Intestinal Health, Theo Niewold

Intestinal health
Intestinal health
Key to maximise growth performance
in livestock
edited by:
Theo Niewold
Wageningen Academic
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DOI: 10.3920/978-90-8686-792-9
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Table of contents
Chapter 1: General introduction – the gastrointestinal tract, the

immune system and the maintenance of health 15
T.A. Niewold
Chapter 2: The composition and role of the microbiota in chickens 21

A.A. Pedroso and M.D. Lee
Abstract 21
2.1 Importance 21
2.2 Composition 23
2.3 Temporal variation 23
2.4 Spatial variation 25
2.5 Role of the microbiome 28
2.6 Short chain fatty acids 33
2.7 Modulation of the intestine using probiotic microorganisms 33
2.8 Conclusions 36
References 37
Chapter 3: Intestinal diseases of pigs 51

S. McOrist and E. Corona-Barrera
Abstract 51
3.1 Introduction and general features 51
3.2 Key intestinal diseases in pigs at or after weaning 53
3.3 Conclusions 68
References 69
Chapter 4: Avian coccidiosis as a prototype intestinal disease – host

protective immunity and novel disease control strategies 71
H.S. Lillehoj, S.I. Jang, S.H. Lee and E.P. Lillehoj
Abstract 71
4.1 Introduction 72
4.2 Chicken immune responses to Eimeria 74
4.3 Prevention and control of avian coccidiosis: alternatives to antibiotics 82
Intestinal health 7
4.4 Passive immunization against avian coccidiosis using hyperimmune antibodies 83
4.5 Immunomodulation with phytochemicals against avian coccidiosis 86
4.6 Novel immunization strategies against avian coccidiosis 93
4.7 Conclusions 101
Acknowledgements 101
References 101
Chapter 5: Intestinal health in carnivores 117

E.A. Hagen-Plantinga and W.H. Hendriks
Abstract 117
5.1 Introduction 118
5.2 Defining ‘gut health’ 119
5.3 Intestinal microbiota of carnivores 121
5.4 Influence of nutrition on canine and feline gastrointestinal microbiota and
gut health 128
References 132
Chapter 6: Pig intestine, weaning and dietary interventions 139

J.P. Lallès and D. Guillou
Abstract 139
6.2 Recent advances in intestinal physiology and pathophysiology of post-
weaning disorders 141
6.3 Dietary nutrients (or precursors), animal proteins and minerals 143
6.4 Dietary feed components 149
6.5 Probiotics 153
6.6 Interactions between feed-added substances and with the rearing environment 155
6.7 Distant effects of early nutritional interventions 156
6.8 Conclusions and perspectives 157
References 158
8 Intestinal health
Chapter 7: Effect of feed contaminants on intestinal health of
monogastric farm animals 169
I. Alassane-Kpembi and I.P. Oswald
Abstract 169
7.2 Mycotoxins in feed 171
7.3 Dioxins in feed 173
7.4 Effects of feed contaminants on intestinal epithelium renewing and
intestinal barrier function 174
7.5 Histo-morphological alterations of intestine induced by feed contaminants 176
7.6 Modulation of digestive functionality of the intestine by feed contaminants 176
7.7 Modification of the intestinal microflora by feed contaminant 179
7.8 Effect of feed contaminant on secretion of some intestinal defence components 180
7.9 Modulation of intestinal immune response by feed contaminants 181
7.10 Conclusions 182
References 183
Chapter 8: Techniques for investigating gut function in vivo, ex vivo

and in vitro in monogastric farm animals 191
J.P. Lallès and I.P. Oswald
Abstract 191
8.2 Intestinal permeability measurement in vivo 192
8.3 In situ and ex vivo intestinal loops 196
8.4 Ussing chambers 197
8.5 Ex vivo GIT tissue explants 202
8.6 In vitro intestinal cell cultures 205
8.7 Conclusions and perspectives 208
References 209
Chapter 9: Intestinal health biomarkers in vivo 219

T.A. Niewold
Abstract 219
9.2 Enterocyte biomarkers 223
Intestinal health 9
9.3 Fecal serum protein 224
9.4 Inflammatory cells 224
9.5 Plasma acute phase proteins 225
9.6 Discussion 225
References 226
Chapter 10: Intestinal health research and proteomics, a well-

matched couple 229
L. Soler and I. Miller
Abstract 229
10.2 Overview of techniques 231
10.3 Proteomics as a useful instrument for developmental studies 239
10.4 How can proteomics help us to improve feeding regimes? 242
10.5 Expression proteomics to unravel the gastrointestinal immune response 243
References 245
Chapter 11: Systems biology – applications in intestinal health 253

D. Schokker and M.A. Smits
Abstract 253
11.2 Intestine as a model 254
11.3 Integration 255
11.4 Perturbations 256
11.5 Mathematical formalisms 260
11.6 Biological networks/graphs 262
References 266
Index273
10 Intestinal health
Acknowledgements
It is an honour to be asked to be the editor of a book, although at the time I did
not realise what came with it. One of the problems is that writing a book chapter is
nowadays not properly rewarded, instead the emphasis is on publications in high
impact factor journals, teaching, supervising research, writing grants etcetera.
Therefore, I am very grateful to the authors who were willing to dedicate precious
time to the writing of their respective chapters. In my career, I have met many
excellent scientists from many different countries, and I feel fortunate for their
willingness to participate in this project. And last but not least, I also like to thank
the publisher, and in particular Mike Jacobs for the support and patience throughout
the long process of finalisation of this book.
I hope the wait was worthwhile.
Theo Niewold
Intestinal health 13
Chapter 1: General introduction – the
gastrointestinal tract, the immune
system and the maintenance of health
T.A. Niewold
Nutrition and Health Unit, Department of Biosystems, Faculty of Bioscience
Engineering, KU Leuven, Kasteelpark Arenberg 30, 3001 Heverlee, Belgium;
theo.niewold@biw.kuleuven.be
The gastrointestinal tract (GIT) is essential in the maintenance of health and

wellbeing in man and animals alike. It forms a protective barrier, and simultaneously
functions for the uptake of nutrients. The GIT is best described as a complex and
dynamic ecosystem. Feed interacts with the microbiota, and with the host mucosa,
and mutual interaction exist between the three components (Figure 1.1). Interactions
of immune cell populations, present in the epithelium of the intestine, and other
components of the intestinal mucosa are essential in the maintenance of equilibrium
with commensals and the defence against pathogens. The mucosa itself is not simply
Mucosa
genetics
immunology
nervous system
Health and growth
Feed Microbiota
nutrients commensals
additives (potential) pathogens
contaminants
Figure 1.1. Schematic representation of the three components of the intestinal ecology important
in determining health and growth in production animals. Mutual interactions exist between the
three components (feed, microbiota, and mucosa), and for each component the major factors of
influence within the component are given.
T.A. Niewold (ed.) Intestinal health

DOI 10.3920/978-90-8686-792-9_1, © Wageningen Academic Publishers 2015
T.A. Niewold
composed of a layer of identical enterocytes, but contains many different cell types of
the latter, as well as immune cells, nerve cells, and many other, forming an intricate
network needed for proper function. Furthermore, there are great functional
differences along the GIT from oral to distal, and there are of course differences
between the monogastric species, in our case between chicken and pigs. The details
of the anatomy of the GIT and the differences between species are not discussed here
because there are sufficient excellent reviews and books available on that matter. In
this volume, the emphasis is on the different components of the intestinal system
and on their mutual interactions. In this, the mucosal immune system plays a central
role, and first a brief description of the main principles involved is given below.
The GIT contains a large mucosal immune system. This immune system is geared
towards tolerance as opposed to the systemic immune system. It is important to
realise that the responses of the immune system are influenced by environmental
as well as by the genetic factors, and by the immunological history of the animal.
The GIT immune system responds to the intestinal contents (microbiota, and feed
components), and this reaction can lead to tolerance (e.g. for commensal bacteria),
or to a defence reaction. In general, the response pathway consists of inducers,
sensors, mediators, and effectors, each important in determining the type of response
(Medzhitov, 2008).
Inducers are exogenous or endogenous signals that initiate the inflammatory response.
The exogenous inducers can be either microbial or non-microbial. These microbial
inducers are virulence factors, and microbial- or pathogen-associated molecular
patterns (MAMP/PAMP) detected by pathogen-recognition receptors (PRRs), such
as Toll-like receptors (TLRs) present on the effectors. The MAMP/PAMP are a limited
and defined set of conserved molecular patterns that are carried by microorganisms
(whether pathogenic or commensal). The virulence factors, however, are restricted
to pathogens, and are not directly sensed by dedicated receptors. The non-microbial
inducers include allergens, irritants, foreign bodies, and toxic compounds (Majno and
Joris, 2004). The endogenous inducers of inflammation, detected by PRRs, are host-
derived danger signals (danger-associated molecular patterns; DAMPs) produced by
stressed, damaged, infected, or otherwise malfunctioning tissues.
The signals elicited by the inducers can be received by an effector site. The effectors
of an inflammatory response are tissues and cells, the functional state of which
is influenced by a host of inflammatory mediators e.g. cytokines, chemokines,
and many more (Majno and Joris, 2004). Different intestinal effector sites can be
1. General introduction
distinguished, epithelial cells, Peyer’s patches, and (mucosal) immune cells of which
the dendritic cells are believed to sense even inside the lumen of the GIT (Rescigno
et al., 2001).
Intestinal epithelial cells (IECs) of the GIT were long thought to play only a
secondary role in mucosal immunity. However, it has become clear that epithelial
cells are key in orchestrating the intestinal immune response of the host to pathogens
(Pitman and Blumberg, 2000). IECs function as sensors detecting MAMP/PAMP,
and DAMP, express TLRs, do secrete cytokines and chemokines, promote adaptive
immune responses (Kagnoff and Eckmann, 1997), function as antigen presenting
cells, and regulate T cell responses in the intestinal mucosa (Snoeck et al., 2005). It
can be concluded that IEC are immunocompetent cells in their own right, as well as
pivotal in the intestinal immune response.
Important effector sites of the mucosal immune system are found in the GALT (Gut
Associated Lymphoid Tissue) examples of those are Peyer’s patches. These organized
sites of lymphoid tissue are found in the small intestine. They contain a host of
different immune cells, and are covered with a specialised lympho-epithelium. This
epithelium, which has no crypts or villi, is responsible for the transport of the luminal
antigens into the lymphoid areas through specialized M (microfold) cells (Jung et
al., 2010), containing numerous vesicles involved in transport of luminal antigens to
the underlying lymphoid tissue (Siebers and Finlay, 1996). The effector immune cells
consist of those released by the Peyer’s patches, the intraepithelial lymphocytes, cells
recruited from the blood, and dendritic cells. Intraepithelial lymphocytes, primarily
T cells have potent cytolytic and immunoregulatory capacities. (Hayday et al.,
2001). They have an important role in local immunosurveillance of the intestinal
epithelial cells, and the regional microenvironment (Hayday et al., 2001; Lefrancois
and Vezys, 2001). Once the local system is activated, and if not contained, it can
lead to recruitment of immune cells from the blood stream. The cell types involved
range from phagocytes (monocytes, macrophages, dendritic cells, mast cells, and
neutrophils), eosinophils, basophils, to natural killer cells. There is a pivotal role
in this for neutrophils for their role in killing invading agents (Medzhitov, 2008;
Nathan, 2006). Unfortunately, they can also cause great damage to the host tissue
itself (Nathan, 2002), and that is most likely the reason for the presence of the
intestinal anti-inflammatory reflex, which is mediated through the nervous system.
The GIT contains a very large nervous system. Recently, it has become clear that
at least part of it is involved in modulating immune responses. The afferent neural
nerve (vagus nerve), present in the intestine (Wang and Powley, 2007), alerts the
T.A. Niewold
central nervous system of an injury, infection, or cytokine excess, and stimulates

by reflex an anti-inflammatory response to prevent excessive inflammation and
associated damage (Tracey, 2002; Tracey et al., 2001). The afferent vagus nerve
will respond by inhibiting the inflammatory reaction by vagal efferents (Luyer et
al., 2005) and nicotinic receptors, the nicotinic acetylcholine receptor α7 subunit
(nAChR α7) (Wang and Powley, 2007) present on macrophages, and other cytokine
producing cells. It thus functions as an anti-inflammatory reflex, protecting essential
physiological systems (Tracey, 2007).
The innate part of the immune system leads to the quick recruitment of immune
cells, and the production of acute phase proteins by the liver, as a first line of defence.
The specific or adaptive part of the immune system leads ultimately to the generation
of cytotoxic immune cells, and the production of antibodies. It is clear that this is
associated with costs to the animal because resources used for defence cannot be
used for growth (Iseri and Klasing, 2013). The magnitude of the actual costs depends
very much on the pathogen in question, but it is apparent that the innate response is
energetically markedly more costly than the adaptive response in most cases (Iseri
and Klasing, 2013).
It can be concluded that especially in production animals inflammation should be

contained as much as possible. As mentioned above, inflammation can be induced
by pathogens, initiating the PAMP pathway, also, feed can contain toxins which can
trigger the DAMP pathway. In addition, (psychological) stress does also induce a
pro-inflammatory state (Niewold, 2010). Furthermore, it has recently become clear
that in humans the consumption of high energy food itself can lead to post-prandrial
inflammation (Margioris, 2009). It is argued that this phenomenon could also exist
in production animals because they receive high energy diets too (Niewold, 2014).
Irrespective of the cause of inflammation, it is very costly for the animals, for a
series of reasons. It leads amongst others to reduced appetence, and catabolism of
muscle resulting in reduced growth. Furthermore, it may enhance the susceptibility
for certain (intestinal) pathogens, leading to further problems.
As stated in the beginning of this chapter, the host and its (intestinal) immune system
do not operate in isolation, but are part of the complex ecosystem in which feed and
the microbiota play a very important role. The composition of the microbiota is at
least in part determined by the host, and in part by the composition of the feed.
The microbial fermentation of feed components gives rise to products which have
effects both on the host and on other microbial populations. A stable composition of
1. General introduction
commensal microbiota may help to exclude pathogens. It is clear that there is ideally
an healthy equilibrium in this ecosystem, and it is equally clear that this equilibrium
can be disturbed by changes emanating from possibly all three components. The
complex relationships within the microbiota and between the different components
of the intestinal ecosystem has hindered our understanding thus far considerably.
Nevertheless, there is already a considerable body of knowledge accumulated in
particular on the microbiota, pathogens and associated diseases in the major
monogastric production species (pigs and chicken) as described in Chapters 2-4.
Also, a chapter (5) on the composition of the microbiota in carnivores is added, for
comparative purposes. The role of the diet, additives and contaminants, as well as
the techniques used to obtain that knowledge is described in Chapters 6-8. Chapters
9-11 describe the newer developments such as the use of biomarkers for intestinal
health, and the application of omics and associated techniques, which are promising
because they are inherently more suited for the analysis of complex interactions such
as exist in the intestinal ecosystem. It is hoped that this provides the reader with an
update on the current knowledge in intestinal research in production animals, as
well as with an insight in the future developments in this field.
References
Hayday, A., Theodoridis, E., Ramsburg, E. and Shires, J., 2001. Intraepithelial lymphocytes:
exploring the third way in immunology. Nature Immunology 2: 997-1003.
Iseri, V.J. and Klasing, K.C., 2013. Dynamics of the systemic components of the chicken
(Gallus gallus domesticus) immune system following activation by Escherichia coli;
implications for the costs of immunity. Developmental and Comparative Immunology
40: 248-257.
Jung, C., Hugot, J.P. and Barreau, F., 2010. Peyer’s patches: the immune sensors of the
intestine. International Journal of Inflammation 2010: 823710.
Kagnoff, M.F. and Eckmann, L., 1997. Epithelial cells as sensors for microbial infection. The
Journal of Clinical Investigation 100: S51-S55.
Lefrancois, L. and Vezys, V., 2001. Transgenic mouse model of intestine-specific mucosal
injury and repair. Journal of the National Cancer Institute. Monographs 29: 21-25.
Luyer, M.D., Greve, J.W.M., Hadfoune, M., Jacobs, J.A., Dejong, C.H. and Buurman, W.A.,
2005. Nutritional stimulation of cholecystokinin receptors inhibits inflammation via the
vagus nerve. Journal of Experimental Medicine 202: 1023-1029.
Majno, G. and Joris, I., 2004. Cell, tissue and disease. Oxford University Press, Oxford, UK.
T.A. Niewold
Margioris, A.N., 2009. Fatty acids and postprandial inflammation. Current Opinion in
Clinical Nutrition and Metabolic Care 12: 129-137.
Medzhitov, R., 2008. Origin and physiological roles of inflammation. Nature 454: 428-435.
Nathan, C., 2002. Points of control in inflammation. Nature 420: 846-852.
Nathan, C., 2006. Neutrophils and immunity: challenges and opportunities. Nature Reviews
Immunology 6: 173-182.
Niewold, T.A., 2010. The effect of nutrition on stress and immunity. In: Garnsworthy, P.C.
and Wiseman, J. (eds.) Recent advances in animal nutrition. Nottingham University
Press, Nottingham, UK, pp. 191-205.
Niewold, T.A., 2014. Why anti-inflammatory compounds are the solution for the problem
with in feed antibiotics. Quality Assurance and Safety of Crops & Foods. 6: 119-122.
Pitman, R.S. and Blumberg, R.S., 2000. First line of defense: the role of the intestinal epithelium
as an active component of the mucosal immune system. Journal of Gastroenterology 35:
805-814.
Rescigno, M., Rotta, G., Valzasina, B. and Ricciardi-Castagnoli, P., 2001. Dendritic cells
shuttle microbes across gut epithelial monolayers. Immunobiology 204: 572-581.
Siebers, A. and Finlay, B.B., 1996. M cells and the pathogenesis of mucosal and systemic
infections. Trends in Microbiology 4: 22-29.
Snoeck, V., Goddeeris, B. and Cox, E., 2005. The role of enterocytes in the intestinal barrier
function and antigen uptake. Microbes and Infection 7: 997-1004.
Tracey, K.J., 2002. The inflammatory reflex. Nature 420: 853-859.
Tracey, K.J., 2007. Physiology and immunology of the cholinergic antiinflammatory pathway.
Journal of Clinical Investigation 117: 289-296.
Tracey, K.J., Czura, C.J. and Ivanova, S., 2001. Mind over immunity. The FASEB Journal 15:
1575-1576.
Wang, F.B. and Powley, T.L., 2007. Vagal innervation of intestines: afferent pathways mapped
with new en bloc horseradish peroxidase adaptation. Cell and Tissue Research 329:
221-230.
Chapter 2: The composition and role of the
microbiota in chickens
A.A. Pedroso and M.D. Lee*

Poultry Diagnostic and Research Center, University of Georgia, 953 College
Station Rd, Athens, GA 30602, USA; mdlee@uga.edu
Abstract
The poultry microbial community has been object of study since the discovery of
techniques to study microbes. Our limited knowledge about the nutritional and
physiological needs of the intestinal community was restricted to the members that
we could easily grow under laboratory conditions. These results masked the true
composition and behavior of the intestinal microbiota of chickens until molecular
techniques were adopted, including PCR, DGGE, T-RFLP, cloning and sequencing
among others. Considerable progress has been made in obtaining a census of the
community once DNA sequencing techniques became more affordable and less time
consuming. The true composition, including uncultivated organisms, was revealed
and new insights about its role in chicken physiology have been revealed. Here
we discuss the microbiota and its effect on intestinal homeostasis, development,
differentiation, and maturation. In addition, we address how modification of the
microbiota may improve absorption of nutrients and change the composition of
the mucin layer that affects intestinal function. Much progress has been made and
future work will illuminate the mechanisms by which the microbiota influence the
bird’s physiology. Future advances in poultry production will include microbial
modulators that have been designed for specific effects.
Keywords: microflora, microbiota, microbiome, intestine, holobiont, homeostasis,

differentiation, mucin, probiotic
2.1 Importance
The term ‘holobiont’ has been coined to describe the vertebrate superorganism that
results from commensalism and mutualism with an assemblage of host-associate
microbes (Singh et al., 2013b; Walter et al., 2013). A perfect balance among the

microbial consortium may be needed for the host to reach its maximum genetic
potential and indeed it appears that each vertebrate species may have coevolved
genomes with its microbiome (Xu and Gordon, 2003). For example, the intestinal
microbiota creates a complex environment within the intestine that affects many
host functions. Tasks under influence of these microorganisms include degradation
of mucin, controlling pathogen abundance and behavior, regulation of inflammation,
and stimulation of intestinal differentiation and development, among others. In food
animal production, many strategies are used to manipulate microbial groups in the
intestinal tract hoping to influence nutrient digestion and absorption. In addition
resistance to infection and colonization with zoonotic pathogens may be influenced
by the microbiome. Common approaches to enhance the abundance of beneficial
organisms include use of direct-fed microbials such as probiotics and of prebiotic
additives which feed the desirable members of the microbiota. Competitive-exclusion
products and antimicrobial agents are used to suppress the abundance of undesirable
organisms. Interest in effectively applying such approaches is on the increase because
of the consumers’ desire for a more ‘wholesome’ source of meat, poultry, and eggs.
A search of current literature reveals an increasing volume of research in the area
of host-related microbial ecology (Figure 2.1). The lessons obtained in laboratory
12,000
Annual publications retrieved by Google Scholar
10,000 Prebiotic
Probiotic
8,000 Intestinal microbiota
6,000
4,000
2,000
0
65
70
75
80
85
90
95
00
05
10
19
19
19
19
19
19
19
20
20
20
Year
Figure 2.1. The number of publications related to the normal flora, prebiotic and probiotic from
1960 to 2010.
2. The composition and role of the microbiota in chickens
and field experiments will allow us to maximize poultry production and animal
health. The combination of molecular tools and traditional culture techniques to
study the intestinal ecosystem is allowing us to reveal the interactions involved in
the functioning of the holobiont.
2.2 Composition
The bacterial composition of the chicken intestinal tract is not static but presents
temporal variations related to the age of the bird and spatial variation observed
within the different compartments of the intestinal tract (Pedroso et al., 2012). Early
on in the bird’s life, the composition appears to be more influenced by host factors
such as age and genetics (Lumpkins et al., 2010) than by external factors such as diet.
Understanding these variations may be important for designing strategies to reduce
the impact of pathogens and enhance production efficiency.
2.3 Temporal variation
2.3.1 Pre-hatched phase
The eggshell evolved to be a barrier against microorganisms (Gast and Holt, 2000).
However, the cuticle that protects against the eggshell can be degraded resulting in
microorganisms penetrating to reach the internal structures of the egg (Cook, 2003).
From a management standpoint, this can occur shortly after the egg is laid, in the nest,
the transport belt or in the harvest platform. Therefore the vertical transmission of
microorganisms is also possible from the hen to the chick albeit in a different way than
mammals. Pathogenic organisms such as Salmonella, Escherichia coli, Mycoplasma
and Campylobacter can be vertically transmitted (Doyle and Erickson, 2006; Methner
et al., 1995; Okamura et al., 2007). Studies of Salmonella and Mycoplasma have
shown that the bacteria can be present within the ovary. It is plausible to accept
the hypothesis that beneficial microorganisms could be also vertically transmitted.
The anatomy of the reproductive tract of the hen suggests that the embryo is likely
colonized with organisms that are deposited prior to formation of the eggshell.
Microorganisms could become established within the gut of the embryos during
development, especially when the embryos begin to ingest amniotic fluid. In addition,
if microorganisms are present in the yolk sack they could be internalized as yolk is
taken into the intestine later in the embryonic development. Embryonic exposure to
bacteria is not necessarily lethal and in fact challenge of chick embryos has been used
to screen for pathogenic strains of bacteria (Maurer et al., 2002). It has been reported
that viable bacteria can be found in embryos although molecular analysis has revealed
that a low diversity indicating a rudimentary microbiota (Pedroso et al., 2006, 2008).
Species related to Clostridium, Propionibacterium and Lactobacillus were detected
indicating that these organisms may be vertically transmitted from hens to chicks
even in commercial poultry conditions.
2.3.2 Starter phase
Immediately after the hatch, chicks have contact with the microbes in their
surrounding environment. The commercial egg incubator is a source of microbial
contamination, allowing chicks to hatch containing microorganisms that may not
have mutualistic effects (Cason et al., 1994; Cox et al., 1991). Consumption of water
and food results in a rapid acquisition of additional microorganisms and indeed
the process of handling, transport, and vaccination contributes to the evolution of
the intestinal microbiota of commercial poultry. At the moment of delivery to the
poultry farm, the hatchling already has a structured microbiota (Pedroso et al.,
2005). The organization of the intestinal microbiota occurs quickly and species
present within the young chicken may be present at the end of the rearing period
(Yin et al., 2010). This is particularly problematic if the early colonizer is also of
food safety importance such as Salmonella and Campylobacter. Many studies have
shown that foodborne pathogens are most successful in colonizing young chicks
that are exposed in the first week of hatching (Nurmi et al., 1992; Wagner, 2006)
when the intestinal environment is rapidly changing and the microbiota is least
diverse and unstable.
At the farm, the use of deep litter systems allows exposure to a high diversity of
environmental and intestinal microbes which largely increases the number of
genotypes within the intestine of the young chick. During this time the cecum
microbiota in young birds is relatively simple and very similar to the microbiota
observed in the small intestine (Lu et al., 2003) indicating that the spatial
environmental compartments have not yet developed in the intestine. At 3 days
of age, the chick’s ileal microbiota contains a large proportion of environmental
bacteria (Lu et al., 2003), especially if the birds are raised on deep litter (Cressman
et al., 2010), while at 7 days of age, chicks contain an ileal mucosal microbiota
primarily dominated by Lactobacillus, followed by unclassified Lachnospiraceae and
Enterococcus (Cressman et al., 2010). After the second week of age, cecum and small
intestine develop distinctly different communities. This can probably be attributed to

the maturation of the spatial environments including differences in pH, atmosphere
(O2, CO2, and H2), surfactants, osmolarity, substrates and bacterial metabolites such
as short chain fatty acids (SCFA).
2.3.3 Grower phase
Many reports now reveal the composition of the microbial community of the
chicken ileum during growout (Cressman et al., 2010; Czerwinski et al., 2012; Lin
et al., 2013; Lu et al., 2003; Pissavin et al., 2012; Sun et al., 2013; Zhao et al., 2013).
For example Nakphichit et al. (2011) observed that the most abundant organism
in the small intestine was Lactobacillus whose diversity increased from 21 to 42
days of age. Lactobacillus salivarius, L. johnsonii, L. reuteri, L. oris, and L. crispatus
were detected at 21 d of age. Three weeks later, sequences related to L. gallinarum,
L. equi, L. salivarius, L. crispatus, L. aviaries, L. johnsonii, and L. reuteri were
observed in abundance. These studies reveal that although Lactobacillus dominates
the small intestinal microbiome, there is a succession of species and strains as the
birds’ age. In contrast, studies using traditional culture techniques have described
a community of Streptococcus, Staphylococcus, Lactobacillus, Escherichia coli,
Eubacterium, Propionibacterium, and Clostridium (Salanitro et al., 1978) illustrating
the limitations of traditional plate methods (Pedroso et al., 2012). Lu et al. (2003)
described an increase in the Clostridia population in the broiler ileum from the
starter phase to the grower phase with increasing richness at the end of the rearing
period (Gong et al., 2008).
2.4 Spatial variation
The chicken intestinal tract is composed of many compartments, and each one has
its own characteristics. Three proximal segments follow the oral cavity; the crop, the
proventriculus, and the gizzard. Once the diet is ingested it reaches the crop, where
it resides for minutes to several hours (Savory, 1999). The crop is a food storage and
fermentation organ, the proventriculus acidifies the food, while the gizzard is used
for grinding (Sekelja et al., 2012). The lower gut consists of the small intestine, the
colon, and two large cecal fermentation chambers (Sekelja et al., 2012). The ceca,
a blind-ended structure, is capable of storing its contents for longer periods than
would be possible in the rapid transit environment of the small intestine (Clench
and Mathias, 1992). As a result these compartments differ in complexity of the
atmosphere, pH, and nutrient availability of the digesta, salt and water levels. These
variations select for different microbiotas along the length of the digestive tube.
The intestinal microbiota of an adult animal contains at least 17 bacterial families
encompassing approximately 500 different microbial species distributed along
the intestinal tract (Lakhan and Kirchgessner, 2010). An increase in diversity and
complexity is observed from the proximal portions of the intestine to the distal (Yan
and Polk, 2004).
2.4.1 Crop/proventriculus/gizzard
The crop, proventriculus and gizzard have a microbiota with low diversity compared
to distal sections of the chicken intestine which contains a complex community. The
composition of the microbiota of the crop, gizzard, and proventriculus is quite similar
and is dominated by Lactobacillus species (Janczyk et al., 2009; Sekelja et al., 2012).
Lactobacillus agilis, L. salivarius, L. johnsonii, L. reuteri, L. helveticus, L. ingluviei,
and L. vaginalis are commonly detected in the crop of chickens regardless of diet
(Hammons et al., 2010). Chickens fed wheat, corn and soybean meal contain an
abundance of L. aviaries, L. salivarius, and a small proportion of bacteria related to
Clostridia including Arthromitus candidatus, the segmented filamentous bacteria
(SFB) (Gong et al., 2007). It is hypothesized that the abundance of Lactobacillus
within the proximal intestine may be due to high availability of amino acids for which
it is unable to synthesize de novo (Bringel and Hubert, 2003). However Lactobacillus
crispatus has been described to be adherent to tissue sections of the chicken crop
(Edelman et al., 2012) and L. acidophilus binds fibronectin (Kapczynski et al., 2000)
an intercellular matrix protein, revealing an additional mechanism by which this
genus prevails in the small intestine. Other studies have observed that the crop is
dominated by Lactobacillus followed by Gallibacterium (family Pasteurellaceae); less
abundant genera included Veillonella and Enterococcus (Videnska et al., 2013). In
addition, Atopobium, Bifidobacterium, and Clostridia species related to Eubacterium
rectale and Clostridium coccoides have been described as associated with the crop
surface of chickens (Collado and Sanz, 2007) indicating that the crop lumen
develops an anaerobic environment. Similarly, the anaerobes Faecalibacterium and
Bacteroides were detected in the proventriculus (Videnska et al., 2013).
2.4.2 Small intestine
The microbiota within the small intestine is sparse in the duodenum and most
abundant within the jejunum and ileum. The small intestine harbors an abundance
of species related to Lactobacillus. One study observed that approximately 90%

of the microbiota of the small intestine of chickens is comprised of Lactobacillus
(Dumonceaux et al., 2006). Another determined that 70% of sequences from
the ileum were Lactobacillus, with the remaining related to Clostridiaceae (11%),
Streptococcus (6.5%), and Enterococcus (6.5%) (Lu et al., 2003). Ninety-nine percent
of the 16S bacterial sequences collected from jejunum were related to lactobacilli
(Stanley et al., 2012). However, the microbiota of the small intestine can respond to
changes in diet. For example, the microbiota of birds fed a diet containing feathers
contained higher counts of Enterococcus faecium, Lactobacillus crispatus, L. reuteri,
and L. salivarius on keratin agar plates compared to the control group (Meyer et
al., 2012). Lactobacilli are not classically keratinase producers therefore the small
intestinal chicken microbiome can express keratinolytic activity sufficient to enhance
growth of nonkeratinase producers. The small intestine is the main zone of digestion
and absorption of nutrients and the composition of the microbiota could positively
contribute to the processes.
2.4.3 Cecum
The cecal environment harbors predominantly the phyla Firmicutes, Bacteroidetes

and Proteobacteria (Qu et al., 2008) most of which are strict anaerobes. While the
microbiota of the small intestine is dominated by Lactobacillus, the chicken’s cecum
harbors an abundance of Clostridia species. 16S rDNA reveals that the majority are
related to Clostridium leptum, Sporomusa spp., Clostridium coccoides and enterics.
Sequences related to Bacteroides, Bifidobacterium infantis and Pseudomonas
represented less than 2% of the total (Zhu et al., 2002).
Analysis of 972 sequences obtained from cecum public databases showed that
92.8% of the sequences represent 10 bacteria phyla. The most predominant phyla
included Firmicutes and Bacteroidetes, accounting for approximately 78 and 11% of
the total cecal sequences, respectively. The cecal sequences from chicken contained
59 bacterial genera, the phylum Firmicutes contained 31 genera, Ruminococcus,
Clostridium, and Eubacterium each represented ≥5%, of the sequences (Wei et al.,
2013). Similarly, in a study using 16s rRNA clone libraries, most of the sequences
retrieved from cecal samples were also related to Clostridia (Cressman et al., 2010;
Gong et al., 2007). The cecal microbiome seems to be dominated by Clostridia, many
of which are poorly characterized.
Most of the studies about the composition of the poultry microbiota use broiler
chickens as a model, however in a recent work using laying hens, most of the sequences
retrieved from cecal samples were closely related to Clostridia (Ruminococcaceae and
Lachnospiraceae) and lactobacilli, giving further insight into the intestinal microbiota
of laying hens (Janczyk et al., 2009). The cecal microbiota of 18-week-old laying
hens was composed of the phyla Bacteroidetes and Firmicutes, with Proteobacteria,
Fusobacteria, Actinobacteria and Deferribacteres also being represented, but only
in low abundance. Butyricimonas spp. and Faecalibacterium spp. were the most
abundant organisms (Nordentoft et al., 2011).
2.5 Role of the microbiome
2.5.1 Intestinal development and homeostasis
The gut microbiota is now considered a key endogenous organ which modulates
host anatomy and physiology (Delzenne and Cani, 2011). Alterations in the
composition or metabolic activity of the microbiota may have repercussions on
host health (Kabeerdoss et al., 2013). This imbalance is frequently termed ‘dysbiosis’
and the specific pathways that are impaired in the holobiont are poorly understood.
However, synergistic activity among the constituents of the vertebrate holobiont
could led an improved performance (Turnbaugh et al., 2006). Evidence for the role
of the microbiota in host development and signally is being revealed in insect and
mouse models.
Microbe-induced signaling pathways that link the microbiome to gut cell

differentiation and physiology is seen with the Drosophila genetic model (Lee, 2008b,
2009). Germ free animals can be associated with a defined number of bacteria and
the holobionts resulting can be maintained under germ-free conditions. Time-
course analyses of the community structure and host have determined the effects of
microorganisms on the community structure and host phenotypes (Bäckhed et al.,
2004; Rawls et al., 2006). This model has revealed that bacteria can modulate intestinal
stem cell development by activating pathways responsive to reactive oxygen species.
Recent studies demonstrated that signals produced by different types of gut-microbe
interactions are involved in determination of intestinal stem cells activation (Buchon
et al., 2009; Chatterjee and Ip, 2009; Cronin et al., 2009; Jiang et al., 2009). Some of
these findings have been substantiated in germ-free mouse models in which bacteria
have been shown to direct postnatal development of the intestine. Research using
Bacteroides thetaiotaomicron, a dominant member of the mammalian intestinal

community, have revealed symbiotic interactions with the vertebrate gastrointestinal
system. For example, when used to colonize germ-free mice, it increases mucosal
expression of terminal α-linked fucose in the distal small intestine (Bry et al., 1996).
The changes in host glycan expression allow the bacterium to expand its colonization
niche along the small intestine. Bacteroides mutants deficient in fucose utilization
colonize germ-free but do not elicit fucosylated glycoproteins indicating that
bacterial metabolites act as signals for host development (Bry et al., 1996; Hooper
et al., 2000) The microbiota also affects development of the submucosal capillary
beds; the intestinal capillary network is rudimentary in adult germ-free mice.
Colonization with B. thetaiotaomicron stimulates angiogenesis so that it resembles
that of conventional mice (Stappenbeck et al., 2003). These findings indicate that the
microbiota or bacterial metabolites may be necessary for complete development of
the intestine to its full absorptive capacity. Mouse models have revealed that specific
members of the intestinal microbiota affect specificity and efficiency of bacterial
digestion of dietary polysaccharides, thereby influencing host calorie harvest and
adiposity (Ley et al., 2005; Samuel et al., 2007; Turnbaugh et al., 2006).
Bacteroides thetaiotaomicron stimulates development of the intestinal mucosal

barrier in mammals by inducing defensin secretion by Paneth cells (Xu et al.,
2003). This function of the microbiome may be important in balancing the mucosal
response to bacteria. Clostridia cluster XIV species can comprise 60% of the bacteria
adhered to the intestinal mucus of birds (Van den Abbeele et al., 2012). Members
of the Clostridia XIVa cluster adapt to different stages of microbial succession and
the pathobiont species (gut symbionts that can cause disease in permissive genetic
or environmental circumstances) are often adapted for early succession (Lozupone
et al., 2012). While toxigenic species such as Clostridium perfringens are considered
primary pathogens (Rood and Cole, 1991), studies have shown an abundance of
Clostridia is also observed in situations of enhanced performance. For example,
chickens fed growth-promoting antibiotics show an increased proportion of
Clostridia (Lu et al., 2008; Singh et al., 2013a). Perhaps one of the mechanisms of
action of antibiotic growth promoters is changing the physiology of select members
of the microbiome. Faecalibacterium prausnitzii, a member of Clostridia cluster
IV (Van Immerseel et al., 2010) and a butyrate-producing bacterium, has a very
intriguing role within the intestinal microbiome. It has been observed in high levels
in intestinal communities of pigs (Haenen et al., 2013) as well as in healthy birds (Lu
et al., 2003, 2008). However, low abundance of F. prausnitzii has been described in
inflammatory bowel disease in humans (Fujimoto et al., 2012; Hansen et al., 2012;
Kabeerdoss et al., 2013). Some members of the intestinal bacterial community

exhibit anti-inflammatory effects on the mucosa (Lin et al., 2009; Neish, 2010) which
suggests that the microbiota may be able to prevent inflammation in the presence of
a specific pathogen (Lee and Lee, 2013).
2.5.2 Role of mucin in intestinal function
The mucus that lines the surface of the gastrointestinal mucosa acts as a lubricant
enhancing the propulsion of the chime. It also modulates nutrient absorption
because of its permeability and helps protect the underlying epithelium from enteric
pathogens (Tsirtsikos et al., 2012). Mucus consists of the mucin protein which is
heavily glycosylated with long carbohydrate chains (Forstner and Forstner, 1994).
The composition of mucin is affected by the interaction with microbiota (Kirjavainen
et al., 1998; Xu and Gordon, 2003), host intestinal glycosylation (Bry et al., 1996;
Freitas et al., 2005) and degradation by the intestinal microbiota (Ruas-Madiedo et
al., 2008). The mucin acts as a colonization matrix for the microbiota; it contains
a diversity of attachment sites and its carbohydrates and amino acids are used as a
nutrient source (Louis et al., 2007; Macfarlane and Macfarlane, 1997; Macfarlane and
Dillon, 2007). The ceca of germ-free mice is frequently distended with mucus due to
the absence of bacteria (Falk et al., 1998).
A strong indication that the mucin production, composition and degradation is

dependent on the microbiome was obtained by evaluating germ free mammals.
They have a thinner colonic musculature, shorter crypts, few goblet cells and a
thin mucus layer (Enss et al., 1992; Hill et al., 1990; Szentkuti et al., 1990). In a
healthy intestine the synergism of the holobiont is enhanced by an intact mucosal
barrier (Becker et al., 2013). The intestinal mucus consists of two layers and under
normal situations, the inner layer is adherent to the surface of the epithelium and
the outside layer is the major habitat for commensal bacteria (Johansson et al.,
2008). Changes in the composition of mucin or reduced production correlates with
increased susceptibility to disease (Byrd and Bresalier, 2004; Corfield et al., 2000).
A thinner or discontinuous mucus layer is also associated with inflammatory bowel
disease and has been hypothesized to contribute to the loss of tolerance of the human
host toward the commensal colonic microbiota (Strober et al., 2007). Helicobacter
pylori inhibits mucus secretion by suppressing both MUC1 and MUC5AC gene
expression in tissue culture, which demonstrates the ability of microbes to directly
alter MUC gene expression (Byrd et al., 2000). However a probiotic cocktail induced
the secretion of MUC2 in HT29 cells (Otte and Podolsky, 2004), but not in LS174T
cells (Caballero-Franco et al., 2007). Probiotic strains Lactobacillus plantarum 299v

and Lactobacillus rhamnosus GG increases expression of intestinal MUC2 (Gum
et al., 1994) when added to colon cells in tissue culture (Mack et al., 1999). Similar
results were obtained in vivo with the administration of Lactobacillus acidophilus
NCFM (Bergstrom et al., 2012). Bifidobacterium bifidum and a complete microbiota
has been described to be effective improving the intestinal production of mucin
(Bergstrom et al., 2012; Khailova et al., 2009). Feed additives also can contribute to
altering the microbiota and consequently mucin dynamics. Propionic, sorbic acid
and plant extracts were shown to decrease the number of Campylobacter and alter
the composition of mucin and Goblet cells (Grilli et al., 2013).
2.5.3 Cleavage and absorption of nutrients
It has been demonstrated that germ free animals have decreased enterocyte turnover
(Abrams et al., 1963; Lesher et al., 1964; Savage and Whitt, 1982) and increased brush
border enzyme activity (Kozakova et al., 2001). In addition, germ free animals have
longer microvilli than conventional animals (Meslin and Sacquet, 1984; Willing and
Van Kessel, 2007). Despite what appears to be a physiological change that would
produce improved intestinal absorption, germ free animals fail to thrive because
the intestine does not develop to its full absorptive potential. The mechanism by
which bacteria induce these morphological and physiological changes in the brush
border is not fully understood (Byrd et al., 2000; Caballero-Franco et al., 2007).
However, the microbiota may influence mitogen-activated protein kinases (MAPK)
pathways which regulate enterocyte proliferation and function. Elevated p42/p44
MAPK activities stimulate enterocyte proliferation, while low levels of MAPK activity
increase expression of sucrase-isomaltase, showing an inverse relation between cell
proliferation and brush border enzyme activity (Aliaga et al., 1999).The intestinal
microbiota could contribute to the enzyme activity in the villi and nutrient cleavage,
explaining the need for higher expression of brush border enzymes in germ-free
animals (Willing and Van Kessel, 2009).
Disaccharides are cleaved on the surface of the epithelium by brush border enzymes
anchored in its surface. However, the uptake of hexoses into the enterocyte is
mediated by specific transporters whose expression can be affected by the intestinal
microbiota. Recently it was demonstrated that Lactobacillus up-regulates glucose
transporter expression by enterocytes (Ikari et al., 2002). There was an increase in
glucose uptake by the enterocyte within 10 min of exposure to bacteria that may
be attributed to either the trafficking of existing transporters from the cytosol to
the brush border membrane or the activation of transporters already present in the
brush border. The microbiota not only elicits an increase in the activity of glucose
transporters and it also modulates the brush border membrane Na+/H+ exchanger 3
(NHE3) (Musch et al., 2001).
2.5.4 Intestinal differentiation, maturation and apoptosis
The microbiota promotes substantial changes in gut morphology, including villi

architecture crypt depth, stem cell proliferation, and blood vessel density (Sommer
and Bäckhed, 2013). It has been demonstrated that germ-free animals have
decreased enterocyte turnover (Abrams et al., 1963; Lesher et al., 1964; Savage and
Whitt, 1982) and increased brush border enzyme activity (Kozakova et al., 2001).
In addition, germ-free animals have longer microvilli than conventional animals
(Meslin and Sacquet, 1984; Willing and Van Kessel, 2007). Despite what appears to
be a physiological change that would produce improved intestinal absorption, germ-
free animals fail to thrive because the intestine does not develop to its full absorptive
potential. Enterocyte proliferation in caudal small intestine can be influenced by
the composition of colonizing commensal bacteria (Willing and Van Kessel, 2007).
The intestinal microbiota can contribute for the maintenance of cell-to-cell junctions
(Cario et al., 2007; Hooper et al., 2001) and promotion of epithelial repair following
injury (Lutgendorff et al., 2008; Rakoff-Nahoum et al., 2004; Sekirov et al., 2010)
and epithelial dead. Programmed cellular dead or apoptosis plays an important
role in determining the architecture of intestinal epithelia (Watson and Pritchard,
2000). In disease pathogenesis, apoptosis has been implicated with pathogens such
as Helicobacter and Shigella flexneri (Pritchard and Watson, 1996). In comparison,
Lactobacillus rhamnosus GG reduces apoptosis in vitro and in vivo systems by
up-regulating a battery of genes with known and likely cytoprotective effects (Lin
et al., 2009). Similarly, the probiotic mixture VSL#3 consisting of L. acidophilus,
L. bulgaricus, L. casei, L. plantarum, Streptococcus thermophilus, Bifidobacterium
breve, B. infantis, and B. longum, has been shown to produce anti-apoptotic effects
by increased cytoprotection of epithelial cells (Venturi et al., 1999). Lactobacillus
rhamnosus GG derived soluble factors regulate cell survival signaling and inhibit
cytokine-induced apoptosis in intestinal epithelial cells (Yan et al., 2007).
2.6 Short chain fatty acids
Short-chain fatty acids (SCFA), plays a major role in the physiology of the intestinal
mucosa. SCFAs, predominantly acetate, propionate, and butyrate, are metabolic
products of carbohydrate fermentation by the microbiota. The majority of SCFAs
are absorbed from the gut and metabolized in various body tissues.
SCFAs have an effect on nutrient acquisition by increasing the expression and

activity of SGLT1 and GLUT2 transporters in the brush border (Tappenden et al.,
1997). Butyrate has been shown to up-regulate expression of the GLUT2 transporter
(Mangian and Tappenden, 2009). Enterocyte surface receptors, such as G-protein
coupled receptor (GPR)43 and GPR41 may function as a sensor of intestinal SCFAs
(Karaki et al., 2008). Butyrate exerts a wide variety of effects on intestinal function
(Hamer et al., 2008). It is the major energy source for the enterocytes and it affects
cellular proliferation, differentiation (Kim et al., 1980) and apoptosis (Alvaro et
al., 2008). Studies have reported that butyrate metabolism is impaired in intestinal
inflamed mucosa of patients with inflammatory bowel disease (IBD). In other
hand, butyrate-producers localized close to the epithelium could enhance butyrate
bioavailability, which could be useful as an energy source for intestinal epithelial
cells and treating intestinal diseases.
Butyrate-producing bacteria are of increasing interest because of their potential

to affect signaling pathways. Clostridia clusters XIVa and IV, abundant organisms
in chickens, contain a range of butyrate-producing species (Collins et al., 1994).
Increasing abundance of bacteria involved in butyrate metabolism could be used as
an indicator of good intestinal health and nutrition in chickens.
2.7 Modulation of the intestine using probiotic microorganisms
Until recently the digestive health of food animals and poultry was managed using
subtherapeutic antibiotics and vaccines targeting specific pathogens. However because
of consumer apprehension, and concerns regarding new drug-use regulations, many
poultry integrators have reduced or eliminated the use of subtherapeutic antibiotics
but have experienced an increase in enteritis. Probiotic bacterial formulations have
been used to facilitate formation of a mature intestine and to prevent development of
enteritis. Microorganisms could be used as a modulator of the microbial ecosystem
and host physiology in poultry.
The health benefits attributed to consumption of probiotic organisms include

prevention of intestinal infections, lowering of serum cholesterol, expression of
anti-cancer activities, stimulation of the immune system, improvement of lactose
utilization, and enhancement of short chain fatty acid (SCFA) levels (Gomez-Gil et
al., 1998). The administration of bacteriotherapy to neonate chicks could provide
pioneer colonizers that enhance intestinal development. Pioneer colonizers can
influence the nutrient foundation by altering the glycome of host glycoproteins and
mucin thereby regulating the succession of species comprising the microbiota of the
developing chick. One of the most important functions of the intestinal microbiota
is to suppress pathogenic populations of bacteria. Until recently, suppression
was assumed to occur because the interaction of bacteria in the intestines is
fundamentally competitive. However, studies have shown that use of a competitive
exclusion product in day of hatch chicks can prevent symptoms of disease weeks
after administration of the product (Hofacre et al., 1998a,b, 2002).
Nutrient utilization, enhanced by complementary interspecies metabolic activity

and production of inhibitory metabolites, can produce a community that is resistant
to colonization by pathogens. However, the resistance to digestive diseases and
colonization with Salmonella may be enhanced in animals by early exposure to
intestinal symbionts that direct the nutrient foundation of the developing intestine
and foster development of intestinal microbiota that suppress the growth or toxin
production of enteropathogens. Fukata et al. (1991) showed that birds which were
reconstituted with either Lactobacillus or Enterococcus were more resistant to
colonization with C. perfringens. This finding indicates competitive suppression,
however Craven et al. (1999) demonstrated that probiotic administration reduced
toxin production by C. perfringens in the chicken intestine. These findings suggest
that probiotics can be effective in reducing the prevalence and severity of disease.
Animal performance and feed efficiency are closely related to the status of the
intestinal microbiota (Huyghebaert et al., 2011). The use of defined and undefined
microorganism as probiotics in animals has been reported to result in improved
health, performance, and weight gain (Kyriakis et al., 1999; Patterson and Burkholder,
2003). Because of the diversity of available products, and the probable diversity of
mechanisms of action, there is no consensus on how these products work. It is
unlikely that colonization of the intestine is a requirement of all the products because
some contain strains that are not of poultry origin. But many products, defined and
undefined, are manufactured to include some microbial metabolites which may be
involved in stimulating intestinal development or suppressing inflammation.
Defined mixtures of certain bacteria and yeasts can be used as a probiotic for poultry
in many countries. Lactobacillus species are probably the most common bacteria
used with probiotic cultures as shown in Table 2.1. Most of these products are used
Table 2.1. Microorganisms used as probiotics for poultry.
Microorganism Genus and species Reference
Bacteria Lactobacillus fermentatum Bai et al., 2013; Yamawaki et al., 2013

Lactobacillus acidophilus Hossain et al., 2012; Rodriguez-Lecompte et al.,
2012; Yamawaki et al., 2013
Lactobacillus plantarum Biernasiak et al., 2006; Hossain et al., 2012
Lactobacillus casei Biernasiak et al., 2006;
Rodriguez-Lecompte et al., 2012
Lactobacillus paracasei Biernasiak et al., 2006
Lactobacillus brevis Biernasiak et al., 2006
Lactobacillus salivarius Robyn et al., 2012; Yamawaki et al., 2013
Lactobacillus reuteri Klose et al., 2006; Robyn et al., 2012
Lactobacillus agilis Meimandipour et al., 2010; Robyn et al., 2012
Lactobacillus helveticus Capcarova et al., 2011; Robyn et al., 2012
Enterococcus faecium Hossain et al., 2012; Robyn et al., 2012;
Rodriguez et al., 2012
Enterococcus faecalis Robyn et al., 2012
Bacillus coagulans Hossain et al., 2012
Bacillus subtillis Chen et al., 2009; Hume et al., 2011;
Jayaraman et al., 2013; Rajput et al., 2013
Streptococcus faecium Rodriguez-Lecompte et al., 2012
Pediococcus acidilactici Lee et al., 2007
Clostridium butyricum Yang et al., 2012
Bifidobacterium animalis Giannenas et al., 2012
Bifidobacterium longum Santini et al., 2010
Bifidobacterium bifidium Talebi et al., 2008; Willis et al., 2007
Yeast Saccharomyces cerevisiae Bai et al., 2013; Chen et al., 2009;
Hossain et al., 2012; Pizzolitto et al., 2012;
Rajkowska and Kunicka-Styczynska, 2010;
Rodriguez-Lecompte et al., 2012
Saccharomyces boulardii Rajput et al., 2013; Lee et al., 2007
to enhance feed conversion or for Salmonella control although off label use for
reduction of dysbacteriosis and similar maladies is common in the poultry industry.
A high level of success can be observed when undefined microbiota, directly

collected from the chicken intestine, is administered to poultry. This concept is
called ‘competitive exclusion’ and has been found to affect a number of production
parameters (Blaszczak et al., 2001; Hofacre et al., 1998a,b; Nurmi et al., 1992).
Competitive exclusion formulations can be produced directly from the ceca of specific
pathogen-free birds or by amplifying cecal contents in commercial bioreactors.
The product can be lyophilized and administered via drinking water, sprayed onto
eggs in the hatchery, or sprayed onto litter at placement. Competitive exclusion
formulations consist of many uncultured and obligate anaerobic bacteria, many of
which are members of Clostridia clusters XIV and IV (Lee, 2008a; Lu et al., 2008).
Because effective products are difficult to define using classical culture techniques,
it is difficult to obtain approval from regulatory agencies to use these preparations
as direct-fed microbials for poultry (Waters et al., 2006). For these reason, the
administration of undefined bacterial flora is not acceptable to regulatory agencies
in some countries (Methner et al., 1997). Unfortunately the greatest successes in the
control of intestinal pathogens, managing gut development and disease resistance
occurs when these products are used in birds (Cox et al., 1992; Hofacre et al., 1998a;
Hollister et al., 1995; Hoszowski and Truszczynski, 1997)
2.8 Conclusions
The literature base is rapidly growing with descriptions of the positive effects of
probiotics, prebiotics, direct-fed microbials, and competitive exclusion on poultry
production and disease control. Until recently, it was costly to apply the molecular
techniques needed to characterize changes in the uncultivable portion that makes up
the majority of the distal intestine of birds. Affordable methods such as denaturing
gradient gel electrophoresis (DGGE) allowed assessment of microbiota changes
which revealed whether changes correlated with production parameters. Rapidly
reducing costs in DNA sequencing has allowed detection of specific organisms
and the ability to correlate their presence with desired parameters. However this
knowledge has not lent itself to rapid translation in the field but has revealed that the
mechanisms involved in the normal function of the poultry holobiont are complex.
Future work is needed to illuminate the mechanisms by which the microbiota directs
development of the intestine, regulates inflammation, enhances nutrient update,

affects energy metabolism so that products can be designed for specific effects.
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Chapter 3: Intestinal diseases of pigs
S. McOrist1* and E. Corona-Barrera2
1Consultant pig veterinarian, Jaffe Road, Hong Kong; 2Universidad de
Guanajuato, División Ciencias de la Vida (DICIVA), Km 9 Carretera Irapuato-

Salamanca, Irapuato, Gto. CP 36824, Mexico; smcori01@hotmail.com
Abstract
Intestinal diseases affect farmed pigs from birth through to their marketing age and
significantly limit the productivity and profitability of pig production around the
world. The main intestinal diseases of weaned pigs in many regions are bacterial –
colibacillosis due to enterotoxigenic Escherichia coli (ETEC) infections, salmonellosis,
swine dysentery due to Brachyspira hyodysenteriae and proliferative enteropathy due
to Lawsonia intracellularis infections. The bacterial strains ETEC, Salmonella and
Lawsonia all appear to be ‘embedded’ in many pig farms, causing endemic intestinal
disease problems. These diseases and swine dysentery are all increasing in incidence
and importance for pigs globally, as political restrictions on the usage of key pig
farm medications, such as antibiotics and zinc oxide, have appeared. Many pigs
across Asia also suffer viral enteritis due to coronaviruses, such as porcine epidemic
diarrhea virus. Piglets globally suffer the host-specific coccidial parasite, Isospora
suis. Parasitic nematode diseases of the pig intestine remain common in some groups
of pigs raised outdoors. In active grower pigs, torsions of the small intestine can
occur, which can quickly result in an extreme intestinal accident, compromised
blood supply and sudden death of the pig.
Keywords: Brachyspira, Lawsonia, Salmonella, coronavirus, colibacillosis
3.1 Introduction and general features
Intestinal diseases affect farmed pigs from birth through to their marketing age and
significantly limit the productivity and profitability of pig production around the
world. This chapter will focus on intestinal diseases of pigs on solid food diets, that
is after weaning at 3 to 4 weeks old.

The clinical signs that may be apparent in pigs affected by intestinal disease can vary
a great deal depending on the location, type, severity and duration of the disease
process. Signs of dehydration should alert the attendant to possible intestinal tract
disease. Diarrhoea is an increased frequency & fluidity of faecal discharge. It is a
very common presenting sign and occurs with inflammation of the small or large
intestine, but also with the ingestion of indigestible or fermentable material and
many other dietary or infectious problems. The presence of noticeable diarrhoea in
pigs has been shown to correlate well with reduction in the dry matter content of
faeces. Dry matter contents below 20% usually correlate with clinical diarrhoea and
those below 10-15% correlate with watery faeces. The passage of darker black-red
tarry faeces due to presence of digested blood in the diarrhoea faeces is known as
dysentery. Abdominal pain or colic may be recognized as a tense abdomen that is
sensitive to touch; it is usually due to distention of bowel.
Digestion and nutrient absorption for the pig occur in the intestine, especially the
small intestine. Mature enterocytes, which cover the villi lining the small intestine,
are responsible for nutrient absorption, by a complex process that combines active
and passive transport, with extensive enzyme action at the brush border. The normal
turnover rate of villus enterocytes on the ‘staircase’ from the bottom of a crypt to
the tip of a villus is about 3-4 days. Regeneration of gut epithelium in response to
injury and disease is therefore rapid but requires surviving crypt cells. In some types
of diseases, only villus cells are injured, so the villi become shortened and fused,
known as villous atrophy. Examples of the porcine agents that selectively destroy
the villus enterocytes are coronaviruses and rotaviruses. The crypts will undergo
marked hyperplasia as a reparative or compensatory response within a few days of
injury to replace the damaged villus enterocytes. The large intestine has no villi and
the colonic crypts are much less active in nutrient absorption, but are responsible
for water and electrolyte absorption.
It is important when investigating intestinal diseases in pigs to recognise that post

mortem changes (autolysis) are often extensive, with gas accumulation, breakdown
and diffusion of blood and yellow bile pigments into the intestinal wall. These can
all serve to confuse and hide the real cause of the disease situation. The breakdown
of haemoglobin by bacteria can cause obvious black pigmentation, initially in the
large intestine. A common mistake is to describe routine, if spectacular, intestinal
blood vessel congestion, as significant haemorrhage or inflammation. For collection
of appropriate samples for fixation for pathology examination requires the samples
to be placed into buffered formalin less than 30 minutes after death. Interpretation
3. Intestinal diseases of pigs
of any gut samples from animals that have been dead for over one hour is highly
problematic. The wall of the large intestine of pigs can often have a scattering of
prominent white nodules, up to 2 cm diameter. These are due to local dilation of
lympho-glandular areas and do not form the basis for any pig health concern.
In the first two weeks of life, piglets may develop infectious or congenital intestinal
diseases. Congenital anomalies are most commonly seen as the inherited condition,
segmental atresia or blockage. At necropsy, the proximal (front) end of the intestine
is grossly distended, due to retention of faecal material. Colibacillosis due to
enterotoxigenic Escherichia coli (ETEC) in suckling piglets often occurs at an early
age, within the first week of life. It is most often associated with litters derived from
gilts, which become infected quickly after birth, due to environmental contamination
and inadequate maternal antibody transfer. Further details on the pathogenesis of
ETEC are given below. Clostridial infections due to toxigenic Clostridium perfringens
or Clostridium difficile are occasionally noted as necrotic enteritis in the intestines
of young piglets. Clostridial strains occur in specific types (A-E) that each contain
a variety of toxins, such as lecithinase, which causes coagulative necrosis of the
mucosa. This disease is much less common in pigs than in chickens, but it is possible
that future shifts in feed components may lead to its emergence in pigs. Vaccination
of late pregnant gilts and sows should be performed routinely to induce lactogenic
immunity for piglets for both ETEC and clostridial infections.
Detailed studies in pigs have shown that a reduced weaning weight directly translates
to a reduced bodyweight at 20 weeks old; compensatory growth does not readily
occur in smaller weaner pigs. In other words, the weight gain over the wean-to-
finish period is directly affected by the initial weaning weight. The total absorptive
capacity of the small intestine at weaning is therefore a key parameter for the later
growth potential of pigs.
3.2 Key intestinal diseases in pigs at or after weaning
3.2.1 Bacterial diseases
Post-weaning Escherichia coli infections
E. coli infections are ubiquitous in animals. Unlike Salmonella, the hundreds of various
E. coli serotypes are numbered (not named) and many non-pathogenic forms occur.
The ETEC possess a combination of attachment factors and enterotoxins, which are
both necessary for full intestinal disease to occur. The attachment factors on ETEC are
specialized fimbrial or pilus proteins, which firmly attach to enterocyte glycoprotein
receptors on the intestinal cells. These receptors are only present for a limited time
– usually until six weeks after birth in pigs. The ETEC attachment factors are now
known as fimbrial antigens F4, F5, F41, etc., although still frequently known by earlier
designations of K88, K99, 987P, etc. This attachment and adherence then allows the
ETEC to resist normal gut movements and so colonize the intestine. The ETEC can
then produce and ‘inject’ their enterotoxins, such as heat-labile or heat-stable toxins,
LT or ST. These toxins act on intestinal cells to cause hypersecretory diarrhoea,
producing fluid outflow into the gut lumen, but without major cell death or damage.
Epidemiology – This is a very common global infection among farmed pigs – ETEC
appear to be ‘embedded’ in most pig farms globally, so elimination is not a current
option. Vaccination of sows or gilts with ETEC vaccines has no effect on post-
weaning E. coli infections. Affected pigs are often derived from gilt litters with low
maternal antibodies to ETEC. After weaning, the loss of sow milk and IgA assists
strains of resident E. coli to proliferate. The common on-farm risk factors for the
occurrence of ETEC intestinal disease are (1) cold accommodation (that is less than
15 °C) due to draughts and inadequate heating of weaner nursery facilities – pigs do
not gain full thermal control until around 10 weeks-old, and (2) poor hygiene, with
inadequate cleaning of the weaner nursery facilities – because a high infectious dose
is generally required for ETEC disease to be a full clinical problem. Therefore many
at-risk buildings are often older and have been used for pig farming for some years.
ETEC disease has had a marked recent resurgence of cases in Europe following
political moves to reduce usage of zinc oxide in pig feeds. This compound has proved
very effective at controlling ETEC disease on pig farms, and outbreaks have now
often followed its removal from the weaner pig feed ration.
Clinical signs – Clinical signs are nearly always in pigs at seven to ten days after
weaning, with an outbreak of yellow-white, creamy-watery, projectile scours. The
incubation period is 10 to 30 hours only; so many pigs will appear to become quickly
affected in a group. This watery fluid diarrhoea has little solid feed evident and pigs
rapidly show dehydration and loss of condition. It is often possible to press the
abdomen of a suspect pig and see whether this diarrhoea is evident. Over a group of
pigs, the diarrhoea can vary in consistency from very watery to a paste with a wide
range of colour from grey white, yellow and green. Fresh blood or mucus is absent.
Affected pigs are often from gilt litters. In severe cases a pig is found dead with
sunken eyes and slight blueing of the extremities. A useful test to help differentiate
between E. coli diarrhoea and virus infections involves the use of litmus paper to
determine whether the scour is an alkaline or an acid consistency. Soak the paper in
the scour, E. coli diarrhoea is alkaline (blue colour change) whereas viral infections
are acid (red colour change).
Diagnosis – The culture of faeces from pigs with diarrhoea is often unrewarding
– the examination of fresh pigs at autopsy is required. The autopsy of ETEC cases
usually shows dilated, congested thin small intestine loops filled with the watery
yellow diarrhoea, with dehydration and reduction of body fat. Histological lesions
are minimal – it is a biochemical lesion, but large numbers of coliform bacteria may
be seen attached to villi.
Bacterial culture can confirm the presence of E. coli which should be checked for
haemolysis, toxins and sensitivity to antibiotics. Fimbrial antigen F4 (K88) is the most
common strain and can colonise the entire small intestine. The F5 and other strains
may only attach to receptors along the jejunum and ileum. ETEC frequently also
have a haemolytic toxin, allowing ready differentiation from non-pathogenic E. coli,
when intestine samples are cultured on blood agar plates, specific agglutination
reagents are available for the attachment proteins.
Management and control of colibacillosis outbreaks – It is important to know the

history of the disease on the farm and antibiotic sensitivities to the ETEC present.
Sick pigs should be treated individually and group treatment applied to the pigs by
water medication. If pigs appear dehydrated, then electrolytes should be provided
in a separate drinker supply. Antibiotics often used for the treatment of E. coli are
apramycin, neomycin, tiamulin and sulphonamides. Re-introduction of in-feed zinc
oxide at 2,500 mg/kg of zinc per tonne is necessary to prevent further outbreaks. It
is necessary for this compound to be placed in feed for at least two to three weeks
after weaning. Vaccines aimed at preventing post-weaning E. coli are gaining wider
usage and may assist control. It is not possible to eradicate ETEC.
The temperatures, air flow, draughts and fluctuations in the weaner housing must be
addressed – 15 °C is not enough for these pigs at 3 weeks-old. Similarly, it is vital to
address hygiene of this housing, related to stocking density – group sizes and mixing
related to proper pen cleaning. Some farms develop poor hygiene practices related
to usage of messy creep feed bowls in weaner pens.
The recent increase in diarrhoea outbreaks due to ETEC has also been accompanied
by an increase in outbreaks of enterohaemorrhagic E. coli (EHEC) infections in pigs.
These are similar E. coli strains, but are often of F18 adhesin type and also contain
specific verotoxins or shiga-like toxins. These toxins enter the bloodstream of the pig
and damage certain extra-intestinal blood vessels, producing neurological signs and
gelatinous oedema of the head, eyelids, larynx, stomach and colon.
Salmonellosis
Salmonella species are ubiquitous bacteria capable of survival in a wide range of

organic matter environments, including the intestines of all animals, soil, water,
vegetation, organic feed components. In pigs, they multiply mainly in the intestines
of young growing pigs and may be shed in faeces for several weeks or months with
no clinical disease at all. Salmonella in the gut of the pig can contaminate carcasses
during the slaughter process and their presence creates potential public health
risks from food poisoning. Pigs also have a specific host-adapted strain, Salmonella
cholerae-suis, which causes a generalized systemic disease, but this syndrome is rare.
Other than this host-adapted strain, most infections are enteric and can be due to any
one of a range of non-specific enteric strains. Most commonly involved in pigs are
S typhimurium because this strain has an actively adaptable and diverse genotype.
Salmonella sp. readily enter epithelial cells of the intestine via induced ruffling of the
membrane and formation of a novel endosome, which develops filaments that switch
off lysosome attachment.
Epidemiology – Many pigs are colonised by Salmonella in the growing phase and
the bacteria may be shed in faeces for several weeks with no apparent clinical disease.
Pigs may also become long-term sub-clinical carriers, the organisms surviving in the
mesenteric lymph nodes. Testing of pig faeces and intestines and mesenteric lymph
nodes taken at slaughter can track the level and occurrence of infections on farms.
Typically, the percentage of infected pigs rises steadily among the group over the
growing and finishing period.
Clinical infection in pigs typically occurs from a combination of a high Salmonella

dose presented to a susceptible young, immune-suppressed pig. Young pigs
commonly suffer from separate virus infections that markedly reduce the immune
function – porcine circovirus type 2, the PRRS virus and classical swine fever (hog
cholera). Therefore the occurrence of Salmonella clinical cases following and during
outbreaks of these primary virus infections has been consistently noted. Also, disease
is dose dependent, that is, a relatively large number of organisms are required before
clinical signs occur. So, occurrence of Salmonella clinical problems is also related to
exposure and dose, therefore pigs raised at a high stocking rate and in areas with poor
hygiene, access to organic bedding and contact with bird and rodent contamination
will often have more severe and clinical problems than pigs kept indoors on well-
cleaned concrete slats. As noted above for E. coli infections, the at-risk buildings are
often older and have been used for pig farming for some years.
Clinical signs – The most common sign is mild to moderate diarrhoea, sometimes
with flecks and spots of jelly-like mucus evident. Deaths are usually associated
with dehydration and severe enteritis and colitis. Salmonellosis can occur at any
age but is most common in growing pigs over eight weeks of age. Affected pigs will
also often be affected with symptoms of one or more of the main viral immune-
suppressive conditions.
Diagnosis – Salmonella are usually easy to grow by bacterial culture of faeces or

intestines or lymph nodes in the laboratory. The main intestinal-associated strains
in pigs are S typhimurium and S derby. Isolates should be checked for serotype and
sensitivity to antibiotics. The autopsy of suspect cases will often show mucosal
necrosis and catarrhal inflammation, particularly in the ileum, caecum and colon.
When infection is more severe, fibrino-necrotic enteritis develops in a segmental or
diffuse manner – often over the lymphoid tissues in the ileum and large intestine,
producing lesions known as ‘volcanos’ or ‘button ulcers’. Histological lesions
include destruction of intestinal epithelium and marked sub-mucosal infiltration
by mononuclear cells, with lymphadenitis and multi-focal hepatitis.
Management and control of salmonellosis – Sick pigs should be treated individually

and group treatment applied to the pigs by water medication. Antibiotics often used
for the treatment are the same ones as for ETEC, such as apramycin, neomycin,
tiamulin and sulphonamides. There is a major difference in the management of
Salmonella infections between the chicken and pig farming industries. Pigs do not
possess stages in their life cycle (such as eggs and hatchery) that can form the basis
of eradication programmes. Therefore the control of Salmonella in pigs, in a broad
sense, relies on general application of hygiene principles. For best Salmonella control,
pigs should be raised indoors on slatted concrete floors in rodent and bird proof
conditions, which are regularly and carefully cleaned. Vaccines are occasionally used
in control of pig salmonellosis, but are not yet a first choice in management due to
efficacy and cost-return issues.
Swine dysentery
Swine dysentery is an important bacterial cause of colitis due to the anaerobic, snake-
like spirochaete bacterium Brachyspira hyodysenteriae. This bacterium lives deep in
the colonic crypts but they do not invade the body. They produce a strong haemolysis
toxin that causes a severe inflammation of the large intestine. Typically, it is a major
clinical problem on an affected pig farm, and can severely limit the profitability and
productivity – farmers often feel its presence is incompatible with normal pig farming.
Epidemiology – Outbreaks often follow the entry of the bacterium onto a clean farm.
This entry usually occurs with purchase of infected stock or entry of contaminated
trucks and equipment. The disease swine dysentery remains common in Europe
and Australia, and is re-emerging in North America after a long absence. It is spread
around a farm via faeces from stock and contaminated boots and equipment. The
bacterium can survive outside the pig for up to seven weeks in cold, moist conditions,
but it dies out in two days in dry, warm environments. So the spread and impact
of the disease often subsides in summer. The high cost of disease is associated with
low mortality, high morbidity, marked depression of growth and feed conversion
efficiency, and the costs of continual in-feed medication. It is less common in some
regions due to the commercial availability of breeding stock sources that are certified
free of the disease. So a farmer may stock a new farm with clean stock and following
good biosecurity can remain free of the disease for years.
Clinical signs – The disease usually occurs in 6 to 20 week-old pigs (12 to 75 kg

bodyweight). Many affected pigs have typical and distinctive light-brown to gray,
mucoid-haemorrhagic diarrhoea with large jelly-like lumps of mucus. In other
pigs, a more severe fluid-watery bloody form of diarrhoea can occur. In the early
stages, pigs will show staining under their tail, increased tail twitching and tail biting
activity. Disease is most common in fattening pigs but cases will also occur regularly
in the gilts in the affected farm system. Pigs will lose their appetite and show a rapid
loss of body condition, with a gaunt appearance, sunken eyes and hollow flanks.
Spread of the disease through the herd is often slow, building up in numbers as
the dose rate of the agent builds up in the environment. The incubation period in
field cases is normally 7 to 14 days but can be as long as 60 days. Pigs may develop
a sub-clinical carrier state initially and then break down with clinical disease when
there is a change of feed. Pigs that recover only develop a mild immunity, but rarely
suffer from the same disease again.
Diagnosis – The clinical features are confirmed by post-mortem examinations and

the isolation and identification of the anaerobic B. hyodysenteriae by the laboratory.
Post-mortem examinations show that the distinctive muco-haemorrhagic lesions are
confined to the large bowel. So the ileum and the stomach are normal. Histological
lesions are colon inflammation with crypt dilation and hyperplasia. Dilated crypts
are filled with mucus, with epithelial hypertrophy and inflammation in deep mucosa.
Non-pathogenic spirochetes are also common in the colon, therefore anaerobic
culture is required for diagnosis, along with histology and PCR assay. There are
several closely-related and similar Brachyspira species. The main feature of the agent
associated with swine dysentery is potent β haemolysin activity. Serology tests are
not available.
Management and control of swine dysentery – Acute cases must be treated via
injections with tiamulin antibiotic and followed up with use of tiamulin in the water
and in the feed medication programme. It is very important when using tiamulin
not to mix its usage with any ionophores, such as monensin or salinomycin, as
there is a severe toxic cross-reaction. Alternatives to tiamulin are dimetridazole
and carbadox, which are very effective but are now not licensed in many countries.
Strains of B. hyodysenteriae are emerging that are resistant to tiamulin, therefore very
few alternatives are available in this situation. Lincomycin used at high dosages by
injection or in the feed can reduce clinical signs.
Swine dysentery vaccines are not available. Prevention on farms with endemic disease
consists of providing one or more of the useful antibiotics in the weaner stages to
reduce or eliminate infection in pigs which may then reach the later grower phase
facilities. It is not possible to eradicate the disease without partial or total depopulation
combined with a major cleaning and disinfection and rodent control programme.
Proliferative enteropathy or ileitis (Lawsonia intracellularis)
Proliferative enteropathy (PE; also known as ileitis) forms a group of acute and
chronic conditions of differing clinical signs but with a single and underlying
pathological change: a thickening of the mucosa of the small intestine and colon.
The cause of PE is the obligately intracellular bacterium L. intracellularis, which
preferentially grows within the cytoplasm of intestinal epithelial cells. This bacterial
growth is invariably accompanied by localized proliferation of infected immature
crypt epithelial cells. It has not yet been cultivated in cell-free media, due to unique
metabolic requirements.
Epidemiology – PE is worldwide in distribution and occurs commonly in all

pig-raising regions and in all pig farm management systems, including outdoor ones.
Estimates via serology and faecal PCR diagnostics show that around 96% of farm
sites are infected, wherein around 30% of weaner-to-finisher pigs have detectable
lesions at some point, causing clear economic losses. L. intracellularis can remain
viable in faeces at 5 to 15 °C for 2 weeks, the infectious dose is relatively low and
faecal excretion may be high in some infected ‘spreader’ pigs. The 4% of farm sites
with no detectable infection are usually isolated breeding farms with closed herds.
There are two main patterns of farm infection, which occur in relation to the
management system and antibiotic usage. On single-site farms with a continuous
pig flow between different farm areas (farrow-to-finish systems), infection usually
occurs a few weeks after weaning, presumably when maternal antibodies fade.
The infection can then amplify via oral-faecal infections over the next few weeks
in groups of weaner-nursery and early grower pigs. On some farms, the varying
periods of oral antibiotic usage may modify this pattern. On farm systems with
distinct age- and site-separation of groups of post-weaning and breeding pigs (multi-
site systems), L. intracellularis infection may only occur rarely in breeding stock
and is usually delayed in grower-finishers until they are 14-20 weeks-old. As with
ETEC, it is likely that the environment of most pig farms contains a sustained level
of L. intracellularis infection ‘embedded’ in the residual faecal material, pens, insects,
etc. in the buildings. Studies tracking PE infection on breeding farms have indicated
that infected gilts or sows do not readily transmit the infection to their progeny in
the farrowing area.
Clinical signs – Clinical cases of chronic PE are observed most commonly in the
post-weaned pig between 6 and 20 weeks of age. Diarrhoea and poor weight gain
are often seen together in a group of pigs, but not necessarily in the same pigs. In
many cases of chronic PE in growing pigs, the clinical signs are mild to sub-clinical,
and little more is seen than increased variation in pig performance. Diarrhoea is
generally moderate, with loose, sloppy to watery stools of normal grey-green colour.
Blood or mucus is not a feature of chronic PE diarrhoea. Some pigs can develop
more severe necrotic enteritis and show marked loss of condition and often scour
persistently. These severe cases may occur more often in pigs on organic bedding,
which facilitates oral-faecal intakes and secondary bacteria, such as Salmonella sp.
Progress to slaughter weight can take place despite extensive lesions, but there will
be a consistent reduction in average weight gain, and a significant extension of the
time and feed pigs need to attain market weight.
Unlike chronic PE, cases of acute haemorrhagic PE occur as a severe clinical problem
in young adults 4 to 12 months old, such as breeding gilts and boars, which present
with black tarry faeces and anaemia.
Diagnosis – Moderate diarrhoea due to chronic PE is common, but mixed infections

of endemic disease agents can occur. Acute hemorrhagic PE is most likely to be
confused with oesophago-gastric ulceration, or acute swine dysentery or blood-filled
intestinal torsion cases. The difficulty in routinely culturing L. intracellularis has led
to use of serology and faecal PCR methods for diagnosis. Pigs with active lesions are
usually found to be excreting the agent over several weeks. Animals 6-10 weeks old
usually have the highest prevalence rates for screening of single-site farms. While
detectable antibody responses relate well to the presence of lesions, exposure may
not induce significant seroconversion in all cases. Although blood collection can
be more time-consuming than faeces collection, the serology tests are cheaper to
perform and more suitable for initial group testing.
At autopsy, chronic PE in growing pigs occurs specifically in the ileum and colon.
The magnitude of the proliferation varies widely, but in the developed lesions the
wall is visibly thickened, thrown into deep folds and the overall diameter increased.
Some subserosal and mesenteric oedema is common, and the normal corrugated
pattern of the serosal surface is emphasized. Histologically, the mucosa is composed
of enlarged, branching crypts lined with immature epithelial cells. Silver staining
or specific immunostaining of affected intestinal sections will reveal numerous
intracellular L. intracellularis in the apical cytoplasm. In acute haemorrhagic PE,
the affected intestine is thickened, dilated and the lumen of the ileum and colon
usually contains one or more formed blood clots, often with no other bloody fluids
or feed contents evident. The rectum may contain black, tarry faeces of mixed blood
and feed. No bleeding points or ulcers are observed.
Management and control of proliferative enteropathy – Controlled field and

experimental evaluations of treatment and prevention measures in commercial pigs
over many years have indicated that macrolides and pleuromutilins are the most
effective antibiotics, when given at an adequate dosage rate per kg of bodyweight.
Apparent medication failures with these drugs are most likely to occur in pigs with
ileitis that are under-dosed on a bodyweight basis, such as breeding pigs with a low
feed intake, or when pigs are medicated before or too long after actual peaks of
exposure. Antibiotics used for control achieve the best results if given early in the
course of infections, and on many single-site farms this is around 8-11 weeks of age.
The endemic nature, major economic impact and variable time of onset of PE have
led to a vaccine approach being widely used. Oral administration of a single dose
of an attenuated live vaccine (Enterisol® Ileitis, Boehringer Ingelheim) to young
pigs provides significant levels of protective immunity against subsequent challenge
with virulent L. intracellularis. Killed or subunit vaccine types are not available. This
attenuated vaccine is particularly important for introduction of replacement breeding
stock into new premises. Previous use of acclimation and medication programs in
this situation led to many failures resulting in severe PE outbreaks. Medication of
older pigs, such as breeding stock, is not likely to eliminate the infection, therefore
partial depopulation and medication-based eradication attempts have been largely
unsuccessful.
Improved farm hygiene measures will reliably reduce levels of PE. Quaternary
ammonium-based compounds have effective anti-Lawsonia disinfectant activity, but
isolates appeared somewhat resistant to phenolic or iodine-based mixtures.
3.2.2 Viral enteritis
Coronaviruses – PED, TGE and HEV
The group of enteric coronavirus agents affecting pigs globally are the closely
related group including RNA viruses known as transmissible gastroenteritis (TGE)
and porcine epidemic diarrhoea (PED) and haemagglutinating encephalomyelitis
virus (HEV). The prevalence and importance of each of these agents has varied
considerably around the world over time. Throughout the 1970’s and 1980’s,
transmissible gastroenteritis was a widespread and pathogenic pig disease problem,
and local outbreaks of PED and HEV were also noted. Then in the 1990’s, a milder
mutant strain of TGE virus appeared, known as porcine respiratory coronavirus
(PRCV), which arose from deletions in the spike protein. The rapid and natural
spread of this new strain caused ‘natural’ TGE vaccination in many areas and a
marked reduction in the incidence of actual TGE disease. More recently, since
around 2000, novel pathogenic strains of PED have emerged, causing numerous
and severe outbreaks of intestinal disease. The TGE, PED and HEV viruses all act
in the same way, the virus enters the mature epithelial cells of the villi in the small
intestine, and causing cell death and villous atrophy, thus reducing the absorptive
surface, with loss of fluid and dehydration.
Epidemiology – Acute outbreaks of diarrhoea occur when the virus is first

introduced into a susceptible population. This introduction can occur from entry
of new breeding stock or from contamination of a farm with faecal material via
dirty trucks – such as those which may be collecting dead piglets from different
farms. After introduction of the virus into a susceptible breeding herd, a strong
immunity can develop over a period of two to three weeks. The newly-appeared
lactogenic immunity then protects newborn litters of piglets on the farm. The virus
can disappear spontaneously from breeding herds, particularly herds with less than
300 sows. In larger breeding herds, not all the females may become infected first time
round and there may be recurrence of cases over a period of time. So recurring farm
outbreaks every six months or so are a regular feature in endemic areas.
Clinical signs – Following the appearance of the infection in the herd, large numbers
of pigs of different ages across the farm will show intestinal disease and diarrhoea.
It is often noted as a rapidly-spreading cause of profuse diarrhoea in young pigs
before and after weaning. The pigs have acute watery diarrhoea with no blood or
mucus. Many piglets will also show vomiting and will rapidly develop dehydration
and loss of body condition. Mortality in young pigs may be high. The incubation
period is approximately 2 days and diarrhoea lasts for 7 to 14 days. A high percentage
of sows may also be affected with mild, sloppy diarrhoea, with some more severely
affected sows showing vomiting and watery diarrhoea. Following two or three weeks
of intensive diarrhoea cases around the farm, the herd immunity will appear and
case numbers will decrease.
Diagnosis – At autopsy of fresh pigs, changes may be limited to dehydration, yellow

fluid diarrhoea, thin-walled dilated intestine and reduced body fat. The stomach may
be full of food (milk). Recognition of the histological changes of villous stunting
requires very fresh samples. No inclusion bodies are seen. Virus diagnosis is by
antigen detection or electron microscopy of faeces. Infections cause the intestines to
become pale, thin-walled and distended with watery brown fluid and gas.
Management and control of coronaviruses – Antibiotics will have no specific effect

on the outbreaks. Affected piglets will require supportive nursing with fluids and
milk to prevent dehydration. Once an outbreak has started, it is often recommended
to increase herd immunity and attempt to shorten the period of clinical problems by
providing access to all pigs on the farm with the virus material. This is best done by
homogenising the intestines from several affected piglets and feeding this material
to all pigs on the farm – a process known as feedback. Full herd immunity is not
achieved by only dosing a limited number of sows with feedback materials. Better
PED vaccines are being sought for recent outbreaks and the experience with PRCV
may indicate that a strategy can be successful if a useful avirulent coronavirus strain
is found.
Rotaviruses
Rotaviruses are ubiquitous agents among pig farms and also replicate in and attack
the mature villous epithelial cells of young animals, causing villous stunting.
Infections of rotavirus type A of various G, P types are the most common type of
rotavirus in many pig herds. Rotavirus infections are considered less pathogenic
than coronaviruses because they attack further up the villi tips, allowing structural
and functional restoration to occur quicker. The viruses move in a ‘wave’ of infection
down the intestine, therefore segmental enteritis can occur at any part of the intestine,
but infection is most often seen in the jejunum.
Epidemiology – Rotaviruses are very stable DNA viruses that can survive in the
environment for long periods (years), creating persistent infections on most farms.
In many farms, piglets can often become infected from their mother with a low
infectious dose and only show mild transient infections for one week or so.
Clinical signs – Mild transient diarrhoea may be the only clinical sign in pigs
around the time of weaning, due to the reduction in maternal immunity protection.
Pigs often recover in less than one week and solid immunity is seen in older animals.
Rotavirus-infected herds may suffer a greater number of cases of other post-weaning
diarrhoea problems, compared to case-control herds, due to the rotavirus damage
to the intestine at weaning.
Diagnosis – At autopsy of fresh pigs, changes may be limited to dehydration, yellow

fluid diarrhoea, thin-walled dilated intestine and reduced body fat. Recognition of
the histological changes of villous stunting requires very fresh samples. No inclusion
bodies are seen. Virus diagnosis is by antigen detection or electron microscopy
of faeces. The ubiquitous presence of this agent means that mixed infections are
common and it can be difficult to confidently ascribe the intestinal disease situation
in pigs solely to a rotavirus problem.
Management and control of rotaviruses – Antibiotics will have no specific effect

and supportive nursing is suggested. Rotavirus vaccines are available for other
animals (humans) and may play a future role in pigs.
Porcine circovirus associated enteritis
Porcine circovirus (PCV) associated disease has been a major global problem since
the 1990’s, specifically due to the emergence of porcine circovirus genotype 2. This
virus causes severe depletion of lymph node tissue and immunosuppression in
growing pigs, with many highly problematic secondary infections occurring. More
recent development of highly effective vaccines has led to a marked reduction in the
global impact of this agent.
Affected pigs will have chronic wasting and secondary infections, particularly
pneumonia. Enlargement of peripheral lymph nodes is a common feature. Post-
weaning mortality in the affected group of pigs is likely to rise to 10% but is
sometimes much higher.
In some cases of PCV associated disease, intestinal disease and diarrhoea are noted,
but this intestinal disease occurs as part of the more systemic disease syndrome.
The small intestines may show thickened granulomatous inflammation and faecal
shedding. Immunohistochemistry can be used to demonstrate PCV in intestinal
tissues.
3.2.3 Protozoal enteritis
Isospora suis coccidial infections
The various protozoa have a direct life cycle, and are common in many species, but
are usually host species-specific. Coccidia, particularly Isospora suis are an important
and global problem in piglets. Isospora are typical coccidia that invade epithelial
cells in the small intestine at various stages of the life cycle, causing the cells to
develop protozoal compartments. Infected cells are released into the lumen, creating
waves of cell destruction, during reproductive phases of coccidial growth.
Epidemiology – Coccidial oocysts are highly resistant and easily transmitted on

equipment and clothing, hence eradication is not possible. Studies of farrowing areas
show the key feature of a wide variation in numbers of oocysts in adjacent pens, with
problems occurring in more highly contaminated pens. Numerous oocysts may be

demonstrated in the faeces of piglets, these are shed as non-sporulated forms; the
oocysts then require moist pen conditions to sporulate to the infective form. As in
all hosts, clinical protozoal disease is more likely where over-crowding and contact
with warm, wet infected faeces occur. Therefore coccidiosis is more problematic in
summer months in humid conditions. However, it also occurs commonly in farms
located in colder climates, because the farrowing areas are often artificially heated.
Clinical signs – Affected piglets have a soft, yellow, toothpaste-like scour at ten to 20
days of age, with weight loss, dehydration and occasional deaths. The sow will appear
normal. As noted for rotavirus, the occurrence of this infection around the time of
weaning is associated with a greater incidence of post-weaning enteric diseases.
Diagnosis – Flotation and smears of faecal samples will show numerous coccidial
oocysts evident. At autopsy, the intestines will have segments of necrotic enteritis
evident in the jejunum. Histology will demonstrate significant intestinal tissue
damage and coccidial elements in the mucosa, seen as small banana-shaped
merozoites within epithelial cells.
Management and control of coccidiosis – Coccidiosis in piglets is managed by

two linked elements with the strategy of reducing deposition and sporulation of
oocysts in the farrowing areas. One element is to conduct rigorous cleaning of the
farrowing pen in the so-called ‘downtime’ between different litters. This requires
products and techniques capable of killing oocysts. The second element is to treat
piglets in the early stages of infection with a suitable product, such as toltrazuril
that targets coccidial stages in the intestine. This strategy has proved a very effective
dual one to control coccidiosis in most situations and is vital in most farms in warm
summer conditions.
3.2.4 Parasitic enteritis
The major intestinal worms of pigs are:

• Ascaris suum – large roundworm;
• Trichuris suis – large intestine whipworm;
• Oesophagostomum sp. – nodule worm.
These worms are now only relatively common in local areas where pigs are kept
outdoors on pasture or in contact with soil from organic bedding. Other worms do
occur in the intestine of pigs, such as the threadworm Strongyloides ransomi and
the acanthocephalan Macrocanthorynchus hirudinaceous, but these are localised and
rare in occurrence.
Ascaris suum are large roundworm parasites that are readily recognised at examination
of the intestines at slaughter. They are common parasites of many outdoor farms and
in indoor pigs exposed to soil via organic bedding. In mild infections, there are few
overt clinical signs. However, more severe exposures may cause reduction of lung,
liver or intestinal function. Reduction of liver and intestinal function will create
an impact on weight gains, feed conversion and the time taken for pigs to reach
slaughter condition. Worm eggs pass to the external environment in faeces. These
eggs are oval and thick-shelled with a sticky external coat that is highly resistant and
can persist on the ground for years. Pigs are infected by ingesting eggs, which have
developed to the larval stage. Once eaten, the larvae pass to the liver within 3 days
then to the lungs then to the intestines. The adult worm matures and begins to lay
eggs approximately 6 to 8 weeks after infection. Therefore animals must be treated
with an effective worm medication, such as fenbendazole or ivermectin, every 4 to
6 weeks to break the life cycle of this worm.
Trichuris suis are whipworms that have a direct life cycle of three weeks duration.
They are not as common as Ascaris and are now most commonly seen in pigs
raised in small and dirty outdoor farms. Clinical signs are of slow and poor growth,
emaciation, with individual pigs showing soft, bloody diarrhoea. Trichuris suis
whipworms cause a muco-haemorrhagic colitis similar to that described for swine
dysentery, but numerous thin, white 4-8 cm long whip-shaped worms are visible
in the inflammed colon mucosa. Ivermectins and piperazine therapies are not fully
effective so this parasitic disease is hard to control where it occurs.
Oesophagostomum sp. worms cause localized 1-2 cm diameter nodules in the wall of
the small and large intestine of pigs. These are now rarely noted and are essentially
non-pathogenic.
3.2.5 Intestinal accidents
Intestinal torsions
The small intestinal tract of the pig is quite long, usually measuring fifteen times
the length of the body. It is connected to the pig’s body by a connective tissue
mesh known as the mesentery, which hangs loose from the roof of the abdomen
in a standing pig. In active grower pigs, this means that twists and torsions of
the small intestine can occur commonly, which can quickly result in an extreme
intestinal accident, compromised blood supply and sudden death of the pig. One
explanation is that the animal does the twisting, such as when the pig steps up to
a feeder and suddenly falls, somersaults, or turns over with a lack of abdominal
press, allowing the fluid filled bowel to stay still while the animal twists around it.
Fighting, horseplay, mounting, etc. may also be a trigger for this twisting action.
The delivery of a single large volume of feed to the intestine may assist the motion
by adding ingesta weight to the bowel. One could note the analogy to twisting a
martini glass around a skewered olive. Therefore it is not uncommon to notice
‘outbreaks’ of deaths due to intestinal torsions in groups of boisterous growing pigs
with step-up feeders filled once per day.
It is therefore very important that any investigation of the cause of sudden death in a
pig includes a palpation of the root of the mesentery, to check for intestinal torsion.
To perform this palpation, once the pig is lying on its left side and the abdomen
is initially opened, the left hand is slid, palm upwards, under the back edge of the
intestines at the root of the mesentery. This mesentery root should normally be felt
as a flat smooth shelf, compared to pigs with an intestinal torsion, where this feature
is tight and cord-like.
3.3 Conclusions
In the pig farming industries of America and Europe and Australia, the main
intestinal diseases of weaned pigs in many regions are bacterial – ETEC infections,
salmonellosis, swine dysentery due to B. hyodysenteriae and proliferative enteropathy
(ileitis) due to L. intracellularis infections. The bacterial strains ETEC, Salmonella
and Lawsonia all appear to be ‘embedded’ in many pig farms, causing common and
endemic intestinal disease problems. These diseases and swine dysentery are all
increasing in incidence and importance for pigs globally, as political restrictions on
the usage of farm medications, such as antibiotics and zinc oxide, have appeared. In
the pig farming industries of Asia, coronavirus infections are more prominent and
are also increasing in incidence and importance.
References
Neumann, E.J., Ramirez, A. and Schwartz, K.J. (eds.), 2009. Swine diseases manual. 4th
edition. American Association of Swine Veterinarians, Perry, IA, USA, 173 pp.
Sims, L.D. and Glastonbury, J.W. (eds.), 1996. Pathology of the pig: a diagnostic guide. Pig
Research and Development Corporation and Agriculture Victoria, Australia, 456 pp.
Zimmerman, J.J., Karriker, L.A., Ramirez, A., Schwartz, K.J. and Stevenson, G.W. (eds.), 2012.
Diseases of swine. 10th edition. Wiley-Blackwell, Ames, IA, USA, 1008 pp.
Chapter 4: Avian coccidiosis as a prototype intestinal
disease – host protective immunity and
novel disease control strategies
H.S. Lillehoj1*, S.I. Jang1, S.H. Lee1 and E.P. Lillehoj2

1Animal Biosciences and Biotechnology Laboratory, Beltsville Agricultural
Research Center, Agricultural Research Service, United States Department

of Agriculture, Beltsville, MD 20705, USA; 2Department of Pediatrics,
University of Maryland School of Medicine, Baltimore, MD 21201, USA;
hyun.lillehoj@ars.usda.gov
Abstract
Poultry meat consumption has increased globally by 50% since 2000, accounting for
greater than 100 million tons in 2012. Multiple challenges confront the rising demand
for poultry food products, including governmental restrictions on the use of antibiotic
growth promoters and novel feedstuffs, high-density production conditions, waste
management, and the emergence of infectious pathogens, particularly those that
cause intestinal diseases. There is little doubt that in-feed antibiotics has dramatically
increased the efficiency of commercial poultry production over the last 50 years.
However, antibiotic usage in chickens has raised concerns regarding chemical
residues in the poultry food products, and has directly led to the appearance of
antibiotic resistance among avian pathogens that has the potential to be transferred
to humans pathogens. Much interest, therefore, has focused on the development of
alternative, antibiotic-free methods of commercial poultry production. Additionally,
identification of new chicken genetic markers opens the door for the development
of novel chicken breeds with increased resistance to infectious diseases through
gene modifications and DNA-based selection strategies. This chapter addresses
alternatives to antibiotics in the context of avian coccidiosis, a prototypical intestinal
disease of chickens. First, the biology of Eimeria, the causative agent of coccidiosis, is
briefly reviewed, followed by a summary of the chicken immune response to Eimeria
infection, and finally an appraisal of recent advances in nontraditional coccidiosis
control strategies.
Keywords: chicken, Eimeria, antibiotic, gut, disease

4.1 Introduction
Avian coccidiosis is one of the most widespread infectious diseases of chickens

(Shirley and Lillehoj, 2012). The etiologic agent of avian coccidiosis is Eimeria, a genus
of eukaryotic obligate intracellular parasites belonging to the phylum Apicomplexa,
along with the genera Plasmodium, Cryptosporidium and Toxoplasma, among others.
All members of the Apicomplexa are characterized by the presence of a cellular
apical complex composed of polar rings, rhoptries, micronemes, often a conoid,
and other organelles (Figure 4.1). While more than 1,700 species of Eimeria have
been described, only seven are known to infect chickens: E. acervulina, E. tenella,
E. maxima, E. brunetti, E. mitis, E. necatrix, and E. praecox. All seven species are
distributed worldwide, with E. acervulina, E. tenella, and E. maxima being the
most common (McMullin, 2008). These parasites infect the intestinal tract and are
transmitted between chickens though a fecal-to-oral route (Figure 4.2). Clinical
manifestations of infection include damage to the intestinal epithelium, decreased
nutrient absorption, inefficient feed utilization, and impaired growth rate, which, in
severe cases, may lead to mortality (Shirley et al., 2004, 2005; Williams, 1999).
The life cycle of Eimeria parasites includes intracellular and extracellular phases,
as well as asexual and sexual stages (Figure 4.3) (Hammond, 1982). The infectious
process begins with ingestion of oocysts, an environmentally-resistant structure
A B
9 10 11
1
8 2
3
7
4
6 5
Figure 4.1. (A) Schematic illustration of an Eimeria sporozoite (not to scale). 1, polar ring;
2, conoid; 3, micronemes; 4, rhoptries; 5, nucleus; 6, nucleolus; 7, posterior refractile body; 8,
posterior ring; 9, alveoli; 10, Golgi apparatus; 11, micropore. Adapted from http://en.wikipedia.
org/wiki/Apicomplexa. (B) Transmission electron micrograph of an E. tenella sporozoite.
4. Avian coccidiosis as a prototype intestinal disease
E. acervulina E. praecox E. nectarix E. maxima E. mitis E. brunetti E. tenella
Figure 4.2. The seven species of Eimeria infected discrete regions of the chicken intestine.
Sporocyst
Sporozoite
Oocyst 6 1
Microgametes
2
5
4
Zygote
Merozoite
Macrogametes
Figure 4.3. The Eimeria life cycle. Infective oocysts containing sporocysts are ingested by chickens
(step 1), leading to the development of sporozoites. Sporozoites invade intestinal epithelial cells
(step 2), where they undergo replication and differentiation into merozoites. Merozoites exit
the infected cells and reinfect neighboring cells (step 3). Merozoites differentiate into male
microgametocytes and female macrogametocytes (step 4). Micro/macrogametocytes develop
into micro- and macrogametes which fuse to form a zygote (step 5). The zygote develops into an
oocyst that is released in the feces. Following excretion, oocysts undergo sporulation to generate
sporocysts, each containing two sporozoites (step 6). Adapted from http://en.wikipedia.org/wiki/
Apicomplexa.
containing four sporocysts, each enclosing two infective sporozoites. Once within
the intestinal lumen, sporozoites invade epithelial cells and differentiate into
merozoites through the process of merogony. Mature merozoites released from host
cells reinfect neighboring cells. The number cycles of parasite release and reinfection
varies from two to four depending on the Eimeria species. The last generation of
merozoites develop into the sexual forms, macrogametocytes and microgametocytes,
that subsequently form macrogametes and microgametes, respectively. Fertilization
of these two parasite stages generates a zygote, which leaves the host cell and matures
into an oocyst to begin the life cycle once again.
Over the past decades, widespread use of in-feed, synthetic anticoccidial drugs during
commercial poultry production has effectively controlled coccidiosis outbreaks by
interrupting the fecal-oral infection cycle. Coccidiostats, however, are costly and
their ubiquitous use has promoted the development of drug resistance (Chapman,
2009; Dalloul and Lillehoj, 2006). Because Eimeria infection leads to a strong,
species-specific, protective immune response, vaccination of poultry flocks offers
an alternative method of disease control. Several coccidiosis vaccines that contain
live, attenuated or nonattenuated parasite mixtures of different Eimeria species
are commercially available. While the use of Eimeria vaccines has been valuable
in reducing the need for in-feed medication, a possible complication of using live
vaccination is an early diminution in chicken growth. Experimental coccidiosis
vaccines based upon recombinant Eimeria genes and proteins have been developed
and shown to be effective in model systems of experimental infection, but are yet to
be commercially marketed on a widespread basis.
4.2 Chicken immune responses to Eimeria
As in mammals, avians possess innate and adaptive, as well as humoral and cell-
mediated, arms of immunity (Dalloul and Lillehoj, 2006; Lillehoj, 1998; Lillehoj
et al., 2004). Eimeria infection of chickens induces a complex immune reaction
encompassing all of these different components, although some responses may not
be as important as others for complete protective immunity. A major challenge for
poultry immunologists studying avian coccidiosis is determining which aspects of
immunity are responsible for conferring protection against Eimeria infection.
4.2.1 Chicken innate immunity
The innate immune system comprises cells and their secreted products that defend
the host against microbial infections in a relatively nonspecific manner. These include
physical barriers imposed by mucosal epithelia, phagocytic and other leukocytes,
cytokines, chemokines, and components of the complement system. More specifically,
pattern recognition receptors (PRRs) on and within epithelial and hematopoietic cells
constitute a critical component of innate immunity in avians and mammals. PRRs
include the cell surface Toll-like receptors (TLRs) and intracellular Nod-like receptors
(NLRs) (Kumar et al., 2011). TLRs and NLRs are type I transmembrane proteins
responsible for recognizing conserved components of pathogens, the pathogen-
associated molecular patterns (PAMPs). Well-defined PAMPs include bacterial
lipopolysaccharide (LPS), lipopeptides, glycolipids, CpG DNA, and viral nucleic acids
(Lemaitre, 2004). Protozoan PAMPs also are potent stimulators of innate immune
responses, although their cognate PRRs are not as well defined, compared with those
that recognize bacterial and viral PAMPs (Gazzinelli and Denkers, 2006).
Ten TLRs have been identified in the chicken: TLR1LA, TLR1LB, TLR2A, TLR2B,
TLR3, TLR4, TLR5, TLR7, TLR15, and TLR21 (Temperley et al., 2008). Phylogenetic
analyses revealed that six of these (TLR2A, 2B, 3, 4, 5, and 7) have orthologs in
mammals and fish, while one (TLR21) is only shared by fish, and three (TLR1LA,
1LB, and 15) are unique to chickens. The role of chicken TLRs in response to
Eimeria infection remains to be established. Sumners et al. (2011) reported increased
expression of TLR3, TLR4, and TLR15 in the intestine of chickens infected with
E. praecox, compared with uninfected animals. Zhang et al. (2012a) demonstrated
increased expression of TLR1LA, TLR4, TLR5, TLR7, and TLR21 at 12 hr post-
infection with E. tenella, and that TLR1LA, TLR5, and TLR21 remained highly
expressed at 72 hr post-infection, compared with uninfected controls. Most recently,
Zhou et al. (2013) showed increased expression of TLR4, TLR15, and the TLR
adaptor protein, MyD88, in chicken heterophils and monocyte-derived macrophages
stimulated in vitro with E. tenella, compared with unstimulated cells.
The TLR agonists that are expressed by Eimeria parasites are now beginning to be
discovered. While chickens do not express the equivalent of the mammalian TLR9,
the TLR9 agonist, CpG DNA, was shown to activate chicken macrophages in vitro
(Xie et al., 2003), and to increase protection against experimental avian coccidiosis
in vivo (Dalloul et al., 2004). Moreover, co-administration of CpG DNA with the
Eimeria recombinant protein, EtMIC2, was associated with increased protection
against E. acervulina and E. tenella infections, compared with EtMIC2 alone

(Dalloul et al., 2005). A similar effect was reported following co-administration
of E. tenella heat shock protein 70 (HSP70) plus EtMIC2 (Zhang et al., 2012b). In
mammals, HPS70 is a ligand for TLR2 and TLR4 (Asea, 2008). Among the TLR
agonists expressed by Eimeria parasites, the most widely studied is profilin, an
actin-binding protein found in all eukaryotes that is involved in the turnover and
restructuring of the actin cytoskeleton. Eimeria profilin is a 19 kDa protein that is
highly conserved among all Apicomplexa protozoa and is expressed during most
stages of the parasite’s life cycle (Fetterer et al., 2004; Lillehoj et al., 2000). Profilin
was originally isolated as a recombinant protein from E. acervulina merozoites and
shown to enhance antigen-stimulated interferon-γ (IFN-γ) production by spleen
cells from E. acervulina-infected chickens, compared with uninfected birds (Lillehoj
et al., 2000). Subsequent studies demonstrated that immunization of chickens with
profilin, either as a recombinant gene or protein, increased protective immunity
following challenge infection by coccidia parasites (Ding et al., 2004; Lillehoj et al.,
2005a,b; Ma et al., 2011, 2012; Min et al., 2001a; Song et al., 2000; Xu et al., 2006).
Although the chicken TLR that recognizes Eimeria profilin has not been identified, the
homologous protein of Toxoplasma gondii, toxofilin, binds to mouse TLR11 inducing
a potent interleukin (IL)-12 response in dendritic cells (Yarovinsky et al., 2005,
2006). Eimeria profilin possesses antiviral and anticancer properties in mammals
through its ability to stimulate proinflammatory cytokine production (Gowen et al.,
2006; Juckett et al., 2008; Julander et al., 2007; Rosenberg et al., 2005). Interestingly,
while profilin-driven innate immune responses were dependent upon MyD88, under
some conditions profilin also suppressed these same responses in a TRIF-dependent
manner (Seregin et al., 2011). Based on these combined studies, Eimeria profilin has
been investigated as a vaccine adjuvant in mammalian model systems of infection.
Hedhli et al. (2009) demonstrated that combined administration of Eimeria profilin
with T. gondii increased protective immunity against experimental toxoplasmosis,
compared with mice given T. gondii alone. Appledorn et al. (2010a) showed that
co-administration of mice with human immunodeficiency virus Gag proteins plus
profilin increased Gag-specific cell-mediated immune responses, compared with
mice given Gag proteins alone. Given that Eimeria profilin exhibited no toxicity in
human and chicken preclinical and clinical trials (Rader et al., 2008; Rosenberg et
al., 2005; Zhao et al., 2013), future use of Eimeria profilin as an adjuvant in human
and veterinary medicine may be realized.
4.2.2 Chicken adaptive immunity
Antigen-specific, adaptive immunity is mediated by T cells expressing the T cell

receptor (TCR), B cells expressing surface immunoglobulins, and their secreted
antibodies, cytokines, and chemokines. In the intestine, the gut associated lymphoid
tissues (GALT) mediates adaptive immunity through three interrelated processes,
antigen processing and presentation, production of intestinal antibodies, and
activation of cell-mediated immunity. In the naïve chicken, infection by coccidia
parasites activates dendritic cells and macrophages within the GALT, leading
to the elicitation of a plethora of cytokines and chemokines (Hong et al., 2006b;
Lillehoj, 1998; Min et al., 2013). These mediators drive not only the generation
and differentiation of effector lymphocytes and other immune cells, but also that
of memory cells, which remain in the host after parasite clearance and respond to
the same pathogen upon reinfection. In immune hosts, parasites that enter the gut
are prevented from further development, suggesting that memory cell-mediated
acquired immunity resulting from the initial pathogen exposure inhibits the natural
progression of parasite development (Trout and Lillehoj, 1996; Yun et al., 2000a).
The following sections describe in more detail the roles of the humoral and cell-
mediated arms of acquired immunity in the host response to avian coccidiosis.
4.2.3 Antibody responses during avian coccidiosis
Three antibody isotypes are recognized in birds, IgM, IgA, and IgY. Although
often incorrectly considered as the equivalent of IgG, chicken IgY differs both
structurally and functionally from mammalian IgG (Larsson et al., 1993; Ohta
et al., 1991). During embryogenesis, maternal IgY is transported to the egg yolk
sack, a process which has been considered in a similar way as passive immunity
in mammals (Wallach, 2010; Wallach et al., 1992; West et al., 2004). While some
studies suggested a lesser role for humoral immunity in protection against Eimeria
infection, compared with cell-mediated immunity (Lillehoj, 1987), more recent
investigations have demonstrated that chicken antibodies produced in response
to parasite infection can block parasite invasion, development, and transmission
and are capable of conferring passive immunity against infection (Smith et al.,
1994; Wallach, 2010). As discussed by Wallach (2010), at least two explanations
may be offered to account for the conflicting literature reports concerning the
role of antibodies in protection against Eimeria infection. First, during secondary
challenge infections were cellular immune responses alone are dominant, there
may be no need for anti-Eimeria antibodies to control infection. Second, while
immunization with live Eimeria vaccines is effective in producing complete and

long-lasting protective immunity, an equivalent level of resistance to infection often
is not possible using antibodies alone.
4.2.4 Cell-mediated immune responses during avian coccidiosis
Pioneering studies by Rose et al. (1979) revealed that T lymphocytes, the

primary mediators of cell-mediated immunity, played a critical role in the avian
response against Eimeria infection. Subsequently, chickens treated with the T cell
immunosuppressant, cyclosporin A, were reported to exhibit signs of reduced
protective immunity against E. tenella infection, compared with untreated controls,
confirming the importance of cellular immune mechanisms (Lillehoj, 1987).
Coccidia antigen-specific proliferation of T lymphocytes in Eimeria-immune
chickens provided direct evidence for the presence of parasite-specific T cells
(Lillehoj, 1986; Vervelde et al., 1996). An increased percentage of intestinal T cells
expressing the γδ-TCR was observed in chickens infected with E. acervulina,
compared with uninfected controls (Choi and Lillehoj, 2000). As in mammals,
chicken T cells expressing the γδ-TCR are at their highest abundance in the
intestinal mucosa, compared with their counterpart αβ-TCR T cells (Lillehoj and
Chung, 1992). Similarly, an increased percentage of CD8+ T cells was observed in
the intestine of chickens infected with E. acervulina, compared with uninfected
animals (Lillehoj and Bacon, 1991). Further, following infection of chickens with
E. acervulina, sporozoites were seen primarily in CD8+ lymphocytes and these cells
also appeared to be in contact with parasite-infected epithelial cells, suggesting a
role for CD8+ T cells in sporozoite transport and host protection (Trout and Lillehoj,
1995). Selective depletion CD8+ T cells led to increased oocyst production following
infection with E. tenella or E. acervulina (Trout and Lillehoj, 1996). The proportion
of CD8+ peripheral blood lymphocytes was increased in chickens at 8 days following
a primary infection with E. tenella, concurrent with greater production of nitric
oxide (NO) and IFN-γ by these cells following stimulation with the T cell mitogen,
concanavalin A, or E. tenella sporozoite antigen (Breed et al., 1997a,b). Although
not as numerous as gut CD8+ T cells, some role for chicken CD4+ T cells in avian
coccidiosis also has been reported (Cornelissen et al., 2009; Vervelde et al., 1996).
Concomitant with T cells, other leukocyte types have been described to play an
important role in the chicken immune response to Eimeria infection. Chicken
macrophages and monocyte expressing the major histocompatibility complex
(MHC) class II and/or K1 surface markers, are involved in different phases of
the host immune response to coccidia parasites (Lillehoj et al., 2004). Greater
numbers of mononuclear leukocytes were observed in the intestinal lamina
propria of E. tenella-infected chickens, compared with uninfected controls, and the
majority of these infiltrating cells were macrophages and T cells (Vervelde et al.,
1996). Similar to macrophages, dendritic cells (DCs) are antigen-presenting cells
that act as messengers between innate and adaptive immunities. Avian follicular
and interdigitating DCs have been isolated and their morphologic, phenotypic,
and functional properties characterized (Del Cacho et al., 2008, 2009). Protective
immunity against E. acervulina, E. tenella, and E. maxima experimental infections
was achieved using purified DCs as well as DC-derived extracellular exosomes (Del
Cacho et al., 2011, 2012). Natural killer (NK) cells constitute another subpopulation
of leukocytes that mediated cellular immunity. NK cells were originally described in
mammals as cells with cytotoxic activity against some virally-infected cells and tumor
cells. Chicken NK cells in the spleen and intestine of Eimeria-infected chickens have
been described (Lillehoj, 1998). NK-lysin, originally described as a bacteriocidal
peptide of possible NK cell origin (Andersson et al., 1995), was recently shown to be
produced by chicken cytotoxic T cells and to exhibit in vitro cytotoxic effects against
chicken tumor cells, as well as sporozoites of E. acervulina and E. maxima (Hong et
al., 2006a).
4.2.5 Cytokine responses during avian coccidiosis
A major mechanism through which immune cells respond to infectious pathogens

involves the elaboration of cytokines and chemokines as soluble mediators of
inflammation. Cytokines encompasses a large and diverse family of peptides and
proteins that are produced by cells of varied origins. Immune-related cytokines serve
to initiate, amplify, and sustain innate and adaptive immune responses. Chemokines
are chemotactic cytokines that induce the directed migration of responsive cells
from distal sites towards the focus of an infection. The major chicken cytokines
that have been described in avian coccidiosis are IFN-γ, IL-1, IL-2, IL-4, IL-5,
IL-6, IL-10, IL-12, IL-13, IL-15, IL-16, IL-17, IL-18, tumor necrosis factor-α (TNF-
α), lipopolysaccharide-induced TNF-α factor (LITAF), TNF-α superfamily 15
(TNFSF15), transforming growth factor-β1 (TGF-β1), TGF-β2, TGF-β3, TGF-β4,
and granulocyte-macrophage colony-stimulatory factor (GM-CSF) (Lillehoj, 1998;
Lillehoj et al., 2004; Lowenthal et al., 1999; Ovington et al., 1995). The major chicken
chemokines that have been reported during Eimeria infection are IL-8 (CXCL8),
lymphotactin (XCL1), macrophage inflammatory protein 1β, (CCL4), K203 (CCL3),
ah221 (CCL9), and K60 (CXCL1) (Dalloul et al., 2007; Hong et al., 2006b; Laurent
et al., 2001). The following section reviews the published literature that documents
the roles of three selected cytokines with well-documented roles in avian coccidiosis,
IFN-γ, IL-2, and IL-17A.
IFN-γ production during avian coccidiosis has been examined by quantitative

RT-PCR (Choi et al., 1999; Lillehoj et al., 2000), and, more recently, by gene expression
profiling (Min et al., 2003). Following experimental infection with E. acervulina
or E. tenella, chicken IFN-γ gene transcripts were detected in the intestinal cecal
tonsils and spleen (Choi et al., 1999). The ability of chicken IFN-γ protein to
activate immune cells against Eimeria parasites has been examined in various model
systems of parasite infection (Lillehoj, 1998). Chicken macrophages that had been
pretreated with IFN-γ blocked the development of E. tenella sporozoites in vitro
through a mechanism involving increased NO production (Dimier et al., 1998;
Lillehoj, 1998). These results provided a rational basis for the use of IFN-γ as an
immunomodulator to augment protective immunity against Eimeria infection in
vivo. Multiple intramuscular injections with chicken IFN-γ recombinant protein
prior to and following infection with E. acervulina increased protection against
parasite challenge, as measured by increased body weight gain and decreased oocyst
shedding, compared with untreated controls (Lillehoj and Choi, 1998). Reduced
NO production by IFN-γ-stimulated chicken macrophages was observed in cells
transfected with a small interfering (si)RNA targeting inducible nitric oxide synthase,
the enzyme responsible for NO production (Cheeseman et al., 2008). Chicken IFN-γ
also has been demonstrated to possess adjuvant-like properties in conjunction
with the profilin subunit protein vaccine through its ability to enhance protective
immunity against experimental avian coccidiosis (Ding et al., 2004; Lillehoj et al.,
2005a; Min et al., 2001a).
Chicken IL-2 is another proinflammatory cytokine that regulates protective immunity

during avian coccidiosis. Li et al. (2002) reported that the SC (Eimeria resistant)
and TK (Eimeria susceptible) chicken strains produced equivalent amounts of IL-2
following primary infection with E. tenella. After secondary infection, however,
SC chickens displayed greater intestinal IL-2 levels, compared with the TK strain.
The kinetics of IL-2 production also were different when comparing primary vs
secondary E. tenella infections. Following primary infection, IL-2 levels in serum
and cell culture supernatants of spleen lymphocytes stimulated with mitogen or
E. tenella sporozoites were greatest at day 7 post-infection, compared with uninfected
controls (Miyamoto et al., 2002). This peak in IL-2 levels coincided with the time
of maximum parasite-derived intestinal lesions. By contrast, following secondary
infection, the highest IL-2 levels were recorded at day 2 post-infection, whereas
intestinal lesions remained most severe at day 7 post-infection. Similar to IFN-γ,
chicken IL-2 has been evaluated as an adjuvant co-administered with a variety of
different Eimeria subunit vaccines, including profilin (Ding et al., 2004; Lillehoj et
al., 2005a), EtMIC2 (Lillehoj et al., 2005b), the E. acervulina sporozoite antigen,
cSZ-2 (Shah et al., 2010a,b, 2011), E. acervulina lactate dehydrogenase (Song et al.,
2010), the E. tenella surface antigen, TA4 (Song et al., 2009; Xu et al., 2008), and the
E. tenella refractile body protein, SO7 (Song et al., 2013).
The IL-17 cytokine family contains the most recently described mediators of
protective immunity against avian coccidiosis (Min et al., 2013). Members of this
family include IL-17A, IL-17B, IL-17C, IL-17D, and IL-17E. Following experimental
infection by E. acervulina or E. maxima, IL-17A mRNA levels in intestinal
lymphocytes were generally increased, whereas these transcripts were decreased
following E. tenella experimental infection (Hong et al., 2006a,b; Kim et al., 2012a).
Compared with chickens infected with Clostridium perfringens, the etiologic agent
of avian necrotic enteritis, chickens co-infected with E. maxima and C. perfringens,
had reduced levels of IL-17A gene transcripts in intestinal lymphocytes (Park et
al., 2008). However, Zhang et al. (2013) reported that infection with E. tenella
increased IL-17A expression by intestinal lymphocytes, compared with uninfected
controls. Shaw et al. (2011) showed that chickens raised on litter contaminated with
E. tenella had greater levels of IL-17A gene transcript in their intestinal lymphocytes,
compared with chickens raised on uncontaminated litter. These studies highlight the
importance of environmental factors in regulating chicken IL-17A gene expression
in the context of avian coccidiosis.
Co-administration of chicken IL-17A in combination with the E. tenella surface

antigen, MZP5-7, reduced fecal oocyst shedding and decreased the severity of
intestinal lesions following experimental E. tenella infection, compared with MZP5-7
alone (Geriletu et al., 2011). Similarly, vaccination of chickens with Eimeria profilin
protein in combination with IL-17A improved protective immunity against E.
acervulina experimental infection, compared with chickens immunized with profilin
alone (Ding et al., 2004). Importantly, co-vaccination with profilin plus IL-17A also
was the most efficacious at increasing protective immunity against E. acervulina,
compared with profilin plus IL-2, IL-6, IL-8, or IFN-γ. However, IL-17A does not
appear to be a universal coccidiosis vaccine adjuvant, because immunization with
EtMIC2 plus IL-17A did not influence the course of experimental E. tenella infection,
compared with EtMIC2 alone (Lillehoj et al., 2005b).
4.3 Prevention and control of avian coccidiosis: alternatives to

antibiotics
The immunobiology of avian coccidiosis involves a complex interplay between the

various life stages of Eimeria parasites and host intestinal epithelial and immune
cells. One of the most immediate and significant outcomes of this host-parasite
interaction is a devastating destruction of the intestinal mucosa as a consequence
of parasite infection of, and replication and development within, intestinal epithelia
(Figure 4.4). This pathological effect, and the associated retardation of chicken
growth, has been the major impetus for development of anticoccidial control
strategies. Synthetic coccidiostat drugs have played a major role in mitigating the
negative consequences of Eimeria infections on commercial poultry farms. It is
also clear, however, that alternatives to antibiotic chemotherapy are becoming an
increasing priority for modern poultry production in order to maintain commercial
profitability, reduce the emergence of drug-resistant parasites, and ensure consumer
confidence in providing safe, consumable foods. The following sections review some
of the more novel and promising antibiotic-free coccidiosis control measures that
may someday find widespread commercial usage.
Figure 4.4. (A) Scanning electron micrograph of Eimeria tenella parasites growing in the chicken
intestinal cecal tonsils. Copyright, Professor D.J.P. Ferguson, University of Oxford. Reproduced
with permission. (B) Gross pathology of the intestinal cecal tonsils at 6 days post-infection with
E. tenella illustrating thickening of the cecal wall, accumulation of blood, and hemorrhage in
the cecal lumen.
4.4 Passive immunization against avian coccidiosis using

hyperimmune antibodies
Passive immunization of protective immunity using hyperimmune, parasite-specific

antibodies is an alternative control strategy potentially applicable to intestinal
diseases such as avian coccidiosis. As opposed to pathogen-specific immunity
achieved by active vaccination with live or inactivated microorganisms, or subunits
derived from these pathogens, passive immunization relies on the transfer of
humoral immunity in the form of active antibodies from one individual to another
(Rosenow et al., 1997). Polyclonal antibodies from mammals, such as rabbits and
goats, have been commonly used for passive immunization, but rising concerns over
animal welfare issues are prompting the pharmaceutical industry to explore less
invasive alternatives for producing therapeutic antibodies. In this regard, chicken
hyperimmune egg yolk IgY antibodies offer a practical alternative to mammalian
serum antibodies because of their feasibility for large-scale commercial production
and the relative noninvasive methods used for their preparation (Schade et al., 2005).
Maternal IgY is concentrated in the yolk sac of eggs during embryogenesis, allowing
it to be easily collected, purified, and used to passively immunize mammals. Passive
immunity against Escherichia coli and Salmonella enteritidis infections in pigs, mice,
and cows has been demonstrated using IgY antibodies from eggs of hens immunized
with the respective bacteria (Ikemori et al., 1992; Yokoyama et al., 1992, 1998).
Other studies involving chicken antibody transfers, either experimentally or

maternally, or exogenous administration of mouse monoclonal antibodies, have
established a role for passive immunity in avian coccidiosis. Rose (1974) demonstrated
that injection of chickens with antibodies against E. maxima reduced disease
severity following experimental parasite infection, compared with controls. Crane
et al. (1988) reported passive protection of chickens against E. tenella experimental
infection using a monoclonal antibody produced against E. tenella sporozoites.
Passive transfer of maternal antibodies from hens infected with E. maxima to their
eggs partially protected offspring against E. tenella challenge infection (Smith et
al., 1994). Mouse monoclonal antibodies reactive with a major protein of the E.
tenella oocyst wall reduced fecal oocyst output following experimental infection
with E. tenella or E. maxima when intravenously injected into nonimmune chickens
(Karim et al., 1996). Recently, the protective effect of the IgY fraction of egg yolk from
hens hyperimmunized with Eimeria oocysts was evaluated in young broilers with
experimental coccidiosis (Lee et al., 2009a). Chickens which had been continuously
fed from hatch with a standard diet supplemented with 10% or 20% (wt/wt) of a
freeze-dried egg yolk powder from hens hyperimmunized with E. acervulina

showed significantly increased body weight gain and reduced fecal oocyst shedding
following experimental E. acervulina infection, compared with control birds given
an unsupplemented diet. Still lower doses of egg yolk supplementation (0.01-0.05%)
also reduced fecal oocyst shedding, but with no differences in body weight gain.
Nevertheless, it is encouraging to note reduced oocyst shedding by chickens fed with
the low-dose egg yolk diets, which suggests that this immunization strategy may
prove beneficial for disruption of the infectious cycle of Eimeria parasites in the field.
In another study, the effect of dietary supplementation with Supracox (IASA,

Inc., Puebla, Mexico), a purified IgY fraction derived from egg yolks of hens
hyperimmunized with Eimeria oocysts, on protection against E. tenella and E. maxima
experimental infection was evaluated (Lee et al., 2009b). Feeding chickens with
Supracox significantly improved body weight gain of birds infected with E. tenella
at 0.02% and 0.05% supplementation, as well as birds infected with E. maxima at
0.05% supplementation, compared with chickens given an unsupplemented diet. In
chickens infected with E. tenella, oocyst shedding was reduced in birds fed with the
0.05% IgY-supplemented diet, compared with controls. In chickens infected with
E. maxima, intestinal lesion scores were lower only in birds given 0.05% Supracox,
compared with unsupplemented controls. From a practical perspective, the infection
doses of E. tenella and E. maxima oocysts (4.0 x 104) that were used in these studies
are likely to be considerably higher than the levels that commercial birds are exposed
to in production facilities (Wallach et al., 1995). These promising preliminary
studies provide the impetus for future field investigations to evaluate the effects of
hyperimmune antibodies at low-dose exposures, and for in vivo mechanistic studies
to determine how passively administered antibodies bestow protection against
coccidia challenge infection. Finally, elucidating the parasite components that are
recognized by antigen-specific IgY antibodies in egg yolk will facilitate the discovery
of novel coccidiosis vaccines.
Belli et al. (2004) identified and isolated two E. maxima recombinant gametocytes
proteins, gam56 and gam82, encoding immunodominant components of the
commercial coccidiosis subunit vaccine, CoxAbic (Phibro Animal Health Corp.,
Teaneck, NJ, USA). CoxAbic was shown to induce partial protection against
E. acervulina, E. tenella, and E. maxima infections in chickens hatched from
vaccinated hens (Wallach et al., 2008). However, the strategy using purified egg
yolk IgY antibodies to induce passive immunity against coccidiosis as described
by Lee et al. (2009b) is different from the CoxAbic vaccine in at least two aspects.
First, the IgY antibodies were obtained from eggs produced by hens which had been
hyperimmunized with multiple species of Eimeria oocysts instead of recombinant
proteins from a single parasite species. Thus, the former would be expected to
induce greater cross-species disease protection, compared with the latter. Second,
protective immunity stimulated by the purified IgY antibodies was conferred for as
long as the birds were fed with the dietary supplement, whereas protection induced
by maternally-derived antibodies wanes with time and disappears within 3 weeks
of hatching. Additional advantages of passively-administered IgY antibodies over
active vaccination by live parasites or recombinant subunit vaccines include (1)
the relative ease of use and noninvasive administration method that is applicable
in commercial settings, (2) the comparatively low cost of production given current
egg-laying and yolk preparation technologies, and (3) the ability to quickly
target unique antigenic variants of Eimeria species that may emerge in particular
geographic locations.
Yet another technology of passive immunotherapy against avian coccidiosis relies on

single chain fragments of the antibody variable region (ScFv) that possess antigen
binding activity (Abi-Ghanem et al., 2008; Kim et al., 2001; Min et al., 2001a; Park
et al., 2005; Réfega et al., 2004; Song et al., 2001; Zhao et al., 2010; Zimmermann et
al., 2009). One advantage of ScFv antibodies, compared with intact antibodies, is
their increased ability to penetrate into tissues due to their relatively small molecular
size. We previously produced a ScFv fragment derived from the VH and VL genes
encoding the 6D-12-G10 monoclonal antibody (Kim et al., 2004). This monoclonal
antibody was reactive with a 21-kDa E. acervulina sporozoite protein located
with the apical complex and thought to be involved in parasite binding to a host
cell receptor (Figure 4.5) (Sasai et al., 1996). The 6D-12-G10 antibody was cross-
reactive with E. tenella, E. maxima, E. brunetti, E. mitis, E. necatrix, and E. praecox,
as well as with Toxoplasma, Neospora, and Cryptosporidium (Matsubayashi et al.,
2005; Sasai et al., 1998). Further, 6D-12-G10 competitively inhibited the binding
to and invasion of chicken cells by infectious Eimeria parasites (Sasai et al., 1996).
A recombinant ScFv antibody gene derived from 6D-12-G10 was expressed in
E. coli and this gene product demonstrated antigen-binding activity equal to that
of the original parent monoclonal antibody by Western blot, immunofluorescence,
and enzyme immunoassays (Min et al., 2001b). These properties were similar to
other recombinant ScFv antibodies against Eimeria that have been produced in our
laboratories (Kim et al., 2001; Park et al., 2005; Song et al., 2001), some of which may
prove beneficial in future passive immunization strategies against avian coccidiosis.
Figure 4.5. Immunocytochemical staining of Eimeria tenella sporozoites with the 6D12 mouse
monoclonal antibody that recognizes a parasite apical complex protein suggested being involved
in binding to a host cell receptor. Monoclonal antibody staining of the parasite apical complex is
indicated in yellow. Sporozoites are counterstained in red. Original magnification, 100×.
4.5 Immunomodulation with phytochemicals against avian

coccidiosis
Phytochemicals are nonnutritive, plant-derived chemicals with disease-preventing

properties. Epidemiological evidence indicates that consumption of phytochemicals
reduces the incidence of human cancer and may also decrease infectious diseases,
hypertension, chronic pain, and respiratory diseases such as asthma. Hippocrates (c.
460 BCE – c. 370 BCE), the father of modern medicine, provided the first written
record of powder made from the bark and leaves of the willow tree (containing
acetylsalicylic acid) to treat headaches, pains and fevers. In modern times, one
of the most widely used anticancer drugs is Taxol (paclitaxel), a phytochemical
initially extracted from the Pacific yew (Taxus brevifolia), that possesses antiviral,
antibacterial, and anticancer properties (Wani et al., 1971). A growing body of
scientific evidence has demonstrated that many of the health-promoting activities
of phytochemicals are mediated through their ability to enhance host defense against
microbial infections and tumors (Lillehoj et al., 2011). Plant-derived chemicals
with potent medicinal properties are currently in clinical trials for treatment of a
variety of diseases. For example, lycopene from tomatoes exhibits potent antioxidant
and antiinflammatory properties and is currently in clinical trials for treatment of

cardiovascular diseases and prostate cancer. By contrast, only a limited number of
literature reports have documented the effects of phytochemicals on avian diseases.
The following sections review recent studies demonstrating the phytochemicals that
regulate intestinal immune mechanisms in the chicken and the avian response to
Eimeria infection (Figure 4.6).
O
O
S S
H S S
O O
Cinnamaldehyde Propyl thiosulfinate Propyl thiosulfinate oxide
HO
OH H
N
O
O
Carvacrol Capsaicin
O O
O
HO OH
OCH3 H3CO
Anethole Curcumin
Figure 4.6. Phytochemicals that have been demonstrated to enhance protective immunity against
avian coccidiosis. Cinnamaldehyde is an active component of cinnamon (Cinnamomum
cassia). Propyl thiosulfinate and propyl thiosulfinate oxide are active components of garlic
(Allium sativum). Carvacrol is an active component of oregano (Origanum vulgare) and thyme
(Thymus vulgaris). Capsaicin is an active component of pepper (Capsicum annuum, Capsicum
frutescens, Capsicum chinense, Capsicum pubescens, and Capsicum baccatum). Anethole
is an active component of anise (Pimpinella anisum), star anise (Illicium verum), fennel
(Foeniculum vulgare), and liquorice (Glycyrrhiza glabra). Curcumin is an active component
of turmeric (Curcuma longa).
4.5.1 In vitro studies
The immune-activating properties of medicinal plants have been evaluated in

vitro using avian lymphocytes and macrophages. Methanol extracts of dandelion
(Taraxacum officinale), mustard (Brassica juncea), and safflower (Carthamus
tinctorius) were tested for their effects on chicken lymphocyte proliferation, NO
production, free radical scavenging activity, and tumor cell growth (Lee et al., 2007).
All three extracts inhibited tumor cell growth and exhibited antioxidant effects,
compared with untreated controls. Further, the safflower extract stimulated chicken
lymphocyte proliferation, whereas the extract from mustard induced NO production
by macrophages. In a separate study, organic phase extracts from milk thistle (Silybum
marianum), turmeric (Curcuma longa), reishi mushroom (Ganoderma lucidum), and
shiitake mushroom (Lentinus edodes) were tested for their effects on chicken innate
immunity and tumor cell cytotoxicity (Lee et al., 2010a). In vitro cultures of chicken
spleen lymphocytes with all four extracts stimulated greater cell proliferation,
compared with the untreated controls. Stimulation of macrophages with extracts of
milk thistle and shiitake and reishi mushrooms, but not turmeric, resulted in robust
NO production similar to that induced by chicken IFN-γ, compared with untreated
cells. All extracts also uniformly inhibited the in vitro growth of chicken tumor cells.
Finally, the levels of gene transcripts for IL-1β, IL-6, IL-12, IL-18, and TNFSF15
were increased in chicken macrophages treated with extracts of turmeric or shiitake
mushrooms, compared with untreated controls.
In vitro co-culture of chicken spleen cells with methanol extracts of persimmon

(Diospyros kaki) and tomato (Lycopersicon esculentum) increased cell proliferation,
compared with untreated controls (Lee et al., 2009c). Stimulation of chicken
macrophages with extracts of raspberry (Rubus crataegifolius), but not persimmon
or tomato, heightened NO production by macrophages to a level similar to that
induced by IFN-γ. All fruit extracts also inhibited chicken tumor cell growth,
compared with vehicle controls. Similar effects on chicken spleen cell proliferation,
NO production, and tumor cytotoxicity were reported using a methanol extract
of safflower (Lee et al., 2008a). Cinnamaldehyde ((2E)-3-phenylprop-2-enal) is a
constituent of cinnamon (Cinnamomum cassia), a widely used flavoring compound,
that has been traditionally used to treat human diseases, including dyspepsia,
gastritis, and inflammation. Cinnamaldehyde was reported to possess antioxidant,
antimicrobial, and anticancer activities (Cabello et al., 2009). In vitro stimulation
of chicken spleen lymphocytes with 25 μg/ml of cinnamaldehyde induced greater
cell proliferation, compared with medium controls (Lee et al., 2011a). At 1.2 μg/ml,
cinnamaldehyde activated cultured macrophages to produce higher NO levels, and

at 0.6 μg/ml inhibited the growth of chicken tumor cells, compared with untreated
controls. Cinnamaldehyde also reduced the in vitro viability of E. tenella sporozoites
at 10 μg/ml, compared with medium controls.
The in vitro effects of two organosulfur secondary metabolites of garlic (Allium

sativum), propyl thiosulfinate (PTS) and propyl thiosulfinate oxide (PTSO), on
chicken leukocytes have been reported (Kim et al., 2012a). Garlicon40 (Pancosma
S.A., Geneva, Switzerland) is a commercial product containing 40% of a mixture of
33% (wt/wt) PTS and 67% (wt/wt) PTSO. In in vitro assays, PTS and PTSO dose-
dependently reduced the viability of invasive E. acervulina sporozoites and stimulated
higher chicken spleen cell proliferation, compared with untreated controls. More
recently, treatment of the HD11 chicken macrophage cell line with the PTS/
PTSO mixture at 10 µg/ml decreased the levels of LPS-induced TLR4 transcripts
and reduce LPS-stimulated nuclear factor-κB1/p105 (NF-κB1/p105) transcripts,
but not NF-κB2/p100 transcripts, compared with vehicle controls (H.S. Lillehoj,
unpublished data). NF-κB1/p105 and NF-κB2/p100, which are initially synthesized
as large precursors which undergo proteolysis to generate the mature subunits, p50
and p52, respectively, constitute members of the NF-κB family along with RelA,
RelB, and cRel. Additional support for the effects of garlic compounds on TLRs and
NF-κB was reported by Youn et al. (2008) showing that an ethyl acetate extract of
the plant inhibited LPS-induced TLR4 dimerization and blocked NF-κB activation.
4.5.2 In vivo studies
The effects of plant extracts on poultry innate immunity that have been demonstrated
by in vitro studies also have been shown to protect against Eimeria infections in vivo.
Dietary supplementation of day-old chickens with a 0.1% (wt/wt) methanol extract
of safflower increased the body weight gain of E. acervulina-infected chickens to
a level identical to that of uninfected controls, and reduced fecal oocyst shedding,
compared with animals that were given an unsupplemented diet (Lee et al., 2008b).
Increased spleen lymphocyte proliferation and higher CD4+/CD8+ T cell ratios were
associated with a safflower-supplemented diet. Finally, higher IL-8, IL-15, IL-17
and IFN-γ transcripts levels were seen in intestinal lymphocytes in chickens fed the
safflower-supplemented diet, but not in the untreated controls.
Dietary supplementation of chickens with plum (Prunus salicina) powder increased

protective immunity against experimental avian coccidiosis (Lee et al., 2008c, 2009d).
Plum increased body weight gain, reduced fecal oocyst shedding, and increased the
levels of mRNAs for IL-15 and IFN-γ in gut lymphocytes, compared with untreated
controls. Chickens fed the plum-supplemented diet exhibited greater spleen cell
proliferation indicating that plum can enhance cell-mediated immunity. In another
study, chickens fed a diet supplemented with cinnamaldehyde at 14.4 mg/kg had up
to 47-fold greater levels of gene transcripts encoding IL-1β, IL-6, IL-15 and IFN-γ in
intestinal lymphocytes, compared with chickens given an unsupplemented diet (Lee
et al., 2011a). Cinnamaldehyde-fed chickens had 17% and 42% increased body weight
gains following E. acervulina or E. maxima experimental infections, respectively,
40% reduced E. acervulina oocyst shedding, and 2.2-fold higher E. tenella-stimulated
parasite antibody responses, compared with unsupplemented controls.
The effects of PTS and PTSO on in vivo parameters of chicken gut immunity during
experimental E. acervulina infection have been evaluated. Chickens continuously
fed from hatch with the PTSO/PTS mixture at 10 mg/kg and orally challenged with
live E. acervulina oocysts had increased body weight gain, decreased fecal oocyst
excretion, and greater E. acervulina profilin serum antibody responses, compared
with chickens fed an unsupplemented diet (Kim et al., 2012a). In uninfected chickens,
in vivo dietary supplementation with the PTS/PTSO mixture increased the levels
of transcripts encoding IFN-γ, IL-4, and the antioxidant enzyme, paraoxonase 2,
compared with chickens given an unsupplemented diet (H.S. Lillehoj, unpublished
data). By contrast, transcripts for peroxiredoxin-6 were decreased in the PTS/PTSO-
treated group, compared with controls. In E. acervulina-infected chickens given the
PTS/PTSO-supplemented diet, transcripts for TNFSF15, catalase, and paraoxonase 2
were increased, while those for IL-10 were reduced, compared with unsupplemented
controls.
Anethole, ((E)-1-methoxy-4-(1-propenyl)benzene) is a phenylpropene compound

that occurs as a major component of the essential oils of anise (Pimpinella anisum),
star anise (Illicium verum), fennel (Foeniculum vulgare), and liquorice (Glycyrrhiza
glabra). In mammals, anethole had demonstrable antitumor, antioxidant,
antiinflammatory, and antimicrobial activities (Camurca-Vasconcelos et al., 2007; De
et al., 2002; Freire et al., 2005). At 10 µg/ml, anethole reduced the viability of invasive
E. acervulina sporozoites after 2 or 4 hr of treatment by 45% and 42%, respectively,
and stimulated 6.0-fold greater chicken spleen cell proliferation, compared with
untreated controls (Kim et al., 2013). Chickens continuously fed from hatch with
an anethole-supplemented diet at 15 mg/kg and orally challenged with live E.
acervulina oocysts had increased body weight gain, decreased fecal oocyst excretion,
and greater profilin serum antibody responses, compared with infected chickens
given an unsupplemented diet. The levels of transcripts encoding IL-6, IL-8, IL-10,
and TNFSF15 in intestinal lymphocytes were increased in parasite-infected chickens
given the anethole-containing diet, compared with unsupplemented controls.
Phytochemicals may exert synergistic effects against experimental avian coccidiosis.

Dietary supplementation of chickens with a mixture of C. longa, Capsicum annuum
(pepper), and L. edodes had improved body weight gains, reduced fecal oocyst
shedding, and higher serum antibody titers against profilin following challenge
infection with E. acervulina, compared with birds given an unsupplemented diet
or a diet containing Capsicum plus Lentinus alone (Lee et al., 2010b). The levels of
transcripts for IL-1β, IL-6, IL-15, and IFN-γ in gut lymphocytes also were greater
in the Curcuma/Capsicum/Lentinus-fed group, compared with the standard diet,
Curcuma only, or Capsicum/Lentinus only groups. In a follow-up report, feeding
of chickens with a combination of carvacrol (5-isopropyl-2-methylphenol), an
active component of oregano (Origanum vulgare) and thyme (Thymus vulgaris),
cinnamaldehyde, and Capsicum oleoresin (a mixture of essential oils and resins),
or with Capsicum oleoresin plus Curcuma oleoresin, increased protective immunity
against experimental E. tenella infection following immunization with profilin,
compared with untreated and immunized controls (Lee et al., 2011b). Birds fed with
either supplemented diet had increased body weight gain, greater profilin antibody
levels, and/or greater lymphocyte proliferation, compared with unsupplemented
controls. Immunized chickens fed the carvacrol/cinnamaldehyde/Capsicum-
supplemented diet had increased numbers of macrophages in the intestine, while
those given the Capsicum/Curcuma oleoresin-supplemented diet had increased
numbers of intestinal T cells, compared with untreated controls.
4.5.3 Genomic approaches to delineate molecular mechanisms of phytonutrient

action
While numerous studies have shown disease prevention or immune enhancing effects
of phytochemicals, few reports have examined the underlying mechanisms that are
involved. Some phytochemicals inhibit innate immune response by targeting PPRs
or their downstream signaling molecules. For example, in mice cinnamaldehyde and
curcumin blocked TLR4 receptor dimerization, while resveratrol, a phenylpropanoid
produced by some plants in response to pathogen infection, inhibited TLR3 and
TLR4 signaling by targeting the TANK-binding kinase-1 and receptor interacting
protein-1 in the TRIF complex (Zhao et al., 2011). In chickens, the effects of carvacrol,
cinnamaldehyde, and Capsicum oleoresin on the regulation of the expression of

genes associated with immunology, physiology, and metabolism were investigated
using high-throughput microarray analysis (Kim et al., 2010). These studies revealed
that Capsicum oleoresin stimulated the greatest number of gene changes, compared
with unsupplemented controls, and many of the altered genes were associated with
metabolism and immunity. The most reliable genetic network induced by dietary
cinnamaldehyde treatment was related to the functions of antigen presentation,
humoral immunity, and inflammatory disease. Further, dietary supplementation
with these three phytochemicals was associated with increased protective immunity
following live E. acervulina challenge infection based on increased body weight
gain and reduced parasite fecal shedding, compared with unsupplemented controls.
Further studies to delineate the intestinal immune pathways affected by phytochemical
feeding were conducted by mRNA microarray hybridization (Kim et al., 2010). When
compared with chickens fed an unsupplemented diet, carvacrol-fed chickens showed
altered levels of 74 gene transcripts in gut lymphocytes (26 increased, 48 decreased),
cinnamaldehyde supplementation was associated with altered levels of 62 mRNAs (31
increased, 31 decreased), and Capsicum oleoresin-fed chickens had altered levels of
254 mRNAs (98 increased, 156 decreased), compared with unsupplemented controls.
Among the transcripts that showed greater than two-fold altered expression levels,
most were encoded by genes associated with metabolic pathways. In the case of
Capsicum oleoresin, these included pathways for lipid metabolism, small molecule
biochemistry, and cancer. In another investigation, global gene expression analysis by
microarray hybridization identified 1,810 transcripts (677 increased, 1,133 decreased)
whose levels were significantly altered in intestinal lymphocytes of anethole-fed
birds, compared with unsupplemented controls (Kim et al., 2013). From these, 576
corresponding genes were identified that were related to the inflammatory response.
The transcriptome-modifying properties an organic extract of C. longa were

evaluated in chickens experimentally infected with E. tenella or E. maxima (Kim et
al., 2012b). Differential gene expression by microarray hybridization identified 601
altered transcripts (287 increased, 314 decreased) in gut lymphocytes of C. longa-fed
birds, compared with unsupplemented controls. Based on the known functions of the
corresponding mammalian genes, the C. longa-altered intestinal transcriptome was
consistent with an antiinflammatory effect in the gut. A similar analysis was reported
for the garlic metabolites, PTS and PTSO (Kim et al., 2012a). In that study, 1,227
transcripts (552 increased, 675 decreased) were identified in intestinal lymphocytes
whose levels were significantly altered of PTS/PTSO-fed birds, compared with

unsupplemented controls. Many of these transcripts were encoded by genes related
to innate immunity, including TLR3, TLR5, and NF-κB.
4.6 Novel immunization strategies against avian coccidiosis
Given that chickens infected with Eimeria develop protective immunity against
reinfection by the homologous parasite, immunization with parasite-derived
vaccines represents a viable method to control coccidiosis (Lillehoj et al., 2000).
Several different live or attenuated parasite vaccines are commercially available
that contain multiple Eimeria species (Table 4.1). However, these vaccines may not
necessarily be effective against antigenic variants of the parasites that may develop
under local, geographically-restricted selection pressures that are not present in
existing formulations. Recombinant DNA or protein vaccines derived from parasite
genes or antigens that are shared by multiple coccidia species have shown limited
success, mainly attributed to their low antigenicity and/or restricted expression
during the parasite life cycle (Ding et al., 2004; Lillehoj et al., 2005a). Each stage of
parasite development is associated with a unique pattern of gene expression, and not
all Eimeria proteins are expressed in all stages. Further, although some progress has
been made in developing subunit vaccines against coccidiosis, limited information
on the immunobiology of host-parasite interactions, the relative lack of Eimeria
genome information, and the complex life cycle stages of coccidia hinders further
vaccine development against avian coccidiosis. In particular, identifying the antigenic
components of Eimeria that are relevant to the development of protective immunity
has been difficult in the absence of defined coccidia genetic knock-out strains. Other
problems have included the lack of immunoassays to identify vaccine candidates,
a comparative absence of information on Eimeria genetics, and the absence of
suitable model systems to analyze the chicken immune response. All of these issues
have been discussed in detail in prior reviews that may be consulted for further
information (Blake et al., 2006; Dalloul and Lillehoj, 2006; Innes and Vermeulen,
2006; McDonald and Shirley, 2009; Peek and Landman, 2011; Sharman et al., 2010;
Shirley and Lillehoj, 2012; Shirley et al., 2007; Wallach, 2010). The following sections
specifically focus on novel adjuvants and dendritic cell vaccines to induce antigen-
specific protective immunity against avian coccidiosis.
Table 4.1. Anticoccidial vaccines currently used or being registered for use in chickens.
Vaccine Manufacturer Eimeria Attenuation Bird type Route Country of

species1 derivation
Coccivac®-D Schering Ea, Et, Em, Eb, Nonattenuated Breeders/ Spray or USA
Plough Eh, Emi, En, Ep layers oral
Coccivac®-B Schering Ea, Et, Em, Emi Nonattenuated Broilers Oral USA
Plough
ADVENT® Novus Ea, Et, Em Nonattenuated Broilers Oral USA
International
Inovocox Embrex/Pfizer Ea, Et, Em Nonattenuated Broilers In ovo USA
injection
Nobilis® Intervet Ea, Et, Em Nonattenuated Broilers Spray or The
COX-ATM International oral Netherlands
Livacox® Q Biopharm Ea, Et, Em, En Attenuated Breeders/ Oral Czech
layers Republic
Livacox® T Biopharm Ea, Et, Em Attenuated Breeders Oral Czech
Republic
Paracox® Schering Ea, Et, Em, Eb, Attenuated Breeders/ Oral UK
Plough Eh, Emi, En, Ep layers
Paracox® 5 Schering Ea, Et, Em, Emi Attenuated Breeders Oral UK
Plough
Immucox® for Vetech Ea, Et, Em, En Nonattenuated Breeders/ Water or Canada
chickens 1 Laboratories layers gel
Immucox® for Vetech Ea, Et, Em, En Nonattenuated Breeders/ Water or Canada
chickens 2 Laboratories layers gel
CoxAbic Phibro Animal Em Em gametocytes Breeders Intra- USA
Health muscular
Corporation
Supercox Qilu Animal Ea, Et, Em Attenuated Broilers Oral China
Phamaceutical
1 Ea: Eimeria acervulina; Et: Eimeria tenella; Em: Eimeria maxima; Eb: Eimeria brunetti; Eh: Eimeria hagani;
Emi: Eimeria mitis; En: Eimeria necatrix; Ep: Eimeria praecox.
4.6.1 Adjuvants that increase the immunogenicity of avian coccidiosis vaccines
Considerable progress has recently been made on the preparation of novel adjuvants
that augment the immunogenicity of protein vaccines, although little information
is available on their use in poultry. Since their first use as immune enhancers
almost 90 years ago (Ramon, 1925), many different types of chemical compounds
and formulations have been demonstrated to be effective in augmenting humoral
and cell-mediated immune responses (Bowersock and Martin, 1999; Gupta et al.,
1995; Newman and Powell, 1995). Among the most frequently used adjuvants for
human and veterinary vaccines are aluminium salts (alum) and oil-based emulsions.
Alum has been incorporated into several human vaccines and is the only adjuvant
approved in the United States. While the exact mechanism of action of alum remains
to be completely understood, physical binding to antigens, retention of antigens at
injection sites, and antigen delivery to lymph nodes are known to play contributing
roles (Kool et al., 2012).
The Montanide ISA series of adjuvants (Seppic, Inc., Puteaux, France) have shown
superior efficacy with a variety of human and animal vaccines (Aucouturier
et al., 2006; Cox et al., 2003). ISA adjuvants are based on mineral oil, other oils,
or a mixture of both, as well as specific surfactant chemistry based on mannitol
oleate. ISA adjuvants may be used to manufacture water-in-oil (W/O), oil-in-water
(O/W), or W/O/W double emulsions. In a recent study, the effectiveness of four
Montanide adjuvants (ISA 70, ISA 71, ISA 201, and ISA 206) in combination with
recombinant profilin antigen vaccination against avian coccidiosis was investigated
(Jang et al., 2010). ISA 70 and ISA 71 are W/O emulsions, while ISA 201 and ISA
206 are W/O/W emulsions. Whereas Eimeria profilin was highly immunogenic and
stimulated protective immunity against experimental avian coccidiosis, profilin
immunization in the absence of adjuvant failed to completely arrest parasite growth
in infected chickens, and fecal oocyst shedding remained detectable, albeit at a
reduced level, in vaccinated birds, compared with unimmunized controls (Ding et
al., 2004; Lillehoj et al., 2005a,b; Ma et al., 2011, 2012; Min et al., 2001a; Song et al.,
2000; Xu et al., 2006). Chickens immunized with profilin plus ISA 70 or ISA 71 had
increased body weight gain, compared with vaccination with profilin alone following
experimental infection with E. acervulina. Profilin plus ISA 71 also reduced fecal
oocyst shedding compared with vaccination in the absence of adjuvant. All adjuvants
tested enhanced profilin serum antibody titers. Increased levels of gene transcripts
encoding IL-2, IL-10, IL-17A, and IFN-γ, but decreased levels of IL-15 mRNAs, were
seen in intestinal lymphocytes of chickens immunized with profilin plus all four
ISA adjuvants, compared with immunization with profilin alone. Finally, increased
infiltration of CD8+ lymphocytes at the site of immunization was observed in birds
given profilin plus ISA 71, compared with profilin alone. In a subsequent study, the
adjuvanticity of ISA 71 in combination with profilin vaccination was extended to
chickens infected with E. tenella (Jang et al., 2011a).
While oil-based adjuvants are effective at increasing protein subunit immunogenicity

under experimental conditions, vaccine delivery to mucosal surfaces under field
conditions is generally more effective using aqueous solutions. Montanide IMS
1313 (Seppic Inc.) is an aqueous-based nanoparticle adjuvant which has been
shown to increase protective immunity against infectious diseases in veterinary
applications (Magyar et al., 2008). IMS 1313 in combination with Eimeria profilin
protein augmented protective immunity in chickens against infection by multiple
species of Eimeria (Jang et al., 2011b). Day-old broilers were immunized twice
with profilin emulsified in either (1) IMS 1313 adjuvant via oral, nasal, or ocular
routes; (2) ISA 71 adjuvant via the subcutaneous route; or (3) complete Freund’s
adjuvant (CFA) via the subcutaneous route, and orally challenged with virulent
E. acervulina parasites. Chickens orally immunized with profilin plus IMS 1313, or
subcutaneously immunized with profilin plus ISA 71, had increased body weight
gain, compared with animals nasally or ocularly immunized with profilin plus IMS
1313, or subcutaneously immunized with profilin plus CFA. Compared with animals
vaccinated with profilin plus CFA, chickens immunized with profilin plus IMS 1313
or ISA 71 had higher post-infection intestinal levels of profilin-reactive IgY and
secretary IgA antibodies. Interestingly, immunization with profilin plus ISA 71 was
consistently better than profilin plus IMS 1313 or profilin plus CFA for increasing
the percentages of CD4+, CD8+, αβ-TCR+, and γδ-TCR+ intestinal lymphocytes.
To better define molecular and cellular pathways responsible for increased protection
against avian coccidiosis following profilin immunization in the presence of the ISA
70 and ISA 71 adjuvants, comparative microarray hybridizations were performed
(Jang et al., 2013). Vaccination with profilin plus ISA 70 vs profilin alone altered the
levels of more total transcripts compared with profilin plus ISA 71 vs profilin alone
(509 vs 296). However, the profilin plus ISA 71 vs profilin comparison was associated
with a greater number of unique genes and a larger number of unique biological
functions, compared with the profilin plus ISA 70 vs profilin comparison. Follow-up
in vivo disease protection studies demonstrated that vaccination with profilin plus
ISA 71 was associated with greater body weight gain following E. acervulina infection,
and decreased parasite fecal shedding after E. maxima infection, compared with
immunization with profilin alone. Further, serum antibody titers against profilin
were greater in E. tenella- or E. maxima-infected chickens immunized with profilin
plus ISA 71, compared with profilin alone. Finally, the levels of IL-2, IL-10, IL-17A,
and IFN-γ gene transcripts in gut lymphocytes were augmented following infection
with E. acervulina, E. tenella, or E. maxima in chickens that had been vaccinated
with profilin plus ISA 71, compared with profilin alone. Taken together, these results
suggest that vaccination with profilin plus ISA 71 augments protective immunity
against experimental avian coccidiosis.
A new adjuvant complex comprising a mixture of a Quil A, cholesterol,

dimethyldioctadecylammonium bromide (DDA), and an acrylic acid polymer
(Carbopol) (QCDC) has been shown to enhance immune responses when used
in conjunction with multiple pathogen vaccines (Dominowski et al., 2009). Many
veterinary vaccines currently in use contain Quil A, a purified saponin fraction
that is derived from the bark of the soap bark tree, Quillaja saponaria (Sun et al.,
2009a). When combined with cholesterol and phospholipids, Quil A, forms an
immunostimulatory complex (ISCOM) that avoids some of the deleterious effects of
Quil A alone (Ozel et al., 1989; Sun et al., 2009b). Addition of DDA to this complex
allows the incorporation of highly hydrophilic proteins and enhances cell-mediated
and humoral Th1-type immune responses (Gall, 1966; Hilgers and Snippe, 1992).
DDA by itself possesses immunostimulatory properties during vaccination against
infectious pathogens, including Eimeria infection of chickens (Lillehoj et al., 1993).
Further addition of a polymer, such as Carbopol, improves the solubility of DDA,
thereby making the final formulation, QCDC, a highly effective adjuvant.
The adjuvant effect of QCDC on profilin-induced protective immunity against avian

coccidiosis was investigated (Lee et al., 2010c). Chickens were immunized with 100
µg of Eimeria profilin protein emulsified in 50 µl of QCDC (12.0 μg/ml of Quil A, 12.0
μg/ml of cholesterol, 0.6 μg/ml of DDA, and 0.75 mg/ml of Carbopol 974P) at 1 and
7 days post-hatch and orally challenged with live E. acervulina at day 14. Chickens
immunized with profilin plus QCDC had increased body weight gain, decreased gut
lesion score, greater profilin serum antibody titer, higher antigen-induced peripheral
blood lymphocyte proliferation, and elevated levels of gut lymphocyte transcripts
for IL-10 and IL-17A, compared with chickens given profilin alone. However, fecal
oocyst shedding in the profilin plus QCDC and profilin alone groups were identical,
and both groups had uniformly increased levels of intestinal lymphocyte transcripts
encoding IL-1β, IL-10, IL-12, IL-15, IL-17A, and IFN-γ. In a succeeding report,
the immunoenhancing effects of the QCDC adjuvant were extended to chicken
embryos vaccinated with profilin and experimentally infected with E. maxima with
comparable effects that were observed in the E. acervulina-infected animals (Lee et
al., 2010d).
Two follow-up studies were performed to evaluate modifications of the QCDC

formulation in an attempt to further increase its adjuvanticity during profilin
vaccination. In the first study, the effects of incorporation of 150 µg/ml of Bay
R1005 into the QCDC adjuvant were investigated (Kim et al., 2012c). Bay R1005
is a synthetic glycolipid analogue, which enables the new adjuvant formulation
(QCDCR) to stimulate both Th1-and Th2-type immunity. Vaccination of chickens
with profilin plus QCDCR reduced intestinal lesion score and increased mitogen-
induced lymphocyte proliferation in chickens infected with E. acervulina, compared
with immunization with profilin alone or with profilin plus QCDC. Immunization
with profilin plus QCDC, or profilin plus QCDCR, increased body weight gain, but
had no effect on fecal oocyst shedding, of infected chickens, compared with birds
vaccinated with profilin alone. Global gene expression analysis by mRNA microarray
hybridization was performed with intestinal lymphocytes from uninfected chickens
to identify the molecular pathways affected by the QCDC and QCDCR adjuvants.
Compared with chickens immunized with profilin alone, chickens given profilin
plus QCDC had 164 altered transcript levels (60 increased, 104 decreased), while
chickens immunized with profilin plus QCDCR had 233 altered transcripts (103
increased, 130 decreased). Compared with chickens vaccinated with profilin plus
QCDC, chickens vaccinated with profilin plus QCDCR had 397 altered transcripts
(193 increased, 204 decreased). Biological function and network analyses revealed
that not only were the majority of altered transcripts encoded by immune-related
genes, but also that immunization with profilin plus QCDCR regulated more
Th2-related genes, compared with profilin plus QCDC.
In the second report, the QCDCR adjuvant was further modified by addition of
10 µg/ml of cytosine-phosphate-guanosine oligodeoxynucleotides (CpG ODN) to
generate the QCDCRT formulation (Lee et al., 2012). Chickens were unimmunized
(control group), or were immunized with profilin alone, profilin plus QCDC, or
profilin plus QCDCRT at 2 and 9 days post-hatch and infected with E. acervulina
at day 16. Compared with unimmunized controls, or with the profilin alone or
profilin plus QCDC groups, the profilin plus QCDCRT group had greater body
weight gain, decreased intestinal lesion score, higher profilin serum antibody titer,
and increased ratios of CD4+/CD8+ and γδ-TCR+/αβ-TCR+ splenocytes following
parasite infection. Future studies to identify particular genes that are activated by
QCDCRT will contribute to a better understanding of the molecular mechanisms of

action of this next-generation adjuvant formulation in the chicken immune system.
Taken collectively, the results of all of these studies suggest that the QCDC adjuvant,
as well as its Bay R1005 and CpG ODN derivatives, may prove beneficial for field
vaccination against avian coccidiosis using Eimeria protein subunit vaccines.
4.6.2 Dendritic cell-derived exosome vaccine against avian coccidiosis
A novel method of vaccinating chickens using parasite antigen-loaded dendritic cells

(DCs) and DC-derived exosomes has been developed. DCs are professional antigen-
presenting cells that regulate the induction and outcome of the immune response.
Mammalian DCs produce antigen-specific cellular and humoral immune responses
by secretion of exosomes, membrane vesicles that are released extracellularly
following fusion of late endosomes with the plasma membrane (Pant et al., 2012).
In mammals, exosomes have been shown to be enriched in molecules involved in
antigen presentation and to stimulate antigen-specific T cells (Viaud et al., 2010).
Given that the major protective immune response which controls avian coccidiosis
primarily relies on cell-mediated immunity, DC-derived exosome vaccines are
promising vehicles for activating parasite-specific effector T cells in chickens. The
methods to isolate avian intestinal DCs, and to load them ex vivo with soluble
Eimeria antigens, have been reported (Del Cacho et al., 2008, 2009). A combination
of cell panning, density gradient centrifugation, and magnetic cell negative selection
produced viable and functional CD45+ DC's from the cecal tonsils from chickens
infected with E. tenella. The isolated DCs expressed on their surface MHC class I and
class II molecules, IgG, IgM, complement factors C3 and B, ICAM-1, and VCAM-1,
but lacked cell surface markers characteristic of macrophages, T cells, and B cells.
Co-culture of the purified chicken intestinal DCs with allogeneic naïve CD4+ T cells
increased proliferation and IFN-γ secretion by the T cells, while co-culture with
allogeneic or autologous B cells increased cell proliferation and immunoglobulin
production.
Intestinal DCs from E. tenella-infected chickens were loaded ex vivo with crude
sporozoites antigens and their extracellular exosomes were purified (Del Cacho et
al., 2011). Chickens immunized with either the parasite antigen-pulsed DCs, or
with their purified exosomes, revealed antigen-staining cells diffusely located in
the lymphoid tissues, with highest concentrations found in the germinal centers of
the intestinal cecal tonsils and spleen. Functionally, DC- and exosome-vaccinated
chickens had elevated numbers of antigen-specific B cells expressing IgG or IgA in
the cecal tonsils and spleen, larger numbers of lymphocytes secreting IL-2, IL-16,
and IFN-γ, and higher proliferation of antigen-specific lymphocytes, compared with
chickens immunized with Eimeria antigen alone. In a following study, an in vivo
vaccination trial was conducted to evaluate the efficacy of parasite antigen-loaded
DCs and DC-derived exosomes on live E. tenella challenge infection. Increased body
weight gain, decreased fecal oocyst shedding, reduced intestinal lesion score, and
lowered mortality were observed in DC- or exosome-vaccinated chickens compared
with animals given parasite antigen alone. Finally, the ability of chicken DCs and
their exosomes to stimulate protective immunity against multiple Eimeria species
was evaluated (Del Cacho et al., 2012). Chicken intestinal DCs were isolated, pulsed
ex vivo with sporozoite antigens from E. acervulina, E. tenella, and E. maxima, and
their purified exosomes were immunized into chickens prior to infection with the
three denoted parasites. Intestinal cecal tonsils and Peyer’s patches of immunized
and infected chickens had greater numbers of cells secreting IL-2, IL-16, and IFN-γ,
higher parasite antigen-stimulated T cell proliferative responses, and increased
numbers of antigen-reactive IgG- and IgA-producing B cells, compared with
unimmunized and infected chickens. In contrast, the numbers of IL-4- and IL-10-
secreting cells were diminished in the immunized and infected chickens, compared
with the unimmunized and infected controls. Chickens immunized with exosomes
that had been loaded with E. acervulina, E. tenella, and E. maxima antigens and
infected in vivo with all three Eimeria species had greater body weight gain, reduced
fecal oocyst shedding, diminished intestinal lesion score, and lower mortality
compared with the unimmunized and infected controls.
In mammals, immunization with antigen-loaded DCs or their exosomes induced

an antigen-specific Th1 immune response, which was characterized by increased
secretion of IL-2, IFN-γ, IgG, and IgA (André et al., 2004; Schnitzer et al., 2010).
Similarly, DC- or exosome-immunized chickens had a preferential Th1 response
characterized by enhanced IL-2 and IFN-γ production (Del Cacho et al., 2012).
Interestingly, greater numbers of antibody- and cytokine-secreting cells and
increased cell proliferation were observed in the cecal tonsils of DC- or exosome-
immunized chickens, compared with the spleen. Given that the cecal tonsils play a
major role in protection against E. tenella infection in vivo, this finding is consistent
with the previous report by Aline et al. (2004) that demonstrated a high percentage of
exosomes migrating to the intestine and lymph nodes after exosome administration
in T. gondii-infected mice. Unlike mammals, chickens do not possess encapsulated
lymph nodes (Olah and Glick, 1985), raising the possibility that primary antigen
processing may occur in the mucosal-associated lymphoid tissues of the avian gut.

Nevertheless, these combined results suggest that it may someday be possible to

produce a DC-derived, exosome-based avian coccidiosis vaccine for immunization
of commercial chickens.
4.7 Conclusions
An increasing body of literature documents the effectiveness of antibiotic-free methods

for reducing bacterial, viral, and parasitic pathogens during food animal production.
Those approaches specifically related to hyperimmune IgY, phytochemicals, vaccine
adjuvants, and DC exosomes for controlling avian coccidiosis have been reviewed
in this chapter. Many of these alternative strategies, and others not mentioned here,
have the potential to be applied to infectious pathogens beyond the Eimeria species
infecting chickens. However, there remains a need to better understand the underlying
immune mechanisms through which these nontraditional approaches operate. As the
demand for animal food products increases to meet the nutritional needs of a growing
world population, it will become ever more imperative to develop and implement
these environmentally sustainable, antibiotic-free strategies to prevent and control
microbial diseases of livestock and poultry.
Acknowledgements
This work has been supported by past and current Trust agreements of the Agricultural
Research Service, United States Department of Agriculture with IASA, Inc., Puebla,
Mexico, Pancosma, S.A., Geneva, Switzerland, and Seppic, Inc., Puteaux, France.
The authors express sincere appreciation to all previous and current collaborators
and colleagues who have contributed to the studies described herein, particularly
Dr. D.K. Kim, Ms. M. Nichols, Ms. S. O’Donnell, Ms. A. Cox, Ms. M.S. Park and
Ms. M. Jeong.
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Chapter 5: Intestinal health in carnivores
E.A. Hagen-Plantinga1* and W.H. Hendriks1,2
1Faculty of Veterinary Medicine, Utrecht University, P.O. Box 80.151, 3508 TD,
Utrecht, the Netherlands; 2Animal Nutrition Group, Wageningen University, P.O.

Box 338, 6700 AH Wageningen, the Netherlands; e.a.plantinga@uu.nl
Abstract
The knowledge on the influence of gastro-intestinal (GI) microbiota on the health

status of humans and animals is rapidly expanding. A balanced microbiome
may provide multiple benefits to the host, like triggering and stimulation of the
immune system, acting as a barrier against possible pathogenic micro-organism,
and providing energy and nutritional support. Both culturing methods and more
modern molecular techniques have provided valuable insights in gut microbiology
of the dog and cat. The major bacterial phyla seem to be similar to those found
in other species, with Firmicutes, Bacteroidetes, Proteobacteria, Fusobacteria, and
Actinobacteria constituting more than 99% of all gut microbiota. However, the
microbiota composition seems to differ substantially on a species/strain level, with
much inter-individual variation. Also, studies with diseased and susceptible subjects
showed clear alterations in gut microbiome, with a reduced richness of species
and dysbiosis as the most commonly found deviations. Several nutritional studies
have demonstrated that modulation of canine and feline gut microbiota may occur
when the amounts of soluble fibres and macronutrients in the diet are changed.
Interestingly, feeding a high protein, low carbohydrate diet to dogs and cats showed
clear shifts in bacterial strains, which are normally associated with negative health
effects in herbivorous and omnivorous mammals. However, no adverse effects of
these bacterial shifts could be noticed in the dog and cat studies. The latter may
indicate that species differences are indeed present, possibly driven by nutritional
strategies during evolution. Further research is warranted to more thoroughly
unravel the mystery of the gut microbiome in general, and that in the carnivorous
dog and cat in particular.
Keywords: dogs, cats, nutrition, microbiome

5.1 Introduction
Currently, there is much interest in supporting intestinal health through nutrition,

not only in production animals, but also in companion animals. It is generally
acknowledged that dietary components or factors affecting gut health will
consequently have a significant influence on the health status and subsequently
wellbeing of the animal as a whole.
The intestinal microbiota comprises all micro-organisms (i.e. bacteria, protozoa,

fungi and viruses) that reside in the gastro-intestinal (GI) tract. Modern molecular
techniques like gene sequencing methods and Fluorescent In Situ Hybridisation
(FISH) have shown that the intestinal microbiota is highly diverse, harbouring several
hundreds of phylotypes (Handl et al., 2011; Suchodolski et al., 2008). The resident
microorganisms can provide many health benefits to the host. The microbiota
utilize dietary and endogenous derived nutrients, thereby aiding in the digestive
process and, in return, provide energy and nutritional support for enterocytes.
Furthermore, the presence of microbiota in the intestines trigger the development
and stimulation of the immune system. Last but not least, gut microbes act as a
barrier against opportunistic pathogens, thereby reducing susceptibility to disease
(Rastall and Maitin, 2002). Gut microbiota composition may vary greatly among
species (Furet et al., 2009) and even among individuals (Ritchie et al., 2010). This
makes assessment of gut health even more challenging. Consequently, measures to
improve gut functioning in herbivorous or omnivorous animals may not necessarily
be effective in carnivorous species such as dogs and cats. A thorough understanding
of ‘normal’ gut functioning in the species of interest is necessary to be able to
develop effective measures to control derailment of the GI system and to improve
gut functioning in that species.
Gut functioning is highly complex, and comprises factors such as gut structure
and integrity, balance of microbiota, immune status and their interactions. These
interactions may, in turn, lead to changes in gene expression, and even endocrine
regulation, which may affect nutrient handling and utilization, organ development,
tissue growth and immune system maturation (Hooper and Gordon, 2001). It needs
to be noted, however, that, although gut health is a major topic in today’s research,
a sound scientific description of ‘gut health’ is lacking and subsequent measures to
define health status of the gut are inconclusive (Bischoff, 2011).

5. Intestinal health in carnivores
The present chapter summarizes our current knowledge on ‘gut health’ and microbiota
composition in carnivores, using data of dog and cats studies, and comparing these
data with other species. Particular emphasis is paid on describing the link between
gut microbiota and nutrition in carnivores.
5.2 Defining ‘gut health’
In order to be able to assess gut health, it is of importance to have a sound definition

of this term. In human medicine, a German scientific committee working on gut
health issues has designated five criteria that might form the basis for an objective
definition of gut health (Table 5.1). These five criteria were further described as to
specific signs that may be monitored to be able to assess normal gut functioning.
Although the criteria formulated by this scientific committee are proposed for human
gut health, these principles may be used to describe gut health in (carnivorous)
animals like dogs and cats as well. However, some criteria cannot be assessed in
animals, as the associated signs are subjective and can only be monitored by
questioning the subject/individual, although behavioural observations may provide
some indication of these criteria.
As can be concluded from Table 5.1, gut health comprises different criteria. First of
all, it needs to be assessed if the gut is functioning properly in terms of digestion
and absorption. In dogs and cats, quality and frequency of the stools, as well as
absence of vomiting, a good appetite, and absence of nutrient deficiency signs
may be helpful in assessing effective digestion and absorption of foods. Absence
of GI illness may be assessed by owner questionnaires regarding stool quality
and frequency, vomiting, diarrhoea, behaviour in terms of food intake, etc., but
its absence can only be ascertained by diagnostic techniques (e.g. endoscopy,
biopsies, complete blood count). To assess an effective immune status, an increasing
number of immunological parameters are available, among which cell counts and
phenotyping, immunohistology, quantification of antibodies and cytokines are the
most well-known. It needs to be noted, however, that sound reference values for
these immunological parameters for dogs and cats may pose a problem.
As for the criterion ‘normal and stable intestinal microbiota’, this may be more difficult
to determine, as gut microbiota composition may vary greatly among individual
dogs and cats (Richie et al., 2010; Suchodolski et al., 2008). Because of a lack of

Table 5.1. Five criteria of gut health and their specific gastrointestinal (GI) signs (Bischoff, 2011).
Major criteria healthy Specific signs GI health

GI functioning
Effective digestion and Normal nutritional status and effective absorption of food, water and
absorption of food minerals
Regular bowel movement, normal transit time and no abdominal pain
Normal stool consistency and rare nausea, vomiting, diarrhoea,
constipation and bloating
Absence of GI illness No acid peptic disease, reflux, or other GI disease
No enzyme deficiencies or carbohydrate intolerances
No IBD, coeliac disease or other inflammatory state
No colorectal or other GI cancer
Normal and stable No bacterial overgrowth
intestinal microbiota Normal composition and vitality of gut microbiome
No GI infections or antibiotics associated diarrhoea
Effective immune status Effective GI barrier function, normal mucus production and no
enhanced bacterial translocation
Normal levels of IgA, normal numbers and activity of immune cells
Immune tolerance and no allergy or mucosal hypersensitivity
Status of well-being Normal quality of life
‘Chi’, or positive gut feeling
Balanced serotonin production and normal function of the enteric
nervous system
reference data, modern molecular techniques, like gene sequencing methods and
FISH, do not yet allow the definition of what can be considered normal or optimal
(Bischoff, 2011). In addition, although modern-day molecular techniques are useful
in characterizing the intestinal microbiome, they have their limitations. Because of
the high bacterial diversity in the gut, groups with a low abundance constitute such
a low proportion of total bacteria that they may escape identification, even when
high through-put sequencing techniques using broad range primers are employed
(Suchodolski, 2011). Also, the use of different DNA extraction methods and PCR
primers will yield slightly different results between studies. At present, no optimal
identification method exists for accurate characterization of all micro-organisms,

making routine individual assessment of a ‘normal’ microbiota nearly impossible.

However, signs like bacterial overgrowth, abnormal stool consistency, and abnormal
transit time may be useful to determine if derailment of microbiota may be present.
Given the importance of a balanced intestinal microbiome in relation to gut health,

the next paragraphs deals with our current knowledge regarding intestinal microbes
in the carnivorous dog and cat and the influence of nutrition on gut microbiota in
these species.
5.3 Intestinal microbiota of carnivores
In dogs and cats, much of the information presently available in the scientific
literature on composition of intestinal microbiota is derived from traditional
culture-based studies (Table 5.2). These studies have provided fundamental insights
into the basic GI ecology of dogs and cats. For example, it was shown that in dogs
and cats, as in other species, an increase in microbiota abundance was seen along
the GI tract, progressing from stomach to colon (Buddington, 2003; Davis et al.,
1977). Anaerobic bacteria predominate the distal part of the GI tract, while a more
equal distribution of aerobic vs. anaerobic can be found in the more proximal
parts of the gut (Mentula et al., 2005). Bacteroides, Clostridium, Lactobacillus,
Bifidobacterium spp. and Enterobacteriaceae were found to be the predominant
culturable bacterial groups in the canine and feline gut (Table 5.2). One culture
based study in cats (Johnston et al., 2001) suggested that the small intestine of
cats harbours relatively high numbers of total bacteria, with a higher portion of
obligate anaerobics compared to humans and dogs. This finding suggests that small
intestinal bacterial overgrowth (SIBO), which is found commonly in humans and
is associated with GI disease, is not a common clinical syndrome in cats. Studies
in dogs initially defined SIBO on the same numerical criteria as humans (bacterial
counts >105 colony forming units (cfu)/g or ml for aerobes and >104 cfu/g or ml
for anaerobes) (Rutgers et al., 1995). However, research showed that in healthy dogs
the bacterial counts exceed the proposed value for humans (German et al., 2003),
making human definitions for SIBO not useful to describe this clinical symptom
in dogs.

Table 5.2. Predominant microorganisms identified in digesta of the canine and feline
gastrointestinal tract based on cultured results.1
Location Bacterial group Log cfu/g (range) dogs2 Log cfu/g (range) cats3
Stomach Total anaerobe 4.3-6.2 N.A.

Bacteroides 4.2* N.A.
Bifidobacteria 3.8* N.A.
Lactobacilli 5.4* N.A.
Clostridia 3.0* N.A.
Total aerobe 4.5-6.1 N.A.
Enterobacteria 5.0* N.A.
Streptococci 5.9* N.A.
Staphylococci 4.9* N.A.
Small intestine Total anaerobe 3.8-6.0 5.0-8.3
Bacteroides 2.3-6.0 4.5-6.2
Bifidobacteria 3.6-5.2 N.D.
Lactobacilli 1.4-5.4 N.D.-6.0
Clostridia 2.5-4.5 3.8-7.5
Total aerobe 5.1-5.8 2.0-7.5
Enterobacteria 3.8-4.9 2.0-6.0
Streptococci 3.0-4.9 N.D.-8.3
Staphylococci 2.7-5.0 N.D.-7.4
Large intestine Total anaerobe 9.7-10.8 N.A.
Bacteroides 7.3-10.6 N.D.-5.6
Lactobacilli 5.6-9.1 N.D.-8.4
Clostridia 5.4-9.0 N.D.-7.7
Enterobacteria 7.0-8.2 4.7-8.2
Streptococci 8.8-9.1 N.D.-8.0
Staphylococci 4.0-5.3 N.D.-5.1

Table 5.2. Continued.
Location Bacterial group Log cfu/g (range) dogs2 Log cfu/g (range) cats3
Faeces Total anaerobe 10.6-11.0 N.A.

Bacteroides 8.6-10.8 10.4*
Lactobacilli 6.8-9.5 8.5*
Clostridia 6.8-10.3 10.0
Enterobacteria 8.3-8.6 8.5*
Streptococci 8.9-10.0 8.8*
Staphylococci 3.8-5.6 5.2*
1 cfu: colony forming unit; N.A.: not available; N.D.: not detected.
2 Data derived from Benno et al., 1992; Davis et al., 1977; Mentula et al., 2005; Simpson et al., 2002.
3 Data derived from Johnston et al., 2001; Osbaldiston and Stowe, 1971; Papasouliotis et al., 1998;
Terada et al., 1993.

* Mean, based on a single reference.
5.3.1 Molecular techniques
Data derived from cultured studies pose a problem, as many anaerobic species
cannot be cultured using these techniques. The unculturable microbiota can only be
characterized by more modern molecular techniques, like FISH and gene sequencing
methods (Greetham et al., 2002; Harmsen et al., 2000). Also, culture-based methods
underestimate total bacterial numbers and do not allow identification of the majority
of bacterial groups in the GI tract (Minamoto et al., 2012). Over the last decade,
however, publications describing gut microbiota with more advanced techniques
have emerged. Various molecular methods are used in the literature to characterize
intestinal microbiota, all with their own strengths and limitations. These techniques
make use of gene probes or gene sequencing to characterize and even quantify
different bacterial groups in the gut. The most targeted gene in this matter is a
small subunit ribosomal RNA (16S rRNA), because it is ubiquitously present in all
bacteria and contain both conserved and variable regions across species of bacteria
and archaea (Clarridge, 2004).

In the companion animal literature, different techniques are used to characterize gut
microbiota. Some of the more common techniques that are used in the literature
are FISH, quantative PCR (qPCR) and gene pyrosequencing. Techniques like FISH
and qPCR are used for quantifying specific bacterial groups. In FISH, group specific
fluorescent probes are used that bind with the 16s rRNA gene of the bacterial cells of
interest. The intensity of the fluorescence is a measure of the quantity of the bacterial
group of interest and can be objectively measured with image analysis (Langendijk
et al., 1995). To date, it is considered the most accurate method for quantification
of specific bacterial groups. In qPCR, group specific fluorescent primers for the
16s rRNA gene are used. Fluorescence is measured after each PCR cycle, and the
number of PCR cycles to reach a certain threshold of fluorescence is a measure
of the quantity of the specific bacterial group (Ginzinger, 2002). For identification
of bacterial diversity in a sample, gene sequencing can be used, in which (a part
of) the nucleotide sequence of 16S rRNA is determined. By using an automated
high-throughput sequencing platform, several thousand sequences can be analysed
within hours, yielding deep phylogenetic information about the bacterial community
(Handl et al., 2011; Ritchie et al., 2010). These sequences can be compared with
existing gene banks to generate the identity of corresponding bacterial strains.
As can be concluded, a combination of techniques is needed to thoroughly describe

quality as well as quantity of the microbial community of the gut. Also, the use of
different techniques in the literature makes comparison of results between studies
unreliable. However, by combining the results of the different studies, a general
overview may be generated on the current knowledge regarding gut microbiota in
dogs and cats.
5.3.2 Dog and cat sequencing data
Table 5.3 summarizes study characteristics of canine and feline studies using
gene sequencing techniques to research the canine and feline microbiota. As can
be concluded from this table, the phyla Firmicutes, Bacteroidetes, Proteobacteria,
Fusobacteria, and Actinobacteria constitute more than 99% of all gut microbiota.
This is in close agreement with human data (Eckburg et al., 2005) and data in mice
(Ley et al., 2005). However, percentage distribution of these phyla between the
different dog and cat studies varies widely, probably caused by discrepancies due to
differences in DNA extraction methods, PCR protocols and/or sequencing methods
(Suchodolski, 2011). Also, the abundance of these bacterial phyla varies along the
length of the GI tract.

Table 5.3. Characteristics of canine and feline gene sequencing studies.1
Primary phyla (%)
Proteobacteria
Actinobacteria
Bacteroidetes
Fusobacteria
Firmicutes
Method3
Primary
Reference N2 Sample families4
Barry et al., 2012 4F Faeces W 7 40 34 13 1 N.A.

Desai et al., 2009 5F Faeces C 32 16 41 N.D. 1 1,2,3,12,14,15
Garcia-Mazcorro 12 C Faeces V 1.6 0.1 96.9 0.1 0.1 3,4,8,11,15
et al., 2011 12 F 4.0 0.1 95.1 0.1 0.1 3,4,8,11,15
Garcia-Mazcorro 6C Faeces V 1.6 0.1 96.9 0.1 0.1 3,4,8,15
et al., 2012
Handl et al., 2011 12 C Faeces V 1.8 2.3 95.4 0.3 N.D. 3,4,8,11,15
12 F 7.3 0.5 92.1 0.04 N.D. 3,4,9,11,15
Handl et al., 2013 22 C Faeces V 2.7 1.2 87.2 6.4 2.6 3,7,8,15,17
Middelbos et al., 6C Faeces V 0.8-1.4 32-34 28 24-40 5-6 N.A.
2010
Ritchie et al., 2008 4F Jejunum V 2.2 1.1 87.6 4.5 4.5 N.A.
Ileum 4.7 17.5 65.4 1.7 10.7 N.A.
Colon 2.2 13.4 75.1 5.0 4.2 N.A.
Ritchie et al., 2010 15 F Faeces V 2.3 2.4 87.3 0.2 7.9 3,6,11,15,18
Suchodolski et al., 6C Duodenum V N.D. N.D. 65.0 1.1 33.9 N.A.
2008 Jejunum N.D. 5.9 49.8 19.9 24.4 N.A.
Ileum N.D. 22.7 26.2 31.2 19.9 N.A.
Colon N.D. 26.1 47.9 17.6 8.5 N.A.
Suchodolski et al., 5C Jejunum V 11.2 6.2 15.0 5.4 46.7 3,5,6,10,
2009 13,16
Suchodolski et al., 32 C Faeces V 1.8 N.D. 96.6 0.1 0.3 3,4,8,15
2012
Swanson et al., 2011 6 C Faeces W 1.0 37-38 35 7-9 13-15 N.A.
Tun et al., 2012 5F Faeces W 1.2 67.5 13.0 0.7 5.9 N.A.
1 N.A., not available; N.D.; not detected.
2 C = canine; F = feline.
3C = Cpn60 gene sequencing; V = V1-V3 region 16S rRNA gene pyrosequencing; W = whole genome
pyrosequencing
4 1 = Bacteroidaceae; 2 = Bifidobacteriaceae; 3 = Clostridiaceae; 4 = Coriobacteriaceae; 5 =
Corynebacteriaceae; 6 = Enterobacteriaceae; 7 = Enterococcacaea; 8 = Erysipelotrichaceae; 9 =

Eubacteriaceae; 10 = Fusobacteriaceae; 11 = Lachnospiraceae; 12 = Lactobacillaceae; 13 = Moaxellaceae;
14 = Prevotellaceae; 15 = Ruminococcaceae; 16 = Spirochetacea; 17 = Streptococcacaea; 18 =
Turicibacteraceae.
Most mammals seem to harbour similar bacterial groups when analysed on a higher
phylogenetic level (phylum and family level) (Ley et al., 2008), but the microbiota
of each individual animal seems to differ substantially on a species/strain level, with
typically only a 5% to 20% overlap between individual animals of the same species
(Handl et al., 2011).
Modern molecular techniques have also provided some insight into the biological
diversity of the intestinal microbiota in dogs and cats. A sequencing study in
cannulated dogs (Suchodolski et al., 2009) has estimated that approximately 200
bacterial species and 900 bacterial strains reside in the canine jejunum. Handl et
al. (2011) reported the presence of several thousand phylotypes in faecal samples
of dogs and cats. In this study, feline faecal bacterial microbiota appeared to be
more diverse compared to dogs. However, less interindividual differences were seen
in cats compared to dogs, in other words more cats than dogs shared the same
bacterial groups.
Molecular fingerprinting techniques have also demonstrated that every individual

dog and cat has a unique microbial ecosystem (Ritchie et al., 2010; Simpson et al.,
2002; Suchodolski et al., 2005). For example, a recent study by Ritchie et al. (2010)
has shown that 84% of cats harboured Bifidobacterium spp. However, only a minor
percentage of cats harboured the same species of Bifidobacteria. Interesting to note
is that, although a large amount of variation exists in the composition of bacterial
strains of the intestinal microbiota between individual animals, the metabolic end
products that are produced seem to be quite similar. Recent metagenomic studies
in dogs and cats, in which the functional capacity of the dog and cat microbiome
was researched, revealed that individual animals have a relatively similar array of
microbial genes present in the GI tract (Swanson et al., 2011; Tun et al., 2012). This
may indicate that a stable GI microbial community harbours a core microbiome.
Although these recent metagenomic studies provide some interesting new insights,
more research is needed to further clarify the significance of the data and the role it
plays in terms of disease.
Last but not least it is of great importance to note that most sequencing studies
in dogs and cats use luminal samples to evaluate gut microbiota. It was already
shown that a large variation exists in microbiota composition of the different
compartments of the gut (Table 5.3). However, it is important to realize that there
also may be significant differences between luminal microbiota versus microbiota
that are present within the mucosa of that same gut compartment (Zoetendal et al.,

2002). As the mucosal microbiota are in the closest contact with the host’s defense
system, it may be crucial to more closely study the gut mucosal microbiota and the
influence of different factors on its composition when researching gut health. The
question thus remains whether sequencing luminal microbiota is a true reflection of
gut microbiome diversity and may be considered a satisfactory measure to research
its composition.
5.3.3 Alterations in microbiota of dogs and cats with gastro-intestinal disease
Because of the high variety in composition of microbiota on an individual level, it

is nearly impossible to generate ‘normal’ reference data for a stable microbiota in
the gut of cats and dogs. However, much can be learned from studies that evaluated
microbiota composition of animals with gastro-intestinal disease. Reduced bacterial
species richness, for instance, was identified in the small intestine of dogs suffering
from inflammatory bowel disease (IBD) (Craven et al., 2009; Xenoulis et al., 2008). In
two separate studies (Janeczko et al., 2008; Xenoulis et al., 2008), dogs and cats with
idiopathic small intestinal IBD showed an increase in Enterobacteriaceae compared
with controls. Similar to humans, IBD dogs showed a reduction in the abundance
of Bacteroidales and Clostridiales compared to control dogs without clinical signs
of disease (Jergens et al., 2010). A recent study by Suchodolski et al. (2012) revealed
a bacterial dysbiosis in faecal samples of dogs with various GI disorders, with a
common decrease of bacterial groups that are known to be important short-chain
fatty acid producers.
In addition, several studies have shown altered immune responses in dogs and
cats with chronic GI disease. For instance, a differential cytokine expression was
shown in cats and dogs with inflammatory enteropathies (Janeczko et al., 2008;
Luckschander et al., 2010; Nguyen Van et al., 2006). Also, an increase in the mucosal
expression of Toll-Like Receptors (TLR) 2, 4 and 9 was found in different dog breeds
with IBD (Burgener et al., 2008; McMahon et al., 2010). Together, these data clearly
demonstrate the complex interactions between the host’s immunity and intestinal
microbiota, and the role these interactions may play in the pathogenesis of canine
and feline GI disease. The question remains, however, what causes these alterations
in both microbiota composition and innate immune system. One factor that may be
important to consider is nutrition.

5.4 Influence of nutrition on canine and feline gastrointestinal

microbiota and gut health
Nowadays, a rapidly growing amount of commercial dog and cat foods are
emerging on the market, which make use of so-called prebiotic fibres, probiotics,
or a combination of both (synbiotics), in order to improve gut health and gut
functioning. The rationale behind addition of these dietary ingredients is to attempt
to increase concentrations of ‘beneficial’ intestinal microbiota. An increasing amount
of studies have indicated that a variety of different dietary ingredients possess the
ability to selectively stimulate these beneficial bacteria, thereby altering microbiota
composition in the dog or cat’s GI tract (Barry et al., 2009; Swanson and Fahey,
2006). A recent meta-analysis by Patra (2011) on the responses of feeding prebiotics
on faecal microbiota composition in dogs concluded that the number of beneficial
bacteria such as bifidobacteria and lactobacilli can be significantly increased with
increasing doses of prebiotics. However, the number of possible pathogenic bacteria,
like Clostridium perfringens and Escherichia coli were not significantly affected by
prebiotic supplementation.
Although these studies undeniably show that pre- and probiotic interventions
may have the ability to alter the canine and feline microbiota in faecal samples,
the methods used in the majority of these studies have many limitations. First of
all, many of these studies are performed with healthy animals rather than animals
that are susceptible to GI microbiota disturbances (neonatal, geriatric or diseased
animals). As healthy animals seem to harbour a rather stable microbiome compared
to diseased animals, the question remains whether the effects found in healthy
subjects can be extrapolated to susceptible subjects.
On top of that, the majority of these studies make use of culturing techniques to
describe quantitative effects of the dietary interventions on the faecal microbiota.
As mentioned above, culture-based methods underestimate total bacterial numbers
and do not allow identification of the majority of bacterial groups in the GI tract.
This implies that only a minority of effects may be researched with culturing
techniques. In addition, most studies have analysed faecal samples, while it was
shown in sequencing studies that variation exists between the composition of faecal
microbiota, and microbiota elsewhere in the GI tract (Table 5.3). The question
remains whether the shift in microbiota found in faecal samples is indeed a measure
for alterations in other parts of the GI tract as well.

Last but not least, the prebiotic dosages and the probiotic strains that are used in the
different studies show much variation, which makes comparison of different study
results difficult to accomplish.
Recently, some canine and feline studies were published using gene sequencing
techniques to describe the effect of pre- and probiotic dietary intervention on canine
and feline microbiota. Middelbos et al. (2010) conducted a study with six healthy
adult dogs in a cross over design. Both groups were fed a control diet and a diet
supplemented with 7.5% of beet pulp. Faecal samples were subjected to 16S rRNA
pyrosequencing of the hypervariable V3 region, which showed that feeding beet pulp
was associated with a general decrease in Fusobacteria and an increase in Firmicutes.
Garcia-Mazcorro et al. (2011) researched the effect of a multi-species commercial
synbiotic on faecal microbiota in healthy dogs and cats. The synbiotic contained 7
strains of bacteria and a blend of fructooligosaccarides and arabinogalactans. Faecal
samples were subjected to qPCR and V1-V3 region 16S rRNA gene pyrosequencing.
The probiotic strains were detectable in the faeces of 10/12 dogs and 11/12 cats, and
disappeared after discontinuation of the treatment. No major changes in bacterial
phyla were observed during treatment and no significant changes in GI function or
immune markers were observed.
In a study by Barry et al. (2012), 4 healthy adult cats were fed diets containing
cellulose, FOS of pectin for 30 days in a replicated 3×3 Latin square design. Faecal
samples were subjected to whole genome pyrosequencing, yielding information on
the total array of microbial genes present in the GI tract of the researched cats.
Although significant percentage shifts were noted with regard to the different
bacterial phyla, the overall gene counts and, thus, the microbiome itself was not
majorly modified by the different fibre sources that were tested. Authors concluded
that it appeared that the microbiome seems to be highly conserved with respect to
microbial function, irrespective of diet. However, the small research population and
large interindividual variation in microbiota composition prevented drawing firm
conclusions from these data.
The research group of Swanson et al. (2011) conducted a metagenomic study in

dogs to study the phylogeny and functional capacity of the canine GI microbiome.
These authors used six healthy adult dogs in a cross-over design, which were fed a
low fiber control diet, or a diet containing 7.5% beet pulp. The control group showed
a greater percentage of Fusobacteria and Proteobacteria, while the treatment group
showed an increase of Firmicutes. This seemed in close agreement with the findings

of Middelbos et al. (2010). As for the functional capacity of the microbiome, feeding
beet pulp had no significant effect on the array of gene products that was present in
the faecal samples, which is in agreement with the data from the study of Barry et
al. (2012) in cats.
In the literature, most research on the effect of nutrition on gut microbiome in dogs
and cats study the effect of dietary plant-derived fibre. However, as carnivorous
species, evolving on a high protein, low carbohydrate diet, the amount of plant-
derived fibre present in the natural diet of dogs and cats is low (Plantinga et al., 2011;
Bosch et al., unpublished data). In that respect, studies researching the effect of high
dietary protein and dietary format may be of importance.
Lubbs et al. (2009) conducted a study with 8 adult cats to study the effect of feeding
a moderate protein (MP, 34% crude protein (CP)) versus a high protein (HP, 53%
crude protein (CP)) dry formula, after feeding a baseline diet for four weeks (37.6%
CP). qPCR to measure E. coli, Bifodobacterium, and C. perfringens and 16S rRNA
sequencing of the V3 region were used to study the effect on the feline microbiota
composition. Bifidobacterium populations were greater in cats fed the MP compared
to the HP diet. C. perfringens populations were increased in the HP fed group
compared to the MP-fed cats. Gene sequencing revealed a shift from carbolytic to
more proteolytic bacteria, and a modest increase in microbial diversity, amongst
others reflected in the appearance of two bacterial strains (Novosphingobiumtaihuense
and Haliangium ochraceam) with capacity to use aromatic end-products of protein
fermentation.
Vester et al. (2009) studied the effect of a moderate protein (MP) versus high protein
(HP) dry diet (34 vs. 53% CP on DM basis) on intestinal microbiota in growing
kittens during weaning. Faecal samples were subjected to qPCR analysis to quantify
Bifidobacterium, Lactobacillus, C. perfringens and E. Coli concentrations. Kittens fed
the HP diet had lower counts of Lactobacillus and Bifodobacterium compared to the
MP-fed kittens, and the bacterial concentrations seemed to be affected by age. The
authors concluded that the relevance of the data required additional in-depth studies.
Hooda et al. (2013), in a parallel study with 14 male growing kittens (7 kittens in each
group), researched the effect of a changes in the dietary protein:carbohydrate ratio
on faecal microbiota during weaning. In this study, 16S rRNA sequencing was used
to measure the effect of on the feline faecal bacterial groups. Feeding a high protein,
low carbohydrate diet (53% CP and 11% nitrogen free extract (NFE) on a DM basis)

was associated with a decrease in faecal Actinobacteria and concurrent increase

fecal Fusobacteria compared to feeding a medium protein, medium carbohydrate
diet (34% CP and 31% NFE). On a genus-level, the most prominent changes were
a significant decrease in Bifidobacterium, Megasphaera, and Lactobacillus, and a
significant increase of Clostridium, Ruminococcus, Faecalibacterium, Eubacterium,
and Fusobacterium.
Bermingham et al. (2013) conducted a study with 16 adult cats, which were randomly
allocated to a wet or dry diet in a 5-week cross-over design. Faecal bacterial DNA was
isolated and 16S rRNA gene pyrosequencing was performed. On a dry matter basis,
the wet diet contained 41.9% CP and 5.3% NFE, while the dry diet contained 32.9%
CP and 45.9% NFE. At a phylum level, feeding the wet diet was associated with a
significant decrease in Actibobacteria, and a significant increase in Fusobacteria and
Proteobacteria. On a genus-level a significant decrease was seen in Lactobacillus,
Megasphaera, Olsenella, Prevotella and Streptococcus, with a concurrent increase in
Peptostreptococcus, Fusobacterium, Clostridium and Bacteroides, when feeding a wet
diet. The data found in this study is in close agreement with the data found by Hooda
et al. (2013).
In a recent study, Beloshapka et al. (2013) aimed to determine the effect of feeding
a raw meat-based diet with or without inulin or yeast cell wall extract on fecal
microbiota composition in 6 healthy adult dogs in a Latin square design. Faecal
samples were subjected to 16 S rRNA gene pyrosequencing, and qPCR was
performed on Lactobacillus en Bifodibacterium strains. Compared to previous
studies conducted in dogs with dry based formulas (Table 5.3), the authors reported
a relative predominance of Fusobacteria and a higher percentage of Proteobacteria,
which is in agreement with the above mentioned cat studies. Feeding a prebiotic
supplement had a small effect on microbiota composition, with a modest increase
in Lactobacillus on the inulin supplemented diets, and an increase in Bifidobacteria
on the yeast cell wall supplemented diets.
In other, more omnivorous species like humans and pigs, the shifts in bacterial genera
seen in the above mentioned studies may have negative health effects. In human
children, it was shown that overweight children harbour significantly less strains of
Bifidobacteria compared to normal weight children, implying a possible protective
effect of high Bifidobacteria against metabolic disease later in life (Kalliomäki et
al., 2008). The genus Megasphaera, a major butyrate producer, has been shown to
have a positive effect on gut health in weaning piglets, recovering from mucosal

atrophy (Yoshida et al., 2009). The presence of higher amounts of Lactobacillus in

weaning piglets reduced the chance to develop GI disturbances related to weaning
stress (Siggers et al., 2008). Although the outcome of these studies may indicate a
detrimental effect of feeding a high protein, low carbohydrate diet, no adverse effects
were seen of feeding a high protein diet to healthy dogs or cats, not even during the
weaning phase. This may indicate that species differences do exist, possibly driven
by nutritional strategies during evolution. It is, therefore, of utmost importance to
exercise cautious using extrapolated data from more omnivorous species to strict
carnivorous species, like the cat. Further research is warranted to gain a better
understanding of the health implications of dietary format and diet composition on
gut microbiota of carnivorous species.
Moreover, although most of the studies on the effect of dietary alteration on canine
and feline microbiota clearly showed that significant shifts in bacterial phyla
and even genera occur during prebiotic or high protein feeding, the finding that
on a functional level the total microbiome seem to harbour a rather stable gene
expression (Barry et al., 2012; Swanson et al., 2011) raises the question whether
dietary intervention is indeed able to lead to significant functional changes in the GI
tract in healthy subjects. Future, more clinically oriented metagenomic studies with
diseased and susceptible subjects may be needed to further unravel the mystery of
the gut microbiome in general, and that in the carnivorous dog and cat in particular.
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Chapter 6: Pig intestine, weaning and dietary
interventions
J.P. Lallès1* and D. Guillou2

1INRA, UR 1341, ADNC, Domaine de la Prise, 35590 Saint-Gilles,
France; 2Lallemand SAS, 19 rue des Briquetiers, 31700 Blagnac, France;

jean-paul.lalles@rennes.inra.fr
Abstract
The post-weaning period continues to cause problem in rearing piglets, despite

sustained research efforts and derived applications in feed industry over the past
decades. The present chapter highlights from the scientific standpoint that a significant
number of alternatives to in-feed antibiotics have a potential for alleviating post-
weaning gut disorders in young pigs and provides possible underlying protective
mechanisms. A limited number of alternatives are already in use (e.g. zinc oxide,
spray-dried animal plasma protein, selected organic acids). However, many studies
were conducted in highly controlled experimental facilities and with low numbers
of observations. Therefore, the robustness of many other potential alternatives needs
to be tested on larger numbers of pigs and in field conditions for confirming their
protective effects. Another important point in this chapter relates to interactions
among feed components or ingredients and with genetic and environmental factors
that are presently poorly understood. The available data reveal the reality and
the complexity of these interactions. Obviously, beneficial effects of two or more
substances/components evaluated individually are not simply additive or synergistic
when combined. Antagonisms have been also disclosed. Therefore, massive work
in this field needs to be implemented in order to define and optimize the rules of
association of such alternatives into starter diets. Long-term studies are also needed
for understanding distant effects of early life events on gut health better.
Keywords: gut function, alternatives to in-feed antibiotics, post-weaning

6.1 Introduction
The post-weaning (PW) period continues to cause problem in rearing piglets, despite
sustained research efforts and derived applications in feed industry over the past
decades. The total ban on anti-microbial growth promoters (AGP) introduced in
Europe on January 1st, 2006, reinforced by the EU Commission action plan against
the rising threats from anti-microbial resistance (15/11/2011) with consequences on
medicated diets prescribed by veterinarians, had left feed industry and pig producers
with little efficient alternatives for facing PW gut disorders and diarrhoea. However,
a major benefit of this legal decision has been to stimulate academic and applied
research on a large array of potential alternatives. The work has included various
approaches from refining the provision of specific nutrients (e.g. L-glutamine
or L-arginine) and protective elements (e.g. zinc, mineral vs. organic forms) to
developing completely new alternatives (e.g. seaweed extracts; SWE). Although
most of these alternatives originate from plants, some products of animal origin
(e.g. spray-dried animal plasma protein; SDAPP) have proven effective and found
their niche in starter diets.
Many reviews have been successively published for analysing the progress made
(and gaps left) in the understanding of PW intestinal alterations at the cellular and
molecular levels, and on the mechanisms of action of such potential alternatives
(Heo et al., 2013; Lallès, 2010a; Lallès et al., 2004, 2007, 2009; Pluske, 2013; Pluske
et al., 1997). The review by Lallès et al. (2009) analysed the literature published
in the 2003-2007 period on organic acids (including sodium butyrate), specific
amino acids (L-glutamine, L-tryptophan, L-arginine, L-threonine), SDAPP and
bovine colostrum, and a number of plant extracts with antibacterial activities. The
major conclusions of this review were that beneficial effects, close to those obtained
with AGP have been consistently reported for some organic acids (e.g. formic
acid, potassium diformate, benzoic acid) and blends, for L-glutamine and SDAPP.
The underlying mechanisms documented included acid-induced bactericidal
action of organic acids in the stomach and proximal small intestine, stimulation
of protein synthesis and defence systems (e.g. inducible heat shock proteins; HSP)
for L-glutamine, and immunoglobulin-mediated neutralization of enteric pathogens
with SDAPP, and of course zinc. Two recent papers on SDAPP revealed improved
ileal and colonic barrier function, together with reduced inflammation and improved
anti-oxidant capacity of the gut (Gao et al., 2011; Peace et al., 2011). Protective
mechanisms of zinc are multiple (Lallès, 2010a) and are reviewed here. Medium-
chain triglycerides/fatty acids have proven their efficacy in protecting pig’s gut against

6. Pig intestine, weaning and dietary interventions
PW disorders (Decuypere and Dierick, 2003; Zentek et al., 2011; Price et al., 2013)
and will not be reviewed here. Among short chain fatty acids, effects of n-butyrate
are contrasted since they seem to depend on the dosage, the duration and period of
administration (Lallès et al., 2009). For example, we showed that pre-weaning supply
with n-butyrate at 3 g/kg milk DM intake stimulated PW body growth and feed
intake. Beneficial effects were associated with delayed gastric emptying, decreased
intestinal mucosa weight and increased feed component digestibility (Le Gall et al.,
2009). Finally, in vivo trials with plant extracts evaluated most often did not show
any improvements in gut health or on performance (Lallès et al., 2009).
In the present chapter, we review data published between 2007 and early 2013, we
summarize the new knowledge gained on PW gut disorders over the considered
period, and we focus on new insights into protective mechanisms of already known
or completely novel alternative substances. Feed-added enzymes are not considered
here because reviews are available on this matter (Kiarie et al., 2013).
6.2 Recent advances in intestinal physiology and pathophysiology

of post-weaning disorders
6.2.1 Basic physiology
As repeatedly reviewed, piglets are submitted to an accumulation of stress of

psychological, environmental and dietary origins that are inherently linked to
industrial pig production practices. These include confined rearing environment,
weaning at an early age, abrupt switch from feeding with highly digestible maternal
milk to less digestible plant-based complex diets often distributed in a dry form, and
finally animal commingling. All these stress strongly impact the gastrointestinal tract
(GIT) through nervous, immune, hormonal, nutritional and metabolic pathways,
with two major consequences: lowered vital gut function and increased sensitivity
to enteric pathogens (e.g. entero-pathogenic and entero-toxigenic Escherichia coli;
EPEC and ETEC, respectively) both leading to decreased voluntary feed intake and
growth check. Most consistent PW GIT alterations include 20-30% reduction in the
weight of the small intestinal mucosa, villous atrophy, compromised barrier function,
reduced digestive and absorptive capacity, and finally stimulated secretory capacity
leading to diarrhoea (Montagne et al., 2007; Pluske et al., 1997). These changes have
also profound implications on gut microbiota composition and activity and on the
development of the mucosal immune system (Lallès et al., 2007).

Recent investigations in the young pig, like previously shown in laboratory rodents
have demonstrated its high sensitivity to stress, and the major role played by the
nervous system, enteric nerves and mucosal mast cells in gut physiology alterations.
Indeed, the hormone corticotropin-releasing factor (CRF) and mucosal mast
cells dependently control both absorptive-secretory and permeability physiology
(Moeser et al., 2007a,b). The disorders involved increased jejunal and colonic
concentrations of CRF-R1 receptor and mast cell degranulation, released tryptase
and inflammation (e.g. TNF-α) (Moeser et al., 2007a,b; Overman et al., 2012; Smith
et al., 2010). Among other gut modifications induced by weaning, genes related to
antioxidant and digestive enzymes were down-regulated while enzymes regulating
reactive oxygen-species generation (e.g. tumor protein 53) tended to increase
(Zhu et al., 2012). The concentration of PPARγ coactivator-1α (PGC-1α), which
protects against oxidative stress by regulating the expression of mitochondrial anti-
oxidants was reduced (Zhu et al., 2012). Finally, blood plasma activity of superoxide
dismutase decreased and the concentrations of malonyl-dialdehyde, nitric oxide
and hydrogen peroxide increased PW, indicating reduced body redox balance
towards more oxidation (Zhu et al., 2012).
6.2.2 Weaning age
Importantly, the younger the pig at weaning, the higher the sensitivity to stress
(Moeser et al., 2007a,b; Smith et al., 2010) and the lower innate immune responses
to a subsequent ETEC challenge (McLamb et al., 2013). Early weaning altered the
expression of cytokine and tight junction protein, and activated mitogen-activated
protein kinases (MAPK) in pigs (Hu et al., 2013). The weaning stress activated
MAPK signaling pathways in the intestine, which may be an important mechanism
of weaning-associated enteric disorders of piglets (Hu et al., 2013).
6.2.3 Fasting and re-feeding
Abrupt reduction in feed intake immediately PW is an important causal factor in

weaning-mediated gut alterations (Lallès et al., 2004, 2007). Bruininx et al. (2001)
described the hourly evolution of eating pattern of group-housed weaned piglets,
and the results are consistent with highly variable fasting time among piglets raised
in commercial conditions. We recently showed that a controlled 36 hour-fasting was
a strong driver of mucosal atrophy and up-regulation of stress proteins, especially
in the stomach and in the colon (Lallès and David, 2011). The activity of many
digestive enzymes was dramatically reduced suggesting altered digestive capacity

(Lallès and David, 2011). Importantly, down-regulation of both gene expression and
activity of intestinal alkaline phosphatase (IAP) has been recently reported PW in
pigs (Lakeyram et al., 2010). This may be involved causally in PW pathophysiology
and inflammation because IAP specifically displays many functions, including the
detoxification of bacterial pro-inflammatory components (e.g. lipopolysaccharide,
LPS) (Lallès, 2010b). Re-feeding piglets for 60 hours nearly restored intestinal mass
and physiological levels of stress proteins, except in the stomach where the recovery
was slower (Lallès and David, 2011). Besides, feed restriction has sometimes been
viewed as a possible strategy for reducing PW disorders and diarrhoea, based on
the idea that it would limit alternate episodes of fasting followed with large feed
intake. This does not seem to be a good strategy, especially in a context of poor
sanitary conditions because it reinforces the negative effects of such conditions on
pig performance and health PW (Pastorelli et al., 2012).
6.3 Dietary nutrients (or precursors), animal proteins and minerals
6.3.1 Crude protein
Reducing crude protein (CP) content of weanling diet has long been recommended
to improve digestive health (Dirkzwager et al., 2005). However, recent research
addressing this issue did not provide clear evidence that such a recommendation
depended on gut physiology. Nyachoti et al. (2012) obtained changes in jejunum
and ileum, but not duodenum, morphology (villous height to crypt depth ratio),
following quadratic or cubic patterns when varying CP between 23 and 17%.
On the other hand, Bikker et al. (2006) did not observe such changes in the mid
jejunum, comparing 21 to 15% CP diets. Hermes et al. (2009) found reductions
of small intestine and large intestine weights, density of goblet cells and increased
intra-epithelial lymphocytes when reducing CP from 20 to 16%. These apparent
discrepancies suggest indirect influence of CP, either through individual amino acid
supply or ingredient-based interactions.
6.3.2 Glucose, lactose and starch
Vente-Spreeuwenberg et al. (2003) compared isocaloric supply of glucose, lactose

and starch. Dietary carbohydrate did not impact organ weights and architecture.
Especially lactose did not improve small intestinal integrity when compared to
glucose or starch.

6.3.3 Fibre, protein to fibre balance
Although early work by Pluske et al. (1996) indicated that non-starch polysaccharide
(NSP) content from grain was related to increased incidence of dysentery in infection
trials with Serpulina hyodysenteria, many authors attempted to demonstrate a
protective effect of increased fibre content in weanling pig diets. Hedemann et al.
(2006) demonstrated the influence of both fibre concentration and type (pectin) on
gut morphology. Fibre concentration decreased crypt depth without altering villus
height in the small intestine, while colonic crypts remained unaffected. Pectin had
negative influence on most indicators in both intestinal segments. Similarly, Gerritsen
et al. (2012) found favourable effects (e.g. increased stomach weight, improved fecal
score) of adding insoluble NSP to a diet with low protein ‘quality’. Bikker et al. (2006)
observed increased intestinal length and decreased maltase activity with increasing
content of fermentable carbohydrate. Lastly, Hermes et al. (2009) found an increased
colonic (but not small intestine) tissue weight (relative to body weight) due to the
higher dietary fibre content.
6.3.4 L-glutamine and precursors
L-Glutamine is an important amino acid in the nutrition and intestinal health of

pigs (Wu et al., 2011). Dietary L-glutamine (10 g/kg diet) provided for 7 days to
piglets weaned at 21 days of age increased intestinal expression of genes involved in
cell growth and anti-oxidant systems and reduced that of genes promoting oxidative
stress and immune activation (Wang et al., 2008). Functionally, L-glutamine
increased intestinal tissue mass, glutathione content and body weight gain (Wang
et al., 2008). In another study, a higher dose of L-glutamine (20 g/kg diet) was
evaluated in 21-day old pigs after 3, 7 and 14 days of supplementation (Zhong et
al., 2011). Average daily gain and feed intake were higher and diarrhoea incidence
was lower in the supplemented pigs. Intestinal mass and villus height-to-crypt
depth ratio were also increased. L-Glutamine supplementation stimulated intestinal
expression of the protective HSP70 in the jejunum but not in the ileum (Zhong et
al., 2011). L-Glutamine was shown to increase protein synthesis by stimulating the
mTOR signaling pathway in pig intestinal epithelial cells (Xi et al., 2012). A third
study investigated even higher L-glutamine level (44 g/kg diet) for 2 weeks, in an
intestinal loop experiment associating or not E. coli challenge in 21-day old piglets
(Ewaschuk et al., 2011). Such a supplementation was able to reduce the level of
intestinal tissue gene expression of both pro- and anti- inflammatory cytokines

(IL-1β, IL-6, TGF-β and IL-10) and to alleviate alterations in gene expression of
tight junction proteins induced by E. coli challenge (Ewaschuk et al., 2011).
α-Ketoglutarate is a key intermediate in the metabolism of L-glutamine. Two

studies were conducted to evaluate the effects of α-ketoglutarate (10 g/kg diet) on
intestinal mucosa of pigs weaned at 21 days of age and challenged chronically with
LPS between day 10 and day 16 (Hou et al., 2010, 2011). LPS challenge increased
phosphorylated mTOR-to-mTOR ratio and the expression of HSP70 (Hou et al.,
2010). α-Ketoglutarate reversed these LPS-induced responses (Hou et al., 2010).
α-Ketoglutarate also reversed the adverse effects of LPS challenge on small intestinal
mucosa, through restoring enterocyte nutrient oxidation capacity and increasing
tissue AMP to ATP ratio and levels of phosphorylated AMP kinase (Hou et al.,
2011). The authors concluded that α-ketoglutarate supplementation stimulated
intestinal protein synthesis and energy status in LPS-challenged piglets. In terms of
mechanisms, α-ketoglutarate was recently demonstrated to contribute to L-glutamine
sparing and to stimulate protein synthesis through mTOR signaling pathway in pig
intestinal epithelial cells (Yao et al., 2012).
Dietary supplementation with N-carbamyl-glutamate (0.8 g/kg diet) for 7 days in

21 day old-pigs resulted in reduced diarrhoea incidence together with increased
body weight gain and intestinal growth, enhanced villus height and crypt depth
and goblet cell counts along the small intestine, an over-expressed gene and protein
HSP70 (Wu et al., 2010). Glycyl-glutamine is a dipeptide allowing overcoming
the question of L-glutamine stability in feed. This dipeptide was added at 1.5 g/kg
to a diet fed for 3 weeks to very young (14 day-old) piglets submitted to an LPS
challenge carried out after 7 and 14 days of treatment (Jiang et al., 2009). Glycyl-
glutamine supplementation improved pig growth performance, gain-to-feed ratio
and intestinal architecture while decreasing inflammation marker levels systemically
(Jiang et al., 2009).
Collectively, these investigations confirm and extend the protective role of

L-glutamine and precursors on the small intestine of weaned piglets. They also
indicate that underlying protective mechanisms involve L-glutamine regulation of
protein turnover in intestinal cells through the mTOR signaling pathway, reduced
tissue inflammation and oxidation stress associated with stimulated expression of
cytoprotective (HSP70) and anti-oxidant (glutathione) components. Such protective
effects were seen with a wide range of L-glutamine doses.

6.3.5 L-arginine
Diet supplemented with L-arginine at 7 or 12 g/kg was fed to small piglets (5 kg

BW) for 10 days (Zhan et al., 2008). The L-arginine-deficient diet increased jejunal
concentration of the stable metabolite of nitric oxide, nitrite and nitrate, intestinal
villus height, tissue immune-reactive vascular endothelial growth factor (VEGF) and
the expression of CD34 (a glycoprotein cell-cell adhesion factor) in intestinal mucosa
(Zhan et al., 2008). The higher L-arginine dosage increased jejunal endothelin-1 but
decreased VEGF concentrations in the duodenal mucosa (Zhan et al., 2008). Thus
L-arginine dose may be critical. In another study, L-arginine added at 5 or 10 g/kg
diet in piglets fed for 16 days limited intestinal structural alterations induced by an
LPS challenge (Liu et al., 2008a). This was so thanks to stimulated intestinal epithelial
cell proliferation and reduced apoptosis. L-arginine supplementation reduced
LPS-induced body weight loss and prevented the increase in jejunal (both doses)
and ileal (L-Arg, 5 g/kg) tissue gene expression of pro-inflammatory cytokines (IL-6
and TNF-α) and increased intestinal gene expression of PPARγ (Liu et al., 2008a).
In a third study, a diet supplemented with L-arginine (6 g/kg diet) was fed to 21
day-old weaned pigs for 7 days (Wu et al., 2010). It enhanced intestinal growth and
stimulated tissue gene and protein expression of HSP70 (Wu et al., 2010). In a fourth
study, dietary L-arginine (10 g/kg diet) supplemented for 7 days to early-weaned pigs
increased pig growth rate and feed efficiency, with no effect of feed intake (Yao et
al., 2011). The relative weight of the small intestine, villus height in all segments and
VEGF expression were all increased (Yao et al., 2011). L-arginine supplementation
reduced plasma concentrations of ammonia, urea and cortisol, suggesting improved
nitrogen metabolism and decreased stress (Yao et al., 2011). Finally, in rotavirus-
infected pig neonates, Corl et al. (2008) showed that L-arginine (0.5 g/kg BW/day)
increased intestinal protein synthesis and improved gut permeability, although
without preventing diarrhoea. Collectively, these data support the notion that
dietary L-arginine displays anti-inflammatory and anti-stress properties in piglets
during the PW period and that the outcomes may dependent on the dose.
6.3.6 L-threonine
A threonine-deficient diet (6.5 vs 9.3 g Thr/kg) fed for 2 weeks to 7 day-old piglets
did not influence their performance or intestinal goblet cell numbers (Hamard
et al., 2007). However, it increased ileal permeability and reduced ileal villus
height-to-crypt depth ratio and amino-peptidase N activity (Hamard et al., 2007).
L-Threonine deficiency also impacted numerous genes (214 over-expressed; 110

under-expressed) involved in immune and defense responses, energy metabolism

and protein synthesis, including tight junction proteins (ZO-1; cingulin) and myosin
light chain kinase (MLCK) (Hamard et al., 2010). This study indicates that dietary
deficiency in L-threonine may affect intestinal key functions in young pigs. However,
the effects of higher L-threonine levels have not been reported.
6.3.7 L-tryptophan
Addition of 5 g/kg L-tryptophan to a basal diet supplying 2 g/kg digestible tryptophan

improved villus height to crypt depth ratio but did not affect intestinal paracellular or
transcellular permeability (Koopmans et al., 2006). In pigs weaned at 21 days and fed
a diet supplemented with L-tryptophan (10 g/kg diet) for 9 days and challenged with
ETEC after 5 days of treatment, Trevisi et al. (2009) reported complex interactions
with pig genetics (susceptibility or not to E. coli adhesion through intestinal receptors
to K88 fimbrae) (see paragraph on interactions below).
6.3.8 Lactoferrin derivatives and lysozyme
Bovine lactoferrin displays anti-microbial properties associated with its digestion

products lactoferricin and lactoferrampin. Lactoferricin and lactoferrampin fusion
protein (expressed in Photorhabdus luminescens) was incorporated at 100 mg/kg
into diets and fed for 3 weeks to piglets weaned at 21 days and challenged with
ETEC (Tang et al., 2009). These polypeptides increased pig growth performance and
intestinal morphology while they reduced E. coli counts and favoured lactobacilli and
bifidobacteria in the ileum, caecum and colon (Tang et al., 2009). Lysozyme, another
small protein with antibacterial activity was incorporated at 1 or 2 g/l of drinking
water (4,000 lysozyme units/ml) and fed to weaned pigs for 7 or 14 days (Nyachoti
et al., 2012). Challenge with ETEC was carried out on day 8. Lysozyme was found to
reduce ileal counts of ETEC and to increase ileal villi at 1 g/l (Nyachoti et al., 2012).
Plasma levels of pro-inflammatory cytokines (TNF-α, IL-6) which were the highest
with lysozyme at 1 g/l were the lowest with 2 g/l. Pig performance was not influenced
by lysozyme supplementation (Nyachoti et al., 2012). These two studies suggest that
bovine milk-derived proteins with antimicrobial properties have beneficial effects
on pig intestine morphology and microbiota taxonomic composition.

6.3.9 Calcium and phosphorus
Low and high levels of Ca and P (65 vs 125 and 115% of requirements for Ca and P,
respectively) were incorporated into diets fed for 2 weeks to weaned piglets (Metzler-
Zebeli et al., 2012). High Ca and P levels reduced duodenal gene expression of the
pro-inflammatory cytokine IL-1β and caecal crypt depth (Metzler-Zebeli et al.,
2012). This may have been due to stimulated activity of IAP as recently reported in
rats (Brun et al., 2012). IAP is a key enzyme detoxifying bacterial LPS and down-
regulating gut inflammation (Lallès, 2010b). Furthermore, it has been shown that
both Ca and P levels need to be high for alleviating chemical-induced colonic
inflammation in rodents (Schepens et al., 2012).
6.3.10 Zinc
Zinc deficiency has detrimental effects on gut permeability and tight junction
proteins (Finamore et al., 2008). Conversely, high levels of zinc have shown consistent
favourable effects on pig growth and gut health. The effects of zinc oxide at two doses
(100 and 3,000 mg/kg diet) fed for 10 days on pig performance and gut mast cells
were investigated (Ou et al., 2007). While the highest zinc dose reduced diarrhoea
incidence, zinc supplementation reduced mRNA and protein levels of the cytokine
stem cell factor (SCF), the number of mast cells in the mucosa and submucosa of the
small intestine, and histamine release from mast cells (Ou et al., 2007). Serosal (but
not mucosal) zinc was able to reduce intestinal chloride secretion (Carlson et al.,
2008), suggesting the role of absorbable zinc in this protective effect. Organic zinc
(Zn chelate) was incorporated at 80 mg/kg into diets fed for 2 or 3 weeks to 21 day-old
piglets (Castillo et al., 2008). Gain-to-feed ratio and pig faecal scores were improved
in the 3-week period (Castillo et al., 2008). In another study, two forms of zinc,
zinc oxide or tetrabasic zinc chloride were incorporated into diets (2000 mg Zn/kg)
and fed for 2 weeks to 24 day-old piglets (Zhang and Guo, 2009). As anticipated,
zinc supplementation increased pig growth rate, feed intake and feed efficiency and
improved PW faecal scores (Zhang and Guo, 2009). Tetrabasic zinc chloride was
found to be superior to zinc oxide in reducing intestinal permeability and increasing
tight junction protein occludin and ZO-1 gene and protein expression in the ileum
(Zhang and Guo, 2009). Zinc improved gut redox status and prevented apoptosis
(Wang et al., 2009). Finally, zinc is also known for preventing E. coli adhesion and
invasion on cultured porcine epithelial cells (Roselli et al., 2003), and recent data
indicate that zinc can reduce intestinal expression of receptors to E. coli K88 in vivo
(Sargeant et al., 2010).

Collectively, the most recent data on dietary zinc indicate that this element is active
on multiple physiological pathways contributing to its overall protective effects in
pigs PW.
6.4 Dietary feed components
6.4.1 Feed components tested in vitro
Various feed ingredients, including wheat bran, casein glycol-macropeptide, mannan-

oligosaccharides, locust bean (carob, Ceratonia siliqua) extract and Aspergillus oryzae
fermentation extract were all found to reduce K88 E. coli binding to porcine jejunal
epithelial cells in culture, with the highest inhibition being observed with casein
glyco-macropeptide and mannan-oligosaccharides (MOS)(Hermes et al. (2011).
Importantly, the highest wheat bran concentration was the only feed component
to down-regulate pro-inflammatory cytokine and chemokine genes in this study
(Hermes et al., 2011).
6.4.2 Cereal β-glucans
The effects of oat β-glucans provided at 89.5 g/kg in diets fed for 2 weeks to weaned
piglets were investigated (Metzler-Zebeli et al., 2012). Oat β-glucans increased colonic
IL-6 and caecal MCT1 transporter gene expressions that correlated with luminal
n-butyrate and total SCFA concentrations, respectively (Metzler-Zebeli et al., 2012).
Barley β-glucans were incorporated at low, medium or high (17, 35.5 and 73.5 g/kg)
levels in a wheat-based diet and fed for 2 weeks to 21-day old piglets (Ewaschuk et
al., 2012). Besides modifying various traits of cellular immunity, barley β-glucans
were shown to increase intestinal tissue electrical conductance and permeability to
the marker mannitol (Ewaschuk et al., 2012). K88 E. coli binding to enterocytes also
increased proportionally to dietary β-glucan incorporation (Ewaschuk et al., 2012).
Therefore, barley β-glucans had detrimental effects on gut function and microbiota,
despite their immune-modulatory properties.
6.4.3 Oligosaccharides
Mannan-oligosacharides (MOS) were incorporated at 2 g/kg diet and fed for

2 or 3 weeks to 21 day-old piglets (Castillo et al., 2008). Gain-to-feed ratio and
pig faecal scores were improved in the 3-week period (Castillo et al., 2008). MOS

decreased enterobacteria counts in the jejunum (Castillo et al., 2008). Besides, chito-
oligosaccharides (COS) were added at different levels (100, 200 or 400 mg/kg) in
diets fed for 3 weeks to 16 day-old piglets (Liu et al., 2008b). COS improved PW
faecal diarrhoea scores. COS at 100 and 200 mg/kg improved pig growth, feed intake
and gain-to-feed ratio (Liu et al., 2008b). Total tract digestibility of feed components
was the highest with 100 (for DM, Ca and P) and 200 (for DM, CP, GE, CF, Ca and P)
mg COS/kg diet (Liu et al., 2008b). COS favoured lactobacilli in faeces and at COS
of 200 mg/kg diet it increased ileal villus height and decreased faecal E. coli counts
(Liu et al., 2008b). In another study, COS was added at a level of 160 mg/kg diet and
fed for 7 and 14 days to 17-old piglets otherwise challenged or not with E. coli K88
(Liu et al., 2010). COS reduced diarrhoea incidence after challenge but had no effect
on performance (Liu et al., 2010). Therefore, COS provided at 100 to 200 mg/kg
diet decreased PW diarrhoea while a dose of 200 mg/kg diet may be optimal for
improving growth performance and gut functioning in very early-weaned piglets.
6.4.4 Rice
Incorporation of cooked rice in weaner diets has proven effective in improving

starch digestibility and reducing faecal shedding of ETEC and diarrhoea in pigs
PW (Montagne et al., 2004; Pluske et al., 2007). Pigs of 37 days of age were fed
complex diets containing cooked and flaked maize (500 g/kg) with a degree of
starch gelatinization of 840 g/kg (Vicente et al., 2009). Maize starch was substituted
with rice with degrees of gelatinization of 110 (raw), 520 or 760 (cooked), g/kg.
Compared with the maize diet, feeding rice improved feed component digestibility
and ileal morphology. The diets containing cooked rice led to the highest dry matter
(but not nitrogen) digestibility (Vicente et al., 2009). The diet with intermediate
rice cooking resulted in highest ileal villus height-to-crypt depth ratio and feed
component digestibility while the highly cooked rice diet had detrimental effects
(Vicente et al., 2009). Thus, moderately cooked rice appeared the best option in this
study. Torrallardona et al. (2012) conducted two trials comparing cereals: in a first
trial, rice, naked oats and barley were compared raw before inclusion in the feed,
while a second trial they compared the same grain extruded. Grain source affected
microbiota intra-group similarity in ileum and colon but not gut morphology in
both trials.

6.4.5 Seaweed components
SWE have been recently studied by an Irish group (Leonard et al., 2011b). The SWE
were fed at 2.8 g/kg diet either to the sows (from day 107 of gestation until weaning
at 26 days) or to the piglets or to both of them. The authors found that offspring born
to sows fed SWE as well as pigs supplemented with these extracts PW transiently
displayed higher colonic MUC2 mRNA abundance and lower colonic E. coli after
11 days (Leonard et al., 2011b). In another study, SWE extracts were used alone or
with a supplement of fish oil (Leonard et al., 2011a). SWE contained laminarin (100
g/kg), fucoidan (80 g/kg) and ash (820 g/kg) and was fed at a level of 10 g/d/sow.
Again, there was a beneficial effect of SWE on offspring growth performance during
the 3 weeks PW and on caecal E. coli counts that were lower and villus height-to-
crypt depth ratios in the ileum and jejunum that were higher (Leonard et al., 2011a).
Furthermore, ileal TNF-α and colonic TFF3 mRNA expression were increased in
piglets born to sows fed SWE (Leonard et al., 2011a). Importantly, seaweeds extracts
were recently shown to display anti-inflammatory properties on pig colon when
co-incubated in vitro with LPS (Bahar et al., 2012).
6.4.6 Oils and fats
Sow’s feed supplementation with linseed oil (rich in alpha-linolenic acid, 18:3 n-3)
during gestation and lactation (5 and 55 g/kg diet, respectively; compared to
lard-containing diets) was shown to increase ileal permeability and modify its
neuro-immune regulation in 28 day-old non-weaned offspring (Boudry et al., 2009;
De Quelen et al., 2011). In sows fed protected fish oil or algal docosapentaenoic
acid (DHA) (10 and 1.4 g/kg diet, during gestation and lactation), the offspring
aged 14-17 days and submitted to a 1-day fasting (to simulate weaning) displayed
higher AMP kinase-activated intestinal sodium-dependent glucose absorption
(Gabler et al., 2007, 2009). Although the PW period was not investigated in these
studies, one could anticipate some long-lasting effects of sow’s dietary LC-PUFA on
offspring intestinal function. Earlier work by Cera et al. (1988a,b, 1990) and Li et
al. (1990) on pigs PW showed an influence of fat sources on gut morphology and
lipase activity. Corn oil inconsistently reduced gut health indexes. Combination of
soy oil with coconut oil was preferred to single additions. Regarding long-chain fatty
acids, fish oil containing 40% eicosapentaenoic acid (EPA) and 25% DHA was fed to
sows at 100 g/day from day 107 of gestation until weaning at 26 days, and offspring
intestines were analyzed. Maternal FO supplementation induced higher growth rate
and feed efficiency in offspring between days 7 and 14 PW, but colonic mRNA levels

of IL-1α and IL-6 were increased (Leonard et al., 2011a). Fish oil (50 g/kg diet) was
fed to weaned pigs for 21 days that were submitted to ETEC challenge at the end
of the trial (Liu et al., 2012). Fish oil improved intestinal morphology and barrier
function through enhancing tight junction proteins (occludin and claudin-1). It also
decreased intestinal inflammation, apoptosis and stress (Liu et al., 2012).
Collectively, the available data show that fatty acid composition of maternal diets
impacts offspring gut function and may be a way of preparing pigs to the weaning
period. However, more work is needed on longer periods of investigation.
6.4.7 Fibre sources
In a study, inulin was provided at a level of 4 g/kg diet for 4 weeks to young pigs (Mair
et al., 2010). Inulin supplementation was found to down-regulate gene expression
of two cytokines (TNF-α, TGF-β) in the colon, with no effects on the small intestine
(Mair et al., 2010). Importantly, antagonistic interactions between inulin and a
probiotic were observed in this study (see paragraph on interactions below).
Wheat bran, which is an insoluble fibre source rich in cellulose and hemicellulose,
was incorporated at a level of 30 g/kg diet and fed to piglets for 37 days. It increased
villus height-to-crypt depth ratio along the small intestine and up-regulated NFκB
mRNA gene expression in the stomach and the jejunum and that of TGF-β, TNF-α
and caspase 3 in the jejunum of piglets fed the wheat bran-supplemented diet
(Schedle et al., 2008). In another study, wheat bran was incorporated at levels of 40
or 80 g/kg diet and fed to piglets for 13 days (Molist et al., 2011). At 40 g/kg diet,
wheat bran had no specific effect while at 80 g/kg it decreased organic and dry matter
digestibility (Molist et al., 2011). Therefore, wheat bran may have some beneficial
effects on pig small intestine at moderate incorporation levels while higher levels
have negative impact on digestibility. As another source of insoluble fibre, pollen
from Chinese Masson pine (Pinus massoniana) was incorporated at 12.7 or 25.5 g/kg
in piglet’s diet and fed for 37 days (Schedle et al., 2008). Pine pollen down-regulated
the expression of many genes (e.g. NFκB, TNF-α, TGF-β, caspase 3, CDK4 and
IGF-1) in the colon, suggesting anti-inflammatory properties for this preparation
(Schedle et al., 2008).

6.4.8 Antioxidant substances
Polyphenol-rich apple or red wine pomace were incorporated into diets and fed from
3 days before to 3 weeks PW in piglets that were slaughtered serially (Sehm et al.,
2007). Both supplements minimized villus alterations and Peyer’s patch enlargement
caused by weaning, and stimulated colonic crypt size development (Sehm et al.,
2007). However, red-wine pomace inhibited jejunum villi growth compared to
apple pomace (Sehm et al., 2007). Similarly, a grape seed and marc extract rich in
polyphenols and incorporated at 10 g/kg diet for 4 weeks was able to improve pig
performance, intestinal integrity and to reduce intestinal inflammation (Gessner et
al., 2013). A blend of antioxidant compounds (6.75 g/kg diet: containing vitamin
C, vitamin E, tea polyphenols, lipoic acid, and microbial antioxidants fermented by
bacteria and yeast) was fed to 21 day-old piglets for 2 weeks (Zhu et al., 2012). This
blend was able to alleviate intestinal digestive enzyme alterations caused by weaning,
to reduce the activity of enzymes involved in ROS production (e.g. tumor protein
53) and to stimulate anti-oxidant factors (e.g. PPARγ coactivator-1α, PGC-1α) (Zhu
et al., 2012).
Therefore, antioxidant compounds seem to be promising in alleviating PW gut

disorders. However, negative interactions with dietary protein digestibility may be
forecasted since some polyphenolic compounds have a high protein-binding capacity.
6.5 Probiotics
6.5.1 Bacteria
The probiotic E. coli Nissle 1917 strain was shown to abolish PW diarrhea through
a reduction of jejunal chloride secretion and to restore ETEC-induced intestinal
permeability alterations (Schroeder et al., 2006). Lactobacillus amylovorus (formerly
named Lactobacillus sobrius) which is autochtonous in pig gut was found to reduce
E. coli infection and to support the growth of infected pigs (Konstantinov et al.,
2008). These beneficial effects might have been due to reduced ETEC adhesion
and improved intestinal barrier as shown on intestinal epithelial IPEC-1 porcine
cell line (Roselli et al., 2007). The probiotic Lactobacillus fermentum (strain I5007)
was fed for 13 days to weaning pigs (Wang et al., 2012). The probiotic decreased
the expression of intestinal proteins involved in apoptosis or stress response and
increased the expression of proteins implicated in detoxification in the GIT (Wang

et al., 2012). Conversely, L. fermentum stimulated the expression of proteins

involved in energy and lipid metabolism, cell structure and motility, protein
synthesis and immunity (Wang et al., 2012). Importantly, such latter effects were
not observed with the AGP (aureomycin) control (Wang et al., 2012). In another
study, a probiotic mixture (containing enterococci, lactobacilli and bifidobacteria)
fed for 4 weeks to weaning pigs stimulated villus height and increased densities
of goblet cells producing neutral mucin in the jejunum (Mair et al., 2010). This
mixture had no effect on pro-inflammatory gene (NFκB, TNF-α) markers in the
small intestine or in the colon (Mair et al., 2010). The effects of a digestion-protected
form of Lactobacillus plantarum, strain Lq80 (at 1010 bacteria/day) provided alone
or in association with Megasphaera elsdenii, strain iNP-001 (109 bacteria/day) was
investigated in 20 to 28 day-old piglets for 2 weeks (Yoshida et al., 2009). M. elsdenii
is a lactate-utilizer and butyrate producer which activity might be enhanced in
presence of lactobacilli. L. plantarum limited PW villous atrophy and tended to
stimulate colonic IgA production (Yoshida et al., 2009). The combination of the two
bacteria stimulated colonic n-butyrate production and mucosa thickness and tended
to increase colonic IgA concentrations (Yoshida et al., 2009). A trial with piglets
supplemented with Pediococcus acidilacti during the suckling period (3 days a week
by gavage) and after weaning at 21 days of age was carried out (Lessard et al., 2009).
The treatment reduced bacterial translocation to the mesenteric lymph nodes after
ETEC challenge carried out at 52 days of age (Lessard et al., 2009). By contrast, a diet
supplemented with Enterococcus faecium failed to influence intestinal expression of
TFF family genes (Scholven et al., 2009). Finally, Lactobacillus rhamnosus GG which
is non-autochtonous to pigs was reported to induce adverse effects PW (Bosi and
Trevisi, 2010).
6.5.2 Yeasts and yeast derivatives
Yeast culture was added at 1.25 g/kg in diets fed for 5 weeks in 27 day-old piglets
(Van der Peet-Schwering et al., 2007). It improved pig growth and gain-to-feed ratio
but it did affect neither feed intake nor jejunal villus-crypt architecture (Van der
Peet-Schwering et al., 2007). Bontempo et al. (2006) obtained different results when
feeding diet with 2 g/kg of another yeast strain (Saccharomyces cerevisiae CNCM-I
1079, ‘boulardii’) during 30 days PW: growth and ileum morphology were improved,
mucus thickness decreased, and mucosal macrophage counts increased. In another
study, the effects of increasing doses (2.5, 5, 10, 20 g/kg diet) of yeasts were evaluated
in 28 day-old piglets fed yeasts for 21 days (Shen et al., 2009). While gain-to-feed was
not affected by yeast intake, pig growth and feed intake were maximized with yeasts

at 5 g/kg and at 5 and 10 g/kg, respectively (Shen et al., 2009). In a second trial with
5 g/kg diet fed for 3 weeks to 21 day-old piglets, yeast supplementation improved
feed digestibility (of DM, CP, gross energy) and intestinal architecture (Shen et al.,
2009). Gut IFNγ and T CD4+ lymphocyte infiltration were also lower after 14 days of
feeding the yeast-supplemented diet (Shen et al., 2009). In the study by Lessard et al.
(2009) mentioned above, S. cerevisiae boulardii supplementation reduced bacterial
translocation to the mesenteric lymph nodes after ETEC challenge carried out at 52
days of age, and increased ileal production of IgA (Lessard et al., 2009). Therefore,
the beneficial effects of probiotics in pigs PW seem to strongly depend on probiotic
strain, its level of provision and the duration and period of administration.
6.6 Interactions between feed-added substances and with the

rearing environment
6.6.1 Interactions between feed-added substances
Some studies have addressed the important question of interactions between

different substances added to weaning diets. It appears that synergies were less
frequent than antagonisms. A synergy between organic zinc (80 mg/kg) and MOS
(2 g/kg) added to a diet fed for 2 weeks in 21 day-old piglets was reported on jejunal
crypt depth that was decreased (Castillo et al., 2008). However, an antagonism was
observed between these compounds for faecal scores that were no longer improved
(Castillo et al., 2008). Also, an antagonism between wheat bran and zinc oxide was
reported on faecal E. coli counts: they were decreased with wheat bran alone but
increased when zinc was added (Molist et al., 2011). Antagonism between inulin
(40 g/kg diet) and a probiotic cocktail (enterococci, lactobacilli and bifidobacteria)
was reported for numerous parameters (Mair et al., 2010). Inulin supplementation
annihilated the beneficial effect of the probiotic mixture. Antagonistic interactions
were observed between SWE (10 g/day/sow) and LC-PUFA (fish oil, 40% EPA, 25%
DHA; 100 g/day per sow) (Leonard et al., 2011b). The beneficial effects of SWE
on E. coli numbers and villous architecture disappeared with FO supplementation
(Leonard et al., 2011b). Finally, little interaction was observed between CP and
fibre content of weaning diets (Bikker et al., 2006; Gerritsen et al., 2012; Hermes
et al., 2009). No additional effect of adding a MOS-rich yeast cell wall product
over yeast culture alone was reported in the study by Van der Peet-Schwering et
al. (2008). No additional effects between P. acidilacti and S. cerevisia boulardii were
observed in the study by Lessard et al. (2009). Interactions between oat β-glucans

and calcium/phosphorus levels were not significant on pig performance but intake
of oat β-glucans tended to reduce duodenal IL-1β gene expression in pigs fed the
low dietary calcium-phosphorus level (Metzler-Zebeli et al., 2012). Manzanilla et
al. (2009) reported that the influence of an essential oil mix was more pronounced
at 18% CP than 20%, and depended of protein source (fish meal vs. soybean meal).
6.6.2 Interactions between diet composition, genetics and environment
Trevisi et al. (2009) reported complex interactions between L-tryptophan

supplementation and pig susceptibility to E. coli intestinal adhesion. They found that
ETEC-susceptible pigs supplemented with L-tryptophan partially compensated the
adverse ETEC effects by increasing feed intake. Also, L-tryptophan supplementation
reduced both small and large intestine length (without affecting total tissue weights)
and increased villus height in the proximal intestine of ETEC-susceptible pigs only
(Trevisi et al., 2009). Poor sanitary environment usually reduces voluntary feed intake
and limits pig performance PW. The interaction between fibre content and rearing
environment was investigated in a recent study (Montagne et al., 2012). Surprisingly,
the poor sanitary condition favoured the establishment of a more beneficial gut
microbiota (Montagne et al., 2012). However, a high (169 vs 121 g/kg in the control
diet) dietary fibre level oriented the microbial ecosystem towards a less beneficial
balance, suggesting fibre to be a substrate for pathogenic bacteria in the context of
poor sanitary condition (Montagne et al., 2012).
Therefore, some beneficial effects of given dietary compounds may disappear or

even become adverse in presence of other compounds with known beneficial effects,
and outcomes may depend on genetic and environment factors too. Clearly, more
investigation is needed specifically on interactions between all these factors. This will
probably help understand apparent discrepancies among published studies.
6.7 Distant effects of early nutritional interventions
Pre-natal and early post-natal events such as unbalanced diets or environmental

stress are known for their effects on early programming, with subsequent long-
term effects on metabolism and function of various organs and tissues. Recent
investigations suggest that the GIT is also submitted to programming although data
are still scarce in the pig species (Lallès, 2012).

The effects of SWE fed to sows during gestation and lactation on their offspring
have been recently studied (Leonard et al., 2011b). It was found that pigs born
to SWE-supplemented sows had higher body weight in the 3-week PW period
(Leonard et al., 2011b). However, supplementing these pigs directly with SWE did
not provide additional beneficial effects. Longer term effects indicated that faecal
counts of Enterobactericae were reduced at 117 days of life in the SWE-supplemented
pigs (Leonard et al., 2011b). Feeding yeast (S. cerevisiae CNCM-I 1079, ‘boulardii’)
included at 2 g/kg to sows had a carry-over effect on piglet’s villus height: crypt depth
ratio PW (Di Giancamillo et al., 2007). The probiotic Lactobacillus brevis (strain
1E1) provided to piglets under the sow was able to reduce E. coli and coliform counts
in the small intestine and to increase villus-crypt ratio in the ileum of piglets aged 9
days and in the duodenum of piglets aged 22 days (Gebert et al., 2011). Clearly, this
field warrants more investigations in the future.
6.8 Conclusions and perspectives
The present chapter highlights from the scientific standpoint that a significant
number of alternatives to in-feed antibiotics have a potential for alleviating PW gut
disorders in young pigs and provides possible underlying protective mechanisms.
However, many of these studies that are conducted in highly controlled experimental
facilities and are often based on low numbers of observations. Apart from a limited
number of alternatives already well recognized (e.g. zinc oxide, SDAPP, selected
organic acids), the robustness of many potential alternatives needs to be tested
on larger numbers of pigs and in field conditions for confirming their protective
effects. Another important point in this chapter relates to interactions among feed
components or ingredients that are presently poorly understood. The few available
data reveal the reality and the complexity of these interactions. Obviously, beneficial
effects of two or more substances/components are not simply additive or synergistic.
Antagonisms have been also disclosed. Therefore, massive work in this field needs
to be implemented in order to define and optimize the rules of association of such
alternatives into starter diets. Long-term studies are also needed for understanding
distant effects of early life events on gut health better.

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Chapter 7: Effect of feed contaminants on intestinal
health of monogastric farm animals
I. Alassane-Kpembi1,2,3 and I.P. Oswald1,2*

1INRA, UMR 1331, Toxalim, Research centre in Food Toxicology, 31027 Toulouse,
France; 2Université de Toulouse, INP, UMR 1331, Toxalim, 31027 Toulouse,

France; 3Hôpital d’Instruction des Armées, Camp Guézo 01BP517 Cotonou,
Bénin; iakpembi@gmail.com, isabelle.oswald@toulouse.inra.fr
Abstract
As the most extensively exposed surface in the body, the intestinal mucosa has to
face important chemical and biological challenges. The intestinal mucosa has three
main physiological functions. It establishes a physical barrier between the internal
milieu and the luminal content. The intestinal mucosa is also responsible for luminal
nutrients digestion and their subsequent absorption. The mucosal epithelium is at
the interface of immune system and luminal contents, including dietary antigens
and microbial products. This implies a local defence mechanisms regulation that
involves integrating all the signals that come from the external and internal world
to preserve immune homeostasis steady-state conditions. Either of these intestinal
physiological functions may be targeted by feed contaminants. These contaminants
may be naturally occurring compounds or substances from anthropogenic sources.
In the present chapter, we present mycotoxins and dioxins, which are representative
examples of both classes of contaminants. Data gathered show clearly that dietary
exposure to realistic doses of these contaminants impairs intestine functionality and
its integrity as well. The mechanisms of action of mycotoxins and dioxins targeting
the gastrointestinal tract are clarified and evidences for their deleterious effects for
monogastrics intestinal health are provided.
Keywords: mycotoxins, dioxins, barrier function, absorption-digestion, immunity
7.1 Introduction
The gastrointestinal tract has the most extensively exposed surface in the body and
is constantly exposed to potentially harmful food-born substances from diverse

sources (Yegani and Korver, 2008). Gut damages caused by these contaminants may
lead to poor intestinal health. Three main functions that may be targeted by feed
contaminants are devolved to the intestinal mucosa (Rescigno, 2011; Turner, 2009).
First, it establishes a physical barrier between the internal milieu and the sometime
hostile luminal content. The intestinal mucosa is also responsible for nutrients
digestion and their subsequent absorption, which require a selectively permeable
barrier. For these two functions, the mucosal epithelium is de facto at the interface
of immune system and luminal contents, including dietary antigens and microbial
products. That raises the third function for intestinal mucosa, which is in charge of
integrating all the signals that come from the external and internal world to preserve
intestinal immune homeostasis steady-state conditions.
Naturally occurring contaminants and pollutants from anthropogenic sources are

the two classes of contaminants that may be present in feed. Natural contaminants
from both plants and fungal specific primary and secondary metabolisms may be
found in feed. These substances may have anti-nutritional or specific toxic effects
for farm animals. Toxins from fungal and plant origin include and are not limited
to mycotoxins, lectins, cyanogens, gossypol, glucosinolates and phyto-oestrogens
(D’Mello, 2004). Environmental naturally occurring contaminants like heavy metals
and metalloids are widespread and can be found from traces to macro levels. Their
significant contamination through soil of seeds and plants fed to animals may also
result in chronic toxicity. Contaminants from anthropogenic source are in general
substances manufactured for industrial use that are not naturally occurring, but
may enter accidentally or deliberately the environment and result in environmental,
agricultural, industrial or other contaminations. In other cases industrial activity
may increase the mobility of naturally-occurring chemicals, or increase their
amount available to circulate in the environment, allowing them to contaminate
feed at higher levels than would otherwise occur. Examples of these anthropogenic
contaminants include persistent organic pollutants (especially dioxins), pesticides
and radionuclides.
In this chapter, we present mycotoxins and dioxins which are illustrative examples
of both feed contaminants classes, and their reported effects as well as mechanisms
of action targeting the gastrointestinal tract and potentially deleterious for the
intestinal health of monogastric farm animals.

7. Effect of feed contaminants on intestinal health of monogastric farm animals
7.2 Mycotoxins in feed
Mycotoxins are secondary fungal metabolites produced under specific environmental

conditions by a variety of mould spoiling agricultural commodities. As secondary
metabolites, they are not essential for life, but may provide the fungus with an
ecological advantage in certain environments. Factors contributing to the presence
of mycotoxins include ecological and storage conditions that are most times beyond
human control. Toxigenic moulds are known to produce one or more of these
mycotoxins and a substrate can be spoiled by more than one mould. Some 300
compounds have been recognized as mycotoxins of which a dozen is considered as
threat to human and animal health. It has been estimated that at least 25% of the
yearly worldwide grain production is contaminated (CAST, 2003). The mycotoxins
of biological and economic significance in animal agriculture, i.e. aflatoxins,
fumonisins, trichothecenes, zearalenone, and ochratoxins have been extensively
reviewed and will be only shortly described in this chapter (Bennett and Klich, 2003;
Bryden, 2012).
7.2.1 Aflatoxins
Aflatoxins were isolated and identified in England in the earlier 1960’s as the cause
of a mysterious outbreak of hepatic necrosis affecting thousands of poultry. Similar
incidents were then reported in pigs. Investigations revealed that toxicity was
associated to the presence of Aspergillus flavus in the feed and further that extracts
of the culture of the fungus were capable of inducing the syndrome. Later many
other species in the sections Flavi, Nidulantes and Ochraceorosei were also identified
as aflatoxin-producers (for detailed review see Varga et al., 2011). Structurally
aflatoxins are difurocoumarin derivatives that fluoresce under ultraviolet light. The
most important aflatoxin in terms of toxic potency and occurrence is aflatoxin B1
which has been classified as a group 1 carcinogen by the International Agency for
Research on Cancer (IARC). AFB1 is hepatotoxic and hepatocarcinogenic, but many
other effects can be associated to its toxicity: immunosuppression, reduced growth
rate and reproduction, lowered milk and egg production (Rawal et al., 2010).
7.2.2 Fumonisins
Fumonisins are products of polyketide and amino acid metabolism and have a
linear structure with amine and tricarballylic ester functions. They are produced by
Fusarium verticillioides and many other Fusarium species. Some twelve fumonisins

have been isolated, but fumonisin B1 (FB1) is the most abundantly produced and
the most toxic. The toxicity of FB1, including its effect on the intestine, is mainly
exerted through the ability of this toxin to disrupt sphingolipid metabolism (Bouhet
and Oswald, 2007). The IARC has classified FB1 as possibly carcinogenic to humans
(group 2B). This toxin may also be implicated in the aetiology of human oesophageal
cancer and neural tube defects. FB1 also causes leukoencephalomalacia in equines,
pulmonary oedema and hydrothorax in swine, nephrotoxicity and hepatotoxicity
in many species.
7.2.3 Trichothecenes
Trichothecenes are closely related sesquiterpenoid mycotoxins with a 12,13-epoxy

ring and a variable number of hydroxyl, acetoxy or other substituents. They are
classified as macrocyclic or non-macrocyclic depending on the presence of a
macrocyclic ester or an ester-ether bridge between C-4 and C-15. Food and feed-
born trichothecenes are mainly non-macrocyclic compounds produced by fungi
of the Fusarium genus. They can be subclassified in type A which have a hydrogen
or ester type side chain at the C-8 position and type B which have a ketone group
instead. Trichothecenes are well-characterized inhibitors of the protein and nucleic
acids synthesis. In acute toxicity tests, type A members such as T-2 toxin have been
found to be more toxic than type B components such as deoxynivalenol (DON)
and nivalenol (NIV). However in practical situations of chronic intake, the effects
and syndromes arising from DON are likely to be more important. In animal
studies, chronic exposure to trichothecenes caused impaired weight gain, anorexia,
haematotoxicity and immune dysregulation.
7.2.4 Zearalenone
Zearalenone (ZEA) frequently co-occurs with certain type B trichothecenes as they

are simultaneously produced by the same fungi. ZEA is a non-steroidal oestrogen and
its alcohol metabolites (α-zearalenol and β-zearalenol) have increased oestrogenic
activity corresponding to their binding affinities for hepatic, uterine, mammary and
hypothalamic oestrogen receptors. Dietary exposure to low doses of ZEA leads to
hyperoestrogenic syndromes and subsequent reproductive dysfunctions in pigs.

7.2.5 Ochratoxins
Ochratoxins are isolated from fungi belonging to Aspergillus and Penicillium geni.
They are chemically described as 3,4-dihydromethylisocoumarin derivatives linked
with an amide bond to the amino group of L-β-phenylalanine. The most commonly
occurring and most toxic member is ochratoxin A (OTA) which toxicological
profile includes nephrotoxicity, hepatotoxicity, teratogenicity and immunotoxicity.
In addition, OTA has been demonstrated to be carcinogenic among laboratory
animals, which justifies that IARC has rated OTA as a possible human carcinogen
(group 2B). Among farm animals, pigs are particularly sensitive to the toxin for its
tissue accumulation, due to a rather long serum half-life and the entero-hepatic
recirculation. The feed occurrence of ochratoxins is not only important from the
animal health and performance perspective, but also from the potential human
indirect exposure through the animal derived foods consumed.
7.3 Dioxins in feed
Dioxins and dioxin-like chemicals are structurally related and environmentally

persistent compounds that share a common mechanism of action, and as a consequence
a common spectrum of biological responses. The term ‘dioxin’ commonly refers
to polychlorinated dibenzo-p-dioxins (PCDDs) and dibenzofurans (PCDFs) that
are trace level unintentional by-products of most forms of combustion and several
industrial chemical processes including chlorine bleaching of wood pulp and the
manufacture of organochlorine pesticides, phenoxyacid herbicides and fungicides.
PCDDs and PCDFs may also be present as contaminants in polychlorinated biphenyls
(PCBs), which are considered dioxine-like compounds. While PCDDs and PCDFs
are produced unintentionally as unwanted by-products, PCBs were manufactured
for use in transformers, insulators, and many others technological applications.
PCB production was banned by the Stockholm Convention on Persistent Organic
Pollutants in 2001. Exposure levels are expressed in toxic equivalents (TEQ) of the
most toxic congener, 2,3,7,8-tetrachloro-dibenzo-p-dioxin (2,3,7,8-TCDD). The
main health issues concerning the dioxins have been reviewed elsewhere (Schecter
et al., 2006). These compounds have a high-affinity binding to the aryl hydrocarbon
receptor (AhR) which is an intracellular ligand-activated transcription factor
involved in regulation of the expression of a large number of genes and which also
interacts with regulatory proteins such as specific cellular kinases, and some of cell
cycle control as well as apoptosis proteins. Even tiny doses of dioxins can cause death

in certain laboratory animals and wildlife species, and 2,3,7,8-TCDD is considered

the most toxic man-made chemical. Its LD50 for guinea pigs is evaluated to 1µg/kg
body weight. The observed effects in humans and experimental animals include
toxicity to the immune system, reproductive and developmental abnormalities, and
endocrine disruption with diabetes and thyroid disorders (Bursian et al., 2013a,b;
Pavuk et al., 2003; Weisglas-Kuperus et al., 2000). The IARC has classified 2,3,7,8-
TCDD as group 1, carcinogenic to humans. Polygastrics grazing pastures that are
close to industrial area are typically considered farm animals exposed to dioxin risk
(Kamphues and Schulz, 2006), but the Belgian PCB/dioxin incident revealed that
poultry and pigs may also be at risk (Bernard et al., 2002; Covaci et al., 2008). For
example, an incident occurred at the end of January 1999 when a mixture of PCBs
contaminated with dioxins was accidentally added to a stock of recycled fat used in
the production of animal feeds. Signs of poultry poisoning were noticed by February,
1999, and it appeared that more than 2,500 Belgian, French and Netherlands farms
could have been supplied with contaminated feeds. Important features of the
crisis were the creation of the Federal Agency for Food Safety in Belgium and the
introduction in 1999 of norms for PCBs in feedstuffs and food in Belgium followed
by the introduction in 2002 of European harmonized norms for PCDD/Fs in animal
feed and food of animal origin. The Belgian crisis was followed few years later by
an Irish pork PCB/dioxin contamination which source was traced to an animal feed
production facility using the hot gases from the combustion of contaminated fuel
oil to dry animal feed (Marnane, 2012).
7.4 Effects of feed contaminants on intestinal epithelium renewing

and intestinal barrier function
7.4.1 Intestinal cell proliferation
The intestinal epithelium is the most vigorously self-renewing tissue of adult

mammals (Heath, 1996). The mammalian intestinal epithelium is renewed every
4-5 days. This high cell turnover allows rapid resealing of the epithelial barrier
following injury or damage in order to maintain an effective barrier function. The
normal homeostasis may be impaired by feed contaminants affecting self-renewing
capacities of the intestinal epithelium. The non-transformed porcine intestinal
epithelial cell lines IPEC-1 and IPEC-J2 (Chapter 8; Lallès and Oswald, 2015) have
been used to assess mycotoxins toxicity on pig intestine (Bouhet et al., 2004; Diesing
et al., 2011; Vandenbroucke et al., 2011). Proliferating intestinal epithelial cells

appears more sensitive to trichothecenes than differentiated cells, suggesting that

exposure to these feed contaminants could dramatically impair the physiologically
constant state of regeneration of the intestinal epithelium. Moreover, co-exposure
to low concentrations of different trichothecenes leads to synergistic cytotoxicity in
intestinal epithelial cells (Alassane-Kpembi et al., 2013).
7.4.2 Intestinal barrier function
Testing high concentrations of DON (2,000 ng/ml) on both intestinal cell lines
resulted in disintegration of tight junction protein zonula occludens-1 (ZO-1),
increase of cell cycle phase G2/M and early activation of caspase-3, while low
concentration (200 ng/ml) showed no effect on these parameters (Diesing et al.,
2011). Reduced expression of other tight junction proteins, claudin 3 and claudin 4,
was also reported in IPEC-1 monolayer epithelium exposed to DON (Pinton et al.,
2009, 2010). Other mycotoxins T2-toxin, FB1 and ZEA, also impaired the integrity
of IPEC-J2 monolayer via altered viability and reduced trans-epithelial electrical
resistance (TEER), and promoted the trans-epithelial passage of the antibiotics
(Goossens et al., 2012). It has also been observed that a prolonged exposure to
FB1 prevents the establishment of the TEER and alters the resistance of an already
established monolayer IPEC-1 epithelium (Bouhet et al., 2004).
The mechanism by which mycotoxins could alter pig intestinal barrier function
has also been investigated using intestinal cells and intestinal explants culture. The
activation of the p44/42 ERK signalling pathway by DON and its acetyl derivatives
inhibits the expression of tight junction claudin-4 protein, which leads to impaired
intestinal barrier function (Pinton et al., 2012). Selective removal of claudin isoforms
was also demonstrated with OTA which decreased intestinal barrier properties by
repressing claudin 3 and 4, but not claudin 1 (McLaughlin et al., 2004). Limited data
related with chicken intestinal barrier function suggest that acute exposure to AFB1
moderately affect TEER (Yunus et al., 2011b).
Beside the mycotoxins, PCBs constitute the other group of frequently occurring
feed contaminants exerting an ascertained disruptive effect on intestinal epithelial
integrity, even though no study to our knowledge assessed the specific sensitivity
of farm animal models to this class of compounds. The disruptive effect of
highly chlorinated PCBs on gut integrity has been demonstrated in human colon
adenocarcinoma cells (Caco-2) and in C57BL/6 mice (Choi et al., 2010). The
authors showed that exposure to each of PCB congeners PCB153, PCB118, PCB104,

and PCB126 increased the permeability of the intestinal barrier to fluorescein

isothiocyanate (FITC)-labelled dextran (4 kDa) and disrupted expression of tight
junction proteins ZO-1 and occludin.
7.5 Histo-morphological alterations of intestine induced by feed

contaminants
A broad spectrum of histo-morphological alteration patterns is related to the

presence of contaminants in feedstuffs. DON and its acetyl derivatives, FB1 and
ZEA are associated with mucosal morphological abnormalities in poultry and pigs.
Villi height decrease or atrophy as well as villi fusion and other regressive lesions
in the gastro-intestinal tract are frequently reported (Awad et al., 2006; Kolf-Clauw
et al., 2009; Sklan et al., 2003; Yunus et al., 2012; Zielonka et al., 2009). However
hyperplasia of goblet cells was observed in 1-day-old broilers fed ad libitum for
two weeks a 300 mg/kg FB1-contaminated diet (Brown et al., 1992). A chronic
exposure of Ross 308 male chicks to increasing levels of AFB1, was associated with a
dose dependent reduction of duodenum and jejunum weight (Yunus et al., 2011a).
Interestingly, a compensatory increase in the length of the duodenum and jejunum
was noted after 4 weeks of exposure to high concentration of toxin. Contrary to the
observations in boilers, a linear increase in the crypts depth in distal jejunum was
noted with increasing levels of AFB1 in the diet of layers (Applegate et al., 2009).
Polychlorinated dibenzo-p-dioxins (PCDDs) are also associated with regressive

intestinal lesions. A survey of environmental contamination with 2,3,7,8-TCDD
and other PCDDs indicated that the intestine weight in two great blue heron (Ardea
herodias) colonies regressed negatively on TCDD level (Sanderson et al., 1994).
7.6 Modulation of digestive functionality of the intestine by feed

contaminants
7.6.1 Absorptive functionality
The frequent regressive intestinal lesions may explain at least in part the reduced
absorption of nutrients observed with exposure to several feed contaminants. Smith
et al. (2012) reviewed evidence from human and animal studies that mycotoxins
may share a downstream pathway for children stunting by targeting the intestinal

tract and inducing Environmental Enteropathy. Aflatoxin reduced iron absorption

in chicken, regardless of the presence of anaemia (Lanza et al., 1981). Surprisingly
broiler chicks fed a 10 mg AFB1/kg diet for one week showed an increase of the both
mediated and passive components of glucose and methionine in vitro absorption,
while lower exposure (1.25 to 5 mg) for longer period (3 week) showed no effect
compared with control birds receiving no aflatoxin (Ruff and Wyatt, 1976).
Glucose uptake was assessed into laying hens jejuna epithelia in presence of DON
(Awad et al., 2007). DON decreased glucose uptake almost as efficiently as phlorizin.
In the presence of phlorizin, pharmacological inhibitor of sodium glucose-linked
transporter 1 (SGLT-1), DON had no additional effect on the glucose uptake. SGLT-1
is the main apical transporter for active glucose uptake in the small intestine. It works
like a symporter that uses the electrochemical gradient of Na+ to drive the glucose
absorption. This similarity between the effects of phlorizin and DON on glucose
uptake evidences their common ability to inhibit Na+-D-glucose co-transport. These
authors also evaluated mRNA expression of SGLT-1 in broiler chicken fed a diet
naturally contaminated with DON (1 and 5 mg/kg), (Awad et al., 2011). After 5
weeks, the mRNA of SGLT-1 was down-regulated in duodenal and jejunal tissues of
DON-supplemented groups. Ussing chamber experiments conducted at the same
time confirmed the glucose-induced inhibition of currents in intestinal tissues.
Taken together, these results suggest that gene expression mediate the inhibitory
effects of DON on intestinal glucose uptake. Based on this transcriptomic approach,
Dietrich et al. (2012) showed that not only DON impairs sugars (glucose and
fructose) intestinal absorption in broilers, but that mycotoxin might also alter the
uptake of palmitate and monocarboxilates in the jejunum at realistic doses of DON
(2.5 to 5 mg/kg).
Conversely, sodium-dependent glucose absorption might be up-regulated in pig

after acute or long term exposure to the mycotoxin FB1 (Lalles et al., 2009; Lessard
et al., 2009). Studies in mice found that exposure to fumonisins in utero increases
the frequency of developmental defects, and administration of folate or a complex
sphingolipid is preventive (Marasas et al., 2004). The folate uptake is mediated by the
folate receptor, which like many glycosylphosphatidylinositol-anchored proteins, is
enriched in cholesterol and sphingolipids. In the human intestinal epithelial Caco-2
model, the fumonisin induced-depletion in sphingolipids was shown to impair the
folate receptor function (Stevens and Tang, 1997). The authors suggested that dietary
exposure to FB1 could adversely affect folate uptake and potentially compromise
cellular processes dependent on this vitamin. In pigs, sphingolipids depletion was

observed in intestinal epithelium after a seven-days-dietary exposure to 1.5 mg/kg

body weight FB1 (Loiseau et al., 2007).
Active intestinal absorption of glucose and leucine may also be impaired after oral
treatment with 2,3,7,8-TCDD or PCBs (Ball and Chhabra, 1981; Madge, 1976a,b).
7.6.2 Activity of digestive enzymes
Modulation of digestive enzyme production and/or activity is one of the biological

effects of food contaminants targeting the gastrointestinal tract. Several studies
concluded that enzyme activity is modulated following mycotoxin consumption, even
though contradictory effects were reported. In pigs the activity of aminopeptidase N,
but not of sucrase, was markedly lowered by consumption of corn culture extracts
containing fumonisins in a nine-day feeding trial (Lessard et al., 2009). A 2-week
feeding study with laying hens showed that the specific activity of the intestinal
maltase increased quadratically by feeding up to 1.2 mg/kg of aflatoxin and declined
at 2.5 mg/kg of aflatoxin (Applegate et al., 2009). Activities of digestive enzymes from
duodenum contents including protease, chymotrypsin, trypsin and amylase were
increased in AFB1-treated group for ducks in a six week- feeding trial (Han et al.,
2008). On the contrary α-amylase and lipase activities in duodenum were lowered
in hens fed the aflatoxin-contaminated diet while pancreas amylase, trypsin and
chymotrypsin activity were increased (Matur et al., 2010). In line with lipase activity
decrease, there was a highly significant increase of lipid excretion in faeces of young
broiler chickens at 1.25 µg/g and above dietary aflatoxin exposure (including a three-
fold increase at 10 µg/g) (Osborne and Hamilton, 1981). The paradoxical upward
trend of pancreatic enzyme secretion might be ascribed to pancreas cells damage as
acute and chronic pancreatitis are associated with great proenzyme release (Han et al.,
2008; Matur et al., 2010). It’s noteworthy that Osborne and Hamilton (1981) reported
pancreatomegaly in chicken for 2.5 µg/g dietary aflatoxin exposure and above.
The intestinal mucosa can also be regarded as the largest endocrine organ, which
utilizes some 100 identified messengers (Ahlman and Nilsson, 2001; Furness et al.,
1999). The enterochromaffin cells constitute the largest endocrine cell population in
the gastrointestinal tract. On their apical side, gut endocrine cells have microvilli,
which may serve to taste the intraluminal milieu (Newson et al., 1982). As an
example cholecystokinin (CCK), also known as cholecystokinin-pancreozymin, is
released from duodenal endocrine cells after a meal. This hormone is produced by
enteroendocrine I cells, which are distributed throughout the proximal intestine

epithelium. It mediates digestive enzymes production from the pancreas and activates
neurons of the gallbladder wall with subsequent emptying of bile for breakdown of
fats and proteins in the meal. CCK also inhibits gastric emptying and initiates a
satiety response via vagal afferents.
Chronic exposure to different types of commonly encountered environmental

polychlorinated aromatic hydrocarbons (i.e. PCBs, dioxins, and
2,3,4,7,8-pentachlorodibenzofuran; PeCDF) may exert an endocrine disruptive
effect on intestinal CCK (Lee et al., 2000). These authors demonstrated that chronic
ingestion of PCB-126, PeCDF, or TCDD alone; a mixture of these three chemicals;
or a mixture of PCB-126 and PCB-153 decreases intestinal stores of CCK peptide
in a specific manner in rats. In addition, TCDD treatment of intestinal CCK cells
lowered levels of prohormone convertase-1 and -2, which are involved in processing
pro-CCK to mature, biologically active CCK.
7.7 Modification of the intestinal microflora by feed contaminant
As other fungi secondary metabolites especially antibiotics, several mycotoxins

have shown their antimicrobial potential (Ali-Vehmas et al., 1998; Burmeister
and Hesseltine, 1966). As a consequence, mycotoxins may modify the intestinal
microbiota. In pigs trichothecenes DON and T-2 toxin modified the dynamics of
the intestinal bacteria communities, causing an increase of the number of aerobic
bacteria (Tenk et al., 1982; Waché et al., 2009). A significant decrease of the amount of
Salmonella Typhimurium bacteria present in the cecum content, and a tendency to a
reduced colonization of the jejunum, ileum, cecum, and colon contents was showed
in presence of 15 and 83μg T-2 toxin per kg feed in pigs (Verbrugghe et al., 2012).
Microarray analysis revealed that T-2 toxin causes an intoxication of Salmonella
Typhimurium, represented by a reduced motility and a down-regulation of metabolic
and Salmonella Pathogenicity Island 1 genes. However, in vitro T-2 toxin promoted
the susceptibility of intestinal epithelial cells to Salmonella Typhimurium invasion
and its translocation over an intact intestinal porcine epithelial cell monolayer. The
enhanced Salmonella invasion in and translocation over the intestinal epithelial
IPEC-1 and IPEC-J2 cells was also observed with non-cytotoxic doses of DON
(Pinton et al., 2009; Vandenbroucke et al., 2011). Besides trichothecenes, the ability
of OTA and patulin to increase the translocation of commensal bacteria across intact
epithelium was also reported (Maresca et al., 2008).

7.8 Effect of feed contaminant on secretion of some intestinal

defence components
7.8.1 Mucus
In order to protect the mucosa, the host produces a complex layer of mucus that
covers the gastrointestinal tract (Johansson et al., 2011; McGuckin et al., 2011). The
mucus is organized in two layers that are organized around the highly glycosylated
MUC2 mucin, forming a large, net-like polymer. The mucus gel provides a matrix
for the retention of antimicrobial molecules in the mucosal environment. In
addition, mucin glycoproteins that form the major macromolecular constituents of
mucus can themselves have direct antimicrobial properties that limit the growth of
microorganisms in the mucus. The mucin glycoproteins are produced by goblet cells.
Feed contaminants may modulate mucus secretion.
ZEA and FB1 were found to exert a proliferative effect or an increasing activity on the
goblets cells and their content of mucinogen vesicles in pigs and chickens (Brown et
al., 1992; Obremski et al., 2005). On the contrary, low doses of mixtures of T-2 toxin,
DON and ZEA reduced the number of goblet cells and the tightness of the intestinal
glycocalyx in pigs (Obremski et al., 2008).
7.8.2 Anti-microbial peptides
The other major secretory cells within the gastrointestinal tract are the Paneth cells,
which are identified by their characteristic intracellular granules containing a range
of antimicrobial molecules that are secreted into the mucus to ensure sterility of
the stem cell niche. Among them are defensins, a family of cationic antimicrobial
peptides containing a specific six-cysteine motif also produced by epithelial cells
(Yang et al., 2007). On porcine IPEC-J2 cells, an up-regulation of porcine beta-
defensins 1 and 2 mRNA expression following exposure to DON, NIV, ZEA,
individually and in mixtures has been observed (Wan et al., 2013). Supernatants
from IPEC-J2 cells exposed to toxins, singly or in combination, however, possessed
significantly less antimicrobial activity against Escherichia coli than untreated
supernatants. The results suggested interactive effects when cells were exposed to
mycotoxin combinations.

7.8.3 Secretory immunoglobulin A
Secretory antibodies, immunoglobulin A (IgA) and IgG that are very important
components of the mucosal barrier, are secreted into the mucus by the epithelial cells
(Strugnell and Wijburg, 2010). These antibodies influence the commensal microbiota
and contribute substantially to the capacity of the mucus to retain and clear potential
pathogens. Specific immunoglobulin (Ig) receptors are responsible for the immune
response by regulating the Ig transport and cellular concentrations in various tissues.
These receptors transport Igs across epithelial tissues to their sites of action. The Fc
receptor (FcRn) is specific for IgG, whereas the polymeric immunoglobulin receptor
(pIgR) recognizes dimeric IgA and pentameric IgM. Expression of the pIgR gene in
epithelial cells of mucosal and glandular tissues is an unconditional pre-requisite
for acquiring mucosal immunity, while FcRn could be important in immune
activation and tolerance (Dickinson et al., 1999; Verbeet et al., 1995). In sheep, the
silage contaminating mycotoxin mycophenolic acid (MPA) lowered FcRn expression
in the liver, which may result in a lower IgG serum-to-bile transport, while pIgR
expression in ileum was stimulated (Dzidic et al., 2004). For viruses that invade
via the mucosal route, both IgA and IgG can provide protection and mediate viral
clearance. In a mouse model, T-2 toxin impaired the gastrointestinal tract clearance
of the enteric reovirus serotype 1 and increased its faecal shedding (Li et al., 2006).
These effects corresponded to a transient suppression of the induction of the specific
IgA in faeces and decreased secretion of reovirus-specific IgA and IgG2a in Peyer’s
patch and lamina propria fragment cultures.
Also in mouse model, a single oral administration of low dose 2,3,7,8-TCDD resulted
in a marked decrease in IgA secretion in the gut, showing that relatively low dose of
dioxins may impair intestine mucosal immunity (Ishikawa, 2009; Kinoshita et al.,
2006). Altogether, these results bring strong evidence that feed contaminants may
modulate the secretory antibody-dependant intestinal immune response.
7.9 Modulation of intestinal immune response by feed contaminants
When keeping the boundaries with the external world, the intestinal mucosa
has to integrate internal and external signals for coordinating an innate and/or
adaptive immune response (Maldonado-Contreras and McCormick, 2011). The
deregulation of this intestinal mucosa immunomodulatory function may lead to
intestinal inflammation or failure to face the continuous challenge of the transient

and/or resident intestinal microbiota. A finely tuned cross-talk between several cell
lineages of the intestinal mucosa determines the homeostatic balance. This cross-
talk is mediated by cytokines and chemokines which are small peptide molecules.
Feed contaminants are able to modulate the production of these molecules. A heat
map for the frequently regulated intestinal cytokines and chemokines in farm or
experimental animals dietary exposed to DON, FB1, AF, T-2 toxin, OTA, alone or in
combination has been proposed (Grenier and Applegate, 2013). A marked intestinal
pro-inflammatory cytokines up-regulation, especially IL-6 and IL-8, is associated
to mycotoxin exposure. The double-mechanism by which DON up-regulates the
chemokine IL-8 production is thought to involve the activation via MAPKinases and
Protein Kinase R pathways of NFκB-dependent transcription of the IL-8 gene, and the
HuR/ELAVL1 RNA binding protein-dependent stabilization of IL-8 mRNA (Pestka,
2010). Conversely, FB1 is known to decrease the basal expression and synthesis
of IL-8 chemokine in pig intestine (Bouhet et al., 2006). It makes sense that the
subsequent reduction in inflammatory cell recruitment during intestinal infections
explains at least in part the higher susceptibility of FB1 dietary exposed pigs (Oswald
et al., 2003). The same picture of down-regulation of the pro-inflammatory response
can be observed in animal models exposed to TCDD or its congeners (Monteleone
et al., 2011, 2012).
Impairment of the intestinal adaptive immunity is also attributed to exposure to

feed contaminants. A decrease in intestinal IFN-γ production, the Th1 signature
cytokine, is observed when trichothecene-exposed animals are orally challenged
with enteric reovirus (Li et al., 2005, 2006). As a consequence, those animals fail to
clear the virus from their intestine.
7.10 Conclusions
Intestinal health is of great interest for all animals, especially monogastrics. Lengthy
literature has been dedicated to the impact of chemicals on intestinal health.
Data gathered show clearly that realistic doses of naturally occurring as well as
anthropogenic feed contaminants impair intestinal functionality via different
pathways. Physical barrier functionality of the gut, its digestive/absorptive role
as well as its local defence mechanisms regulation assignment are sometimes
simultaneously affected in numerous situations of dietary exposure to contaminants.

Moreover several contaminants may be present at the same time in the feed
and interact together. More attention needs to be paid to the toxicological effect
of contaminant mixture to determine whether they act additively, in synergy or
antagonism. There is a growing body of evidence pointing that mycotoxins may
impair in a synergistic manner intestinal integrity (Grenier and Oswald, 2011;
Wan et al., 2013; Alassane-Kpembi et al., 2013). The characterization of these
toxicological interactions deserves to be extended to other contaminant mixtures
to improve our understanding of intestinal health risk associated with the presence
of feed contaminants.
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Chapter 8: Techniques for investigating gut
function in vivo, ex vivo and in vitro in
monogastric farm animals
J.P. Lallès1* and I.P. Oswald2,3

1INRA, UR 1341, ADNC, 35590 Saint-Gilles, France; 2INRA, UMR 1331
ToxAlim, Research Center in Food Toxicology, 31027 Toulouse cedex 03,

France; 3Université de Toulouse, UMR 1331, Toxalim, 31076 Toulouse, France;
jean-paul.lalles@rennes.inra.fr
Abstract
Intestinal health is a highly complex notion covering the diverse functions of

the gastrointestinal tract, including absorptive, secretory, barrier and immune
functions. The gastrointestinal tract is composed of successive segments that display
specific functional properties. As many of these functions are almost impossible to
investigate unequivocally in vivo, researchers have developed a set of complementary
tools for investigating various facets of gut function: permeability in vivo, isolated
loops (without or with excision), tissues mounted in Ussing chambers, cultured
tissue explants and cultured epithelial cells. These techniques allow investigating
gut electrophysiological properties, absorptive and secretory capacity, permeability
to generic or specific molecules of varying sizes, together with the underlying
endocrine, nervous and immune regulations. The ex vivo or in vitro techniques
can be used as ‘screening’ devices or as techniques for investigating mechanisms
that could contribute to explain in vivo results. Intestinal cell cultures are by far the
most used worldwide, followed by Ussing chambers, explants and loops. In terms
of disciplines and domains, these devices are essentially used for studies dealing
with physiology and disease states. This chapter is not an exhaustive review of the
literature. Rather, our aim is to remind the basic principles and limitations and to
illustrate the broad diversity of use of these techniques for investigating different
aspects of gut function in pigs and poultry.
Keywords: gut, intestinal loop, Ussing chamber, explant, cell culture

8.1 Introduction
Intestinal health is a highly complex notion covering the diverse functions of

the gastrointestinal tract (GIT), including absorptive, secretory, barrier (of ions,
substances and bacteria) and immune functions. These functions are controlled by
the endocrine, immune and nervous systems. They vary along the GIT compartments
or segments considered (stomach, duodenum, jejunum, ileum, caecum, colon and
rectum) and with age, diet and environment. Without the help of expert surgery,
many of these functions are almost impossible to investigate unequivocally in vivo.
This may be due to confounders (e.g. intestinal permeability as influenced by gastric
physiology) or because of the intrinsic impossibility to carry out a precise measurement
(e.g. segmental permeability) in the whole animal. Therefore, investigators have long
been facing the absolute necessity of developing complementary ex vivo (e.g. GIT
sheets in Ussing chambers or GIT explants in culture) or in vitro (e.g. intestinal cell
cultures) systems that could help dissect global intestinal health into ‘elementary’
functions while limiting the use of experimental animals. However, as all ex vivo
or in vitro techniques, the ones developed for investigation GIT functions have
advantages and limitations. These techniques can be used as ‘screening’ devices
or as techniques for investigating mechanisms that could contribute to explain in
vivo results. Thus, it must be borne in mind that these ‘reductionist’ approaches
cannot fully stand by themselves; they need to be associated with in vivo trials for
full validation of data and conclusions. This chapter is not an exhaustive review of
the literature. Rather, our aim is to remind the basic principles and limitations and
to illustrate the broad diversity of use of these techniques for investigating different
aspects of GIT function in pigs and poultry. Intestinal cell cultures are by far the most
used worldwide, followed by Ussing chambers (UC), explants and loops (Figure 8.1).
In terms of disciplines and domains, these devices are essentially used for studies
dealing with physiology and disease states (Figure 8.2).
8.2 Intestinal permeability measurement in vivo
The gastrointestinal tract is composed of successive segments, including the stomach,

the small intestine with the duodenum, the jejunum and the ileum, and finally the
large intestine with the caecum, the colon (proximal, median, distal) and the rectum.
Each segment displays specific permeability to ions, molecules and maybe bacteria
(so called bacterial translocation). Permeability is a complex notion supported
by diverse underlying mechanisms. In brief, gut epithelium is not completely

8. Techniques for investigating gut function in monogastric farm animals
2,000
Intestinal cell cultures
Intestine, Ussing chambers
1,600 Intestinal tissue explants
Experimental intestine loops
1,200
800
400
0
Human Rodents Swine Chicken
Figure 8.1. Numbers of publications dealing with intestinal investigations using different devices
(Pubmed search – February 2013).
Intestine and...
Intestine and diseases and...
Intestine and infections and...
Intestine and nutrition and...
Intestine and immunology and...
Intestine and nervous system and... Cell cultures

Explants
Intestine and hormones and... Ussing
0 500 1000 1,500 2,000 2,500 3,000
Figure 8.2. Number of publications on intestinal investigations and domains of research

(Pubmed search – February 2013).
impermeable and allows the passage of small or slightly larger, charged or neutral
(e.g. ions, small peptides or other molecules) compounds through populations of
pores differing in their size (e.g. 4-5, 10-15 and >20 Å) and localization along the
crypt-villus axis (Camilleri et al., 2012). The para-cellular route is controlled at the
level of tight junctions through neuro-immune mechanisms (Shen et al., 2012). By
contrast, macromolecules were shown to cross epithelial cells (trans-cellular route)
(Heyman et al., 1989, 1992; Van Niel and Heyman, 2002).

Importantly, permeability measurements are usually carried out after overnight

fasting and additional fasting after marker administration (e.g. 2-4 hours). This
is so for avoiding interference between marker and dietary components and with
diet-dependent gastro-intestinal motility and transit time (Bjarnason et al., 1995;
Hollander, 1999). Permeability can be assessed by administering a single marker (or
labelled bacteria) or a combination of markers either in the gut lumen or in blood
circulation (Szabo et al., 2006). Then, appropriate biological fluid is sampled in the
‘diffusion’ compartment reflecting marker passage, e.g. blood plasma or urine after
luminal administration, or gut fluid lavage after systemic marker administration.
Sampling can be spot (e.g. blood plasma, urine) or total for a determined period of
time (e.g. urine, gut lavage fluid). Kinetic sampling can be carried out in order to
get an idea of marker appearance dispersion over time (and calculation of the ‘area
under the curve’, e.g. in blood plasma or lavage fluid) or for estimating segmental
permeability. For example, quantitative collection of urine after administration of
a non-digestible marker (e.g. Cr-EDTA) over 3-5 hours and 5(3)-24 hours allows
estimating permeability in the small and large intestine, respectively. However,
it must be borne in mind that these are only estimates because oro-caecal transit
time as well as urine volume stored in the bladder vary widely across individuals.
Another approach for evaluating regional permeability is by using more specific
probes (Table 8.1). Sucrose is used for determination of gastric permeability because
it is not absorbed in the healthy stomach and is then hydrolyzed in the upper small
intestine (Sutherland et al., 1994). This technique has been successfully used in horses
for gastric ulcer detection (Hewetson et al., 2006). Small intestinal permeability
measurements most often involve small sugars (or combinations of mono- and di-
or tri-saccharide: mannitol, lactulose, stachyose, etc.) that are not digestible but that
are fermented in the large intestine, or non-digestible and non-fermentable probes
such as Cr-EDTA, polyethylene glycols of small-to-medium molecular weights
and fluorescent probes like fluorescein sodium (Table 8.1) (Bjarnason et al., 1995;
Hollander, 1999; Szabo et al., 2006). Marker assay in blood plasma or in urine takes
place a short period of time after its administration (e.g. 1-2 h for plasma, 3-5 hours
for urine). A limitation with sugar probes may be intestinal bacterial overgrowth
that may degrade the marker. One exception is sucralose, an artificial sweetener
that is not digested in the small intestine nor fermented by bacteria (Anderson et
al., 2005). Therefore, it is a marker of choice for colonic permeability measurement
in vivo. For pigs around weaning, the use of lactulose has been proposed recently for
determining small intestinal permeability (Wijtten et al., 2011a,b).

Table 8.1. Permeability markers and compounds most often used for investigating permeability
in vivo and/or ex vivo.1
Marker MW Chemistry, property Use Route
Permeability markers used in vivo

Mannitol 182 Da monosaccharide, intestinal permeability (dual-sugar para-cellular
fermentable test)
Cr-EDTA 341 Da metal, intestinal and colonic permeability para-cellular
non-fermentable
Sucrose 342 Da monosaccharide, gastric permeability para-cellular
fermentable
Lactulose 342 Da disaccharide, intestinal permeability (dual sugar para-cellular
fermentable test)
NaF 376 Da chemical, fluorescent intestinal permeability para-cellular
Sucralose 398 Da disaccharide, colonic permeability para-cellular
non-fermentable
Stachyose 667 Da disaccharide, intestinal permeability (dual-sugar para-cellular
fermentable test)
Permeability markers used ex vivo (in Ussing chambers or in isolated loops)
Gly-Sar 146 Da Glycyl-sarcosine, intestinal active transport (via trans-cellular
hydrophobic PepT1)
Mannitol 182 Da monosaccharide, intestinal and colonic permeability para-cellular
fermentable
Cr-EDTA 341 Da metal, intestinal and colonic permeability para-cellular
non-fermentable
Na-F 376 Da chemical, fluorescent intestinal and colonic permeability para-cellular
FSA 478 Da chemical, fluorescent intestinal and colonic permeability para-cellular
FD4 4 kDa polysaccharide, intestinal and colonic permeability para-cellular
fluorescent
Inulin 5 kDa polysaccharide, intestinal permeability para-cellular
fermentable
LPS 2-20 kDa lipopolysaccharide intestinal or colonic passage trans-/para-
(G- bacteria) cellular
HRP 40 kDa protein, enzyme intestinal and colonic permeability trans-cellular
FD70 70 kDa polysaccharide, intestinal and colonic permeability para-cellular
fluorescent
1 Cr-EDTA: chromium ethylenediamine tetracetic acid (complex); NaF: sodium fluorescein; Gly-Sar: glycyl-sarcosine;
PepT1: peptide transporter 1; FSA: fluorescein sulfonic acid; FD4, FD70: fluorescein isothiocyanate (FITC)-labelled
dextrans (of 4 and 70 kDa); LPS: lipopolysaccharide; G-: Gram-negative (bacteria); HRP: horseradish peroxidase.

8.3 In situ and ex vivo intestinal loops
8.3.1 In situ perfused intestinal loops
This technique called small intestine perfusion system (SISP) was developed in the
Netherlands in the 90’s (Nabuurs et al., 1993). Pigs are anaesthetized, their abdomen
is opened laterally and intestinal loops are prepared along regions of interest (e.g.
proximal, medial or distal intestine). Each loop (10 to 20 cm long, depending on the
objectives of the study and on the size of the pig) is inserted with a fine silicon tube
cranially and a larger tube caudally, in order to be able to inject solutions in and to
collect the fluid secreted from the loops quantitatively. Series of loops are separated
with short (e.g. 2-5 cm) or longer (e.g. 20 cm) segments, with control and treated
loops being positioned at a short distance. Cranial tubes are connected by infusion
pumps to vials containing control or experimental (e.g. bacteria, bacterial culture
medium, other substances) fluid while caudal tubes are allowed to drain into bottles
located slightly below pig’s abdomen. Sterile solutions with minerals and nutrients
or test solutions (with e.g. pathogens, toxins or other substances added) are kept at
37 °C and are infused (e.g. 8-10 ml/h) permanently over 8 to 10 hours. Quantitative
collection of drained fluid allows calculating net fluid movement (secretion or
absorption) by difference with infused volumes. At the end of the experiment, pigs
are euthanized and loops excised. Loop surface area is determined as the product
between loop circumference and length determined under constant tension, and
data are standardized per surface unit. One advantage of this technique is that loops
are irrigated with blood and lymph and submitted to neuro-immune and hormonal
regulation. Conversely, all the work is carried out under anaesthesia.
8.3.2 Ex vivo non-perfused intestinal loops
The ex-vivo non-perfused intestinal loop technique has been used rarely for studies
with pigs (Hansen et al., 1996) and poultry (Gratz et al., 2005). However, it might
be a valuable, cost effective approach when UC are not available. This technique has
been successfully used with mouse intestine (Segawa et al., 2011; Ueno et al., 2011).
The small intestine is cut into loops that are filled to moderate distension with culture
medium without/with test substances or bacteria (e.g. 1 ml/5-6 cm in mice). Loops are
placed in organ culture dishes or flasks and kept in temperature-controlled 5% CO2
incubators for various times (e.g. 30 minutes to 2 hours). Permeability measurements
are carried out by putting a permeability marker into the loop, alone or in presence
of pro-oxidant or pro-inflammatory substances (e.g. mono-chloramine). The outer

culture medium is sampled kinetically (e.g. every 30 minutes) and marker fluxes are
calculated as for UC. Although potentially possible, this device has not been used for
evaluating intestinal absorptive and secretory capacity.
8.4 Ussing chambers
8.4.1 Measurement of active movements of ions
The Ussing chamber (UC) device was set up by the Danish investigator Hans H.
Ussing in the 50’s as an attempt to distinguish between passive and active (energy-
dependent) movements of ions across an epithelium (frog’s skin at that time)
(Boudry, 2005; Clarke, 2009). This question was solved by putting the same buffer
containing the same concentrations of ions on both sides of the tissue in order to
eliminate passive diffusion. Additionally, the spontaneous trans-electrical potential
difference (PD) which is a characteristic of living polarized epithelia was annihilated
by clamping PD to zero by applying an external current across the tissue. This current
is called short-circuit current (Isc) and equals the algebraic sum of electrogenic ion
active movements. In these conditions, H.H. Ussing made it possible to investigate
active transport of ions. His investigations led to the ‘two-membrane’ epithelial
model with a primary active transport of Na+ and K+ at the basolateral membrane
(by the Na+-K+ ATPase) providing the electrochemical gradient for secondary active
transport by Na+ channels and Na+-coupled transporters at the apical membrane.
This methodology has spread to all type of epithelia as well as to epithelial cell
cultures. During confluence, these epithelial cells develop electrical polarity
reflecting physical and functional differences between the apical and the basolateral
membranes, as delineated by intercellular tight junctions. Afterwards, intestinal
mechanisms for electrogenic chloride anion (Cl-) secretion, electro-neutral NaCl
absorption and Na+-dependent glucose absorption have been elucidated. Associated
with these discoveries, a set of transporters were characterized functionally and then
identified at the molecular and gene levels. Sodium-dependent glucose absorption
through the transporter SGLT-1 has become the prototype for Na+-dependent
nutrient absorption.
Methodologically speaking, small segments of small intestine, caecum or colon are

collected from animals and transported to the laboratory in cold buffer (Ringer
bicarbonate). Most often, smooth muscle layers are stripped off in order to limit
tissue thickness and muscle diffusion barrier to substances and oxygen in UC.

Muscle layer removal from the small intestine is done before cutting the segment
along the anti-mesenteric side while it is done after cutting a piece of whole tissue
from the large intestine. Depending on the objective of the study, Peyer’s patches
can be excluded from or included into the tissue zone to be mounted in UC (see
e.g. Green and Brown, 2006). Then, the mucosa sheet is mounted between two half
chambers, thus determining two compartments (lumen side with surface epithelium
vs. interior milieu) associated with tissue electrical and functional polarity (see
figures in Boudry, 2005; Clarke, 2009). Each half chamber is connected to two sets
of electrodes: one set has its ends close to mucosal sides and is used for measuring
and electrical clamping of trans-mucosal PD while the other set is distant and
serves to inject a small electrical current at regular intervals. In this setup, the gut
mucosa behaves as an electrical resistance through which passes this small current,
thus allowing tissue trans-epithelial resistance (TEER) or tissue conductance
(Gt=1/TEER; synonymous with para-cellular ion permeability) to be determined
easily using Ohm’s law (V=TEER×Isc, or V=Isc/Gt).
Each half chamber is connected to a reservoir containing the same volume of buffer
with the same mineral composition (usually Ringer bicarbonate; composition most
often given in publications using UC). The reservoirs are maintained at the desired
temperature with warm water circulating in the jacket surrounding the reservoirs.
Finally, both reservoirs are bubbled with 95%:5% O2:CO2 mixture which serves
for tissue oxygenation and as gas lift for buffer circulation in each chamber. The
UC device allows gut tissue to survive for e.g. 2-3 hours in these conditions. The
electrodes are made of calomel or Ag-Ag Cl connected by salt bridges (e.g. 3% agar
melted in 3 M KCl) to each half chamber. Electrodes are connected to the electric
clamp device and a computer records electrical data permanently. While older UC
devices had glassware reservoirs, more recent devices have been miniaturized and
display reservoirs, connections and half chambers within a small acrylic block. Gut
mucosal sheets are secured on one half chamber with small pins that enter small
holes in the second half chamber. UC equipment is available from various companies
(Physiologic Instruments: www.physiologicinstruments.com; Warner Instruments:
www.warneronline.com; World Precision Instruments: www.wpiinc.com). However,
some laboratories have designed their own ‘Ussing’ chambers (e.g. TNO transport
chambers; Spreeuwenberg et al., 2001).
Before running UC with tissues, it is essential to ‘calibrate’ the chambers in order to

take account of the contributions of the buffer and electrodes to electrical phenomena.
After 10-15 min equilibration of the UC device without tissue, the voltage difference

between the two measuring electrodes is made equal to zero by applying an offset
current. The buffer resistance is electrically compensated in order to get the actual
tissue resistance when it is in place. Electrodes with problems are changed during the
calibration procedure but not afterwards. Then, the voltage clamp system is left in a
stand-by position and the buffer and the half chambers are removed for mounting
the tissue (see before). After tissues have been mounted and left to equilibrate for
e.g. 20-30 min with Ringer-glucose buffer on the serosal side and Ringer-mannitol
buffer on the mucosal side for balancing for osmolarity, investigations can start. UC
measurements allow measuring basal electrical parameters (Isc, PD, TEER or Gt).
These parameters are determined again if conditions are changed in the chambers
(for testing tissue functioning e.g. at various pH, or in oxidative condition after
adding H2O2 or mono-chloramine). For measuring tissue Na+-dependent-glucose
absorption, glucose is added on the luminal side and the change in Isc is recorded.
The maximum Isc difference (delta Isc=ΔIsc) reflects epithelial glucose absorption
capacity. The same approach is used for other nutrients with electrogenic absorption
(e.g. amino acids). Similarly for determining tissue secretory capacity (essentially
Cl- secretion), a secretagogue (e.g. carbachol, theophylline, etc.) is added. ΔIsc
response is measured and corresponds to epithelial secretory capacity for a given
secretagogue. Sufficient time depending on the kinetic response must be left
between two measurements (e.g. 10-15 min after glucose addition). The UC device
is also useful for investigating absorptive and/or secretory mechanisms (agonists
and antagonists of transporters or of enteric nervous system; mast cell stabilizers
or degranulating substances; etc.). Each time, changes in short-circuit current
are taken as a quantitative (positive or negative) response to the added substance.
Therefore, many aspects of gut mucosa electrophysiology can be investigated in UC
chambers. However, protocol diversity is limited by tissue survival time, number
of chambers and water-solubility of added substances. These can be solubilized in
organic solutions (e.g. ethanol, dimethyl-sulfoxide) or emulsified with bile salts (e.g.
for fatty acids) but vehicle solutions must be added to the chambers with control
tissues too. Finally, for those interested in measuring unidirectional (mucosal-to-
serosal or serosal-to-mucosal) ion fluxes this is possible with radioisotopes (22Na+;
36Cl-) placed in either side of the tissue in separate UC modules. In this case, tissues
with similar resistance need to be paired (Clarke, 2009). When only net fluxes are
measured, caution must be exercised in interpreting the data (Lucas, 2009).

8.4.2 Measurement of epithelial (mucosal) permeability
Later on, the use of UC was extended to investigations on intestinal trans-cellular and
para-cellular permeability routes (Camilleri et al., 2012). Using UC for this purpose
has been made possible thanks to small molecules (radioactive or fluorescent, e.g.
51Cr-EDTA or 3H-mannitol or FD4) that are added most often to the luminal side of
the tissue and which appearance on the serosal side is monitored kinetically in order
to calculate marker flow across the mucosa (Ducroc et al., 1983 for calculation).
Chamber buffer volume is kept constant after every sampling by adding an equal
volume of glucose-Ringer buffer. Therefore, it is possible to investigate gut mucosa
for its permeability characteristics. Gut permeability is a major functional property
of epithelia and any deviations from normal values indicate gut pathophysiology or
disease states (e.g. inflammatory bowel diseases; obesity) (Camilleri et al., 2012).
Although apparently simple, these phenomena and underlying mechanisms are
rather complex so that there is no absolute consensus for the type of permeability
markers to use (Table 8.1). Also, increasing numbers of studies focus on the ‘passage’
of specific compounds (e.g. lipopolysaccharide, LPS; Mani et al., 2013) or entities
(e.g. bacteria; Roberts et al., 2013) of interest instead of using generic permeability
markers. Correlations between in vivo and ex vivo measurements of gut permeability
are cruelly lacking.
8.4.3 Examples of investigations with Ussing chambers in pigs and poultry
Ussing chambers can be coupled with in vivo studies (Table 8.2). In brief, experimental
animals of different ages, breeds, body weights (e.g. intra-uterine growth retardation)
are allocated to in vivo treatments (e.g. nutritional, environmental). At the completion
of the experiment, the animals are sacrificed and portions of one or more segments
of the small or large intestine are collected and mounted in UC. Basal physiological
characteristics (e.g. electrophysiology, permeability) of the studied tissues are
determined. Additional treatments (e.g. bioactive substances including toxicants or
drugs) car be carried out on these tissues in UC in order to investigate particular
physiological or metabolic features (e.g. involvement of enteric immune or nervous
system; sodium-dependent nutrient absorption capacity; susceptibility to oxidative
stress) allowing to tentatively explain the effects of the treatments applied in vivo.
This is a good combination of in vivo and ex vivo approaches for getting functional
information in addition to more usual biochemical analyses of tissues.

Table 8.2. Examples of Ussing chamber studies coupled with in vivo treatments in pigs and
poultry.1
Intestinal Treatment in Main outcomes References

site vivo
Weaned pig
Small Diet Barley-derived dietary β-glucans increased para-cellular Ewashuk et
intestine permeability permeability al., 2012
Jejunum Diet Fumonisin B1 mycotoxin increased Basal basal Isc, Lessard et
glucose absorption and theophylline-induced chloride al., 2009
secretion
Jejunum Diet Dietary phytic acid reduced basal Isc and tended to Woyengo et
reduce PD al., 2012
Jejunum Diet infection Infection decreased basal Isc, glucose and phosphorus Walsh et al.,
active transport, but increased glutamine transport. 2012
Dietary microbials and organic acids increased basal Isc
after infectious challenge
Ileum Ischemia2 Lubiprostone reduced and PEG 3350 increased ischemia- Moeser et
induced paracellular permeability alterations al., 2008
Jejunum Weaning age Early weaning increased basal Isc and paracellular Smith et al.,
permeability. Permeability alteration depended on CRF 2010
and mast cells
Milk-fed pig
Jejunum Diet birth Paracellular permeability greater in low-birth weight Boudry et
and ileum weight piglets fed the high protein milk formula. This formula al., 2011
disturbed nervous regulation of permeability in low
birth weight pigs
Ileum Diet ischemia Ischemia-induced decrease in TEER reversed by ARA and Jacobi et al.,
EPA. ARA decreased ischemia-induced para-cellular 2012
permeability
Chicken
Jejunum Diet Dietary inulin reduced TEER but did not influence basal Rehman et
Isc nor glucose active transport al., 2007
Jejunum Immunity Intestinal Isc and chloride secretion increased in response Caldwell et
to antigen challenge in previously sensitized birds al., 2001
1 Isc: short-circuit current; PD: electrical potential difference; PEG: polyethylene glycol; CRF: corticitrophin-
releasing factor; TEER: trans-epithelial electrical resistance; ARA: arachidonic acid; EPA: eicosapentaeinoic
acid
2 Lubiprostone and PEG 3350 treatment in Ussing chambers.

Ussing chambers can also be used independently from in vivo investigations, for
mechanistic studies or product screening (Table 8.3). In this case, fresh gut tissues
are collected from ‘control’ animals (e.g. young or older animal; different segments
of the gut; ileal sheets without or with Peyer’s patches) and mounted in UC. Ex vivo
experiments are thus designed for the purpose of interest. In the case of UC use for
screening of bioactive products, acute dose-response studies as well as mechanistic
investigations can be carried out (Boudry and Perrier, 2008; Lallès et al., 2009)
(Table 8.3).
Collectively, the high numbers of published data obtained with UC illustrate the
versatility of the device and its great contribution potential in a large array of
scientific fields and situations (Figure 8.1 and 8.2).
8.5 Ex vivo GIT tissue explants
The explant system offers all of the advantages of an in vitro system, whilst retaining
the relationships of the tissues, and potentially maintaining the complex patterns
of differentiation seen in vivo. In this system, all the usual cell types of the organ
are found, the tissue architecture is maintained, as well as the interactions between
the different cells and, more importantly, metabolic and transport functions
are preserved. The explant system also offers a more controlled environment for
experimental manipulation, compared with in vivo models, and the possibility of
harvesting multiple explants from a single donor, thus increasing the statistical power
of any investigation. The ability to sensitively control and manipulate the immediate
environment of the explant also lends itself perfectly to a detailed investigation of
the intestinal response (Randall et al., 2011).
Concerning the intestine, explant culture has been demonstrated with organs from
human, and different animals including rodent, pigs and poultry. In the original
report, biopsies, approximately 3 mm in diameter were taken from the duodeno-
jejunal junction but today explants have been isolated from different part of the
intestinal tract. Nevertheless, published data suggest significant variability in the
length of time that explants from different regions of the intestine, irrespective
of species, can be maintained as viable cultures. The large intestinal explants, by
comparison, seem more tolerant to culture conditions. The disparity in the periods of
apparent morphological viability, between the small and large intestine, may be due,
in part, to the lower rate of cell turnover in the latter. The greater rate of cell turnover

Table 8.3. Recent examples of physiological studies or screening tests with Ussing chambers and
intestinal tissues from (control) pigs or birds.1
Animal Intestinal Treatment in Main outcomes References

species site Ussing chambers
Pig Jejunum Thymol Thymol increased Isc, chloride and Boudry and
bicarbonate secretion via activation of Perrier, 2008
nervous nicotinic receptors
Cinnamaldehyde Cinnamaldehyde increased Isc, chloride
and bicarbonate secretion via activation
of nicotinic receptors on enterocytes
Pig Jejunum Fumonisin B1 Fumonisin B1 increased TEER and Lallès et al.,
transcellular permeability 2009
Pig Jejunum Quinoa hull meal Glucose absorption and permeability Carlson et al.,
increased and theophylline- induced 2012
ionic secretion decreased with quinoa
hull meal
Pig Jejunum Deoxynivalenol Deoxynivalenol increases transcellular Pinton et al.,
permeability 2009
Pig Ileum CRF inhibitors Serosal CRF increased FD4 fluxes with no Overman et
change in TEER. Effect blocked by CRF al., 2012
receptor antagonists, mast cell stabilizer,
anti-TNF-α antibodies and neural
blocker
Pig Ileum Mucosal LPS Cod liver oil and fish oil decreased while Mani et al.,
emulsified oils coconut oil increased LPS transport (no 2013
effect of olive and vegetable oils)
Laying Jejunum Deoxynivalenol Deoxynivalenol increased TEER and Awad et al.,
hen decreased glucose transport 2005
Chicken Colon NH4+ NH4+ is excreted through apical Holtug et al.,
H+-ATPase and Na+/K+ATPase 2009
Chicken Jejunum Aflatoxin B1 Aflatoxin B1 increased basal Isc and TEER Yunus et al.,
and decreased glucose and carbamoyl- 2010
choline active transport
Chicken Jejunum Salmonella and its Salmonella and its endotoxin decreased Awad et al.,
Caecum entotoxin mucosal ionic permeability 2012
1 Isc: short-circuit current; TEER: trans-epithelial electrical resistance; CRF: corticotropin-relasing factor; FD4:
fluorescein isothiocyanate (FITC)-dextran (MW 4 kDa); TNF: tumor necrosis factor; LPS: lipopolyssacharide;
ATPase: adenosine triphosphatase.

and a defined structure such as a small intestinal villus means that any perturbation
of the balance of cell replacement or differentiation has a more profound impact
on morphology than that seen in the flattened structure of the large intestine. The
difference in survival times also suggests that the large intestine explants may be
inherently more resistant to the anoxia and associated oxidative stress present in the
culture conditions; the explants are solely reliant on the gaseous diffusion of oxygen,
rather than vascular perfusion present in vivo, leading to problems of ischaemia and
necrosis when large explants are cultured (Randall et al., 2011).
The age of the donor also seems to influence the explant culture. Explants harvested
from embryos appear to be much more tolerant of culturing. Clearly the harvesting
of embryological tissues presents more technical challenges than harvesting the
intestines from adults but, for the longer-term culture of small intestine, foetal
animals appear to provide a more successful source. Neonatal gastro-intestinal
explants may offer a compromise source of material. Indeed neonatal piglets were
used to investigate the attaching effacing lesion induced by some strains of Escherichia
coli (Batisson et al., 2003, Girard et al., 2005). We also observed that explants from
4-5 week-old pigs were better preserved than those of 9-13 week-old animal after 8
h in the absence of any further treatment, as assessed by morphological scores and
by villi lengths (Kolf-Clauw et al., 2009).
Ex vivo cultures are a physiologically relevant model to reproduce early infectious

disease processes and to study pathogenic mechanisms and the innate immune
response during infections, opening up many possibilities for biochemical,
morphological, functional and genetic studies, for humans or for animals, among
others. This model is also useful for searching new drugs and aspects of efficacy
and cytotoxicity of compounds prior to clinical trials. Table 8.4 illustrates the use
of intestinal explants for pigs and poultry. In these animal species tissue explants
have been mainly used in infectious disease studies. In these experiments the main
parameters investigated are bacterial adhesion, lesion and histological changes as
well as the local immune response. Recently our group has used the explant model
in toxicology studies to investigate the effect of food contaminant on the intestine
(Cano et al., 2013; Kolf-Clauw et al., 2009; Pinton et al., 2012).

Table 8.4. Examples of studies using intestinal explants from pigs or birds.
Animal Intestinal Treatment of the explant Parameter measured References

species site
Pig Ileum Escherichia coli Attaching and effacing Batisson et al., 2003;
lesion Girard et al., 2005
Bacterial adhesion Mundy et al., 2007
Pig Jejunum Salmonella enterica and Proteomic analysis of Collins et al., 2010
Lactobacillus plantarum pig tissue
Pig Ileum Yersinia enterocolitica Bacterial colonization McNally et al., 2007
Pig Colon Entamoeba histolitica Histology and lesions Girard-Misguich et
al., 2011
Innate immune Girard-Misguich et
response al., 2012
Pig Jejunum Deoxynivalenol Histology and Kolf-Clauw et al.,
morphometry 2009
Mitogen-activated Pinton et al., 2012
protein kinases
activation
Inflammatory response Cano et al., 2013
Chicken Duodenum S. enterica Bacterial adhesion Allen-Vercoe and
Woodward 1999
E. coli Bacterial adhesion La Ragione et al.,
2000
Caecum Brachyspira pilosicoli and Bacterial development Mappley et al., 2011
Lactobacillus spp.
8.6 In vitro intestinal cell cultures
The gastrointestinal tract is lined with a continuous monolayer of epithelial cells.

A primary function of these intestinal epithelial cells is to act as a physical barrier,
separating the contents of a harsh luminal environment from the layers of tissue
comprising the interior milieu. As a consequence of their exposed location, intestinal
epithelial cells have developed a variety of mechanisms besides the maintenance of
barrier function, which act to reduce the risk of infection by invasive foreign agents.
Such mechanisms include those, which act directly to inhibit bacterial colonization

along the exposed surface of the monolayer and those, which function through an
interactive process with components of the underlying immune system (Oswald, 2006).
To our knowledge no intestinal epithelial cell line from bird origin has been
developed. By contrast three intestinal epithelial cell lines of porcine origin (namely
IPI-2I, IPEC-1 and IPEC-J2) have been characterized and are used. IPI-2I cell line
was established from the ileum of an adult boar (d/d haplotype) and immortalized
by transfection with an SV40 plasmid (Kaeffer et al., 1993). IPEC-1 and IPEC-J2
cells are non-transformed intestinal columnar epithelial cells isolated from neonatal
piglet. IPEC-J2 cells were isolated from jejunum whereas IPEC-1 cells were isolated
from a mixture of ileal and jejunal tissue (Berschneider, 1989). These two cell lines
form polarized monolayers with high transepithelial electrical resistance when
cultured on pore-size filters. They are unique in that they are derived from small
intestinal tissue (compared to the common human colon-derived lines HT-29, T84,
and Caco-2) and are not transformed (compared to the porcine small intestinal
line, IPI-2I).
Although first employed in transepithelial ion transport and cellular proliferation

studies, these primary cell line have been increasingly used to characterize epithelial
cell interactions with feed compounds/contaminants or enteric microorganisms. As
shown in Table 8.5, these three cells lines are used to study the initial host responses
to pathogenic and nonpathogenic (e.g. commensal or probiotic) microorganisms. Of
note, IPEC-J2 cells are increasingly being used in microbiological studies to examine
the interactions of various animal and human pathogens, especially Salmonella
enterica and pathogenic Escherichia coli, with intestinal epithelial cells. The IPEC-J2
cell line has also been employed in some probiotic studies, in which the cells have
been used as an initial screening tool for adhesiveness and anti-inflammatory
properties of the potential probiotic microorganisms (Brosnahan and Brown 2012,
Table 8.5). IPEC-J2 and IPEC-1 cell lines were also used to characterize the effect
of dietary contaminants such as mycotoxins, trace element such as zinc or essential
oils on cell proliferation, cytokine and tight junction protein expression (Table 8.5).
Cell lines allow researchers to characterize cell response at the most basic level,
which can inform higher-level studies involving tissue explants, whole organ
systems, and living organisms. Interestingly, the results obtained with IPEC-1 and
IPEC-J2 cells have strong reproducibility in mucosal explants and in vivo exposed
to microorganisms or feed contaminants (Brosnahan and Brown, 2012, Loiseau et
al., 2007, Pinton et al., 2009, 2012).

Table 8.5. Examples of studies using intestinal epithelial cells from pigs.
Cell line Treatment of the cell line Parameter measured1 References
IPI-2I Escherichia coli Cytokine expression Pavlova et al., 2008

Salmonella enterica β-defensin expression Veldhuizen et al., 2006
S. enterica Cytokine expression Volf et al., 2007
Salmonella typhimurium TLR and cytokine Arce et al., 2010
expression
Saccharomyces cerevisiae and Cytokine expression Zanello et al., 2011
E. coli
Entamoeba histolytica Cytokine expression Meurens et al., 2009
Arcobacter spp. Interleukin-8 expression Ho et al., 2007
IPEC-1 Fumonisin B1 Cytokine expression Bouhet et al., 2006
Fumonisin B1 Sphinganine /Shingosine Loiseau et al., 2007
ratio
Deoxynivalenol (DON) and Claudin expression, Pinton et al., 2012
acetylated derivative MAPKinase Trans
electrical epithelial
resistance (TEER)
Deoxynivalenol, de-epoxy-DON, Cell proliferation Dänicke et al., 2010
and DON-sulfonate
Carvacrol Cell proliferation Bimczok et al., 2008
Lactobacillus sobrius and E. coli Tight junction localization/ Roselli et al., 2007
expression. Cytokine
expression
Saccharomyces cerevisiae Cytokine expression Zanello et al., 2011
Proteins from Trichuris suis Cytokine expression Parthasarathy and
Mansfield, 2005
Campylobacter jejuni Bacterial persistence Naikare et al., 2006
E. coli Bacterial adherence Koh et al., 2008
IPEC-J2 Soyabean allergen Endocytosis Sewekow et al., 2012
Zinc TEER, Hsp70 expression Lodemann et al., 2013
Deoxynivalenol and T-2 toxin TEER, antibiotic passage Goossens et al., 2012
Deoxynivalenol Microarray transciptomic Diesing et al., 2012
analysis
Mycotoxins β-defensins expression Wan et al., 2013
Vesicular stomatitis virus Viral inhibition Botić et al., 2007

Cell line Treatment of the cell line Parameter measured1 References
IPEC-J2 Rotavirus Liu et al., 2010

Sapelovirus Microarray transcriptomic Lan et al., 2013
analysis
Tritrichomonas fœtus Parasite adhesion Tolbert et al., 2013
Lactobacillus species Attachment of E. coli Larsen et al., 2007
Lactobacillus plantarum Cytokine expression Paszti-Gere et al., 2012
LPS from S. typhimurium TLR and cytokine Arce et al., 2010
expression
S. enterica Cytokine expression Skjolaas et al., 2006
TLR expression Burkey et al., 2009
Bacterial internalization, Brown and Price 2007
TEER
E. coli Microarray transcriptomic Zhou et al., 2012
analysis
Bacterial adherence Duan et al., 2012; Koh
et al., 2008; Yin et al.,
2009
1 TLR: toll-like receptor; MAP kinase: mitogen-activated protein kinase; LPS: lipopolysaccharide;
Hsp: heat shock protein
8.7 Conclusions and perspectives
Nowadays, researchers in gut physiology and pathology have access to a wide array
of tools, including cultured intestinal epithelial cells (alone or combined with other
cell types), gut tissue explants, isolated gut segments and whole organisms. All these
techniques have been used in complementarity to investigate gut functioning and
provide valuable information on underlying cellular and molecular mechanisms on
gut responses to all sorts of stimuli, including nutrients, toxicants, commensal or
pathogenic bacteria and environmental factors (e.g. stress). However, while carrying
out experiments with ‘reductionist’ systems, their inerrant limitations must be
borne in mind and the resulting data confronted with (other) in vivo data whenever
possible, in order to validate the coherence of approaches and the biological
meaning of the whole set of information. Finally, these devices allow better scientific

exploitation of living organisms, increase the statistical power of experiments by

allowing repeat measurements within individuals, and finally contribute to reduce
the use of experimental animals. Therefore, all these techniques will continue to
complement each other and in vivo approaches for an always better understanding
of gut function in health and disease.
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Sutherland, L.R., Verhoef, M., Wallace, J.L., Van Rosendaal, G., Crutcher, R. and Meddings,
J.B., 1994. A simple, non-invasive marker of gastric damage: sucrose permeability. Lancet
343: 998-1000.
Szabo, A., Menger, M.D. and Boros, M., 2006. Microvascular and epithelial permeability
measurements in laboratory animals. Microsurgery 26: 50-53.
Tolbert, M.K., Stauffer, S.H. and Gookin, J.L., 2013. Feline Tritrichomonas foetus adhere to
intestinal epithelium by receptor-ligand-dependent mechanisms. Veterinary Parasitology
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Ueno, N., Fujiya, M., Segawa, S., Nata, T., Moriichi, K., Tanabe, H., Mizukami, Y., Kobayashi,
N., Ito, K. and Kohgo, Y., 2011. Heat-killed body of lactobacillus brevis SBC8803
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and Haagsman, H.P., 2006. Differential regulation of porcine beta-defensins 1 and 2 upon
Salmonella infection in the intestinal epithelial cell line IPI-2I. Veterinary Immunology
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Response of Porcine Cell Lines to Salmonella enterica serovar Typhimurium and its hilA
and ssrA mutants. Zoonoses Public Health 54: 286-293.
Walsh, M.C., Rostagno, M.H., Gardiner, G.E., Sutton, A.L., Richert, B.T. and Radcliffe,
J.S. 2012. Controlling Salmonella infection in weanling pigs through water delivery of
direct-fed microbials or organic acids: Part II. Effects on intestinal histology and active
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Genomics 13: 330.

Chapter 9: Intestinal health biomarkers in vivo
T.A. Niewold
Nutrition and Health Unit, Department of Biosystems, Faculty of Bioscience
Engineering, KU Leuven, Kasteelpark Arenberg 30, 3001 Heverlee, Belgium;
theo.niewold@biw.kuleuven.be
Abstract
There is a definite need for biomarkers for intestinal health in vivo, which can be
determined in samples obtained in a non-invasive or minimally invasive way. In
humans, there are approximately fifteen biomarkers suggested, and test and reagents
are available, whereas this is hardly the case for pigs and chicken. Here we review
(possible) biomarkers for intestinal health, and their presence or absence in pig and
chicken. There appears to be a striking lack of information on intestinal biomarkers
in both species. It is also clear that certain biomarkers may not be present in all
species, which is particularly true for chicken, or if present, are immunologically
different. This usually means that reagents for assays are not available. For the pig
there are at least some biomarkers and assays available such as for intestinal fatty acid
binding protein (I-FABP), but essentially none are available for chicken. Given the
importance of intestinal health in production animals, it is hoped that more effort
is invested in this field.
Keywords: enterocyte, inflammation, integrity, urine, faeces
9.1 Introduction
A major problem in trying to determine intestinal health in vivo is the relative

inaccessibility of large parts of the GI-tract. Endoscopic techniques (except
wireless capsule endoscopy) in general do not reach further than proximally to
the duodenum, and from the posterior, to the colon. Although these techniques
enable visual inspection, and to take mucosal biopsies, they are cumbersome,
require sedation, do not allow for larger scale screening, and leave large parts of the
GI-tract uncovered. Stomata can give access to the latter (e.g. jejunum, ileum, and
colon), but require surgical intervention, which by itself may influence the GI-tract.
Furthermore, the presence of stomata itself may interfere with normal intestinal

T.A. Niewold
function, and sampling through them may alter normal (an)aerobic conditions.
It is also evident that these techniques can only be applied under experimental
conditions, and are not suited for larger scale routine evaluation of intestinal health.
As a consequence, usually animals are sacrificed to allow for sampling of intestinal
tissues. This also means that in order to follow the development of intestinal health
over time more animals need to be sacrificed, which raises costs, and raises questions
about representiveness.
Hence there is a definite need for biomarkers for intestinal health, which can be
determined in samples obtained in a non-invasive or minimally invasive way,
meaning from blood (plasma or serum), faeces, saliva, urine, or other bodily fluids.
Suitable candidates should be compounds derived from GI-tract itself directly or
related to it in a specific way. They should be stable which is in particular relevant
in case of excretions such as in the faeces. Furthermore, they should be validated as
indicators of intestinal health, and reagents and assays should be available. And last
but not least in animal husbandry, costs should not be prohibitive.
As in other cases, much can be learned about intestinal biomarkers from human
medicine, for a most part because the greater availability of research funding.
Several biomarkers of intestinal inflammation or dysfunction have been proposed
and validated in humans and experimental animals. It is also evident that due to
species differences all parameters may not be present in production animals, or that
immunological reagents are not cross-reactive. With the increasing availability of
genomic and proteomic information, it is nowadays a relatively easy task to search
for at least the presence of homologous sequences in the genome of most husbandry
species. Whether or not the homologous protein is indeed expressed, or has the
same function and validity as a biomarker must always be established. The greater
the evolutionary distance the more differences can be expected in homology, gene
function, and immunological cross-reactivity, in other words more differences with
chickens than with pigs. As a striking example of the latter, whereas the acute phase
protein haptoglobin is highly homologous in all mammals, the homologous gene
is disused in the branch of birds to which chickens belong, and a totally unrelated
protein (PIT54) has taken over its haemoglobin binding function (Wicher and
Fries, 2006). Another peculiarity of chickens is the different composition of the
excretions, containing a lot of uric acid, which may complicate certain assays. In
this chapter, the current status of biomarkers for intestinal health is reviewed. An
inventory of possible biomarkers has been made, their relevance for intestinal
health and their current or potential applicability for use in pigs and poultry is

9: Intestinal health biomarkers in vivo
evaluated and discussed. Most knowledge is available for the pig, because it is often
used as a model for humans in particular for the gastrointestinal tract.
A distinction should be made between methods which use spontaneous markers,

and added markers. In this chapter, we deal with the biomarkers sensu strictu,
but first briefly describe the dual sugar permeability tests used to assess intestinal
permeability by using dietary probes of different molecular sizes, which can be
measured in plasma and or urine. The method is based on the assumption that
larger molecules pass through the epithelium by the paracellular pathway, via the
tight junctions. Smaller molecules should pass by the transcellular pathways. In case
of diseased or damaged mucosa, the permeability is expected to increase, resulting in
increased levels of the larger molecules. The probes used should be non-fermentable,
not metabolized in the body, and present in urine or plasma in proportion to the
amount absorbed. The probes can be administered alone or in a combination of a
larger and a smaller molecule, in which case usually the ratio of both is determined.
Disaccharides (such as lactulose) are used as large molecules, and monosaccharides
(such as mannitol) are used as small molecules. Varying results have been obtained
for intestinal permeability using these methods, and it is very difficult to interpret
results. This is in part due to a lack of understanding of the mechanisms of intestinal
permeability (Derikx et al., 2010). This may also explain the variable results obtained
in pigs (e.g. Wijtten et al., 2011). To our knowledge, the lactulose/mannitol test has
not been applied in chicken.
As mentioned above, the integrity of the intestinal barrier is very important. The
intestinal wall is a physical and immunological barrier, and permeability should
be controlled. Tight junctions between enterocytes are pivotal in this respect.
Furthermore, enterocytes and inflammatory cells excrete defensins in order to
protect the gut wall against invasion by bacteria and other potential pathogens.
Inflammation in the intestinal mucosa is tightly controlled too, because it may cause
enhanced permeability and damage by itself. As follows from the above, biomarkers
for damage to intestinal health could be either constituents of enterocytes such as
tight junctions proteins, and intracellular products and proteins, either constitutive
or induced. Also, products from inflammatory cells should be useful indicators.
Furthermore, plasma or salivary acute phase proteins are possible parameters for
intestinal inflammation. Last, constituents of blood which can extravasate to the
lumen of the intestine can be useful markers of intestinal integrity (Table 9.1).

T.A. Niewold
Table 9.1. Intestinal health biomarkers, their specificity, presence in species, sampling method,
and the availability of reagents.1
Marker Specificity Species other Sample Test reagents/assay1,2

than human
Enterocytes
• Intestinal fatty acid small intestine porcine • blood Imm: porcine, chicken
binding protein enterocyte damage • urine
(I-FABP) • faeces3
• Claudin 3 tight junction loss, • blood Imm: porcine, chicken
intestine permeability
• Pancreatitis small intestine porcine • urine Imm: porcine
associated protein inflammation • faeces
(PAP, Reg3)
• Citrulline small intestine porcine, • blood Imm: porcine
epithelial loss absent in
chicken
Inflammatory
• Myeloperoxidase intestine inflammation absent in • faeces Imm: / Biochem:
(MPO) chicken porcine
• S100 calmodulin intestine inflammation • faeces Imm: porcine, chicken
• Calprotectin intestine inflammation • faeces Imm: porcine
• Lactoferrin intestine inflammation • faeces Imm: porcine
• HMGB1 intestine inflammation • faeces Imm: porcine, chicken
• Lipocalin 2 intestine inflammation • faeces Imm: porcine
• Neopterin intestine inflammation • faeces Imm: all Biochem: all
• Acute phase inflammation porcine • blood Imm: porcine,
proteins • saliva Biochem: all
(haptoglobin)
Fecal serum protein
• α1-antitrypsin intestine permeability porcine, • faeces Imm: porcine
chicken
1 In italics: claimed but not proven.
2 Imm(unoassay), biochem(ical assay), for species other than human
3 In pig faeces (T.A. Niewold, unpublished data).

9.2 Enterocyte biomarkers
Intestinal fatty acid binding protein (I-FABP, or FABP-2) is an endogenous cytosolic

enterocyte protein of the small intestine which has been shown to be a useful marker
of enterocyte damage in several species. It can be measured in blood, urine and
faeces. It is removed very efficiently by the kidneys leading to a half-life of ca 10
minutes, which makes I-FABP a very useful marker of actual enterocyte damage.
Thus far, using a commercially available human ELISA kit, I-FABP protein has been
demonstrated in pigs (Niewold et al., 2004), but not in chicken. The chicken I-FABP
gene is 71 to 72% homologues to human, mouse, and pig I-FABP genes. The chicken
I-FABP gene was expressed only in intestinal tissues (Wang et al., 2005). This means
that I-FABP could be a good biomarker in chicken too, however, reagents are not
available as yet.
Pancreatitis associated protein (PAP) is also known as regenerating islet-derived 3

alpha (Reg3α). PAP is a C-type lectin, with anti-bacterial and anti-inflammatory
properties, and originally described as a marker for pancreatitis in humans.
However, it became clear that PAP is also produced in the small intestine (Carroccio
et al., 1997), and also in pigs. No PAP expression is found in the jejunum of normal,
non-infected pigs (Niewold et al., 2010). PAP is induced in the jejunum of pigs
by both Gram positive and negative organisms such as Lactobacillus plantarum,
enterotoxigenic Escherichia coli and Salmonella (Niewold et al., 2010). In human
PAP can be demonstrated in plasma and urine, and in faeces of rats (Van Ampting
et al., 2009). Levels reflect the severity of small intestinal damage, but unfortunately
reagents are not cross-reactive with pig PAP. This is most likely caused by the fact
that pig PAP is another isoform than in other mammals (Reg3γ instead of Reg3α
(Soler et al., 2012)). No reports are as yet available on the presence or absence of PAP
or analogues in chicken.
Claudin-3 is the major intestinal sealing tight junction protein. In a rat model
and in human patients with active inflammatory bowel disease (IBD), the
immunohistochemical loss of claudin-3 of from intestinal tissue coincided with the
appearance of this protein in the urine (Thuijls et al., 2010). Thus far, no similar
studies are available on pig or chicken.
Citrulline. Differentiated small intestinal enterocytes produce citrulline (an amino

acid not incorporated into proteins) from glutamine, and produce the major part of
the total amount of circulating citrulline (Curis et al., 2007). This means that levels

T.A. Niewold
of circulating citrulline is a parameter for functional enterocyte mass in mammals,

but not in chicken (Wu et al., 1995). In pigs it has been tested, but is was also noted in
that study that the glutamine level in feed may interfere because glutamine is needed
as the precursor (Berkeveld et al., 2008).
9.3 Fecal serum protein
α-1-antitrypsin (AAT) is a serum trypsin inhibitor and an abundant serum protein

in humans. Intestinal inflammation causes extravasation of AAT into the lumen.
It is resistant to intestinal proteolysis, and is excreted intact in the faeces, and used
in humans for the evaluation of intestinal damage (Kosek et al., 2013). Because the
levels of AAT are very similar in pigs (Takahara et al., 1983), and chicken (Hercz and
Barton, 1978), the technique could be useful in those species too. No reports on the
faecal detection of AAT in pigs or chicken exist as yet.
9.4 Inflammatory cells
Myeloperoxidase (MPO) is an enzyme found in inflammatory cells, mainly in

neutrophiles. It has been used extensively to determine the degree of inflammation
in homogenates of tissues samples, reflecting the amount of inflammatory cells
present. MPO, or rather the total peroxidase (PO) activity, can be quantified by a
simple biochemical assay for PO activity. MPO can be distinguished from total PO
activity by using specific antibodies. Faecal MPO is used to establish the degree
of intestinal inflammation in humans (Kosek et al., 2012). In chicken the method
does not work on tissues and faeces because heterophiles do not contain MPO or
equivalent activity (Brune et al., 1972).
Other faecal inflammatory cell protein markers. In the literature on inflammatory

bowel diseases a whole panel of diverse faecal biomarkers for intestinal inflammation
can be found. Among those are calmodulin also designated S100, and calprotectin,
(D’Haens et al., 2012; Foell et al., 2009), lactoferrin (Sherwood, 2012), lipocalin 2
(Chassaing et al., 2012), and HMGB1 (Vitali et al., 2011). Analogues of most if not
all of these proteins are present in pig. The cross-reactivity of reagents with the pig is
not known. It is much less clear if these biomarkers are all present in chicken. When
working with suckling piglets, it is good to realise that milk-derived lactoferrin and
calprotectin can be found in faeces of breastfed children (Kosek et al., 2013).

Neopterin is a catabolic product of guanosine triphosphate (GTP), a purine

nucleotide. Neopterin is produced by macrophages and dendritic cells upon
stimulation with interferon-gamma (IFN-γ) produced by activated T lymphocytes,
and faecal neopterin is a marker of intestinal inflammation (Kosek et al., 2013). The
advantage of neopterin is that is identical in all species, and thus requires no species
specific reagents. Application in chicken excreta would be hampered by the fact that
neopterin excreted by the kidneys can be derived from inflammatory processes other
than the intestines.
9.5 Plasma acute phase proteins
In the case of IBD in humans, acute phase proteins (APP) such as C-reactive protein
(CRP) and haptoglobin are used as markers of intestinal inflammation. Plasma levels
do correlate well with severity of disease in for instance the disease of Crohn, but
not as well with ulcerative colitis (Vermeire et al., 2006). Furthermore, it is evident
that APP are only good markers for intestinal inflammation in the absence of
other inflammatory processes in the body. What makes APP such as haptoglobin
particularly interesting as biomarkers is that they can actually be measured in saliva
in pigs, and do correlate with severity of disease (Gutiérrez et al., 2009). Furthermore,
haptoglobin is also locally produced in intestinal disease in humans (Jiang et al.,
2013), and in pigs (T.A. Niewold, unpublished data). It is still an open question
whether or not locally produced APP also wind up in the circulation, and if so also
in saliva.
9.6 Discussion
What is striking is the relative paucity of information on intestinal biomarkers in

husbandry species in general. As stated in the introduction, biomarkers may not
be present in all species, as is particularly true for chicken (citrulline, MPO, and
haptoglobin). More importantly, reagents are not available, or not validated. A prime
example of the latter is PAP in the pig. Pig PAP is another isoform than in other
mammals Reg3γ instead of Reg3α which is absent in pigs (Soler et al., 2012), which
explain the lack of cross reactivity with available Reg3α antibodies. Nevertheless,
antibodies are sold with alleged specificity for pig Reg3α (and Reg3γ) without
scientific publications to back this up.

T.A. Niewold
There is a definite need for further research in this field, and initiatives such as the
EU COST Action 1002 Farm Animal Proteomics (www.cost-faproteomics.org) are
very welcome in this respect. For now there are at least some biomarkers available
for pigs such as I-FABP, whereas essentially none are available for chicken. Given the
importance of intestinal health in production animals, it is hoped that our knowledge
in this field will continue to expand.
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Chapter 10: Intestinal health research and
proteomics, a well-matched couple
L. Soler1* and I. Miller2

1INRA, UMR85 Physiologie de la Reproduction et des Comportements, 37380
Nouzilly, France; 2Institute of Medical Biochemistry, Department for Biomedical

Sciences, University of Veterinary Medicine, Vienna, Veterinärplatz 1, 1210
Vienna, Austria; lsolervasco@tours.inra.fr
Abstract
Proteomics can be defined as a field of systems biology oriented at the study of

the proteins present in an organism, tissue or cell under determined conditions. A
wide variety of techniques can be employed in proteomics, with different objectives,
advantages and disadvantages. The use of proteomics is nowadays becoming very
popular in animal sciences, and proteomics is considered a useful tool to explore
at the protein level the changes that occur under different conditions, at particular
events and/or challenges in the intestinal system. In this chapter, we will give an
overview of the proteomic techniques that can be used in intestinal studies in farm
animals. Furthermore, possible applications of these methods will be outlined by
summarizing the most important examples extracted from literature.
Keywords: intestine, proteome, host-pathogen interaction, feed testing, development
10.1 Introduction
The intestinal system is highly complex and its status is influenced by the interaction
of multiple factors. Those factors are related to host (local tissue and components
of the systemic immune system and autonomous nervous system), bacteria (both
commensal and pathogenic) and food (nutrients, functional molecules, toxics). The
variety and intricacy of these interactions result in a highly organized, complex and
dynamic organ (Diekgraefe et al., 2000; Hooper and Gordon, 2001; Niewold, 2005;
Xu and Gordon, 2003). Because of the complexity of the interactions, it is usually
hard to single out effects of specific factors and attribute them to molecular targets.
The recent efforts in characterization of structural and functional pig genomics have

offered solutions to the latter problem, allowing for analysis of multiple responses
captured in gene expression profiles (Van Ommen and Stierum, 2002), mainly
through the use of DNA microarrays and sequencing (Niewold et al., 2005; Wintero
et al., 1996). These techniques, together with the development of bioinformatic tools,
have brought animal research to the molecular level. Furthermore, the advances
in genome sequencing in some species like pig, cow and chicken have greatly
contributed to the representation of farm animal data in public databases (Soares et
al., 2012). In spite of those advances, a complementary analysis is necessary to fully
understand the functions and interactions of the ultimate gene products: proteins.
Many cellular mechanisms are governed by transcriptional regulatory factors, post-
transcriptional and post-translational modifications, protein interactions and protein
abundance changes, which are not directly reflected in genomics (Baggerman et al.,
2005; Eisenberg et al., 2000). As a result, the genomic approach is only sufficient to
determine which set of biomarkers is suitable to predict a given condition. Proteomics
addresses the problems detailed above and is the ideal complementary technique for
genomics. Proteomics is an essential component of systems biology and refers to a
group of techniques aimed at studying the proteome, which is the set of proteins
present in a cell, tissue, organism or population at a certain time point (Wasinger
et al., 1995; Wilkins et al., 1996). Its main objective is describing the structural and
functional properties of a cell/tissue associated with defined conditions by means
of exploring the quantitative and qualitative (e.g. post-translational modifications)
changes in its proteome or identifying the existing protein-protein interactions
(Amoresano et al., 2009; Heck, 2008; Markiv et al., 2012).
In animal sciences, proteomics is a useful tool to connect biological and productive

aspects, since the ultimate goal of farming is the production of proteins in form of
e.g. meat, eggs, milk or wool, in good quality (Bendixen et al., 2011). However, the
use of proteomics in farm animal sciences has been limited thus far; on the technical
side this is probably due to the need of sophisticated equipment and complementary
technical and analytical skills by animal scientists, as well as a lack of awareness of
the possibilities this technique offers (Bendixen et al., 2011; Soares et al., 2012).
Nowadays, the advances in the implementation of simpler, more sensitive, and higher
throughput approaches have made it easier for farm animal researchers to find in
proteomics a useful tool. There is, therefore, more understanding on which questions
may be addressed by proteomics and in which way. As a result, proteomic results are
considered valuable in breeding and farming (for instance, to find relations between
protein pattern, traits and quality), and the number of publications is exponentially
growing (Bendixen et al., 2011; De Almeida and Bendixen, 2012).

10. Intestinal health research and proteomics, a well-matched couple
The application of proteomics in intestinal studies is especially interesting for farm

animal industry. The use of this technique in adequate intestinal models will help
dissecting and describing the molecular processes related to intestinal development,
physiopathology, nutrition or toxicology. Through proteomics, the effect that
changes in nutrition or general handling procedures, preventive (dietary) treatments
or the contact with intestinal pathogens may be properly explored (De Almeida and
Bendixen, 2012; Oozeer et al., 2010). Moreover, the implementation of proteomic
approaches in intestinal research in farm animals, and especially in pigs, is of great
interest for biomedical research. Pigs are ideal biomodels for intestinal research,
since they resemble humans in anatomy, genetics and physiology. In consequence,
there is a wide interest in increasing the molecular biology information in this
species (Rothkotter et al., 2002; Wernersson et al., 2005).
In this chapter, we will outline the technical approaches available for intestinal
proteomics in farm animals and will provide an overview of their possible applications.
10.2 Overview of techniques
Modern proteomics is not a single technique but rather a family of different

highly specialized methods. Here, we will focus on expression proteomics rather
than on other functional, structural or biophysical aspects. The workflow for
proteome-wide characterization of a complex mixture basically begins with a
separation step, either on the protein (Miller, 2011) or on the peptide (Manadas
et al., 2010) level. The patterns obtained can be then compared in order to detect
concentration differences and can be traced back to the proteins which are affected
in the selected investigation. Methodologically, proteomics may be performed as
gel-based and gel-free approaches, and analysis techniques (multi-dimensional
separation, characterization, identification, quantification of proteins/peptides and
comparison of samples) will depend on this. Advantages, properties and drawbacks
of gel-based and gel-free techniques have been widely reviewed (Abdallah et al.,
2012; Baggerman et al., 2005; Monteoliva and Albar, 2004), and main features of the
most representative techniques are detailed in Table 10.1.
Gel-based proteomics: The oldest and most widely used technique in proteomics
is two-dimensional gel electrophoresis (2DE), which involves the electrophoretic
separation of the proteins extracted from the tissue of interest in two steps, first
by their isoelectric point (by isoelectric focusing in gel strips) and then by their

232
Table 10.1. Main features of the most used expression proteomic approaches.
Method Description Advantages Disadvantages
Gel-based Proteins are separated in two-dimensional High resolving power at protein level Labor- and time-consuming, low automation
gels according to isoelectric point (pI) Simultaneous detection of protein Limitations for hydrophobic proteins or
and molecular weight (Mw) and then species (isoforms, fragments, extreme size/pI
identified by MS or MS/MS aggregates) Spot overlap (comigration) impairs
Separation conditions (e.g. pI range) quantification
adaptable to sample composition
2DE One sample/gel Low cost Strict standardization to ensure
Visualization by staining (variable) Dyes with different sensitivity allow reproducibility
variable protein loads and detection Small concentration changes not detectable
limits Low dynamic range for some dyes
(colorimetric dyes)
2DE-DIGE Two or three samples/gel Multiplexing Higher cost
Internal standard for normalization Lower technical errors allow detection Specific equipment
of small concentration chances Higher protein load due to combination of
Sensitive, large dynamic range samples
Intestinal health

Method Description Advantages Disadvantages
Gel-free Proteins are digested into peptides, which High performance Increased complexity of samples due to
are separated by multidimensional liquid Detection of natural peptides possible digestion
Intestinal health
chromatography and then identified and (without digestion) No information on intactness of or variation in
quantified by MS/MS Hydrophobic proteins original protein
Use of multiple/different enzymes to Evaluation of post-translational modifications
generate peptides requires specific approaches
Dependence on quality and completeness of
databases
Label-based Peptides/proteins are labeled in a way that High sensitivity Expensive reagents
does not change chromatographic and Possible multiplexing (up to 4-8 High amount of starting sample
ionization properties, but creates a mass- samples) Multiple preparation steps (sample loss/
shift signature at MS or MS/MS level variability)
Chemical labeling Proteins/peptides are labeled by a chemical Lower costs Sample variability due to labeling
(proteolytic reaction High MS sensitivity Interaction of labels
labeling, ICAT, ICPL,
iTRAQ, TMT)
Metabolic labeling Labels are incorporated at the time of High MS sensitivity Cumbersome
(SILAC, 14N/15N) protein synthesis Low technical variation Expensive
Variable labeling efficiency between
organisms
Label-free (spectral Number of MS/MS spectra matched Simple and cost-effective Prone to analytical variability
counting, spectral to a specific protein are compared or
peak intensities, precursor ion retention times and m/z
data-independent ratios are accurately generated for all MS/
analysis) MS spectra of each protein identified in a
sample, creating a 2D map and matching
peptides across samples
233
molecular weight (by sodium dodecyl sulfate polyacrylamide gel electrophoresis,

SDS-PAGE; Figure 10.1). As a result, the protein mixture is resolved in a
two-dimensional spot pattern known as the proteome map of that tissue (Westermeier
and Görg, 2011). The comparison of the proteome maps of one tissue from different
sources helps identifying the protein spots affected by varied parameters (e.g.
healthy/diseased animals, control/exposed cells). Reproducibility of spot pattern is
one of the key points for reliable interpretation; therefore, sets of fluorophores were
developed that allow pre-electrophoretic labeling of proteins and 2DE separation
of several samples on one gel. This new approach, called 2DE differential gel
electrophoresis (2DE-DIGE) permits relative quantification of regulated spots and
a more accurate comparison of spot patterns, which makes DIGE more and more the
method of choice for 2DE quantitative studies (Friedman and Lilley, 2008; Marouga
et al., 2005). Evaluation relies also on an internal standard on each gel, a pool of all
samples in the respective experiment, for improved pattern matching and relative
quantification (Friedman and Lilley, 2008; Minden, 2012).
Protein identification may be achieved in mainly two different ways: If the protein
of interest is known and a specific antibody available, immunoblotting may be
performed and overall protein pattern correlated to specifically stained spots
(Miller et al., 2009). A more general approach, for identification of unknown
proteins, applies mass spectrometric analysis (MS) on the excised spot, after tryptic
digestion of the protein to peptides (Gevaert and Vandekerckhove, 2000). Protein
identification is based on very accurately determined peptide masses, which are
then attributed to amino acid sequences of known proteins by using bioinformatic
tools and by search in publicly available protein or gene databases (Gevaert and
Vandekerckhove, 2000). Likelihood of identification is increased by determining
also the fragmentation products (MS/MS pattern) combined with a database search
or with de novo sequencing (determination of amino acid sequence of the respective
peptide, for not yet catalogued proteins) (Gevaert and Vandekerckhove, 2000).
Gel-free proteomics: The progress of mass spectrometry has allowed the development
of gel-free techniques for proteomic analysis in the last years, which are also known as
‘shotgun’ or LC-MS proteomics. In this case, the proteins of the (complex) sample are
first trypsin-digested and then subjected to high resolution liquid chromatographic
(LC) separation, often in a multi-dimensional way (typically strong cation exchange
and reverse phase chromatography), prior to MS identification (Figure 10.2; Abdallah
et al., 2012; Monteoliva and Albar, 2004). Although the MS part is based on the
same principles as described for peptides eluted from 2DE spots, due to the higher

Gel-based expression proteomics workflow
Sample Sample
A B
Possible sample treatment

(depletion, fractionation, enrichment) Sequence assignment and
database search
2DE and
Relative abundance
pl image analysis
Mw
Protein
identification
m/z
MS/MS
Relative abundance
Spot
excision
Database
search
Trypsin
MS m/z
digestion
Figure 10.1. Schematic overview of expression proteomics analysis through two-dimensional

gel electrophoresis. Proteins of samples A and B are resolved first by isoelectric focusing (taking
original sample or after treatment to fractionate or deplete/enrich some proteins) and then by
sodium dodecyl sulfate polyacrylamide gel electrophoresis. 2DE gel images are digitized and
analyzed with specific software to identify differentially expressed spots. These spots are excised
from the gels, trypsin-digested into peptides and analyzed by MS. Proteins are identified by
comparing the fragment mass or peptide mass fingerprint of the obtained tryptic peptides with
public databases containing the theoretical fragment masses of known proteins. In some cases, an
additional step is needed to obtain a reliable identification, consisting in peptide fragmentation
by MS/MS and further peptide de novo sequencing.
complexity of the sample and the coupling with LC refined bioinformatic tools had
to be developed. For relative and absolute quantitation, gel-free proteomics relies on
the use of chemical or in vivo labeling (isotopic non-isobaric or isobaric labeling),
or lately also on label-free approaches (Fenselau, 2007; Timms and Cutillas, 2010).

Sample Sample
A B
Denaturation, reduction, digestion

(eventual labeling and sample combination)
Peptide separation by nanoLC
MS/MS and spectra analysis
Protein abundance Protein identification
Gel-free expression proteomics workflow
Figure 10.2. Schematic overview of gel-free expression proteomics analysis. Proteins A and B are
reduced, alkylated and trypsin-digested. If a label-based approach is used (e.g. iTRAQ), peptides
are then labeled and samples combined prior to analysis. Other types of labels (e.g. SILAC) are
already introduced in the intact proteins, before digestion. Peptides are first separated by HPLC
and then analyzed by MS/MS. Peptides are identified by comparing the MS/MS spectra with
public databases. In label-based approaches, relative quantification is achieved directly, since
labeling will cause a known mass-shift of peptides. In label-free approaches, protein abundance
can be calculated by different MS spectra analysis as reviewed by Timms and Cutillas (2010).
Besides these main types, also hybrid forms exist, for instance a combination of
one-dimensional SDS-PAGE to achieve a pre-fractionation and LC-MS of the
tryptic peptides eluted from single gel slices. In addition, various possibilities for
pre-fractionation of the complex protein mixtures or for removal of abundant
proteins have been developed, to be applied also prior to gel-based separations, all to
achieve a higher sensitivity for detection of low abundant or trace proteins (Righetti
et al., 2005; Tichy et al., 2011).

All proteomic methods described in the previous paragraphs are non-targeted

approaches, aiming at detecting differences between protein or peptide patterns
and only in a second step identifying the proteins involved. In contrast to this, the
objective of targeted proteomics is to analyze only a selected list of (low abundant)
proteins in complex samples (Picotti and Aebersold, 2012). The system relies on
the use of liquid chromatography coupled to sophisticated high-resolution mass
spectrometers and an MS technique termed multiple/selected reaction monitoring
(MRM/SRM). The method needs very careful design: peptides specific and
unique for the protein of interest have to be selected and tested in advance, and
the separation parameters carefully established to retain only the selected peptides.
Then, the respective protein can be quantified with high sensitivity in comparison
to labelled peptides which had been spiked into the original sample (Calvo et al.,
2011). Originally, a similar technique has been widely employed for the detection
of drugs and small molecules and only recently has been adapted for proteomic
applications. Typically, targeted proteomics is used to verify and validate markers
which have been discovered by expression proteomics, or for biomonitoring, and it
is yet a field to explore in animal sciences (Whiteaker et al., 2011; Ye et al., 2009).
Presently still more commonly applied for validation is immunoblotting, which
utilizes immunologic reactivity of specific antibodies. Unfortunately, this method
depends very much on the availability of species-specific immunoreagents of good
quality (Hause et al., 2011).
The choice of a particular proteomic approach has to be made very carefully by

taking into account the complexity of the sample and the biological question to be
addressed, due to the big differences in reproducibility, resolution power, dynamic
range, sample throughput, complexity and cost of each technique (Abdallah et al.,
2012; Domon and Aebersold, 2010). It is often mentioned in the literature that
gel-based and gel-free methodologies are complementary, the first approach being
more useful to detect post-translational modifications and the latter having typically
a higher yield in membrane proteins (Baggerman et al., 2005; Leroy et al., 2011; Wu
et al., 2006). As already mentioned, the proteome analysis provides a rich source
of information for describing the status of a tissue or organism. For example, it is
estimated that the 35,000 genes of the human genome give rise to more than 1,000,000
functional entities at the protein level (Wang et al., 2006). Different biochemical
events such as post-translational modifications or protein interactions contribute to
raise the level of complexity and microheterogeneity of the proteome. Mainly, these
additional protein products derive from alternatively spliced genes or represent
isoforms, post-translational modifications (e.g. glycosylation, phosphorylation),

folding states, and breakdown products. 2DE gel patterns are further complicated
as due to the separation conditions (reduced and denatured protein state) proteins
may appear as multiple spots or chains, resulting in complex patterns with hundreds
of spots, depending on resolution and gel size (Miller, 2011). As an example, Figure
10.3 shows a 2DE image of scrapings from pig intestinal mucosa. Similar to 2DE,
also in MS methods sample pretreatment increases complexity of the specimens: in
this case, tryptic digestion creates multiple peptides per protein, thus multiplying the
number of peaks to detect (Soares et al., 2012). All this explains the high technical
demands for proteomic pattern analysis. About ten years ago it was estimated
that our detection systems are not able to monitor concentration ranges of more
than three orders of magnitude (Anderson and Anderson, 2002). With the further
developments of the methods and refined prefractionation steps this has most likely
improved in the meantime, but is still far beyond the range in nature (about 12
orders of magnitude in plasma (Anderson and Anderson, 2002)).
A great variety of human intestine-related proteomic studies are available in the

literature, including research on host-pathogen interactions (Elmi et al., 2012),
cancer (Wang et al., 2012a), inflammatory bowel disease (Felley-Bosco and André,
2004), toxicology (Wang et al., 2012b), parasitic diseases (Wang et al., 2012c) or celiac
94
67
45
kD
30
20
14
4 pl 10
Figure 10.3. Two-dimensional gel electrophoresis of scrapings from pig intestinal mucosa.
Samples were homogenized in lysis buffer (8 M urea, 4% CHAPS, 30 mM Tris-HCl, pH 8.5);
50 µg protein were separated in 2DE gels according to Miller (2012) followed by silverstaining.
pI = isoelectric point.

disease (Bertini et al., 2009). In the majority of these studies, samples were obtained
from hospital patients after surgery. Mouse models of human diseases were also
widely used for in vivo experiments or developmental time-course studies, mostly
using conventional mice, although gnotobionts (Alpert et al., 2009; Roy et al., 2008)
or knock-out (Cooney et al., 2012; Werner et al., 2009) mice have been employed
alternatively. A smaller number of studies have used in vitro models for proteomic
analysis. In these models, besides treatment or exposure-induced protein changes,
also cell-specific alterations were visible, when epithelial cells undergo differentiation
or adaptation to different culture systems (Buhrke et al., 2011; Pshezhetsky et al.,
2007; Stierum et al., 2003). Similar to other applications of cell culture models, the
limited translatability of the results obtained in vitro to in vivo conditions concerning
data interpretation, as well as between different cell lines are important factors to
account for (Lenaerts et al., 2007a).
Despite the benefits and adequacy of this technique, the use of proteomics to evaluate
the influence of different challenges in the intestinal cells is scarce in animal sciences,
and practically limited to pigs. In the following sections we will show the possibilities
of this technical approach to solve current intestine-related problems in animal
sciences. We will review how researchers have applied proteomics to explore (1)
how the intestine develops in the early stages of life; (2) how proteomics can help in
feed testing; and (3) how this technique may be applied to unravel the mechanisms
underlying host-pathogen interactions at the intestinal level.
10.3 Proteomics as a useful instrument for developmental studies
The whole intestine, but in particular small intestinal enterocytes, undergoes

dramatic changes during the early stages of life. Key events like birth (where bacterial
colonization occurs and diet changes from amniotic fluid to milk) and weaning
(where diet changes from milk – a fatty acid based diet – to solid food – a complex
carbohydrates based diet) induce dramatic changes in structure and functionality in
intestinal epithelial cells (Hansson et al., 2011). Those changes are highly determined
by the interaction of diet, bacteria and intrinsic factors. The understanding of these
relations will help defining which handling procedures are more effective to promote
an optimal intestine development in farm animals, mostly those intensively produced,
where a stronger adaptive stress is induced at intestinal level (Hansson et al., 2011).

Recently, the power of proteomics to explore those changes has been illustrated in
mouse models for humans. In humans, the study of intestine development is basically
oriented to finding preventive or therapeutic strategies to several pathologies
observed in newborns. Proteomics has been employed to identify the time-course
modifications occurring due to the dietary changes taking place after birth and
weaning, to determine which modifications appear before the actual dietary change
to prepare the intestinal tract and which are dependent of the diet change itself.
Additionally, the proteomic effect of different postnatal diet regimes and bacterial
colonization (in germ-free or gnotobiotic mice) has also been studied. In contrast
to humans, the objectives of developmental research in intensive farming animals
(except for pigs used as biomodels) are optimizing intestine development for optimal
exploitation of the digestive system as well as the promotion of health in early stages
of life (De Lang et al., 2010; Lallès et al., 2007). There is a small but interesting
collection of publications that describe the modifications induced by development
in the gut of farm animals (mostly pigs) and how several factors can influence it.
In pigs, the major proteome change in epithelial cells after colostrum consumption is
the uptake of colostral IgG or IgA as determined by 2DE. This uptake is significantly
reduced in the inflamed enterocytes, illustrating how important it is to avoid
inflammation in the newborn intestine in order to promote the acquisition of passive
immunity (Danielsen et al., 2006). The importance of immunoglobulins and other
protective or antimicrobial proteins present in colostrum is also evidenced by their
selective resistance to digestive degradation. In contrast, other colostrum constituents,
like caseins, were digested prior to their arrival in the small intestine (Danielsen et
al., 2011). Bacterial colonization is a very important step in intestinal development.
Bacteria assist in making nutrients available, promoting immune and intestinal
development and by having a protective role. The use of germ-free or gnotobiotic
animals has helped understanding those processes by simplifying the model system
and reducing the number/strains of bacteria in the animal. In pigs, the colonization of
different non-pathogenic bacterial species (Lactobacillus fermentum and Escherichia
coli) in germ-free piglets was monitored by shotgun proteomics, showing results
similar to those in laboratory animals (Danielsen et al., 2007). In this study it was
described that bacterial colonization (regardless of the colonizing bacterial species)
affected the metabolic status of enterocytes, especially its proteolytic and lipid
metabolism status. However, some of the proteome changes identified in this study
were species-specific: E. coli induced epithelial proliferation, whereas L. fermentum
induced the development of an immune response. The last finding clearly illustrates

the need of commensal bacteria to build up a protective system at the intestinal level,
and explains why the intestinal immune system in germ-free animals is immature.
Intrauterine growth restriction (IUGR) is a well-known cause of intestinal

development impairing (Wang et al., 2010). This condition is a common problem
in pigs (and to a lesser extent in other farm animals and man), and results in a
lower nutrient utilization efficiency, lower growth rates and health status, as well as
higher mortality. A proteomic analysis described the alterations produced by IUGR
in the abundance of proteins related to cell proliferation, metabolism and innate
immune development after birth and during the postnatal period. These findings
were identified as the cause for the abnormally low absorption of nutrients and were
expected to provide new treatment strategies to diminish the impact of IUGR in
piglets’ mortality and growth (Wang et al., 2010).
Preterm piglets have also been used as models to unravel the pathology of some
human intestinal development problems, such as necrotizing enterocolitis (NEC).
This is a frequent feeding-induced inflammatory disorder in premature newborns
that occurs because of the immatureness of their digestive system. To find therapeutic
solutions, the influence of enteral formula feeding and bacterial colonization in the
disease development were studied by proteomics (Jiang et al., 2008, 2011a). It was
determined that the development of NEC in preterm piglets was characterized by
proteome changes related to oxidative stress, apoptosis and proteolysis, and that
those changes were accentuated with bacterial colonization, which enhanced the
enteral stress response. It was later reported that the proteome changes occurring
during the development of NEC were independent of the birth transitions, although
those pigs enterally fed in utero did not show some changes related to inflammation
observed in piglets fed after birth (Jiang et al., 2011b). The use of antibiotics seemed
to have a preventive effect towards the development of NEC, as demonstrated by
the promoted proteome changes related to antioxidant and anti-inflammatory
effects (Jiang et al., 2012). Moreover, the molecular basis of necrotizing enterocolitis
predisposition in preterm caesarean-delivered pigs was compared with those of
pigs delivered spontaneously at term (Jiang et al., 2013), demonstrating that the
predisposition of preterm piglets is determined by alterations in epithelial integrity,
stress response and impaired cell metabolism.
The proteome changes of the chicken small intestine during adaptation in the early
posthatch period revealed differences between different genetic lines of broilers
(Gilbert et al., 2010). This study also contributed to a better understanding of digestion

and absorption in this species, by describing the enzymatic, proliferative, metabolic

and stress-related changes that the enterocytes undergo when changing from a lipid-
based diet inside the egg to a carbohydrate and protein-based diet (Gilbert et al., 2010).
10.4 How can proteomics help us to improve feeding regimes?
Research in novel health promoting feed additives or the effect of particular feed
compounds on the health status is a field of interest in animal production (De
Lang et al., 2010). However, the approach in animal feed testing is often limited
to evaluating the effectiveness of such compounds for their attributed function
through physiological or productive measures (growth-to-feed and/or growth rates,
body composition, etc.), and the investigation on the effect of those substances
locally on the intestine at the molecular level is relatively rare. Among the
techniques for the latter, proteomics is one of the most useful tools, allowing for
the global exploration of the effects of any substance, and thus investigating their
mechanism of action and toxic effects at the same time (Fuchs et al., 2005). Yet,
sometimes it is not easy to discern which effects derive from the studied substance’s
mechanism of action and which from toxicity; this usually needs more detailed
studies. Proteomics has been successfully employed in human studies to evaluate
the gut proteomic effect of feed components like wheat amylase trypsin inhibitors
(Yang et al., 2011), glutamine (Deniel et al., 2007; Lenaert et al., 2006), antioxidants
(Thébault et al., 2010; Kaulmann et al., 2012), arginine (Lenaerts et al., 2007b) and
other substances in the intestine both in vivo (using rodents) and in vitro. On the
other hand, proteomics has also been applied to investigate the bacterial proteomic
profile of strains with a particular probiotic activity in order to find protein markers
to help screening probiotic bacterial strains within bacterial species (Ashida et al.,
2011; Gilad et al., 2011).
The effect of feed components on the proteome of intensive farm animal intestine
has been scarcely studied. The search for reliable and effective alternatives to the
banned antimicrobial growth promoters is a hot topic in animal nutrition. Among
the proposed alternatives, probiotics have been extensively studied and some
research has been carried out on the proteomic aspect. In fact, the protective role of
Lactobacillus plantarum to Salmonella typhimurium infection was recently described
by proteomics (Collins et al., 2010). It was reported that L. plantarum promoted an
increased acidic mucin secretion, cytoskeletal rearrangements and overexpression
of proteins with immune functions that showed an important defensive role to

the invasion of S. typhimurium (Collins et al., 2010). Additionally, the mechanism

of action of the probiotic L. fermentum I5007 was compared with the antibiotic
aureomycin by 2DE and subsequent MS-based identification. Both (L. fermentum
and aureomycin) improved oxidative and stress resistance of the small intestine
mucosa cells, but diverged in their effect on other metabolic and immune-related
networks. In fact, L. fermentum showed some extra protective effects by promoting
energy and protein metabolism, enterocyte proliferation and immune response
(Wang et al., 2012d).
Zinc oxide has also been identified as a promising growth promoter in different
studies. It was recently demonstrated by proteomics that this substance improved
the redox state and reduced the oxidative stress in jejunal cells, while protecting
them against apoptosis, thus alleviating weaning-associated intestinal dysfunction
(Wang et al., 2009).
The investigations detailed above illustrate the utility of proteomics to explore the
beneficial effects of some feed additives, but it is also useful to determine in which
way a feed toxic or an allergen can affect the intestinal physiology. For instance, it is
not clear how some plant-derived compounds, like soybean-derived β-conglycinin,
which are present in pig’s daily rations, can have negative effects on digestion. In a
recent study conducted by proteomics it was confirmed that β-conglycinin induces
apoptosis, intestinal cell-growth depression and cytoskeleton damage at gut level,
and that such substances should therefore be kept at low levels in animal feeds (Chen
et al., 2011).
10.5 Expression proteomics to unravel the gastrointestinal immune

response
As diarrhea or intestinal parasitosis are important (intestine-related) causes

of economic losses in intensive farming, a great interest is put in unraveling the
mechanism of intestinal disease in search of new preventive or therapeutic targets
(Biron et al., 2011). The intestinal defensive system is highly complex. In addition
to the physical protection offered by the tight junctions of epithelial cells, the mucus
layer and peristaltic movements, the immunocompetent enterocytes, local immune
cells and Peyer’s patches are involved in the mucosal immune system. Furthermore,
the nervous system (mediated by the vagus nerve) and the systemic immune system
also mediate in the intestinal response against pathogens, representing a highly

intricate system. Host-pathogen interaction significantly alters protein abundance,

expression and interactions both from the infected tissue and the infectious agent.
Proteomics is a valuable tool in this respect, since it is able to explore the proteome
changes occurring during infection in a global way.
To date, large efforts have been made to use proteomics to describe the proteins
essential for pathogen invasion or to determine virulence factors of bacterial/viral
species, also in search of proteins relevant to vaccine production. Important pig
intestinal pathogens such as Trichinella spiralis (Wang et al., 2012c), porcine circovirus
type 2 (Fan et al., 2012), pig rotavirus (He et al., 2013), Yersinia enterocolitica (Gu et
al., 2012; Matsumoto and Young, 2006), Campylobacter jejuni (Elmi et al., 2012; Liu
et al., 2012), S. typhimurium (Sun and Hahn, 2012), enterotoxigenic E. coli (Roy et
al., 2010) or Shigella dysenteriae (Kuntumalla et al., 2009; Pieper et al., 2009), among
other, have been extensively studied through proteomics.
In farm animals, substantial efforts are made in the specific breeding of resistance
towards intestinal diseases and in developing effective vaccination strategies. The
understanding of the mechanisms involved in the host response towards those
pathogens is crucial, and proteomics is nowadays recognized as a valuable tool
for this purpose. Unfortunately, up to now there is only one proteomic study on
the host response to pathogens in farm animals. In this publication, the response
of pig ileal cells to S. typhimurium was characterized and the pathogen-driven
changes ‘triggering’ bacterial internalization mechanisms as well as the cellular
innate response were identified (Collado-Romero et al., 2012). Regulation of
different networks associated with innate immune response at the expense of the
specific immune response, anti-apoptosis signaling, anti-inflammatory responses
and dendritic cell maturation was detected. Salmonella are invasive bacteria, and
cytoskeleton proteome changes related to bacterial internalization and also to an
increased phagocytosis were found. Authors combined this proteomic approach
with RT-qPCR analysis and proved by both proteomic and genomic analysis that
S. typhimurium inhibited the Th2 and Th17 response at mucosa level. The latter
study demonstrates that proteomics is a useful instrument to globally evaluate the
mechanisms involved during early stages of intestinal infection.

10.6 Conclusions
Proteomics is an expanding field of study in intensive farm animals. The properties

of this technique make it very adequate to study complex systems, like the
intestinal system. Different studies have applied proteomics to investigate intestinal
development, host-pathogen interactions and the local effect of various substances. A
general picture of the molecular events involved in the latter processes was given, and
potential biomarkers of intestinal health were identified. In conclusion, proteomics
can be considered a valuable tool to explore the intestinal health in search of ways
of promoting animal production.
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Chapter 11: Systems biology – applications in
intestinal health
D. Schokker1 and M.A. Smits2,3*

1Wageningen UR, Livestock Research, Droevendaalsesteeg 1, 6708 PB
Wageningen, the Netherlands; 2Wageningen UR, Central Veterinary Institute,

Postbus 65, 8200 AB Lelystad, the Netherlands; 3Wageningen UR, Host
Microbe Interactomics, Postbus 338, 6700 AH Wageningen, the Netherlands;
mari.smits@wur.nl
Abstract
Animal intestinal health is a complex trait and is determined by the interplay of animal
nutrition, microbiota and host genetics. Complex traits can be better understood by
using a systems biology approach. The ultimate goal of such an approach is to generate
a solid knowledge-base and predictive frameworks on the different functionalities of
the gut along its various spatial, temporal and environmental dimensions. Systems
based knowledge and in silico models of the intestinal tract will be pivotal to fully
exploit the biological potential of host-feed-microbe interactions in the gastro-
intestinal tract of livestock species to improve health traits. This chapter provides a
brief introduction in the upcoming area of intestinal systems biology.
Keywords: intestinal health, mathematical models, host, nutrition, microbiota,

interactions
11.1 Introduction
Systems Biology is the field which focuses on complex biological systems and the
respective interactions within such systems using a holistic approach. Here, we
will focus on two different strategies to get more insight into complex systems: (1)
mathematical models; and (2) networks/graphs. Recent studies increasingly use high
throughput -omics technologies, that generate thousands of data points. With these
technologies it is possible to generate genome-wide global views of the molecular
structures and molecular compositions of biological samples. Such studies have
revealed the complex interaction between microbes and their virulence strategies

on a number of immunological strategies used by host, and on several mechanisms

by which the host-microbe interaction is influenced by external factors, such as
nutrition and stress. However, relative simple interactions of a system can lead to
complex behaviour, in other words the emergent properties of a system are important
to characterize. Moreover, biological functions do not simply occur themselves,
but arise from dynamic interactions of system components. To understand the
genetics and physiology of intestinal health, it is required to get data information
from different timeframes and different scales. Unfortunately, to analyse such multi-
scale systems as a whole is still difficult. The models generated by a Systems Biology
approach will provide a framework to predict (aspects of) the outcome of intestinal
health, when the environment, the host (genetics), or pathogenic pressure changes.
The availability of Systems Biology models may provide a solid foundation for
improving intestinal health in livestock, e.g. prevention of infections/disease and
(early) intervention by feed (additives).
11.2 Intestine as a model
Future livestock systems require robust animals that are resilient to challenges
and are able to maintain production performance under a variety of conditions.
Robust animals are less susceptible to diseases and use lesser quantities of medicines
(antibiotics), which is beneficial for efficient animal production and animal welfare
and limits the transmission of pathogens and antibiotic resistance genes to the human
population. The robustness of production animals is dependent on the ability of their
immune system to respond to challenging conditions in an appropriate manner and
to evoke an efficient immune response cascade towards an antigenic stimulation, i.e.
by displaying the right balance between inflammation and immune tolerance. This
ability is frequently denoted as ‘immune competence’. A significant portion of the
immune system is associated with mucosal surfaces, especially the mucosal barrier of
the gastro-intestinal tract. Therefore, the gut is frequently regarded as the gatekeeper
of health and as a major contributor to a sustainable and socially acceptable animal
production system. Mucosal health is a very complex and therefore a pre-eminent
multifactorial trait. The epithelial layer is a physical barrier and the first line of
defence, which is strategically placed between the luminal content and the underlying
mucosal immune cells. Components of the luminal content, dietary components,
host encoded proteins and metabolites, and microbiota signal via the epithelium
cell layer and the follicle-associated epithelium of Peyer’s patches to mucosal
immune cells to keep up an appropriate immune homeostasis. This signalling is

11. Systems biology – applications in intestinal health
affected by host genetics, maternal effects, neonatal conditions, animal nutrition,

environmental conditions and to a significant extent by the composition and diversity
of the intestinal microbiota. In turn, this microbial composition and diversity is
affected by the host genotype, maternal, neonatal, nutritional, and environmental
conditions, and the use of antibiotics, pre- and probiotics, and feed composition.
Intestinal microbiota consists for a large part of bacteria that belong to the phyla
of Firmicutes, Bacteroides, Actinobacteria, Proteobacteria and Verrumicrobia. They
contribute to the immune signalling, but also to nutrient processing and production
of metabolites with essential functions, such as vitamins and short-chain fatty acids.
The complex interplay in the gut enforces the development and maintenance of an
immune system that has the ability to avoid excessive inflammation and to retain the
capacity to defend against challenges. Disturbances of homeostasis result in growth
retardation, decreased feed conversions and increased susceptibility to infections,
the latter with consequences for the use of antibiotics. Furthermore, it is known
that the pattern of early microbial colonization of the gut shapes the future immune
competence and that variations in microbial gut colonization severely impacts
immune competence and the susceptibility to (infectious) diseases (Russell et al., in
press). Nowadays, specific action in this area are undertaken to develop approaches
for improved immune competence, health and welfare in livestock species in order to
identify routes for the implementation of this knowledge in improved management
of immune competence in livestock species and concomitant robustness of animals.
Understanding the gut as a system requires system approaches to position the
available quantitative data on host-microbe-feed interactions with spatial, temporal
and environmental dimensions into a conceptual framework, providing mechanistic
dynamic models that allow for predictions under a wider range of conditions.
11.3 Integration
To describe complex systems, it is important to identify the different components

and often these components act at different biological levels. The intestine consists
of different components, like cell types, including epithelial, goblet, immune cells,
but also blood vessels, connective and nerve tissue are present. All these different
biological components act at various biological levels. -Omics technologies
allows one to pursue all constituents considered collectively, for example with
transcriptomics it is possible to measure gene expression (activity) of the whole
genome by microarray technology. Various -omics technologies exist and all focus
on different aspects of a system (Table 11.1), generating a flood of data. The challenge

Table 11.1. Various -omics and their corresponding datasets.
-omics Collection of
Epigenomics Epigenetic modifications

Genomics Genes (DNA sequences/chromosomes)
Interactomics All interactions
Lipidomics Lipids
Metagenomics Genetic material found in an environmental sample
Phenomics Phenotypes
Physiomics Physiology of an organism
Proteomics Proteins
Transcriptomics mRNA transcripts
now is to integrate these large datasets in order to understand the underlying

biological mechanisms. Examples are data integration of gene-gene interactions or
protein-protein interactions into a graphical interface. These graphs can be either
pathways or networks, describing cellular processes. It is also possible to test and
simulate different outcomes of these cellular events, by connecting these graphs to
Systems Biology tools/software via the Systems Biology Markup Language (SBML).
However, the question is: do these simulations of cellular processes have enough
detailed information to predict the systems behaviour accurately? Although, efforts
are made to complement these cellular processes at different levels, it is still difficult
to predict the outcome. This is because the data is not binary (i.e. the interaction exist
or not), and direction, location, timing, strength are important as well for a proper
prediction in a model.
11.4 Perturbations
The gastro-intestinal tract is an ecosystem where the state is determined by internal

and external conditions, and these conditions are dynamic. If there would be only
one steady state in the system, the system will always settle back to essentially the
same state after perturbations. However, one could imagine that the gut-ecosystem
has alternative stable states, meaning that if a sufficient severe perturbation to
the ecosystem state occurs it may lead to another state of the system. This way of
thinking was visualized by Scheffer et al. (2001) by generating a stability landscape

(Figure 11.1). Where valleys are stable equilibriums and hills depict the unstable
middle section of the folded equilibrium curve. If the size of the attraction basin is
small, resilience is small and even a moderate perturbation may bring the system
into the alternative basin of attraction.
11.4.1 Impact of perturbations
To translate this landscape into terms regarding the host, we can think of temperature
or pH that can differ in the gut or low/high concentration of nutrients, which shows
that these genotype x environment interactions are highly dynamic. This may result
in constant extracellular changes, as well as changes in the intracellular environment,
like DNA damage, fluctuations in the concentration of RNA/protein. To cope with
this ever changing environment, cells have to respond accordingly by regulating the
expression of their genes (Miller-Jensen et al., 2007). In the gut, multiple processes
run simultaneously, including metabolic (nutrient absorption), immunologic (barrier
function/recognition), and structural (differentiation/apoptosis) processes, and as
mentioned earlier, these processes are regulated by the expression of genes. These
genes form networks/pathways, e.g. gene regulatory networks (GRNs) or signalling
cascades, which consist of genes (nodes) and their corresponding interactions
(edges). When visualizing such networks, it becomes apparent that transcription
Figure 11.1 Metaphorical stability landscape.

factors (TFs) are the key players in these networks or signalling cascades, because
they influence many genes at once. Examples of important TFs in the gut are nuclear
factor-kappa B (NF-κB) and peroxisome proliferator-activated receptor-γ (PPARγ).
NF-κB controls the regulation of pro-inflammatory cytokines and chemokines
that are necessary for mounting an immune response against pathogenic invaders
(Kawai and Akira, 2007). The cellular response is depending on the recruitment
of the adapter proteins MyD88, MAL, TRIF, and TRAM that form a complex with
the C-terminal domains of different TLRs (Akira and Takeda, 2004). PPARs act as
fatty acid activated TFs and belong to the nuclear hormone receptor superfamily
(Kersten et al., 2000). Expression of PPARγ is restricted, with highest levels
expressed by adipocytes and macrophages (Bookout et al., 2006; Escher et al., 2001).
PPARγ contributes in the regulation of inflammation by contributing to epithelial
mechanisms (Shibolet and Podolsky, 2007), and a direct link exists between NF-κB
and PPARγ, because PPARγ promotes export of the p65 subunit of NF-κB from the
nucleus, thereby limiting inflammatory cascades (Kelly et al., 2004). To get more
insight in these networks, both the ‘normal’ and the ‘perturbed’ state of the system
must be investigated. Perturbations are disturbances of the functions of a (biological)
system, and can be induced by external (e.g. drugs) or internal (e.g. autoimmune
disease) mechanisms. Here, we will highlight three of such perturbations: (1) (auto-
immune) disease; (2) microbiota; and (3) nutrition, and their respective impact on
the system.
11.4.2 Disease as perturbation
Disease can be used as a perturbation of the system, because the ‘normal’ (healthy)
state of the system is shifted towards a ‘diseased’ state (Del Sol et al., 2010). Many
studies have contributed to this topic, including cancer studies (Taylor et al., 2009;
Volinia et al., 2010; Wu et al., 2010), diabetes (Newgard and Attie, 2010; Zelezniak
et al., 2010) and autoimmune diseases (Baranzini, 2009). In these studies different
network approaches are performed, e.g. protein-protein interaction, gene-gene
interaction, metabolic networks, to gain more insight in the ‘perturbed’ state of
these ‘cellular’ networks, in other words investigate and characterize the differences
of genes (nodes) and corresponding interactions (edges) between the ‘normal’ and
‘perturbed’ state of the system. To understand the whole system, i.e. the ‘blueprint’ of
the cellular network, is a challenging task, because preferably time-series data needs
to be available and simultaneously multiple (biological) levels (integrated network
approach) need to be measured to get the global view of the system.

11.4.3 Early life factors as perturbation
Another example of a perturbation that affects the system of the gut is from the
Kelly group (Mulder et al., 2009, 2011), where they investigated the microbiota
and host gene expression of pigs that grew up in different environments. These
environments included indoor, outdoor, and individual isolator housing (high-
hygiene). In the latter group, some pigs were also daily administered an antibiotic
cocktail. The results showed that high-hygiene status has a negative impact on
‘normal’ microbial colonization and promotes innate immune activity. Nevertheless,
microbial exposure is necessary throughout the developmental phase in pigs in order
to promote the homeostatic effects of the resident/colonizing microbiota. In these
piglets it has also been observed that high abundance of Lactobacilli may promote
immune homeostasis and consequently reduce pathogenic pressure by competitive
exclusion. Another comparable study by Schokker et al. showed that stress and/or
antibiotic administration in early life of piglets impacts the microbial colonization
and immune development in the gut (Schokker et al., 2014).
11.4.4 Nutrition as perturbation
Nutrition is another important factor that has the potential to modulate intestinal
homeostasis. As explained elsewhere, host immunity is a complex, multi-
dimensional system, and its functionality is dependent on interactions that exist
between host genotype, microbiota, nutrition, and environment via direct and
indirect pathways and signalling cascades. Although the interactions between
these different components are very complex, it has already been shown that feed
(ingredients) can steer the composition and diversity of the microbial community
in the gut (Jensen et al., 2011; Jozefiak et al., 2011). However, the feed composition
also plays an important role in the (overall) performance of the host (Kim et al.,
2007). Different feed groups can be distinguished; for example (1) macronutrients
(including proteins, lipids and carbohydrates); (2) micronutrients (including
minerals and vitamins); and (3) immune-modulatory compounds (including fibres
and anti-oxidants). Furthermore the life-history of each individual is important in
the light of immune competence and different diets, because diet alone is not capable
of modulating all immune system components at once (Cotter et al., 2011).

11.4.5 Microbiota is a super organ
The intestinal microbiota is an important ‘super organ’, changes in the composition

of the microbiota can lead to an imbalance of gut homeostasis. This imbalance could
lead to an infection of pathogenic invaders (parasite), in turn these invaders may be
countered by an immune response of the host. The stability landscape can give more
insight in the mechanisms of transition from one to another stable equilibrium, an
example can be that a shift in the microbiota composition, due to the administration
of pre/pro/synbiotics, could result in an alternate stable equilibrium. By identifying
and characterizing all these equilibriums and how these can be perturbed, it could
be possible to generate an informative map of this landscape. However, one must
keep in mind that this map also needs input from several different (biological) levels;
e.g. gene, protein, microbiota, environment, and time, to have a high accuracy and
specificity. But it is, for example, already known that the gut composition of the
microbiota can be modulated by applying different bacterial strains, for example
Lactobacilli, Bifidobacterium, and others (Gareau et al., 2010; Quigley, 2010). It is
thought that by administrating pre/pro/synbiotics the microbiota composition will
change, in such a way that the numbers of beneficial/commensal bacteria increase
and the numbers of pathobionts/harmful bacteria decrease.
11.5 Mathematical formalisms
The systems biology field can use a variety of modelling approaches to position data
on the interaction map between feed, microbiota, and host genotype with spatial,
temporal, and environmental dimensions into a conceptual framework. The various
modelling approaches include: statistical, graph, (signalling) pathway, immune
response, reaction kinetic, Boolean, agent-based, and constraint-based models
(Martins dos Santos et al., 2010, 2011).
11.5.1 Top-down and bottom-up approaches
Top-down methods are used to map the interactions and general structure of a
system and pinpoints to important components of the system that need to be studied
in more detail to answer particular biological question. What follows is an iterative
process of an ever-more refinement model of the particular (sub)system under study.
Top-down approaches are essential in helping to organize, structure, and interpret
the wealth of information generated with the currents –omics measurements.

Bottom-up approaches are used to build cellular and interaction networks based
on (post)genomic information. They enable to generate testable hypotheses and to
make predictions of, for example, the effects of perturbations.
11.5.2 Boolean models
Network models that attempt to represent interactions in a system by assuming that

individual ‘components’ of the system are either on or off are denoted as Boolean
models. They generate network dynamics much similar to electronic circuits (De
Graaf et al., 2009). Such models require limited experimental detail and knowledge.
In mammalian systems, Boolean modelling has for example be used to link protein
signalling networks with signal transduction complexes such as Toll-receptor and
T-cell receptor signalling (Saez-Rodriguez et al., 2009). Such models, therefore,
enable to describe the cross-talk of different signalling pathways. It is expected that
they will become important in the future to handle immune competence in the gut
in response towards nutrition and gut microbiota.
11.5.3 Agent-based models
Agent based modelling (ABM) is a modelling technique based on the rules and
interactions between the components of a system, simulating them in a ‘virtual world’
to create an in silico experimental model. ABM is an approach that has been used in
a number of relevant fields, for example inflammatory cell trafficking. ABM allows
dynamic knowledge representation and conceptual model verification and facilitates
the development of aggregated modular multi-scale models. Series of linked ABMs
have been used to represent multiple levels of biological organization in the context
of inflammation. For example, an ABM model of gut epithelial permeability was
linked to an endothelial/inflammatory cell ABM to produce an organ model of the
gut (An, 2008). This gut ABM was subsequently linked to a pulmonary ABM to
simulate the gut-pulmonary axis in the pathogenesis of multiple organ failure. Thus,
although not mechanistic as detailed as true dynamic models, such ABM models
are useful tool to connect various levels of biological organization and to couple
different scales of systems.
11.5.4 Kinetic models
Kinetic models, for example pathways, describe the interactions (edges) between
molecular components (nodes) of the system and thus provide mechanistic insights

into the system. Such models are usually built from differential equations and solved
through numerical and computational analyses such as metabolic control analysis
(De Graaf et al., 2010; Roling et al., 2010). A major problem of these models is that
they require detailed knowledge of the underlying molecular mechanisms and of
the respective model parameter values. These are in practice often difficult to obtain
in vivo.
11.5.5 Ordinary differential equations
Ordinary differential equations (ODE) are often used to describe temporal dynamic
events in systems. They consist of one independent variable, for example time,
and one or more derivatives to the independent variable. Different variables can
be expressed by one or more equations. ODE-based models and more enhanced
models are already applied in the field of microbial communities competing for
food (Kaunzinger and Morin, 1998), host immune response (Schokker et al., 2013)
and in host-pathogen interactions (Fenton and Perkins, 2010; Hethcote and Van
den Driessche, 2000). Partial differential equations (PDEs) are able to describe
multi-scale systems. When a system consist of different types of components, it is
sometimes necessary to distinguish between these components by the use of PDEs.
11.6 Biological networks/graphs
A biological network is an interconnection (edge) of sub-units (nodes) of a

biological system, that can be either undirected or directed. In undirected
networks it is known that an edge exists between two nodes, only the directionality
is unknown. Whereas, in directed networks it is known that node A modulates
node B, including inhibition, stimulation, or complex formation. Each biological
system can be transformed to a network, with its corresponding interactions, the
level of detail will determine its complexity. A plethora of these databases already
exist, containing interaction data from different levels, e.g. chemical, gene, protein.
Pathguide (www.pathguide.org) is a repository containing over 500 pathway
and interaction resources, both commercially and freely available as well as in
standardized formats (BioPAX, CellML, PSI-MI or SBML).

11.6.1 Interaction and regulatory networks
Pathways describe events occurring in the cell, signalling pathways for example show
a cascade of interactions between genes and/or chemicals. A harmful substance
in the intestinal lumen can be recognized by receptors in the cell membrane of
epithelial cells, which in turn will send a danger signal to other cells. However, this
is the output the cell generates, first the signalling cascade of gene-gene interactions
will take place, meaning that the signal will be transferred from cell membrane to
the nucleus. Second, in the nucleus transcription factors will be activated (or higher
expressed) which will result in expression of (response) genes, this can be to protect
the cell itself or inform adjacent cells.
11.6.2 Repositories
The Kyoto Encyclopedia of Genes and Genomes (http://www.genome.jp/kegg/) has

various manually drawn pathway diagrams for different species and are categorized
into 7 themes: (1) metabolism; (2) genetic information processing; (3) environmental
information processing; (4) cellular processes; (5) organismal systems; (6) human
diseases; and (7) drug development. All these themes are subdivided into more
specific signalling pathways and can be used to understand high-level functions
and utilities of biological systems (e.g. cell). Although KEGG portrays pathways as
separate units, Oda and Kitano (2006) have generated a detailed map of the Toll-like
receptor (TLR) and Interleukin 1 receptor (IL1R) signalling cascades. This map shows
that myeloid differentiation primary response gene 88 (MyD88) is an essential core
element, and stresses the fact that the system is fragile towards removal or mutation
of the MyD88 gene, which may result in different responses for different stimuli. Thus
by integrating data from the KEGG database and literature it is possible to generate
even larger networks and combined with network analyses it is possible to determine
the fragility of the network. Another repository is REACTOME (manually curated
and peer-reviewed) which contains different bioinformatic databases, including
Ensembl, UniProt, HapMap and Gene Ontology. In REACTOME networks are based
on interacting entities (e.g. nucleic acids, proteins, complexes and small molecules)
that participate in biological reactions/pathways. Networks or pathway maps are
easily downloaded in various standardized formats, including SBML, SBGN, PSIMI,
BioPAX level 2 or 3, Protégé, Word/PDF (curators and cited literature). InnateDB is a
dedicated repository to genes and proteins involved in signalling pathways associated
to innate immunity of microbial infections (human, mice, bovine). Manually curated
data of approximately 18,685 interactions and can be used as knowledge database

as well as systems level analysis. These different (highlighted) repositories allow to

generate maps and subsequently superimpose gene expression data in conjunction
with CytoScape (Shannon et al., 2003). Cytoscape can be used to visualize complex
networks and is able to integrate these networks with any type of attribute data.
Already a legion of ‘Apps’ are available for various kinds of bioinformatics analyses,
network generation and statistics (Lotia et al., 2013). It is possible to visualize
differentially expressed genes (up- or down-regulated) by different colour intensities.
When longitudinal data is available it is even possible to generate different snapshots
in chronological order to create a kind of movie.
11.6.3 Inferring networks
Besides the above mentioned repositories are based on known literature and
experimentation, it is also possible to infer networks from experimental data.
Different algorithms already exist and are embedded in the scripting language R or
stand-alone programs, including weighted correlation network analysis (WGCNA)
(Langfelder and Horvath, 2008), least absolute shrinkage and selection operator
(LASSO) (Friedman et al., 2008) and Mutual Information NETworks (minet) (Meyer
et al., 2008), GeneNet (Opgen-Rhein and Strimmer, 2007; Schafer and Strimmer,
2005), Algorithm for the Reconstruction of Gene Regulatory Networks (ARACNE)
(Margolin et al., 2006), Time-Delay ARACNE (Zoppoli et al., 2010), and Short Time-
series Expression Miner (STEM) (Ernst and Bar-Joseph, 2006). Some of the above
mentioned algorithms can specifically deal with time series data, which is common
in the experimental design of gene expression studies, especially when investigating
intestinal development or the immune response. Generation of gene-to-gene
networks is possible, but the interpretation of these resulting networks is difficult.
For example short-cuts can exist, meaning that if gene A and D are differentially
expressed it may still be possible that gene B and C are part of the signalling cascade.
Petri nets (directed bipartite graphs) comprises places, transitions, and arcs. Places
are a set of states (P), transitions consist of a set of transitions (T), and arcs are a set of
flow relations (F) between P and T and vice versa. A Petri net can also be described
by the following formula, a triple N = (P, T, F). In the associated graphs (or nets)
places may contain a certain discrete number of tokens and arcs run from places to
transitions or vice versa, however never between places or between transitions. In
biological terms, places can be substances/products from metabolism, transitions
may be enzyme-mediated reactions, arcs action parameters, and tokens the number
of reactants. The resulting Petri net represents the state of the biological system at a

certain time (Pinney et al., 2003; Zevedei-Oancea and Schuster, 2011). BioNetSim
(Gao et al., 2012) is a modelling tool, based on Petri nets, to perform simulations of
biochemical processes. This tool connects to KEGG and the BioModel database and
based upon these data generate Petri nets. Furthermore this tool provides qualitative
analysis and creates a graphical view making it possible to trace each substance
during simulation (Gao et al., 2012).
11.6.4 Example of experimental gut data to gene association networks
In our group we have already performed the generation of a gene association networks
(GANs) from experimental data (Schokker et al., 2011). Time-series gene expression
data consisted of the following sample points; 0.33 (8 h), 1, 2, 4, 8, 12, 21 days post
hatch, and was available for both control and Salmonella infected chicken. For both
situations, control and infected, these GANs were generated with 759 ‘selected’ nodes
and based on the top 1,000 edges, furthermore not all the nodes were connected. In
the ‘control’ GAN, 240 nodes were not connected and 519 nodes were connected
and formed the corresponding network. For the ‘infected’ GAN 164 nodes were not
connected and the remaining 595 probes were included in the network. Subsequently
hubs (key regulatory nodes) were identified for both GANs, by taking into account
each GAN separately again. The observed differences between these GANs were
shown in both network characteristics as well as in the associated biology of these
GANs. Despite these differences in network characteristics, both GANs show that
hubs play an important role in signalling cascades and that small alterations to hub
gene expression can lead to changes which impact the system (tissue). Both GANs
exist of different hub, only one overlapping hub could be detected. In the Salmonella
infected state, for example, more hubs were associated to processes of defence/host
response and of communicative nature, whereas in the control hubs the majority
were involved in transcriptional regulation and developmental processes. This
network approach also showed that not only immune genes and associated processes
are affected by an early Salmonella infection. These hubs are potential candidates for
markers of intestinal health and development.
11.7 Conclusions
The interplay of animal nutrition, microbiota and host genetics is complex and
can only be understood using a systems approach. Major challenges for the future
are thr integration of heterogeneous data and on the modeling of all the relevant

functions at all the different biological levels. The ultimate goal is to generate a
solid knowledge-base and predictive frameworks on the functionalities of the gut
along its various spatial, temporal and environmental dimensions. This will enable a
better understanding of how specific host factors, nutrients, diets and environmental
conditions influence the signalling and function of cells and tissues and how this
affects immune competence and intestinal health. System based knowledge and
in silico models of the intestinal tract will be pivotal to fully exploit the intrinsic
biological potential of host-feed-microbe interactions in livestock species, in order
to optimize and ‘customize’ animal feeds and management procedures in order to
improve health and efficiency traits associated with the gut.
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Index
A chito-oligosaccharide – See: COS
AAT 224 cinnamaldehyde 87
acid citrulline 223
–– fatty 140, 151, 199, 239 claudin 152, 175, 223
–– organic 140, 157, 201 Clostridia cluster XIV 29
acute phase protein 18, 225 Clostridium 24, 81, 121, 128
aflatoxin 171 –– difficile 53
α-1-antitrypsin – See: AAT –– perfringens 53
α-ketoglutarate 145 coccidiosis
anethole 87 –– coccidiostats 74
antibiotic 29, 33, 51, 55, 63, 68, –– infection 65
82, 101, 120, 139, 157, 179, 207, 241, –– vaccine 93
254 coronavirus 52
anti-inflammatory reflex 17 COS 150
antioxidant 86, 142, 153, 241 CP 130, 143, 156
apramycin 55 crude protein – See: CP
arginine 140, 146, 242 curcumin 87
Ascaris suum 66 cytokine 16, 32, 75, 79, 100, 119, 127,
142, 182, 206
B
Bacteroides 121 D
Bacteroidetes 27 2DE differential gel electrophoresis –
β-glucans 149, 201 See: 2DE DIGE
Bifidobacterium 2 6, 121, 130, 147, 260 2DE DIGE 234
biomarker 220, 230 denaturing gradient gel electrophoresis
Brachyspira hyodysenteriae 58 – See: DGGE
butyrate 33, 131, 140, 149, 154 dendritic cell 17, 76, 99, 225
deoxynivalenol – See: DON
C DGGE 36
Caco-2 model 177 DHA 151
Campylobacter 23, 31, 207, 244 diarrhoea 52, 58, 62, 119, 140, 145,
capsaicin 87 150
carbohydrate 30, 117, 130, 143, 239, dioxin 173
259 DNA microarray 230
carvacrol 87 docosapentaenoic acid – See: DHA
cell culture 205

Index
DON 172, 182 H

dysbiosis 28, 117, 127 haemagglutinating encephalomyelitis
virus – See: HEV
E haptoglobin 220
eicosapentaenoic acid – See: EPA HEV 62
Eimeria 72 HSP70 144, 207
enterobacteria 150
–– Enterobacteriaceae 121, 157 I
Enterococcus 24 IBD 33, 120, 127, 200, 223
enterocolitis, necrotizing 241 IEC 17
enterotoxigenic Escherichia coli – I-FABP 223
See: ETEC IFN 76, 88, 95, 100, 155, 182, 225
EPA 151 IgA 181
Escherichia coli 2 3, 130, 144, 153, 207, IgG 181
240 IgY 77
–– post-weaning infection 53 ileitis 59
ETEC 53, 141, 147, 153 immunoglobulin 77, 140, 181, 240
explant 175, 191 inflammation 16, 30, 52, 58, 79, 88,
140, 152, 181, 220, 240, 254
F –– intestinal 152
FABP-2 – See: I-FABP inflammatory bowel disease – See: IBD
fatty acid 140, 151, 199, 239 integrity 118, 143, 153, 169, 175, 221,
fibre 117, 130, 144, 259 241
Firmicutes 27, 117, 255 interferon – See: IFN
FISH 123 interleukin 76, 207, 263
FOS 129 intestinal epithelial cell – See: IEC
fumonisin 171 intestinal fatty acid binding protein –
See: I-FABP
G intestinal loop 196
GALT 17, 77 inulin 152
GAN 265 Isospora suis 65
gene association network – See: GAN
glutamine 140, 144, 223, 242 K
Gut Associated Lymphoid Tissue – KEGG 263
See: GALT Kyoto Encyclopedia of Genes and
Genomes – See: KEGG

Index
L O
Lactobacillus 24, 31, 121, 132, 147, occludin 152
154, 205, 223, 240, 259 ochratoxin 173
–– plantarum 242 Oesophagostomum sp. 66
lactoferrin 147 oil 151
Lawsonia intracellularis 59 oligosaccharide 149
LC-MS proteomic 234 organic acid 140, 157, 201
lipopolysaccharide – See: LPS
long-chain fatty acids 151 P
loop, intestinal 196 PAMP 16, 75
LPS 75, 79, 143, 195, 200 pancreatitis associated protein –
lysozyme 147 See: PAP
PAP 223
M pathogen-associated molecular pattern
MAMP 16 – See: PAMP
mannan-oligosacharide – See: MOS pathogen-recognition receptor –
MAPK 31, 142, 182, 205 See: PRR
mass spectrometric analysis – See: MS pathway 263
microbial-associated molecular pattern PCV 65
– See: MAMP PED 62
mitogen-activated protein kinases – permeability 30, 142, 151, 176, 192,
See: MAPK 195, 200, 221
MOS 149 peroxisome proliferator-activated
MPO 224 receptor-γ – See: PPARγ
MS 234 Peyer’s patch 17, 100, 153, 181, 198,
mucin 21, 30, 34, 154, 180 243, 254
mucus 30 polyphenol 153
Mycoplasma 23 porcine circovirus – See: PCV
mycotoxin 171 porcine epidemic diarrhoea – See: PED
myeloperoxidase – See: MPO post-weaning Escherichia coli infection
53
N PPARγ 142, 153, 258
necrotizing enterocolitis 241 prebiotic 22, 128
neomycin 55 probiotic 22, 31, 128, 152, 153, 206,
NF-κB 89, 258 242, 255
non-starch polysaccharide – See: NSP proliferative enteropathy 59
NSP 144 Propionibacterium 24
nuclear factor-kappa B – See: NF-κB propyl thiosulfinate 87

Index
–– oxide 87 threonine 146

proteomic 230 tiamulin 55
PRR 16 tight junction 142, 152, 175, 193, 206,
222, 243
Q tissue explant 202
qPCR 124 tissue trans-epithelial resistance –
quantative PCR – See: qPCR See: TEER
TLR 16, 75, 89, 127, 207, 258, 263
R TNF 79, 142
Reg3α – See: PAP toll-like receptor – See: TLR
rotavirus 52 transforming growth factor – See: TGF
16S rRNA 123 transmissible gastroenteritis – See: TGE
Trichothecenes 172
S Trichuris suis 66
S100 224 tryptophan 140, 147, 156
saccharomyce 35, 154 tumor necrosis factor – See: TNF
safflower 89
Salmonella 23, 34, 51, 56, 68, 83, 179, U
203, 223, 265 UC 197
–– typhimurium 242 Ussing chamber – See: UC
SCFA 25, 33, 127, 255
ScFv antibody 85 V
seaweed 151 vagus nerve 17
Serpulina hyodysenteria 144
short-chain fatty acid – See: SCFA Z
shotgun 234 zearalenone 172
SISP 196 zinc 148, 206
small intestine perfusion system – –– oxide 55, 139, 243
See: SISP
Streptococcus 25, 122, 131
sulphonamide 55
synbiotic 128
systems biology 253
T
TEER 198
TGE 62
TGF 79, 145, 152

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Chapter 1: General introduction – the gastrointestinal tract, the

Chapter 2: The composition and role of the microbiota in chickens 21

Chapter 3: Intestinal diseases of pigs 51

Chapter 4: Avian coccidiosis as a prototype intestinal disease – host

Chapter 5: Intestinal health in carnivores 117

Chapter 6: Pig intestine, weaning and dietary interventions 139

Chapter 8: Techniques for investigating gut function in vivo, ex vivo

Chapter 9: Intestinal health biomarkers in vivo 219

Chapter 10: Intestinal health research and proteomics, a well-

Chapter 11: Systems biology – applications in intestinal health 253

I hope the wait was worthwhile.

The gastrointestinal tract (GIT) is essential in the maintenance of health and

Health and growth

T.A. Niewold (ed.) Intestinal health

central nervous system of an injury, infection, or cytokine excess, and stimulates

It can be concluded that especially in production animals inflammation should be

A.A. Pedroso and M.D. Lee*

Keywords: microflora, microbiota, microbiome, intestine, holobiont, homeostasis,

T.A. Niewold (ed.) Intestinal health

8,000 Intestinal microbiota

2.3 Temporal variation

2.3.1 Pre-hatched phase

2.3.2 Starter phase

intestine develop distinctly different communities. This can probably be attributed to

2.3.3 Grower phase

2.4 Spatial variation

2.4.2 Small intestine

of species related to Lactobacillus. One study observed that approximately 90%

The cecal environment harbors predominantly the phyla Firmicutes, Bacteroidetes

2.5 Role of the microbiome

2.5.1 Intestinal development and homeostasis

Microbe-induced signaling pathways that link the microbiome to gut cell

Bacteroides thetaiotaomicron, a dominant member of the mammalian intestinal

Bacteroides thetaiotaomicron stimulates development of the intestinal mucosal

Kabeerdoss et al., 2013). Some members of the intestinal bacterial community

2.5.2 Role of mucin in intestinal function

A strong indication that the mucin production, composition and degradation is

cells (Caballero-Franco et al., 2007). Probiotic strains Lactobacillus plantarum 299v

2.5.3 Cleavage and absorption of nutrients

2.5.4 Intestinal differentiation, maturation and apoptosis

The microbiota promotes substantial changes in gut morphology, including villi

2.6 Short chain fatty acids

SCFAs have an effect on nutrient acquisition by increasing the expression and

Butyrate-producing bacteria are of increasing interest because of their potential

2.7 Modulation of the intestine using probiotic microorganisms

The health benefits attributed to consumption of probiotic organisms include

Nutrient utilization, enhanced by complementary interspecies metabolic activity

Table 2.1. Microorganisms used as probiotics for poultry.

Microorganism Genus and species Reference

Bacteria Lactobacillus fermentatum Bai et al., 2013; Yamawaki et al., 2013

A high level of success can be observed when undefined microbiota, directly

development of the intestine, regulates inflammation, enhances nutrient update,

Guanajuato, División Ciencias de la Vida (DICIVA), Km 9 Carretera Irapuato-

Keywords: Brachyspira, Lawsonia, Salmonella, coronavirus, colibacillosis

3.1 Introduction and general features

T.A. Niewold (ed.) Intestinal health

It is important when investigating intestinal diseases in pigs to recognise that post

3.2 Key intestinal diseases in pigs at or after weaning

3.2.1 Bacterial diseases

Post-weaning Escherichia coli infections

Management and control of colibacillosis outbreaks – It is important to know the

Chapter 1: General introduction – the gastrointestinal tract, the

Chapter 2: The composition and role of the microbiota in chickens 21

Chapter 4: Avian coccidiosis as a prototype intestinal disease – host

Chapter 6: Pig intestine, weaning and dietary interventions 139

Chapter 8: Techniques for investigating gut function in vivo, ex vivo

Chapter 10: Intestinal health research and proteomics, a well-

Chapter 11: Systems biology – applications in intestinal health 253

4.3 Prevention and control of avian coccidiosis: alternatives to

4.4 Passive immunization against avian coccidiosis using

4.5 Immunomodulation with phytochemicals against avian

4.5.3 Genomic approaches to delineate molecular mechanisms of phytonutrient