countries has risen (Loftus, 2004).
In many countries, the incidence rate is rising faster than can be explained
by genetic factors alone, which suggests that the aetiology of CD is also likely
to be to caused by result of environmental conditions (Loftus, 2004). Environmental factors that are specific to modernization play an important role
in the development of IBD, although despite numerous studies that have
evaluated environmental risk factors, only a handful of disease determinants have been reproducibly confirmed and many others remain controversial. A paradoxical relationship between smoking and IBD has been
consistently demonstrated. A meta-analysis concluded that active smokers
were less likely to develop UC, while more likely to develop CD (Calkins,
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1989). Additionally, the contraceptive pill was implicated in CD and UC
(Godet et al., 1995), whereas appendectomy was shown to be protective in
UC (Koutroubakis et al., 2002). Also, IBD occurs more commonly in urban
centres (Ekbom et al., 1991). Diet has been extensively studied in relation to
IBD (Wild et al., 2007), but has yielded variable results. An analysis of the
association between CD onset and refined sugars gave inconsistent findings
(Riordan et al., 1998), but a study in Japan showed that high fat diets may
increase the risk of CD (Shoda et al., 1996), whereas breastfeeding may protect against CD (Klement et al., 2004). Finally, helminth infections, which
are more common in developing countries, may protect against CD in particular (Weinstock et al., 2004). However, a recurrent environmental factor
implicated in CD has been microorganisms such as MAP or E. coli derived
from food-chain contamination. The preceding risk factors have not
completely explained the occurrences of IBD, and risk factors associated
with industrialization and urban predominance have been incompletely
investigated.
Microbiota
Genetic and environmental studies implicate commensal gut microorganisms in the induction and perpetuation of IBD, as well as many of its
clinical complications (e.g. abscesses, phlegmon, fistulae) (Sartor, 2008).
Genes associated with IBD affect innate and adaptive immunity, as well as
epithelial barrier function, which are key determinants of the composition
of the commensal flora and its interactions with the host (YamamotoFurusho and Podolsky, 2007; Gibson et al., 2008; Lundin et al., 2008). A
variety of genetically engineered rodents spontaneously develop chronic
intestinal inflammation that mimics IBD in the presence of commensal
bacteria, whereas germ-free animals remain disease free (Rath et al., 2001).
Surgical diversion of the ileum from the faecal stream prevents postoperative recurrence of CD, whereas reinserting gut content from the
ileostoma into the diverted intestine results in rapid-onset inflammation
(Harper et al., 1985). Additionally, acute gastroenteritis and antibiotic
exposure are associated with increased risk of developing IBD (Garcia
Rodriguez et al., 2006). Furthermore, probiotic bacteria have been used to
treat active IBD (Fedorak and Madsen, 2004). Finally, candidate organisms in the pathogenesis of CD include adherent-invasive strains of E. coli
and MAP.
Despite several lines of evidence implicating the importance of gut microbiota, the mechanism by which these microbiota influence IBD remains elusive. We hypothesize that environmental factors affect the commensal intestinal
microbiota in genetically predisposed individuals, resulting in the initiation
and perpetuation of IBD. Alternatively, specific bacterial species may play a
more direct role in triggering, maintaining or modulating the disease, as has
been proposed for candidate organisms in the pathogenesis of CD (MAP and
adherent-invasive strains of E. coli).
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Infection with MAP
CD was described by Dalziel (1913), who wrote: ‘Tissue characteristics from
Johne’s and Crohn’s patients are so similar as to justify a proposition that the
diseases may be the same’. Since 1952, researchers have tried to grow Mycobacteria from surgically removed CD tissue. Chiodini et al. (1984) cultured MAP
from the gut wall of children with CD. Further corroboration of such findings
in early-onset CD has been provided recently by an Australian group (Kirkwood
et al., 2009). Additionally, MAP and E. coli strains have been demonstrated to
be present in the blood and intestinal tissue of CD patients (Selby, 2004;
Kotlowski et al., 2007). However, as already mentioned, despite several lines of
evidence implicating the importance of gut microbiota, the mechanism by
which they influence IBD remains elusive. The challenge with defining a causative relationship between MAP (and other pathogens) and CD is temporal:
does the presence of MAP in a subset of CD patients provide evidence that
MAP infection results in CD, or does the development of CD predispose to a
benign colonization of MAP in CD patients? This is reminiscent of the debate
surrounding Helicobacter pylori and peptic ulcers in the 1980s.
Does MAP Cause Crohn’s Disease?
Recent studies have shown that a high percentage of people with CD are
infected with MAP compared with a low percentage of people who do not
have the disease (e.g. Sanderson et al., 1992; Sechi et al., 2001; Kirkwood
et al., 2009). This seems to be a finding specific to CD, as MAP is not significantly associated with UC. Furthermore, it seems to be an association specific to MAP, as other environmental Mycobacteria are found as often in
people with CD as in patients with other inflammations of the bowel. In a
study of 28 CD patients, MAP was cultured from blood samples in 50%,
while this was the case in only 22% of patients with UC, and none from
people who did not have either disease (Naser et al., 2004). CD has been
consistently demonstrated to be more predominant in urban centres. In
contrast, rural regions and farmers have greater exposure to MAP, but
increased rates of CD have not been observed in these populations. However,
not all studies have documented such urban–rural differences (Armitage
et al., 2004).
Case–control studies exploring an association between MAP and CD have
yielded heterogeneous results. While several studies have demonstrated a relationship between MAP and CD, a population-based matched case–control
study from Canada demonstrated no differences in rates of serology for MAP
between CD, UC, randomly sampled controls and unaffected siblings; in all
four groups, one-third of participants were seropositive (Bernstein et al.,
2004). Overall, three recently published meta-analyses and systematic reviews
summarized the results of relevant studies on the association between MAP
(as assessed by ELISA or PCR) and CD, and concluded that an association is
evident (Feller et al., 2007; Abubakar et al., 2008; Waddell et al., 2008). These
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studies, however, stated that the evidence of an association between MAP and
CD does not prove that MAP causes CD. The higher prevalence of MAP in CD
may be secondary to the damage inflicted to the intestinal wall, which places
these patients at higher risk for infection or colonization with MAP after the
disease had developed. This issue can only be resolved by therapy to eradicate
MAP in patients with CD associated with presence of MAP.
If MAP infection causes CD, as is known for JD, then treating MAP should
intuitively treat CD. Many open-label studies evaluating different regimens of
antimicrobial agents targeting MAP have been published, with varying results.
The inconsistency in the data motivated the development of a randomized
controlled trial whereby 213 active CD patients were randomized to 2 years of
maintenance treatment with anti-MAP antibiotics or placebo, in combination
with a 16-week course of prednisolone for induction of remission at the onset
of the study (Selby et al., 2007). The trial used a combination of three antimycobacterial agents – clarithromycin, rifabutin and clofazimine – justified on
the basis of proven efficacy against the Mycobacterium avium complex (MAC),
intracellular penetration, tolerability, safety and redundancy of effect to avoid
resistance. More patients achieved remission in the prednisolone and antibiotics group at 16 weeks than with prednisolone and placebo (66% versus 50%).
During the maintenance phase of the trial, the proportion of antibiotic-treated
patients that relapsed tended to be less than in the placebo group: at 52 weeks
39% in the antibiotics group versus 56% in the placebo group; and at 104
weeks 26% in the antibiotics group versus 43% in the placebo group. In the 12
months after treatment ceased, there was no significant difference in the proportion of patients that relapsed (59% in the antibiotics group versus 50% in
the placebo group). In summary, the only statistically significant benefit of
anti-MAP antibiotics in this trial was the 16% absolute benefit seen in achieving steroid-induced remission at 16 weeks. While it remains possible that this
early benefit is attributable to specific effects on MAP, this should have been
more readily apparent in the maintenance phase, as effective treatment of
atypical Mycobacteria generally requires prolonged antibiotics. Rather, this finding is likely to be a non-specific effect of the antibiotics, or even a consequence
of the direct anti-inflammatory or immunomodulatory effects that have been
described for macrolide antibiotics in particular. Considering that the study
did not specifically enrol CD patients shown by molecular detection to have
‘MAP-associated’ disease, it may not have been adequately powered to discern
a benefit in a subset of patients that presumably harbour MAP. Publication of
the Selby trial (Selby et al., 2007) led to a vigorous debate and correspondence, including reanalysis of the data, which provided support for a favourable influence of combination antibiotics on CD. A replication of this study in
MAP-positive CD patients is required.
Ultimately, the predominant treatment strategy for CD is suppression of
the immune system with drugs such as corticosteroids, azathioprine and
monoclonal antibodies against tumour necrosis factor (TNF) (e.g. infliximab). If human infection with MAP was the dominant pathogenesis for the
development of CD, then suppression of the immune system should result in
widespread infection of MAP. Take for example infliximab, which has been
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clearly demonstrated to reactivate tuberculosis in CD patients with latent
infections. While MAP has been shown to cause disease in HIV patients, similar reports of MAP-related disease have not been observed in CD patients
receiving long-standing anti-TNF therapy, which argues against a clinically
relevant role for MAP in CD. It is possible that immunosuppression favourably affects the inflammation associated with microorganisms, but the persistence of microorganisms relates to inevitable relapse following
discontinuation of immunosuppression in the majority of patients. In addition, several drugs used in the treatment of CD, such as azathioprine,
6-mercaptopurine (Shin and Collins, 2008), 5-aminosalicylic acid (Greenstein
et al., 2007), cyclosporin, rapamycin and tacrolimus (Greenstein et al.,
2008) and thalidomide (Greenstein and Brown, 2009) have all been shown
to inhibit MAP growth, raising the possibility that immunosuppressive
drugs used in CD may have anti-MAP efficacy. In addition, antibiotic studies in CD not specifically directed against MAP had variable results in
often uncontrolled trials.
Overall, numerous studies have demonstrated an association between
MAP and CD, although there are no conclusive data yet to support the idea
that human infection with MAP causes CD. An alternative concept is that MAP
may contribute to the development of CD in a subset of patients, though the
effects of MAP may not be due to direct infection. Increased understanding of
the genetics of CD suggests that defects in innate immunity and autophagy
predispose to the development of CD. Among genetically susceptible individuals with defects in immune response, exposure to MAP may incite a dysregulation of inflammation. Additionally, intestinal inflammation may be a secondary
or terminal manifestation of systemic MAP infection, and difficulties in discerning a causal role could reflect that fact that most studies of MAP have
focused on the intestinal mucosa rather than on distant sites (e.g. circulating
macrophages, regional lymph nodes, mesenteric fat) (Behr, 2010). Future
studies exploring gene–MAP interactions will be necessary to elucidate the
mechanisms by which MAP exposure influences the development of CD.
MAP strains
The broad host range and zoonotic potential of MAP have been suggested
numerous times. This is based on the isolation of MAP from many different
host species, including humans. The isolates from CD patients have commonly
been reported to be of restricted genetic heterogeneity. However, the genotypes of only a limited number of such human isolates have been studied
(Francois et al., 1997; Pillai et al., 2001; Bull et al., 2003; Ghadiali et al., 2004;
Overduin et al., 2004).Therefore, it is currently impossible to conclude whether
the association of MAP with CD is restricted to a limited number of specific
MAP genotypes. The main reason for the small numbers of human isolates
in these studies and in public collections is that MAP strains are very difficult
to isolate from humans, and may require several months to years to produce
colonies. Future comprehensive comparative studies will require larger
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numbers of human isolates and typing techniques than can be applied to
poorly growing isolates. In most comparative studies, the genotypes attributed
to the human isolates are the same as for cattle isolates (Chiodini et al., 1990;
Pavlik et al., 1995; Whittington et al., 2000). This is in contrast with only one
study in which the seven human MAP isolates analysed were unique and did
not cluster with either the bovine or ovine strains.
Evidence of strain sharing between cattle and humans is of special interest
because it would imply the existence of a potential animal reservoir for CD.
Recently, new genotyping techniques have been developed for MAP that have
a high enough discriminatory index to further investigate the transmission
between animals and humans. Currently, a combination of a fingerprinting
and PCR-based techniques is necessary to achieve a high enough discriminatory index (Möbius et al., 2008; Sevilla et al., 2008). Future studies using techniques with high resolving power might uncover interesting associations
between animals and human sources. This has already been demonstrated in
a recent study, when two MAP strains that shared both a rare combination of
short repetitive sequences and fingerprinting patterns were isolated from
humans and cattle from the same geographical origin, raising the question of
a common source (Thibault et al., 2007). Although this suggests a close association of the human and animal strains, it does not provide direct evidence
for zoonotic transmission, nor a causal role of MAP in CD.
In summary, solid evidence of host specificity of MAP isolates is lacking.
Thus, the true degree of host adaptation or preference of MAP isolates
remains unknown.
Possible Transmission Pathways for Crohn’s Disease: Prevalence
in Milk, Beef and Water
The incidence of CD has been correlated with increased intake of meat and
milk protein (Shoda et al., 1996). Live MAP bacteria have been found in a
small proportion of retail milk samples in different countries (Grant et al.,
2002; Ayele et al., 2005; Ellingson et al., 2005), while in all studies a significant
percentage of those samples contained genetic material (DNA) of MAP (e.g.
Gao et al., 2002). Although in some studies a small number of retail milk samples contained live MAP, this does not explain the high number of CD patients
in these countries. Furthermore, Sweden does not have JD (Lewerin et al.,
2007), but has a fair number of CD patients (Lapidus, 2006). It must be noted,
however, that Sweden imports milk from other countries (Wahlström, 2002).
As regards beef, slaughterhouse prevalence studies have proven that infected
cattle are processed for human consumption. A recent study from Canada suggests that some MAP will survive cooking of meat to a medium-rare condition
(63°C), but that their numbers will be greatly reduced, while cooking to a welldone condition (71°C) can be expected to render meat free from viable MAP
(Mutharia et al., 2010).
If people are infected with MAP coming from cows, then it is likely that
pathways other than the consumption of contaminated milk or meat are also
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207
involved. CD is more often found in people living in cities than in rural populations, and farmers do not have an increased risk of CD (Jones et al., 2006),
despite a relatively high proportion of farming families that consume unpasteurized milk (e.g. Hegarty et al., 2002). However, in Europe, robust evidence
for transmission of MAP between wildlife and domestic ruminants has been
described, which provides support for food-chain contamination (Stevenson
et al., 2009). People may become infected while swimming in infected water
(children often swallow water when swimming) or drinking infected water
(Pierce, 2009). Additionally, MAP is not the only organism that has been mentioned as a possible causal factor of CD – e.g. invasive E. coli has also been
described (Packey and Sartor, 2009); for these bacteria, other transmission
pathways need to be examined.
Conclusions
The suggestion that MAP plays a role in CD is nearly 100 years old. MAP may
play a role in the pathogenesis of CD; however, this relationship is not proven.
The pathogenesis of CD is likely to be multifactorial in light of the different
susceptibility genes and phenotypes of the disease. Consequently, MAP may
only influence a subset of CD patients. Transmission of MAP to people through
milk and meat is possible, but the impact of water is not well studied and may
play a larger role than infection by means of animal products. While a final
consensus has not been reached regarding the association of MAP with CD,
the evidence to support this association is increasing. Future studies are
needed to determine whether MAP is an innocent bystander, an infectious
cause in a subset of CD, or an influence on the dysregulation of immune
response through gene–MAP interactions. Further combination antibiotic
therapy in CD patients shown to have MAP requires well designed and adequately powered trials. Understanding the exact nature of the involvement of
MAP in human disease is important because of its potential consequences on
public health. Finally, irrespective of the relationship of MAP with CD, it causes
JD, which is a serious threat to the cattle industry. Consequently, studying MAP
in CD and controlling outbreaks of JD should be a top priority.
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11
Transmissible Spongiform
Encephalopathies as a Case
Study in Policy Development
for Zoonoses
MICHAEL TREVAN
Introduction
When, on 20 March 1996, the UK government announced the discovery of ten
recent cases of a variant form of Creutzfeldt–Jakob disease (vCJD) in young
people that may have been caused by eating bovine spongiform encephalopathy
(BSE)-contaminated beef products, the public felt betrayed (Phillips, 2000)
and the media were outraged. Several weeks later, supermarkets in the UK
heavily discounted the retail price of beef, and the inexorably growing mountain of unsold beef was cleared from the shelves in less than 48 hours! It would
appear that the public’s capricious response to risk could be encapsulated by
the attitude that we have been eating this risky beef for years anyway, and that
piece of prime sirloin is a real bargain.
Transmissible Spongiform Encephalopathies
BSE is one of a group of diseases known as transmissible spongiform encephalopathies (TSEs). These are diseases that, over time, produce debilitating
and eventually mortal destruction of normal brain tissue in an individual. The
best known TSE is a disease of sheep and goats called scrapie, first identified
in the UK in 1732. The symptoms of scrapie include nervousness, itching and
motor dysfunction. The disease rarely occurs in sheep of less than 2 years of
age, and in its 280-year history has never been shown, or suspected, to be
transmissible to any other species through either the food chain or direct contact. Other known TSEs have been discovered more recently: chronic wasting
disease (CWD) of deer and elk in the USA in 1967; kuru, a disease prevalent
among the ritually cannibalistic Fore people of the mountains of New Guinea;
Creutzfeldt–Jakob disease (CJD), a form of dementia seen in humans over the
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(eds D.O. Krause and S. Hendrick)
TSEs in Policy Development
215
age of 40; and vCJD, first recorded in young people in the UK in 1995. Several
other mammalian species, e.g. cats and mink, also exhibit TSE diseases.
The generally accepted cause of TSE diseases, first proposed by Prusiner
in 1982, is that proteins normally present in nervous tissue are stimulated to
refold into an abnormal conformation as a result of the introduction of abnormally folded versions of the same protein. This disruption to the molecular
architecture of the neural tissue presents a histological pattern of sponge-like
holes in the brain, sometimes associated with the appearance of amyloid
plaques. Prusiner coined the term ‘prion protein’ for the proteinaceous
infective particle. TSE diseases are different from all other infectious diseases
in that the putative infective agent, a small protein, contains no DNA or RNA
and, significantly, induces no immune response.
It is well known that any single protein molecule can fold into a number
of different 3-D shapes, depending upon the environment in which the
molecule is placed (Poltorak et al., 1999a,b). The normal prion protein (PrPc)
is a relatively small, globular, heat-labile protein molecule containing significant amounts of α-helix in its secondary structure, whereas the abnormal
prion (PrPsc or PrPres), despite having an identical sequence of amino acids,
contains a high proportion of β-sheet secondary structure and, consequently,
is extremely heat stable and highly resistant to proteolysis. PrPres is stable
for up to 15 min in dry heat at 600°C; this is significant because it means that
the protein will not be denatured during normal rendering, cooking or canning processes. Prions are glycoproteins that have bound to them a variable
percentage of monosaccharides and disaccharides. This variation in sugar
content seems to give rise to distinct ‘strains’ of prion even within the
same animal species; these different strains can be characterized by Western
blotting techniques. For example, prions extracted from the brain tissue of
classical sporadic CJD, or iatrogenic CJD, can be identified as prion types 1 to
3 by Western blotting, whereas vCJD prions from brain tissue are identified as
type 4, and the prions isolated from tonsils of vCJD sufferers (type 4t) show a
similar, but not identical, Western blot pattern. The glycosylation pattern of each
of these five types is different. Thus, types 1 to 3 have twice the amount of monoglycosylation to diglycosylation, type 4 has slightly more disaccharides than
monosaccharides, and type 4t is about 60% diglycosylated and only 27% monoglycosylated (Wadsworth et al., 2003). Within the same species, these different
‘strains’ of prion create different clinical and histopathological symptoms.
Symptomatic differences occur between the various forms of TSE, even
within a species. For example, the three best characterized TSEs of people,
sporadic CJD, vCJD and kuru, differ significantly in the outward display of
symptoms. Sporadic CJD patients are usually between the ages of 45 and 75
(the clinical definition of CJD was in part that the patient would be at least 40,
which made the diagnosis of any form of CJD in those less than 40 years old a
significant challenge), and the principal symptom is dementia, which increases
rapidly, with death occurring within a few months. In vCJD, however, the first
symptoms are usually pain in the limbs, slurred speech, tingling sensations,
involuntary movements and memory loss. Death often does not occur until
after 12 months following the first onset of clinical signs, and dementia may
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occur as a final symptom. The first symptoms of kuru are an unsteady gait,
slurred speech and tremor, while dementia is rarely present; mood changes
are often seen in the latter stages of disease. These differences in symptoms
are probably a result of which area of the brain is most infected. In kuru, it is
known that the cerebellum, which controls motor function, is the principal
site of infection (NINDS, 2010).
For the most part, TSEs are transmitted within a specific host species
either horizontally to other members of the population or vertically from dam
to progeny, e.g. scrapie. It is well established that some TSEs are acquired
when an individual eats material contaminated with abnormal prions. For
example, CWD appears to be spread horizontally, most likely through healthy
individuals eating plants contaminated with urine, faeces or saliva from diseased individuals. Kuru is known to have spread in the Fore peoples of New
Guinea through the ritual practice of eating tissue, including brain tissue, of
deceased relatives. It reached epidemic levels in the 1950s and 1960s, and
following government campaigns to discourage the practice, the incidence of
kuru (which means ‘shiver’ in the Fore language) has declined substantially.
BSE – the UK Experience
The advent and course of BSE and its relationship to vCJD in the UK have
been extensively described in a report to the UK government that was compiled by Lord Phillips (2000). The full report runs to several thousand pages;
the first chapter alone is 308 pages. Much of the detail that follows is taken
from that report. In 1985, the first case of BSE in the UK was reported, but was
not diagnosed as a TSE. It was not until the end of 1986 that the Pathology
Laboratory of the Central Veterinary Laboratory (CVL) in the UK reported
that the first two cases of a new TSE in cattle had been diagnosed. However, an
embargo on immediate publication led to a 6-month delay in this diagnosis
being communicated. By the end of 1987, John Wilesmith, the Head of Epidemiology at CVL, had concluded that this new disease had not been transmitted by contact between individuals. He reached this conclusion because of the
enigma that the first 200 index cases of BSE had all occurred in unconnected,
and mostly closed, herds across the country (an index case is the first diagnosed case in any one herd). Wilesmith perceptively and rapidly realized that
the only possible connection between these index cases, which had occurred
almost exclusively in dairy cows, must be their feed. At that time, beef cattle
were mostly fed on a regime of grass and grain supplements, whereas dairy
cattle required an additional protein supplement, and Wilesmith correctly
identified the practice of feeding rendered meat and bone meal (MBM),
largely from cattle, as the probable cause. The ban on the feeding of MBM
produced from ruminants to ruminants followed quickly (1988), and was the
most decisive measure introduced that eventually brought the burgeoning
epidemic under control.
With the appearance of BSE, and the knowledge that it had probably
spread within the cattle population through the consumption of contaminated
TSEs in Policy Development
217
feed, the question that most concerned those responsible for advising government was: how likely was it that this new TSE could cross a species barrier?
More explicitly, could eating BSE-contaminated beef cause a new TSE in people? When these first cases of BSE were recognized in 1987 in the UK, the CVL
concluded that although there was a possible risk of transmission to people,
the risk was extremely low because BSE was thought to have entered the bovine
herd through the consumption of scrapie-contaminated feed, and scrapie had
never been transmitted to people. John Wilesmith assumed that the first cases
of BSE reported were index cases, and he incorrectly hypothesized that the
infective agent was probably the scrapie prion from sheep, and that it was
changes in the rendering process for animal carcasses introduced into the UK
in the early 1980s that had permitted the survival of the scrapie prion in the
feed chain. Consequently, this new disease was rightly viewed as a major hazard
to bovine health, but it was not seen as a problem for human health, because
scrapie had not been shown to be transmitted to humans in its over 200-year
history, and if BSE had been caused by scrapie it was unreasonable to suppose
that it would be transmitted to people.
It is now clear that the cause of the 1986 cases of BSE was bovine prioncontaminated feed, and that rendering processes had never been capable of
disabling prion proteins. It is still unclear where the disease first originated.
The exact relationship between the (abnormal) prion protein and the route
of infection has been the subject of much controversy. The only certainty is
that for clinical disease to develop the prion protein must be expressed. Mice
devoid of the prnp gene do not develop clinical symptoms even when inoculated with abnormal prions. The key works are summarized in Baron and
Biacabe (2007), but see also Bueler et al. (1993), Prusiner (1998), Chesebro
(1999), Lasmézas et al. (1997), Nonno et al. (2003) and Lezmi et al. (2004).
By the end of 1987, government officials in the UK Ministry of Agriculture,
Fisheries and Food (MAFF) had become concerned that BSE might be transmissible to humans through eating contaminated beef. However, they did not
report this to the Ministry of Health until March 1988. When they did, the
Chief Medical Officer promptly set up a working party, chaired by Sir Richard
Southwood, to investigate the matter. By June 1988, Southwood’s committee
recommended banning BSE-infected cattle from human feed. They did this
on the basis of no evidence that there was potential for transmission between
species, but on the precautionary principle that if there was a possibility of
transmission, the consequences could be catastrophic.
In August 1988, compulsory slaughter for herds where BSE had been
diagnosed in an individual was introduced and compensation was paid to
farmers. In January 1989, the Southwood Committee reported to government
knowing that their report would be made available to the public. It subsequently appeared that the wording of the report was carefully considered so
that it would not cause an alarmist response by the public. Thus, they stated
that ‘it was most unlikely that BSE would have any implications for human
health’. They based this statement on the following assumptions: BSE was
derived from scrapie and would behave like scrapie; scrapie was known not to
affect humans; and any specific medical or occupational risks that might occur
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M. Trevan
could be dealt with by the relevant authorities. But they did not make clear in
their report this basis for their conclusions. When, subsequently, the assumptions they had made turned out to be invalid, their conclusions still held
weight because they had not reported their assumptions and therefore there
was nothing to challenge. Nevertheless, precautionary measures were put in
place and in 1989 a ban on the inclusion of specified bovine offal (SBO) in
human foods was introduced.
In May 1990, a cat in the UK was diagnosed with ‘scrapie-like symptoms’.
The fact that a carnivore could be susceptible to a TSE appeared to cause
surprise, despite the fact that a TSE of farmed mink had been recognized in
North America since the 1930s. It also concerned the public, whose fears
about eating British beef had been growing since the initial announcement of
BSE, and because of the suggestion of a possible link to CJD. Twenty Local
Education Authorities had already banned beef from school dinners. The
government launched a campaign of reassurance that, infamously, showed
the then Minister of Agriculture, John Gummer, feeding a beefburger to his
4-year-old daughter at a boat show in his constituency on 16 May 1990 (BBC,
1990), just 6 days after the news of TSE in a cat had broken and 6 months after
the government’s own ban on bovine offal. John Gummer was later publicly
ridiculed for this act (see Fig. 11.1). Over the next 6 years, the principal
government policy appears to have been to contain public fears with reassurances that beef was safe to eat. In March 1993, the new Chief Medical Officer,
Kenneth Calman, repeated his predecessor’s assurances. By July of that year,
the 100,000th BSE case was recorded, although the rate of infection of cattle
had started to decline. The government campaign culminated in a statement
by Prime Minister John Major in December 1995 that ‘there is no scientific
Fig. 11.1. Cartoon of John Gummer, then UK Minister of Agriculture, and the then UK Prime
Minister John Major (wearing underpants) (copyright Steve Bell, 2000, originally published in
The Guardian newspaper).
TSEs in Policy Development
219
evidence that BSE can be transmitted to humans or that eating beef causes it
in humans’.
In 1990, the UK government established the CJD Surveillance Unit (CJDSU)
as a precautionary measure to actively look for any new spongiform encephalopathy disease within the human population that might, epidemiologically, be
linked to BSE. The CJDSU identified the first possible cases in farmers in 1992,
but these turned out to be cases of classic CJD. The UK government had also
established a Spongiform Encephalopathy Advisory Committee (SEAC). In
1994, SEAC reported that, because of the precautionary regulations that had
already been taken, the risk to humans from BSE was very low.
Appearance of Variant Creutzfeldt–Jakob Disease in the UK
The political time bomb was primed in 1995 when the CJDSU reported the
first two cases of CJD in young people, at a time when only four other cases of
CJD in young people had been reported worldwide. The diagnosis of CJD had,
hitherto, been almost exclusively confined to individuals over the age of 40. By
March 1996, ten cases of this new variant form of CJD (nvCJD or vCJD) had
been found in the UK population. BSE was thought to be the cause because
exposure through food to the BSE agent would have been greatest in the
mid-1980s, and the sudden emergence of cases of CJD in young people was
consistent with a 5–10-year incubation period. This, of course, was a somewhat
circular argument, because if BSE had appeared only 10 years previously, the
incubation period for transmission to people could only be a maximum of 10
years. It did not explain why there had not been a sudden appearance of this
vCJD across the whole population, and only in those under 40 years of age.
The suggested, and entirely subjective, reason was that it was the consumers
under 40 who ate most beefburgers! On 8 March 1996, the CJDSU informed
SEAC of these ten cases. SEAC duly notified the Secretary of Health, Stephen
Dorrell, that it believed that these ten cases of vCJD had been caused through
BSE-infected beef entering the human food chain. Their advice made it clear
that there was no scientific evidence for this view, but there was a lack of any
other credible explanation for the appearance of this new vCJD. The Royal
Society repeated this in its statement made on 23 July 1996:
Is the new variant form of CJD caused by BSE transmission to humans? No
explanation other than the ingestion of ‘BSE-prion’ contaminated food has
come to light, and this explanation must still be considered the most likely cause
at the present time. There is insufficient evidence on the use of bovine offal …
to know with any precision where it might have entered the human food chain.
It is believed however that mechanically recovered meat may have contaminated
some of the relevant bovine offal.
The UK government’s policy response to the BSE crisis was hampered by
miscommunication and limited knowledge. At the time, it was usual for abattoirs to remove the last traces of meat from a carcass mechanically, using high
pressure to produce what was known as mechanically recovered meat (MRM),
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M. Trevan
Table 11.1. Abbreviated chronology of events in the UK related to the occurrence
and control of bovine spongiform encephalopathy (BSE) and variant Creutzfeldt–
Jakob disease (vCJD) (source: Hueston and Bryant, 2005).
1988
1989
1989
1990
1991
1994
1995
1996
1996
1997
1999
2001
2001
Restricted ruminant protein from ruminant feed
EU banned export of UK cattle born before feed ban (July 1988)
Banned specified bovine offal (SBO) from human food
Ban SBO from animal food and export of SBO to EU
Banned export of SBO to rest of world
Restricted all mammalian protein from ruminant feed
Banned mechanically recovered meat (MRM) from the vertebral column
Banned use of cattle >30 months old (except for leather)
Restricted all MBM from animal feed/fertilizer
Sale of beef on the bone banned in UK, but is lifted later
Destroyed all offspring of BSE cattle born after 1 August 1996
Banned all MRM from sheep, cattle and goats
Brain examination of all slaughtered cattle >30 months old
a mince-like slurry that was used principally in the cheaper brands of processed meat products, e.g. burgers, frozen mince and meat pies. At its peak,
the UK produced 5000 t of beef MRM a year. MAFF first questioned the use of
MRM in human food in 1989, because some of the MRM would have come
from the spinal column of the carcass, and raised the issue with SEAC in 1990.
However, MAFF believed, erroneously, that SEAC’s view was that a small
amount of spinal cord contamination in MRM was of no concern; SEAC
believed, incorrectly, that all spinal cord material could be excluded from
MRM. This miscommunication was compounded because in 1990 there was
no evidence that spinal cord tissue contained infective particles. Consequently,
it was not until December 1995 that the use of MRM in human food products
was banned. Despite the best efforts of various agencies, even by 2002 it was
not clear how much MRM or infective spinal cord had entered the UK human
food supply between 1980 and 1995 (UK Food Standards Agency, 2002). The
Phillips report also noted that the measures government had introduced were
reasonable in guarding cattle from a known hazard, and people from an unknown
hazard, but some officials lacked rigour in turning policy into action. This was, in
part, because of the belief that persisted that BSE was not a threat to human
health, and that bureaucratic processes lead to unacceptable delays. Consequently, these ‘sensible measures’ were delayed, inadequately implemented and
not appropriately enforced.
Table 11.1 summarizes the main measures implemented in the UK
concerning the control of BSE and vCJD.
Not Just in Food
Similar confusion to that documented above occurred between government
departments in the UK when it came to the issues around non-food products
TSEs in Policy Development
221
made from cattle, or the use of bovine parts. At the heart of the matter was the
subdivision of responsibility between government departments. For example,
MAFF first discussed the practice of dissection of bovine eyeballs in schools in
September 1989, shortly after the ban on SBO was enacted. The close relationship between eye and brain was thought to pose a possible risk of transmission
of the BSE prion to pupils and teachers. In February 1990, the theoretical risk
was raised with the Medical Adviser to the Department of Education and
Science (DES). In June of the same year, SEAC issued its advice that only
eyeballs taken from cattle less than 6 months old should be used in school
biology classes, and this advice was relayed to the DES Medical Adviser. A recommendation to the Minister responsible for the DES that dissection of bovine
eyeballs should be discontinued in schools was first drafted in August 1990. By
February 1992, the submission to the Minister in the DES was still in its third
and final draft form, but the DES, which is not usually responsible for pupil/
teacher safety in schools (that remit belonged to the independent Health and
Safety Executive), apparently felt that it must consult widely across other
departments, particularly with the Department of Health (DH) and MAFF. By
October 1992, in response to pressure from the DH, the DES finally agreed to
make the submission to its Minister recommending the banning of bovine
eyeball dissection in schools. By the beginning of 1993, three and a half years
after the issue was first raised by MAFF, guidance to schools in England and
Wales was finally issued! Scotland, which, through partial devolution of government in the UK had independent control of its school system, had issued
similar guidance to its schools in February 1990. The cause of this delay
appears to have been the result of the interaction of a number of factors. First,
the DES was not primarily responsible for pupil/teacher safety and it felt
‘distant’ from the BSE crisis, but was receptive to the repeated advice from
government that beef was ‘safe’. Second, other issues at the time seemed more
important to the major responsibilities of key civil servants in the DES and the
DES Medical Adviser, having agitated for the guidance to be issued, appears to
have concluded in May 1992 that such guidance was no longer timely. Third,
the longer the delay in issuing guidance, the less attractive the proposition,
presumably for fear of attracting adverse comment on the delay itself. In Lord
Phillips’ words, ‘Here as in other areas, excessively reassuring language about
the risk from BSE sedated those who needed to act’.
Cosmetic goods were another type of product through which people could
be exposed to ‘hazardous’ bovine material. Cosmetics are usually employed by
application to the skin, lips and eyelids, and bovine products occur in many
types of cosmetic preparations. The assessment of the potential hazard was
informed by the source of bovine material and the extent of processing
required to obtain the final preparation. For most regular cosmetics, both the
type of bovine material and the heavy processing required suggested that there
was minimal risk. However, some premium brands of anti-ageing and antiwrinkle creams contained only lightly processed brain, placenta, spleen and
thymus materials. The safety of cosmetics was governed by the 1987 EU
Cosmetics Directive and Regulations (part of the Consumer Protection Act),
but the implementation of these regulations was essentially a voluntary
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M. Trevan
undertaking by industry, as there was no requirement for cosmetics to be
licensed. In the UK, the cosmetics industry was the responsibility of the Department of Trade and Industry (DTI). Responsibility for enforcement lay with
Trading Standards Officers who worked for local government bodies, who could
only take action following a complaint of harm caused by a cosmetic product.
The Tyrrell Committee, chaired by Dr David Tyrrell, Director of the MRC
Common Cold Unit, was established in 1988 to advise MAFF and the DH on
research priorities related to spongiform encephalopathies. In June 1989 it
reported that it had identified a potential hazard of introducing BSE prions to
people through the use of cosmetics, but neither the DH nor MAFF proactively informed the DTI of this potential hazard. It was not until January 1990
that the DTI sought advice about the matter from the DH. On receiving advice
that there was a potential hazard, the DTI promptly informed the Cosmetic,
Toiletry and Perfumery Association (CTPA), which in turn informed its members, advising them either to reformulate the products to avoid bovine material, or else to source that material from outside the UK. The question remains:
why did neither the DH nor MAFF think to inform the DTI of the potential
hazard? It appears that those responsible in MAFF considered this to be a
human health issue and therefore the responsibility of the DH. For their part,
the officials in the DH appear to have believed that the risk of transmission
through cosmetic products was so small that there was no point in alerting the
DTI to the possibility. Later, the DTI recognized a flaw in their system: that
despite having been prompt in delivering the warning on bovine products to
the CTPA, who were equally prompt in advising their members, not all cosmetic producers were members of the CTPA. In addition, no organization
seems to have had the knowledge of what bovine products were incorporated
into which cosmetics, and no one was clear as to which body should be taking
the lead. CPTA was asked to collect information from its members, but its
attempts to do so produced little result. In 1993, the EU Working Party on
Cosmetics became involved, and progress towards European guidance became
ensnared by bureaucratic procedures, infrequent meetings of the relevant
parties and national differences of position. Thus it took until March 1994 for
the EU Health Council to proclaim that existing measures in respect of cosmetics were sufficient to protect public health. Subsequent to the emergence
of vCJD in 1996, the EU Cosmetics Directive was amended. Meanwhile, the
CTPA, in consultation with the French cosmetics industry, issued guidance to
its members in March 1994 that was essentially the same as that issued by the
World Health Organization (WHO) in 1991.
The EU Perspective
On 25 March 1996, 5 days after the UK government’s announcement that BSE
might be the cause of a new human vCJD, the EU Commissioner for Agriculture
and Rural Development, Franz Fischler (Fischler, 1996), announced a ban on
the export of all beef from the UK. By the beginning of April, the UK government had introduced a ban on any cattle over the age of 30 months entering
TSEs in Policy Development
223
the food chain. The reasoning and timing of this were mostly to do with the
EU ban, and with public reassurance. It was also based on the principle that
BSE took 4–5 years to incubate in cattle, therefore there was a high likelihood
that cattle under 30 months of age would be disease free.
Over the next few months, the UK worked hard to get the EU ban relaxed:
it signalled that it could remove its opposition to ratification of the Europol
police cooperation convention if there was movement on the restrictions on
British beef; it threatened to disrupt EU business; it refused to give assent to
new EU bankruptcy rules; it instituted a policy of non-cooperation with the
rest of the EU; and it blocked a trade pact with Mexico. By 5 June 1996, the UK
had vetoed 40 measures as part of its non-cooperation protest against the beef
ban, and Prime Minister John Major issued a statement that the policy of noncooperation would continue unless and until the EU produced a timetable to
lead to the eventual lifting of the ban on British beef. By July, accusations surfaced that the EU had attempted to cover up the BSE crisis by asking the UK
not to publish the results of its research into BSE, and that the European Court
of Justice had upheld the EU’s worldwide ban on British beef exports at the
same time that the EU beef management committee had approved an
?850 million package of special aid to beef farmers. The accusations and acrimony rumbled on until December 1996, by which time the Dutch had banned
the import of beef from Switzerland, and the French had requested a review of
EU aid for veal producers in the wake of an incident in which French cattle
breeders hijacked Dutch meat trucks and burned the contents in protest
against EU policies and actions. The EU ban on UK beef exports, which had
cost the industry over £670 million, was eased in 1999 to allow de-boned beef
and beef products to be exported, and was eventually lifted on 8 March 2006,
when the number of new cases of BSE had fallen to below 200 per million live
cattle. The easing of the ban on de-boned beef and beef products in 1999 was
not uniformly or immediately implemented. France continued to ban British
beef imports, citing concerns over human health, despite an EU court ruling
that their ban was illegal. In October 2002, the French government announced
that it was lifting the ban, on advice from its experts that British beef was (now)
safe, just a few days before it faced fines of £100,000 per day if it persisted.
The Epidemic Under Control
Although the epidemic of BSE in the UK peaked in around 1992, the number
of new cases being reported was still high. This was to be expected because of
the long and variable incubation period, which shortens as the age at infection
decreases and as the infective dose increases. The number of new cases of BSE
did not fall as rapidly as might have been expected after the peak in 1992, suggesting that the MBM ban of 1988 had not been completely effective, given an
average incubation period of 4 years. Had the 1988 ban been totally effective,
it would be reasonable to expect that the number of new cases of BSE would
have fallen to a few hundred by 1996, whereas some 10,000 cases were reported
that year (see Fig. 11.2, next section). Leakage of farm feed not intended for
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M. Trevan
ruminants into cattle feed was suspected, and in 1996 a reinforced feed ban
was imposed prohibiting all MBM from being used in any animal feed, or for
fertilizer. As Stevenson et al. (2005) reported, there was cross-contamination of
feed. Before the reinforced feed ban of 1996, the regional density of cases of
BSE correlated better with the number of pigs in the region than it did with
cattle density. However, it became apparent the cattle born after the 1996
reinforced feed ban were still contracting BSE (called BARB cases – for Born
After the Reinforced Ban). The number of such cases increased from 3 in
2000/1 to almost 100 by June 2004 (Table 11.2). A number of cases were
probably missed before the introduction of active surveillance in 2001.
In response to these continuing cases, the UK Department for Environment,
Food and Rural Affairs (DEFRA) commissioned a report from Professor
William Hill (2005) of Edinburgh University, with the remit to investigate the
likely causes of these BARB cases of BSE and to identify whether or not this
might be a new and different strain of BSE, or whether any significant new risk
factors might have arisen. Hill concluded that this was not a new strain of BSE,
nor was its occurrence caused by differences in genetic susceptibility of cattle.
He found no evidence of vertical or horizontal transmission, nor that any but
a few cases could have arisen spontaneously, and was drawn to the inevitable
conclusion that the most likely explanation was either deliberate or accidental
failure to remove all traces of contamination from the feed bins on farms.
Accidental importation of contaminated feed seemed an unlikely cause
because of the random distribution of the BARB cases across the country. At
the same time, authorities elsewhere in Europe were reporting the appearance of a variant form of BSE, one in which amyloid plaques were found in the
brain on autopsy, and where the glycosylation patterns of the PrPres were
different. This new bovine TSE disease, termed bovine amyloidotic spongiform encephalopathy (BASE), had a lot of the characteristics of classic CJD
in humans, and might well have been the result of sporadic mutations. Hill
concluded his report:
Conclusions: Elimination of feed borne sources is now, as before, the key to
elimination of BSE. The incidence of the disease can be greatly reduced but not
readily eliminated in any country by adequate imposition of controls, particularly
on animal feed. As the level of incidence falls both in the UK and internationally,
the risks of contamination through feed, or indeed through any other source,
fall whether or not controls in the UK and abroad are further tightened. With
the current expertise in DEFRA and the VLA [Veterinary Laboratories Agency],
GB is well placed to keep on top of and promote developments.
Recommendations: It is essential that appropriate, risk based, controls and
monitoring should be maintained on animals and feed until no cases of BSE are
found, and controls tightened up where feasible, both in the UK and elsewhere
that the UK can influence. In view of the very long incubation period of BSE in
some animals, long-continued vigilance is necessary. It is not evident, however,
that specific new measures are needed. Basically it is necessary to ‘keep taking
the medicine’. Nevertheless, in view of new discoveries on the nature of the
disease and the possibilities of new or changed TSEs arising, relevant research
capacity in GB should be maintained.
Year
from July
BSE
by year of
birth
BARB
by year of
birth
BARB
by year of
diagnosis
TSEs in Policy Development
Table 11.2. Numbers of cattle that went on to develop bovine spongiform encephalopathy (BSE), by year of birth; and cases of BARB (BSE
cases in cattle born after the reinforced feed ban of 1996), both by year of birth (as for BSE) and by year of diagnosis (source: Hill, 2005).
1992/3 1993/4 1994/5 1995/6 1996/7 1997/8 1998/9 1999/2000 2000/1 2001/2 2002/3 2003/4 2004/5 Total
3493
2960
2128
1059
62
42
9702
30
15
5
0
1
3
14
93
35
28
13
93
225
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M. Trevan
Did BSE Cause vCJD?
Did BSE cause this new variant of CJD? Until at least 1997, the evidence was at
best highly circumstantial. Here was a potentially new hazard to human health
with a risk of unknown proportion. Epidemiologists were soon to predict a
devastating epidemic of vCJD on the basis of almost no numerical information. They estimated that the number of new cases would rise to hundreds of
thousands a year in the UK alone – a disease with a mortality rate of 100% and
no known cure. This was not the universal opinion, but it was the mood that
prevailed and influenced policy. More than 6 years of reassurance by government ministers and senior scientists that British beef was safe to eat now
appeared to be, at best, plain wrong, and, at worst, a conspiracy to hide the
truth. Witness the headline of the period in the national Daily Mirror newspaper, ‘We’ve already eaten 100,000 mad cows’. The consequences of this loss of
confidence in the government’s mechanisms for securing food safety were
severe. It was perhaps the major cause of public scepticism over the introduction of genetically modified (GM) crops into the UK. The continual and
repeated support of the new Labour government of Prime Minister Tony
Blair (elected in 1997) for GM crops in all probability hardened the negative
attitude of the British public towards these crops. The public had lost all
confidence in MAFF and in agricultural practices, and shortly after the
general election in 2001, the re-elected Labour Government disbanded MAFF
and vested most of its responsibilities in the new DEFRA. Responsibility for
food safety was vested in a new arms-length non-governmental organization,
the Food Standards Agency (FSA), under the chairmanship of Sir John Krebs.
This agency, through an agenda of promiscuous transparency, did much to
restore the public’s faith in its food supply.
In 1997, the first laboratory reports of BSE transmission to mice were
presented as evidence that BSE had indeed caused vCJD. The conclusion of
the authors, Bruce et al. (1997), was that the RIII strain of experimental mice,
when inoculated with brain tissue from humans who had died of vCJD, showed
a histopathology similar to that when the mice were inoculated with brain
tissue either from BSE-infected cattle or cats with feline spongiform encephalopathy. The same mice, when inoculated with brain tissue from humans who
had died of classic CJD, or with brain tissue from sheep with scrapie, showed a
different pathology. Bruce et al. (1997) concluded that their observations
‘provide compelling evidence of a link between BSE and vCJD’. The other
conclusion to be drawn, however, is that, if BSE and vCJD are linked through
the pattern of disease that they cause in this strain of mice but scrapie causes
a different pattern, then scrapie is unlikely to have been the cause of BSE.
Wadsworth et al. (2003) demonstrated that prion proteins from patients who
had died of classic CJD and vCJD appeared to exhibit different strains of prion
protein. Prions from cases of classic CJD were classified as types 1–3, and
characteristically had a ratio of monoglycosylation to diglycosylation > 1, while
prions from vCJD patients of types 4 and 4t (from tonsils) had a ratio of < 1.
Assante et al. (2003) reported the effect of inoculation of transgenic mice with
prions from BSE or vCJD. The transgenic mice had their own PrPc gene
TSEs in Policy Development
227
removed and replaced with a human PrPc gene, homozygous for either
methionine or valine at amino acid residue 129. Three strains of mice were
studied: Tg35, Tg45 and Tg152. Tg35 mice expressed human PrPc homozygous for methionine at twice the level found in human brain; Tg45 mice
expressed the same prion at four times the level found in human brain; Tg 152
mice expressed human PrPc that was homozygous for valine at residue 129.
Both vCJD and BSE prions caused symptoms similar to vCJD in a small minority
of the Tg 35 and 45 mice, but some mice that at death had the PrPres prions in
their brain tissue had shown no clinical symptoms of disease. Tg152 mice that
died of old age had no PrPres in their brain tissue, suggesting that the homozygous 129 valine prion may be protective against BSE prions. Both the BSE
and vCJD inocula produced ‘florid’ plaques in mouse Tg35 brains, but inoculation with classic CJD did not produce florid plaques. This was cited as additional evidence that BSE and vCJD were related, and different from classic
CJD, and therefore that BSE could be the cause of vCJD. Inconveniently, it was
also observed that the same mice inoculated with scrapie prions exhibited the
same form of florid plaques. This might have suggested that scrapie had been
the cause of BSE, had not the earlier work of Bruce et al. (1997) suggested the
exact opposite. It was these key scientific papers that convinced governments
that BSE had indeed caused vCJD. Yet still the questions remained: why only
in young people? And did the fact that prions from BSE-infected cattle and
vCJD-infected humans cause similar symptoms when inoculated into mice
actually prove that BSE had been transmitted to humans through the food
chain to cause vCJD? It is, of course, possible that any patient presenting with
CJD-like symptoms, but who was over the age of 40, could be diagnosed with
classic CJD even if suffering from vCJD. The evidence was at best still circumstantial – correlation does not prove cause and effect; but that did not prevent
authority from proclaiming it as a fact – and this inconvenient truth was all
that various jurisdictions needed to justify trade barriers to British beef.
By 2000, when the Phillips report had declared that ‘BSE has caused a
harrowing fatal disease for humans’, this universal truth was embedded in the
belief structure of a nation. But from 2001, the number of annual cases of
vCJD started to decline, leading the epidemiologists to revise their doom-laden
predictions. For the first 2 or 3 years of the new millennium, it appeared that
reports of new cases of vCJD had plateaued. This led many commentators to
speculate that vCJD had simply been a disease that had always existed, but had
not been diagnosed until the scientific community set out to search for it. An
existing rare, but previously unrecognized, disease would show an appearance
of cases very similar to that seen for vCJD between 1995 and 2000 – an initial
rapid rise followed by a steady state. Latterly, though, the statistics have shown
something different. The first question must be: why had this ‘new’ disease
only appeared in significant numbers in the UK? Despite active surveillance,
vCJD has not appeared in substantial numbers in any other country, in particular in those countries where BSE had become established, which does not
fit well with the idea that vCJD was an existing, but hitherto undiscovered,
disease. Equally, if BSE had become established in a country, the lack of
vCJD cases could indicate that BSE did not cause vCJD. However, where BSE
228
M. Trevan
40
35
No. of cases
30
25
20
15
10
5
0
1985
1990
1995
2000
2005
2010
Year
Fig. 11.2. UK cases of bovine spongiform encephalopathy (BSE) (1000s) (■) and
deaths from variant Creutzfeldt–Jakob disease (vCJD) (♦) from the late 1980s to
2009 (sources: The National Creutzfeldt–Jakob Disease Surveillance Unit (NCJDSU),
2010; World Organisation for Animal Health (OIE), 2010).
subsequently appeared in other countries, particularly in the EU, all of these
countries had adopted the UK’s precautionary measures that were predicated
to prevent the transmission of BSE across the species barrier to humans.
Figure 11.2 shows the UK figures for the annual cases of both BSE and
vCJD. If one assumes an incubation period for vCJD of 8 years (not that different from the official figure of 10–12 years assumed by SEAC), then the curves
for the appearance of BSE and vCJD are almost identical and suggest a low
infectivity rate of one human case of vCJD per 1000 cases of BSE in cattle. But
the simple correlation between these figures for BSE and vCJD takes no
account of the UK government’s precautionary measures that were instigated
to prevent the possibility that BSE might cause a similar disease in humans. In
other words, the similarity of shape of the BSE and vCJD curves is misleading
because, assuming that vCJD is caused by the consumption of BSE-infected
beef products, cases of vCJD should not mirror cases of BSE, but instead represent the presence of BSE infectious material in the human food chain. In
addition, two potentially confounding events occurred: the banning of SBO
from human food in 1989, and the ban on the use of MRM in food products
in late 1995. The effect of these events on the apparently simple relationship
between BSE in UK cattle and vCJD in UK people should depend upon which
of these two measures (if either) was the sole, or major, action that kept
BSE-infected material out of human food. It is difficult to say with any certainty
which, if either, of these two measures had the greatest effect, although it is
likely that the SBO ban was the most significant because it removed from
human food items known to contain prion-infected materials. MRM was a
product that could, by chance, pick up prions from associated spinal cord
material. One would expect that all the time the number of cattle infected
with BSE was rising exponentially and infected material was entering the
human food chain (assuming that the virulence of the infective agent, or its
TSEs in Policy Development
229
concentration in those foods, did not change over the period) that the number of subsequently detected cases of vCJD would rise exponentially, which is
what we observe. Then again, the sudden removal of the major carrier of
infection, SBO, from human food would be expected to result in a precipitous
decline in observed new cases of vCJD, which did not happen.
Experimental models have shown that the length of the incubation period
for an acquired TSE is inversely proportional to the infective dose acquired.
Thus, the declining tail of new cases of vCJD shown in Fig. 11.2 could be due
to variation in incubation period among the individual cases. All this is, of
course, highly speculative and, given the small total numbers of vCJD cases,
impossible to verify. Despite that, it remains very important, because the peak
of 28 vCJD cases in 2000 (see Fig. 11.2) ought to relate to 1989 when SBO was
removed from human food. This would put the incubation time for vCJD to
an average of 10 years, and the ratio of cases of vCJD to BSE at about 28:7228
or approximately 1:260, rather than the 1:1200 suggested by a simple analysis
of Fig. 11.2, implying a 4.5× higher rate of transmission. Is this difference significant? Probably not, because we are considering a population of 60 million,
of whom at least 40 million could have been regular consumers of beef products. But what if those responsible for advising government on policy options
had taken the view that BSE could not be transmitted to humans, and, as a
result, SBO and MRM had remained part of the food supply? It is certain that
the actions taken to eradicate BSE from the cattle herd would have been
implemented irrespective of the possible risk to people, because BSE was causing dairy and cattle producers significant economic losses. If nothing else had
changed, then we might have expected to see a peak of 135 cases of vCJD
occurring in about the year 2000, and a total number of UK cases of vCJD
close to 1000. However, the optics that figures such as these place upon the
influencers of government policy are out of all proportion to real risk: 135
deaths represents less than 0.02 deaths per 10,000 of the UK population. It is
common, though, for the perception of a hazard to be significantly heightened by its novelty. It should be noted that the only other country in which
there have been significant numbers of cases of vCJD identified is France,
where 25 cases have been reported. Finally, we must consider the possibility
that the decline in reported vCJD cases in the UK from the year 2000 might be
the result of decreased diagnosis because it was assumed that the BSE epidemic was well under control and, therefore, no longer a risk to human health.
To quote a more ancient authority, it could be a case of you find what you are
looking for: ‘Ask, and it shall be given you; seek, and ye shall find; knock, and
it shall be opened unto you’ (King James Bible (1611), Matthew 7:7).
North America
The first case of BSE in North America occurred in 1993 in Canada in a cow
that had been imported from the UK. This isolated case was dealt with swiftly:
the entire herd and any traced outsourced animals were eradicated (Kellar
and Lees, 2003). Over the next decade, both Canada and the USA enacted
230
M. Trevan
measures modelled on the UK’s experience and designed to prevent the
ingress and spread of BSE in North America. Table 11.3 shows some of the
comparable actions taken in the EU, Canada and the USA.
Up until 2003, both Canada and the USA had regarded the risk of BSE
becoming an issue in their cattle herds as being practically zero, and not without cause. Many of the measures introduced to prevent the importation of
animal diseases, e.g. foot-and-mouth disease, would also have been effective
against the importation of BSE. A ban on beef products from other EU countries not considered free of BSE was implemented in 1991. Canada banned
the importation of UK cattle in 1990 in response to the growing BSE epidemic
in the UK, and officially monitored all 182 animals imported from the UK
since 1982. Of these, 14 were still in quarantine at the time and were not
released, 68 had died or been slaughtered, and, of the remaining 100, one
developed BSE in 1993. Ten of the 68 animals no longer alive in 1990 were
later traced back to farms in the UK where BSE had subsequently occurred,
and it is likely that it was through (some of) these cattle being incorporated
into the feed chain that BSE entered Canada (Health Canada, 2005). Similar
circumstances pertained in the USA.
It must be assumed that BSE-infected feed entered the complex animal
feed chain in North America. However, it is now known that the susceptibility
of cattle to infection with BSE increases with decreasing age – younger cattle
Table 11.3. Actions taken to prevent spread and possible effects of bovine spongiform
encephalopathy (BSE) (source: Hueston and Bryant, 2005).
Action taken
Banned import of live ruminants (and products) from UK
Started active surveillance for BSE
Banned mammalian rendered meat and bone meal (MBM)
from ruminant feed
Banned export of UK cattle and milk
Banned import of live ruminants and ruminant products
from EU
Banned use of specified risk material (SRM) from sheep
and cattle
Banned import of all rendered animal proteins
Banned all use of mammalian protein in feed for livestock
Introduced routine testing of advanced meat recovery (AMR)
products for spinal cord
Banned ‘downer’ cattle SRMs from over 30-month-old
animals from human food
Banned MSM (mechanically separated meat) from
human food
Introduce enhanced surveillance programme
Banned SRM/MSM products from cosmetics and diet
supplements
EU
Canada
USA
1989
–
1994
1990
–
1997
1989
1989
1997
1996
n/a
n/a
–
n/a
1997
2000
–
–
2000
2001
–
2000
–
–
–
–
2002
–
2004
2004
–
–
2004
–
–
2004
–
2004
2004
TSEs in Policy Development
231
are more susceptible. The North American practice of slaughtering cattle at a
younger age, when, even if infected with BSE, they would not have had time to
develop high levels of infectivity, would also have prevented BSE from becoming an established endemic disease. However, from 2004, 17 cases of BSE have
been recorded in Canadian cattle (the latest in March 2010). While hardly an
epidemic, this does raise the question of where the latter of these cases have
come from, given that most were born well after the 1997 feed ban was
imposed. Given that it is still uncertain how BSE arose in UK cattle in the first
place, and that the original hypothesis that it came from scrapie-infected meal
has been discredited by the science that shows distinct differences in patterns
of infectivity in ‘humanized’ mice when inoculated with scrapie or BSE prions,
then the hypothesis that BSE is a disease akin to classic CJD in humans needs
to be re-examined. Could BSE have been a sporadic TSE of elderly cattle that
was amplified through the rendering of cattle remains to cattle feed? If so,
then given current surveillance levels for BSE, it would be surprising if, in a
national herd of 10–20 million cattle, the very occasional case of BSE was not
detected.
The first US case of BSE was announced on 23 December 2003 in a dairy
cow in Washington state. Almost immediately, 53 countries, including the
USA’s major markets of Canada, Mexico, Japan and South Korea, banned the
import of US beef products and cattle. This reaction was not surprising, given
that the beef economies of the countries involved suffered from US competition, but it was against the recommendations of the World Organisation for
Animal Health (OIE). The USA had already banned the import of cattle from
Canada following the first recognized case of endogenous BSE in Canada in
May 2003, and had not itself endorsed or operated the OIE recommendations. However, after significant political pressure, the US Department of
Agriculture (USDA) put in place regulations (effective March 2005), to allow
importation of cattle under 30 months of age from ‘minimal risk’ countries
such as Canada. That, however, did not stop the US industry group RanchersCattlemen Action Legal Fund (R-CALF) from successfully gaining an injunction in 2004 from a court in Montana (presided over by the aptly named Judge
Cebull) blocking the federal measure (R-CALF, 2004). R-CALF’s argument
was that it was concerned with the implications of BSE for human health,
despite the fact that most authorities had by then recognized that this was at
best highly tenuous, if not illusory. The economic issue was the potential losses
that might be sustained through competition from Canadian cattle producers.
Marsh et al. (2005) had estimated that Canadian imports would reduce the
price of US feeder cattle by US$4.75 per cwt. The costs of subsequent export
restrictions on US beef, following their own first case of BSE, probably lie in
the range of US$3–5 billion. This, of course, does not include the negative
effect on processors. US beef processors relied on both US and Canadian beef
supply to make their activities economically sustainable. The sudden reduction in supply of cattle because of the import ban on Canadian animals had a
significant negative effect on their businesses.
Testing for BSE has been another source of controversy. Before the
first case of BSE in the USA, testing for BSE had been below international
232
M. Trevan
standards. For example, in 2003 the USDA tested 20,000 cattle compared with
the EU’s 8 million (Fox and Peterson, 2004). As a result of the discovery of the
Washington case of BSE, the USDA announced an enhanced surveillance programme, targeted at high-risk cattle populations. The USDA’s calculation was
that by testing 268,000 cattle on a selective basis, they could predict with 99%
confidence the occurrence rate of a 1:1,000,000 incidence of BSE. Obviously,
the lower the detection rate, the less the negative effect on US beef prices.
Mitchell (2004) criticized the choice of test used by the USDA as being one
that reported a high level of inconclusive results, thus minimizing positive
results and insulating the US market from the over-reporting of BSE cases.
Some producers argued for more extensive voluntary testing. Coffey et al.
(2005) suggested a net premium between US$27.50 and US$48.50 per head in
sales of beef to the Japanese and South Korean markets if voluntary testing
were instituted. However, in 2004 the USDA rejected a request from a Kansasbased company, Creekstone Farms, to test all slaughter cattle for BSE in an
attempt to regain the Japanese export market. R-CALF continued to lobby the
USDA to restrict imports of Canadian-bred beef, latterly on the basis of inadequacies in the Canadian BSE testing regime compared with that described in
the EU Report on the monitoring and testing of ruminants for the presence
of transmissible spongiform encephalopathies (TSEs) in the EU in 2007 (EU,
2009). In their letter of July 2009 to the USDA, R-CALF states:
The 2007 EU Report provides valuable statistical data concerning the status of
the EU’s monitoring and testing program for bovine spongiform encephalopathy
(“BSE”) and other transmissible spongiform encephalopathies (“TSEs”). When
these EU data and their results are compared to and contrasted with the
monitoring and testing program (hereafter “testing program”) that Canada
practices for BSE, it is abundantly clear that Canada’s BSE testing program is
woefully inadequate to: 1) reliably determine the prevalence and evolution of
BSE in Canada; and, 2) protect the food supply, both in Canada and the U.S.,
from contamination by beef from BSE-infected animals.
Concluding Remarks
What these preceding accounts show is how much local and national food
safety issues are as much governed by politics and economics as they are
informed by science, and that the outcome is as much determined by a
government’s coordinated response to risk management as it is by the quality
of scientific advice.
As a final comment, it would be well to remember that the formulation
of policy is the remit and responsibility of politicians. Science can provide
information to guide the derivation of policy, it can never supplant the process. The corollary is that it ill-behoves politicians (and scientists or media
reporters) to pretend that their pronouncements are supported by scientific
evidence when they are not. To do so inevitably renders science an impotent
force in society, which in the end benefits nobody.
TSEs in Policy Development
233
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Index
Page numbers in bold refer to tables; page numbers in italic refer to figures.
aerosols 61
antibiotics
in aquaculture 121–122
in livestock production
content in animal feed 92
extent of use 120
regulations 120–121
resistance to antibiotics see
resistance, antimicrobial
sources of antibiotic residues and
resistance genes 122–124
antibodies, egg yolk 150
avian influenza see influenza
avoparcin 131
Bacillus anthracis 72
bacteria
foodborne diseases 26, 86
transmitted in food and water 23–24
see also individual species
bacteriophages 150–152
bio-aerosols 61
Brucella melitensis 33–34, 72
BSE (bovine spongiform
encephalopathy) 3
association with vCJD 226–229
Canada 229–231
UK
EU ban on UK beef 222–223
events and measures taken
216–219, 220, 223–224
non-food hazards 220–222
number of cattle affected 225
USA 231–232
CAFOs (concentrated animal farming
operations) 121, 122–124
Caliciviridae 35
Campylobacter spp. 30, 66–67, 84–85, 105
bacteriophage treatment 151
in organic animal products 174
vaccines 148–149
cancer, associated viruses 70
carcasses, contamination 64–65, 84,
140–141
cestodes 43–45
transmitted in food and water 25
Chlamydophila psittaci 72–73
chlorate, sodium 143–144
CJD see Creutzfeldt–Jakob disease
Codex Alimentarius 2, 3, 11
colostrum, bovine 104–105, 110
composting 154, 155
conjugation, genetic 126
237
238
Index
consumers
changes in habits 5
perceptions of organic food 168,
175–176
cosmetics 221–222
Coxiella burnetti 32–33, 72
Creutzfeldt–Jakob disease
association with BSE 226–229
hazards of non-food products
220–222
symptoms 215–216
vCJD in the UK 219–220
Crohn’s disease
association with meat and milk
protein intake 206–207
association with Mycobacterium avium
subsp. paratuberculosis (MAP)
203–205
clinical trials 204
course and pathogenesis 199–200
environmental factors 201–202
genetic susceptibility 200
role of intestinal flora 202
Cryptococcus neoformans 72
Cryptosporidium spp. 40–41, 67–68
Cyclospora spp. 41
cysticercosis 44
dairy products
presence of antibiotic resistance
genes 130
see also milk; milk, raw (and raw milk
products)
databases 4
decontamination
of animal feed 91–92
of manure 73–74
diarrhoea, mortality in developing world
21
diphyllobothriasis 44–45
direct-fed microbials (DFM) 145–146
Echinococcus spp. 45
encephalitis, tick-borne 109
Entamoeba spp. 42
Enter-Net 4
enterococci, antibiotic-resistant 130, 131
enteroviruses 35–36
Erysipelothrix rhusiopathiae 72
Escherichia coli
in animal feed 87, 89
bacteriophage treatment 152
disease types and outbreaks 28
dissemination within a beef
production system 142
prevalence in bovine products 63,
105, 106–109
prevalence in non-bovine milk 110
routes of food contamination
63–64, 85
vaccines 149–150
virotypes and virulence 28, 63
farming
concentrated animal farming
operations 121, 122–124
swine production 126–129
see also organic farming and food
fascioliasis 46
feed, animal
decontamination 91–92
implicated in BSE 217–218,
223–224
as potential disease vector 86–87
risk of ingredient contamination
88–89
and salmonellosis in humans 89–91
treatments to reduce pathogen
content 141
antimicrobial additives
143–145
prebiotics 146–147
probiotics 145–146
use of antibiotics 92
zoonotic pathogens in 87–88
flukes see trematodes
FoodNet 4
foot-and-mouth disease 3, 12–13
fruit
pathogens in fruit juices 5
raw produce as infection source 4,
61
gastrointestinal system
intestinal flora 59, 202
see also inflammatory bowel disease
genes
antibiotic resistance 124–126
239
Index
in contaminated food produce
129–130
knowledge gaps 133
microbial ‘perfect storm’ 131,
132
on swine production farms
128–129
and susceptibility to inflammatory
bowel disease 199–200
Giardia spp. 40, 68
globalization, key drivers 2
governance, global: of food safety
1–2
H1N1 virus 189, 190, 191
H3N2 virus 189–190
H5N1 virus 186–188
H7N7 virus 188–189
HACCP (Hazard Analysis and Critical
Control Point) 8
Helicobacter spp. 71
hepatitis, viral
hepatitis A 35–36
hepatitis E 36, 69–70
hydatid disease 45
immunization
passive 150
vaccination 148–150, 198–199
immunodeficiency 5–6, 205
and listeriosis 27
inflammatory bowel disease
characteristics 199–200
environmental factors 201–202
genetic susceptibility 200–201
role of intestinal flora 202
influenza 69
avian influenza
highly pathogenic outbreaks in
poultry 187
history and threat 182–183
pathogenesis 183–184
transmission 184–185
viruses 183
genetic shift 185
new strains 184
in wild versus domestic birds
185–186
zoonoses 186–189
swine influenza
characteristics 189
viruses 189–190
zoonoses 191–192
integron gene cassettes 124–125
Johne’s disease 197–199
kuru 214, 216
Listeria monocytogenes
characteristics and incubation 26
isolation rates in raw milk 104, 105,
106, 110
listeriosis
at-risk groups 27
control 13–14, 27–28
importance of surveillance 7
outbreaks 14, 27
sources of infection 65–66, 85
USDA–FDA study of risk in
foods 27
mannan-oligosaccharides (MOS)
146–147
manure
contributes to antimicrobial
resistance 70–71, 122–124,
153, 154–155
production and use 60
as source of infection 60–61,
169–170
Campylobacter spp. 66
control measures 62
disease types 62
Escherichia coli 63
listeriosis 66
other zoonoses 71–73
protozoa 67–68
Salmonella spp. 64–65
viruses 69–70
treatments to reduce risk 73–74
mastitis, pathogens 108, 109–110
milk
nutritional significance of bovine
milk and milk products
103–104
240
Index
milk continued
pasteurization
influence on nutritional
qualities 102–103
times and temperatures (US
FDA) 102
milk, raw (and raw milk products)
controversy 99
legislation and regulations 112–113
perceived benefits 101–102, 103
prevalence of pathogens in bovine
milk
Asia, Middle East, South
America, Caribbean 106
Escherichia coli 105, 106–109
Europe, Africa 107
Listeria monocytogenes 104, 105,
106
mastitis pathogens 108,
109–110
other pathogens 109
overview 104
Salmonella spp. 104–105, 106
USA and Canada 105
prevalence of pathogens in nonbovine milk 108, 110–111
sales and consumption 100–101
threat to consumers 111–112
mills 89
morbidity
due to zoonotic bacteria 86
foodborne disease in USA 22
mortality
diarrhoea in developing world 21
foodborne disease in USA 22
mozzarella cheese 110–111
Mycobacterium avium subsp.
paratuberculosis (MAP)
association with Crohn’s disease
203–205
causative agent of Johne’s disease
197–198
clinical trials 204
prevalence of infection in cattle 198
strains and identification 205–206
vaccination 198–199
Mycobacterium spp. 72, 109, 110–111
nematodes 42–43
transmitted in food and water 24–25
Nipah virus 6–7
noroviruses 35, 69
Norwalk-like viruses 35, 69
OIE (Office International des
Epizooties) see World
Organisation for Animal Health
oilseed 88, 89
oligosaccharides 146–147
organic farming and food 11–12
advantages 175
consumer perceptions 168, 175–176
history and characteristics 167–168
manure-associated risks 169–170
regulations 168
risks of animal-derived products
172–174
risks of plant products 170–172
wild–domestic animal interactions
169
parasites
associated with vegetation 5
complex epidemiology 37
increasing risk of human exposure
38
see also cestodes; nematodes;
protozoa; trematodes
pasteurization
influence on milk nutritional
qualities 102–103
times and temperatures (US FDA)
102
pathogens
emerging strains and diseases 6–7,
22
methods of detection 6
transmitted in food and water 23–25
‘perfect storm’, microbial 131, 132
picornaviruses 35–36
plasmids 124, 125
poultry
campylobacteriosis 30
other zoonoses 72–73
salmonellosis 29
prebiotics 146–147
PrimaLac 145
prions 215, 217, 226–227
probiotics 145–146
241
Index
production, food
and antimicrobial resistance
129–130
implications of antibiotic use in
livestock see antibiotics
safety implications 4
protozoa
in manure 67–68
transmitted in food and water 24,
38–39
see also Cryptosporidium spp.;
Cyclospora spp.; Entamoeba
spp.; Giardia spp.; Sarcocystis
spp; Toxoplasma gondii
pseudotuberculosis 31
psittacosis 72–73
PulseNet 4
Q fever 32–33, 72
regulations
antibiotics in livestock production
120–121, 168
organic farming 168
residues, antibiotic see antibiotics
resistance, antimicrobial 14–15
case studies of spread
contaminated food produce
129–130
swine production farms
126–129
causative factors 119–120
implications for humans and
environment 131–133
manure as a source of resistant
organisms/resistance genes
70–71, 153, 154–155
transfer of genes at microbial level
124–126
risk assessment 8–9
foods and listeriosis 27
rotaviruses 36
roundworms see nematodes
Salmonella spp.
in animal feed 87
evidence for disease
transmission 89–91
and antimicrobial resistance 14–15
bacteriophage treatment 151, 152
disease outbreaks 29–30, 85
epidemiological importance 64
isolation rates in raw milk 104–105,
106
in manure and carcasses 64–65
in organic animal products 173–174
serovars
distribution in feed and human
disease 90–91
of significance to human health
90
vaccines 148, 149
zero tolerance policy 84
Sarcocystis spp. 41–42
scrapie 217, 226, 227
shedding 85
shellfish 5
sodium chlorate 143–144
SPS (Sanitary and Phytosanitary)
Agreement 1–2, 3
standards, food: international
agreements 9–10
Streptococcus suis 72
surveillance
of disease outbreaks
role and importance 7
systems 8
of food products 3–4
swine influenza see influenza
Taenia spp. 43–44
tapeworms see cestodes
Tasco-14™ 144–145
tetanus 71–72
Toxoplasma gondii 39, 68
in organic animal products 172–173
traceability 2
transduction, genetic 126
transformation, genetic 126
transmissible spongiform
encephalopathies (TSE)
association between BSE and vCJD
226–229
bovine (BSE) 3
Canada 229–231
EU ban on UK beef 222–223
number of UK cattle affected
225
242
Index
transmissible spongiform
encephalopathies
(TSE) continued
bovine (BSE) continued
UK events and measures taken
216–219, 220, 223–224
USA 231–232
caused by prions 215
Creutzfeldt–Jakob disease (CJD)
hazards of non-food products
220–222
symptoms 215–216
vCJD in the UK 219–220
disease spectrum 214–215
incubation 229
symptoms 215–216
transmission 216, 221–222
transposons 124, 125
trematodes 46
transmitted in food and water 25
trichinellosis 42–43
vaccines 148–150, 198–199
vegetables
contamination from manure
169–170
raw produce as infection source 4,
61
risks of organic products 170–172
virotypes 28
virulence
and combined gene pools 131–132
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genes and factors 28
viruses 37
new human pathogens from animal
sources 34–35
tick-borne encephalitis 109
transmission in food and water 23,
34
transmission in manure 69–70
see also enteroviruses; hepatitis, viral;
influenza; Nipah virus;
noroviruses; rotaviruses
VTEC (verocytotoxigenic E. coli) 28, 63,
85
waste, animal
composting 154, 155
importance of management 152,
154–155
see also manure
water
source of pathogens 23–25, 61,
63–64, 67, 69
treatments to reduce pathogen
content 143
WHO-Global Salm-Surv 4
World Organisation for Animal Health
(OIE) 3, 10–11
World Trade Organization (WTO) 1
Yersinia enterocolitica 31–32, 71, 85, 109
Yersinia pseudotuberculosis 31