THE POTENTIAL
ROLE OF GLUTEN
IN EQUINE
INFLAMMATORY
SMALL BOWEL
DISEASE
RESEARCH PROJECT, veterinary medicine.
Lotte Anna van Putten
3155919
The potential role of gluten in equine inflammatory small bowel disease.
Department of Equine Sciences, Medicine Section, Faculty of Veterinary Medicine, Utrecht
University
The VUmc Research Institute for Cancer and Immunology & the Department of Gastroenterology
and Hepatology
SUPERVISOR:
Dr. J.H. van der Kolk
EXTERN SUPERVISOR: Prof. dr. C.J.J. Mulder
Dr. B.M.E. Blomberg
(Dr. M.W.J. Scheurs)
DATE OF SUBMISSION: 01-10-2010
UTRECHT UNIVERSITY
Research Project
Veterinary Medicine
Oct2010- March2011
Research supervisors:
UU:
Dr. J.H. van der Kolk
Dr. G.C.M. Grinwis
VUmc:
Dr. B.M.E. von
Blomberg
M. Reijm
Dr.N.C.T. van Grieken
Prof. Dr. C.J.J. Mulder
Lotte van Putten, 3155919
Motivation
Equine inflammatory small bowel disease (ISBD) is an
idiopathic pathologic condition of the small intestine
characterised by substantial reduction of the available
absorptive surface area. Signs include a dull hair coat,
weight loss, recurrent colic and at times soft faeces,
depression and oedema. The condition is regularly
associated with anaemia. ISBD is a rare disease that in
recent years seems to be increasing in frequency. Different
etiologies are suggested in today’s literature, such as
abnormal host inflammatory reaction to intestinal bacteria,
parasitic antigens and dietary components. Could gluten,
being the known cause of coeliac disease in humans and
gluten-sensitive enteropathy in Irish setter dogs, be
involved in the aetiology of this dreadful disease in horses?
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
ABSTRACT
Background
Equine inflammatory small bowel disease (ISBD) is an idiopathic pathologic condition of the small
intestine characterised by substantial reduction of the available absorptive surface area. Signs
include a dull hair coat, weight loss, recurrent colic and at times soft faeces, depression and
oedema. ISBD is a rare disease that in recent years seems to be increasing in frequency. Different
aetiologies are suggested in today’s literature, such as abnormal host inflammatory reaction to
intestinal bacteria, parasitic antigens and dietary components.
Aims
To clarify the role of gluten in equine ISBD, aiming at improved diagnostic and t reatment
modalities. To find out if there is an analogy between coeliac disease in humans and ISBD in
horses.
Methods
Comparing different tests: the blood samples of 3 different groups (ISBD patients (n=12), gluten rich controls (n=22) and gluten-free controls (n=25)) were ELISA tested for IgA antibodies against
E. coli, human recombinant tissue-transglutaminase (rh-tTGA), guinea pig tissuetransglutaminase (gp-tTGA), antigliadin, deamidated-gliadin-peptides (anti-DGP) and, by using
IFT, antibodies against endomysium (EmA). Differences between groups were statistically
compared by means of the Mann-Whitney U-test. ρ values < 0,05 were considered significant.
One patient was followed after adjustment to a gluten-free diet period for 6 months.
Results
Both ISBD patients and the gluten-rich controls had significant ( ρ < 0.0004) higher rh-tTGA than
the gluten-free controls. Concentrations of anti-DGP antibodies were significantly ( ρ = 0.024)
higher in ISBD patients then in the gluten rich controls. The ELISA’s for gp-tTGA, antigliadin
antibodies and the IFT for EmA’s were inconclusive as no clear relation was found between these
and the use of gluten or ISBD. The patient, followed after a gluten-free diet period of 6 months
showed reduced concentrations of all measured serological coeliac disease parameters.
Conclusion
This is the first study to investigate the potential role of gluten in the aetiology of equine
inflammatory small bowel disease. On the basis of these data it is not possible to draw clear
conclusions on the role of gluten in equine inflammatory small bowel disease yet. But, the finding
that rh-tTGA and DGP antibody concentrations are raised in horses fed a gluten-rich diet suggest an
immune-stimulatory effect of gluten that could play a role in the aetiology of ISBD. The case, showing
both clinically as well as serological improvement after a gluten-free diet supports this possibility.
However, further research is needed to clarify the specifics.
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
PREFACE
This research project was carried out within the study of Veterinary Medicine at Utrecht
University. The study was performed in joint venture with the VU University Medical Center’s
Research Institute for Cancer and Immunology, the Department of Pathology and the
Department of Gastroenterology and Hepatology, Amsterdam, The Netherlands.
The research was executed to get more knowledge about the potential role of gluten in the
aetiology of equine inflammatory small bowel disease.
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
CONTENTS
Content
Abstract
Preface
Contents
Part 1: Introduction and background
1.1 Equine inflammatory small bowel disease
1.2 Coeliac disease in humans
1.3 Gluten-sensitive enteropathy in Irish Setter dogs
1.4 Gluten
1.5 Aim of the study
1.6 Hypothesis
Part 2: Materials and methods
2.1 Animal selection
2.2 Sampling
2.3 Adaption of human coeliac related ELISA’s for testing equine ISBD patients
2.4 Histopathology
2.5 Statistical analysis
2.6 Clinical trial: measuring the effects of a gluten-free diet
Part 3: Results
3.1 Adaption of human coeliac related ELISA’s for testing equine ISBD patients
Page
2
3
4
5
5
7
10
11
12
12
13
13
14
14
16
16
16
17
17
Goat anti horse IgA:HRP testing 17
ELISA: IgA antibodies against E. coli 18
ELISA: Antibodies against recombinant human and guinea pig tissuetransglutaminase 19
ELISA: Antibodies against gliadin 22
ELISA: Deamidated gliadin antibodies 23
IFT: Anti-endomysium antibodies 24
3.2 Histopathology
3.3 Clinical trial: measuring the effects of a gluten-free diet
Part 4: Discussion
Part 5: Conclusion
Acknowledgments
References
Abbreviations
Annexes 1-9
25
26
28
31
32
33
34
34
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
PART 1: INTRODUCTION AND BACKGROUND
Inflammatory small bowel disease is a dreadful disease, both for horses as well as for humans. The
number one cause of inflammatory small bowel disease in humans is known to be an intolerance to
gluten, the main substance in wheat, barley and ray. Since the uniqueness of man, in comparison to
non-human mammals, is becoming more foggily every day, except maybe for their “sophisticated
spoken language” (Jane Goodall), why could not gluten be a cause of this idiopathic disease in
equines? Earlier research suggests a dietary component as a possible cause.7
Originally, horses are steppe animals that used to spend most of the day searching for grasses and
herbs to eat. This feeding pattern is still visible in the way the equine digestive system is build: the
relatively small stomach, the highly developed small intestine and the large caecum. Nowadays
these horses have become major athletes whose energy requirements are met by a diet of highly
concentrated power feed and hay. Researchers have shown that almost all variants of concentrate
contain seeds of the agricultural grown cereals; wheat, barley and rye. 5 And these components
contain gluten proteins. Gluten is the known trigger of, next to human coeliac disease, glutensensitive enteropathy in the Irish setter dog. In analogy with both diseases this study was carried
out to learn more about the potential role of gluten in equine inflammatory small bowel disease.
A rare idiopathic disease that, in recent years, seems to be increasing in frequency.
Because of their broad knowledge about coeliac disease and their wide range in human coeliac
disease diagnostic tests this study was carried out at the VU University Medical Center in
Amsterdam.
So, what is known already?
1.1 Equine inflammatory small bowel disease
Epidemiology
Inflammatory small bowel disease (ISBD) is an idiopathic pathologic condition of the small
intestine characterised by substantial reduction of the available absorptive surface area. 5,6 ISBD
is a rare disease that, in recent years, is diagnosed more frequently in the Equine Clinic of the
Utrecht University. Different aetiologies are suggested in today’s literature, such as abnormal
host inflammatory reaction to intestinal bacteria, parasitic antigens and dietary components. 7
Causation and types of inflammatory bowel disease in the horse
The disease is characterized by villous blunting to atrophy and crypt hyperplasia.
Histopathologically different types of ISBD are distinguished depending on the cell type that
infiltrates the gut:
Eosinophilic enteritis and multisystemic eosinophilic epitheliotrophic disease
Eosinophilic enteritis is recognized as diffuse inflammatory cell infiltration of the small intestinal
mucosa with eosinophils and lymphocytes. Horses of any sex, age, or breed may be affected with
eosinophilic enteritis, but it seems most common in young (2-4 years of age) Standardbred and
Thoroughbred horses. Recurrent colic is a common clinical sign with this form of inflammatory
bowel disease, and the disease may be diffuse or focal. Circumferential mural bands can form
because eosinophilic enzymes stimulate mural fibrosis that partially obstructs the lumen of the small
intestine. This obstruction can lead to low-grade chronic colic or result in an acute episode if
significant obstruction and distension occurs. Other findings include mucosal ulceration, enlargement
of ileal Peyers patches, and mesenteric lymphadenopathy.7
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
The cause of this form of inflammatory bowel disease is thought to be a type I hypersensitivity
reaction possibly caused by inhaled, dietary, or parasitic antigens. Therefore elimination of these
antigens is a necessary part of the treatment plan. Diet change, anthelmintics, and corticosteroids
are all used for treatment of eosinophilic enteritis. Treatment options are discussed in more detail in
a later section. Most of the current literature reports that treatment usually is unsuccessful, but
occasionally horses do respond favourably.7
A subset of affected horses also suffer from eosinophilic infiltration of other organs and tissues
including the skin, liver, pancreas, oral cavity, oesophagus, lungs, and mesenteric lymph nodes.
This more severe form of the disease is known as “multisystemic eosinophilic epitheliotrophic
disease”.7
Granulomatous enteritis
Granulomatous enteritis is characterized by lymphoid and macrophage infiltration of the mucosal
lamina propria with variable numbers of plasma cells and multinucleated giant cells. There usually is
marked villous atrophy, and the ileum typically is the most severely affected portion of the
gastrointestinal tract. This condition is similar to Crohn’s disease in humans and Johne’s disease in
cattle. The cause of Johne’s disease in cattle is Mycobacterium avium ssp. paratuberculosis.7
Horses diagnosed with granulomatous enteritis can be of any age, sex, or breed, but young
Standardbred horses, 4 years old or younger, are over-represented, and there seems to be a familial
predisposition. One study showed that more than 80% of the cases of granulomatous enteritis in
horses were in Standardbreds. As with the other forms of ISBD, the cause is unknown.1,7
Possible causes include an abnormal host inflammatory reaction to intestinal bacteria or dietary
components. Aluminum exposure via invading microorganisms, particularly parasites, also has been
linked to granulomatous enteritis in horses.7, 13
Lymphocytic-plasmacytic enteritis
Lymphocytic-plasmacytic enteritis is characterized by excessive infiltration of lymphocytes and
plasma cells in the lamina propria of the gastrointestinal tract with the absence of granulomatous
change. There is no age, breed, or sex predilection for this form of ISBD. It has been suggested that
this condition may be an early stage of intestinal lymphosarcoma.7
Lymphosarcoma
Intestinal lymphosarcoma can affect horses of any breed, sex, or age and has been reported in a
horse with selective IgM deficiency. Lesions can be primary or secondary metastases from another
site, most commonly mediastinal lymphosarcoma. Horses present with recurrent colic and weight
loss, which can be acute despite the progressive nature of the disease process. The presentation of
the disease may range from discrete tumors to diffuse infiltrates (Fig. 1). Additionally, enlarged
mesenteric lymph nodes infiltrated by malignant cells may be detected. Mucosal ulcers may
contribute to protein loss in horses with lymphosarcoma. Anaemia and thrombocytopenia are
common, and lymphocytosis is rare. The prognosis for this form of IBD is poor, because horses
usually present in an advanced stage of the disease.7, 16
Fig. 1. Small intestinal lymphosarcoma. A discrete nodule within
the small intestine of a 5- year-old Warmblood gelding. The horse
presented with acute colic necessitating surgical intervention. The
affected portion of small intestine was resected. No additional
lesions were found during surgery, but biopsy of a mesenteric
lymph node revealed metastasis had already occurred. 7
Fig. 2. Abdominal ultrasound identifying thickening of the wall
of the small intestine. 8
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
Clinical aspects
Clinical signs of ISBD include a dull hair coat, weight loss (despite a good appetite), oedema and
at times soft faeces, depression and occasionally (recurrent) colic. The condition affects both
young and adult horses. 5
Diagnosis
The tentative diagnosis is based on the clinical signs and physical examination. By rectal
exploration small bowel loops will be palpable. Haematology may show anaemia and slight
increases or decreases in white blood cell counts. Blood biochemistry shows a normal to
decreased total protein concentration and decreased albumin concentration. Additionally a
percutaneous ultrasound can be made to determine the thickness of the wall of the small
intestine usually revealing its thickening (Fig. 2). One can also perform an intestinal absorption
test or glucose tolerance test. 5
The diagnosis is confirmed by either a trans-endoscopic duodenal or a laparoscopic biopsy and
subsequent histopathology. The type of ISBD is based on the degree of inflammation and the
predominant type of infiltrating leucocyte. 7
In the same animal the staging of the pathological changes may differ in different regions of the
small and/or large intestines, thus influencing the severity of clinical signs and absorption
findings.5
Common treatment
If the diagnosis is confirmed there may be different treatment options. Focal lesions can be
surgically removed and multifocal or diffuse changes can be treated with corticosteroids or
metronidazole, an anti-microbial agent with anti-inflammatory effects (Table 1.). It is also
recommended to adjust the diet to short-fibre roughage instead of long-fibre, because of the
altered motility of the intestine. 5,7
In general, long-term treatment of ISBD in horses is reported to be unsuccessful. 7
Table 1. Reported treatment options used in the management of infiltrative intestinal disease in horses. 2,7
Drug
Dexamethasone
Prednisolone
Metronidazole
Dose
0,02-0,2 mg/kg
0,2-4,4 mg/kg
15 mg/kg
Route
PO, IM, IV
PO
PO
Frequency
q 24 h
q 12-24 h
q 6-8 h
Prognosis and complications
The prognosis of a patient with ISBD varies by type. Eosinophilic granulomatosis has the most
favourable prognosis, i.e. 60-70% chance of recovery. Granulomatous enteritis has less than 50%
chance of healing and lymphocytic plasmacytic enteritis has a very poor prognosis. 5
1.2 Coeliac disease in humans
Epidemiology
A possibly similar disease to equine ISBD in humans is coeliac disease. Coeliac disease (CD) is a
chronic inflammatory disorder of the small bowel induced in genetically susceptible people. CD is
characterized by flattened villi on the small bowel mucosa and nutrient malabsorption, and is
induced by proline-rich and glutamine-rich proteins (gluten) in wheat, rye, and barley and in
combination with cofactors related to drugs (interferon α), intestinal infections and infant
feeding practices. Epidemiological studies in first world countries report that the prevalence of
CD disease is around 1:100. 4
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
Causation
Genetic linkage studies show that the disease is strongly associated with HLA -DQ genes. Most
patients carry the DQ2 variant and others carry a variant of DQ8 (HLA-DQ genes are part of the
major histocompatibility complex type 2 on antigen presenting cells). Other environmental
cofactors known to enhance gluten immunogenicity are interferon alpha and rotavirus
infections.4,14
Pathophysiology
The coeliac lesions develop by a T-helper-cell type 1 response. After gluten peptides crossed the
epithelium into the lamina propria, they are deamidated by tissue transglutaminase and then
presented by DQ2+ or DQ8+ antigen-presenting cells to pathogenic CD4+ T cells. The CD4+ cells
drive the T-helper-cell type 1 response which leads to intraepithelial and lamina propria
infiltration of inflammatory cells, crypt hyperplasia and villous atrophy .4
[Intermezzo: T-helper-cell type 1 response: CD4+ cells produce interferon gamma which leads to macrophage
activation and the CD40 ligand produced causes B cell proliferation and immunoglobulin A secretion as well
macrophage activation.]
Gut permeability is enhanced in coeliac patients and gluten can reach the lamina propria through
different routes. Investigators have described a paracellular route, as a consequence of impaired
mucosal integrity, a transcellular route, based on interferon-ɣ-dependent transcytosis and a
protected transport pathway, which is driven by retrotranscytosis of secretory IgA through
transferring receptor CD71, that promotes the influx of intact and thus harmful, gluten
peptides.4
Tissue transglutaminase is a calcium-dependent, ubiquitous enzyme that catalyses the posttranslational modification of proteins and is released during inflammation . It could have at least
two crucial roles in coeliac disease: as the main target autoantigen for antiendomysial antibodies
and hTTG (human tissue transglutaminase) antibodies, and as a deamidating enzyme that raises
the immunostimulatory effect of gluten. Expression and activity of tissue transglutaminase are
raised in the mucosa of patients with CD, where, by deamidating glutamine to glutamic acid, this
enzyme makes gliadin peptides negatively charged and therefore more capable of fitting into the
pockets of the DQ2/DQ8 antigen-binding groove. 4
In the mucosa of patients with active coeliac disease, gluten-reactive CD4+ T cells produce
several pro-inflammatory cytokines, interferon ɣ being dominant, that trigger various effector
mechanisms including raised secretion of tissue-damaging matrix metalloproteinases and
heightened cytotoxicity of intraepithelial lymphocytes against enterocytes with increased
enterocyte apoptosis and villous flattening. 4
Some gluten peptides can directly induce mucosal damage via a non-T-cell dependent pathway
(innate response). 4
Clinical aspects
Although CD affects approximately 1% of the population of first world countries, most affected
individuals remain undiagnosed. This probably reflects the fact that patients with CD can
manifest a spectrum of intestinal and/or extraintestinal symptoms. In some cases, they can be
relatively asymptomatic, with their disease first being detected by antibody screening because
they were identified as being at high risk of developing CD (for example, by being a family
member of an affected patient). 14 Symptomatic patients complain about dyspepsia, abdominal
discomfort and bloating, unexplained anaemia or it manifests with evident symptoms of
malabsorption such as diarrhoea, steatorrhoea, weight loss, cramps, tetany and peripheral
oedema.4
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
Diagnosis
Coeliac disease is diagnosed based on the symptoms, serology and histology. Endoscopic two
major changes can be identified, namely the disappearance or reduction of the Kerckring folds
and the scalloped configuration of reduced folds. 20
Serological there are different enzyme-linked immunosorbent assay’s (ELISA) available with high
sensitivity and specificity. The VU University Medical Center Immunology diagnostics lab uses inhouse ELISA’s to test for either IgA or IgG antibodies against recombinant human tTG (specificity
98-100% and sensitivity 92-96%), gliadin antibodies (showed a sensitivity and specificity range
from 31% to 100% and 57% to 100% 21) and an in-house for IgA antibodies against E. coli to rule
out a IgA deficiency, which has a prevalence of around 1:400.17 Additionally there is an ELISA
available to test for antibodies against deamidated gliadin. A variant to the recombinant human
tTG antibody ELISA is one that uses guinea-pig tTG derived from the liver. The VUmc also has an
IgA-class anti-endomysium immunofluorescence antibody test on hand, by which an indirect
immunofluorescence analysis is performed using unfixed cryostat sections of monkey
oesophagus (showing a sensitivity and specificity range from 85% to 100% and 95% to 100% 21).
Histological the diagnosis of CD is based on the presence of characteristic lesions in smallintestinal biopsy samples. Major features of mucosa suggestive of coeliac disease are: proximal
small bowel involvement, decreasing distally and in some cases patchy distribution. Mucosal
architectural changes include villous atrophy, crypt hyperplasia, thickening of the basement
membrane under the surface epithelium and a reduced numbers of goblet cells. Mucosal
inflammation is revealed by increased intraepithelial lymphocytes and an influx of immune cells
in the lamina propria. Enterocyte changes include cuboidal morphology, loss of basal nuclear
orientation and cytoplasmic vacuoles. Biopsy samples are usually taken from the distal
duodenum and the Marsh-Oberhuber classification is used to grade the severity of the lesions
(Table 2 & Fig. 3). 4,19
Table 2. The modified Marsh–Oberhuber classification.19
IEL count*
Crypt
hyperplasia
Villous
atrophy
Marsh 0
Marsh 1
Marsh 2
>30/100
+
Marsh 3
3a
>30/100
+
<30/100
−
>30/100
−
3b
>30/100
+
3c
>30/100
+
−
−
−
Mild
Moderate
Total
Pre‐infiltrative
Infiltrative
Infiltrative‐hyperplastic
Flat destructive
IEL= intraepithelial lymphocytes; *= Number of intraepithelial lymphocytes per 100 enterocytes.
Minor lesions, such as increased intraepithelial lymphocytes, have a low specificity for diagnosis
of CD since a wide variety of immunologic stimuli can raise intraepithelial lymphocyte numbers.
Therefore diagnosis of CD is confirmed only by clinical symptoms, a positive serology, or
histological improvement after commencement of a gluten-free diet. 4, 26
Fig 3. Marsh–Oberhuber type 1 and type 3 lesions.19
Additionally, HLA typing for HLA-DQ2/DQ8 can be performed which is, when negative, almost
absolute in ruling out coeliac disease in high-risk individuals such as first-degree relatives and
patients with type-1 diabetes. 4
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
Treatment
The only proven treatment for coeliac disease is strict and life-long adherence to a gluten-free
diet. 4
Complications
Known complications to coeliac disease are non-hodgkin lymphoma, refractory coeliac disease,
ulcerative jejuno-ileitis, and enteropathy-associated T-cell lymphoma. The latter is of
importance, given the fact that the histopathology is similar to the alimentary form of malignant
lymphoma in the horse. So this may be another interesting research topic. 4
1.3 Gluten-sensitive enteropathy in Irish Setter dogs
Epidemiology
Gluten-sensitive enteropathy in dogs has some similarities to coeliac disease, but the severity of
intestinal damage and clinical signs tends to be less marked then in humans. Wheat sensitivity
has been confirmed by morphologic and biochemical examination of jejunal biopsies obtained
during challenge studies performed on a litter bred from affected dogs and reared on a normal
wheat-containing diet. 9
Causation
The disease is characterized by partial villous atrophy and increased intraepithelial lymphocytes.
The studies described above introduced the possibility that there might be an age-related delay
in the expression of aminopeptidase N, the most abundant brush border peptidase. This could be
important, since severely reduced levels of this enzyme might allow high concentrations of
potentially toxic gluten peptides to reach the lamina propria due to defective degradation,
resulting in development of hypersensitivity. A permeability disorder could have similar
consequences. 9
A study performed on a litter of affected animals has shown that the dogs reared and kept on a
cereal-free diet until 12 months of age appeared to be resistant to gluten challenge. The litter
mates kept on a normal diet until 12 months of age, followed by a period of a cereal-free diet
and then back on a normal diet 6 weeks after the introduction of a cereal-free diet failed the
gluten challenge. It resulted in clinical relapse manifested with diarrhea and weight loss. This
suggest that there might be a critical age for the introduction of gluten to the diet beyond which
gluten sensitivity does not develop. 9
Clinical aspects
Affected dogs typically present with poor weight gain or weight loss, in most cases accompanied
with chronic diarrhea, and clinical signs are often first observed at approximately 6 months of
age. 9
Diagnosis
Dietary sensitivity may be suspected from the clinical history, which may suggest an association
between a specific dietary antigen, such as gluten, and the presence of clinical signs. Subsequent
investigations should exclude intestinal parasites and pathogens, partial intestinal obstruction,
systemic disease, exocrine pancreatic insufficiency, and bacterial overgrowth. Assessment of the
intestinal mucosa is then needed to show that a specific dietary antigen causes intestinal
damage, which is likely to be the most severe in the proximal small intestine, where antigen
concentration is the highest. The assessments of intestinal damage need to be repeated
following exclusion diet and challenge in order to make a definitive diagnosis. 9
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
Treatment
An exclusion diet consisting of a selected protein source should be used as trial therapy in
suspected cases, and should be fed for a period of at least 6 weeks. Boiled white rice or potato is
generally a suitable carbohydrate source, and lamb or chicken is often used as a protein source.
Oral prednisone, 0,5 to 1 mg/kg twice daily for two to 4 weeks, followed with a reducing dose at
2-weeks intervals, may assist some cases if dietary sensitivity in the initial response to exclusion
diet is disappointing. 9
Prognosis
After being put on a strict long-term gluten-free diet the dogs should improve dramatically
within a few weeks. The diarrhea should resolve and they should gain weight. Within the year no
recurrence of diarrhea has been reported. 15
1.4 Gluten
The history of gluten
The discovery in the Neolithic age, beginning about 9500 BC, of ways to produce and store food
has been the greatest revolution mankind ever experienced. Archeological findings suggest that
this revolution was initiated by the observations made by woman. The woman carried the daily
burden of collecting seeds, herbs, roots and tubers. During the excavation of roots and tubers
she probably observed the fall of grain seeds on the ground and their penetration into the soil
with rain. Finding new plants in the places which she herself digged with a stick, lead to the
connection between fallen seeds and new 'cultivated' plants. 18
The origin of farming practices should be located in South East Asia. In the highlands of this area
the neo-thermal switch caused abundant rainfall. In all of this area existed, and still exists, a
wide variety of wild cereals, sometimes in natural extended fields, induced by the rainfalls.
Triticum Dicoccoides (wheat) and Hordeum Spontaneum (barley) were common and routinely
collected by the local dwellers. The wild cereals had very few seeds (2-4) that fell easily on the
ground on maturation. 18
The people from the Uadi el-Natuf Tell of South East Asia (7800 B.C.) provided the first traces of
the gradual shift from hunters to grain cultivators. Their economy was based on the hunt of the
gazelle, but their diet also included collected grain seeds. These gradually came to form a
substantial proportion of their energy input, as cultivation practices ensued. Around 5000 B.C.
wild animals, more rare due to incoming drought, formed only 5% of the daily diet, while cereals
and farmed animals became a sizeable part of it. The farmer's expansion lasted from 9000 B.C.
up to the 4000 B.C. when they reached Ireland, Denmark and Sweden covering most cultivable
lands in Europe. The expansions followed the waterways of Mediterranean and of Danube across
the time of Egyptians, Phoenicians, Greeks and Romans. 18
Early bread making activities pushed towards grains that contained greater amounts of a
structural protein, which greatly facilitated the bread making: the gluten. Over the last 200 years
of our modern age active genetic selection, and genetic manipulation, have changed the aspect
of the original Triticacee enormously: from few grains and little gluten to great wheat harvests
very enriched in gluten (50% of the protein content), well adapted to cultivation practices and
ready to be handled by monstrous machineries. 18
The chemistry of gluten proteins
Gluten can be defined as the rubbery mass that remains when wheat dough is washed to remove
starch granules and water-soluble constituents. These gluten proteins have different solubility’s
in alcohol-water solutions and, thus, can be roughly separated into two fractions - gliadins and
glutenins. Gluten proteins have a complex chemistry and are responsible for determining the
unique baking quality of wheat by conferring water absorption capacity, cohesivity, viscosity and
elasticity on dough. 10
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
Analysis of gliadin has identified more than a hundred components that can be grouped into four
main types (ω5-,ω1,2-, α/ß-, ɣ-gliadins). The immunogenicity and toxicity of several gliadin
epitopes has been established for humans. A distinction exists between a peptide being
immunogenic or toxic. Glutenins can be divided into groups of high molecular weight and low
molecular weight. Immunogenicity and toxicity in the high-weight group has been shown.
Storage proteins (prolamines), with a similar amino acid composition to the gliadin fractions of
wheat, have been shown in barley (hordeins) and rye (secalin), and show a close relation to the
taxonomy and toxic properties of wheat cereal that affect people with coeliac disease.
Although several gluten epitopes are immunostimulatory, some are more active than others. 4
Gluten in the equine diet
To meet the energy requirements of today’s equine athlete concentrate feeds are given. As
mentioned before most regular concentrate feeds contain wheat, barley and rye, the main
carriers of gluten. Some specialised concentrate feeds are known to consist of up to 25-30% of
barley. Horses that are housed in a field year round or are fed a diet consisting of only hay or
silage are thought be gluten-free (or at least gluten-poor), since it are mainly the seeds of
agricultural grown cereals that contain gluten.
1.5 Aim of the study
1. To clarify the role of gluten in equine inflammatory small bowel disease, aiming at improved
diagnostic and treatment modalities.
2. To find out if there is an analogy between coeliac disease in humans and inflammatory small
bowel disease in the horse.
3. To develop ELISA tests as additional diagnostic tool in inflammatory small bowel disease.
4. If the study reveals a correlation between gluten and ISBD in horses this could provide some
further understanding of the characteristics of the disease for both human and veterinary
medicine.
1.6 Hypothesis
The hypothesis of this study is that: equine patients suffering from ISBD will have higher levels of
human recombinant tissue-transglutaminase (rh-tTGA), guinea pig tissue-transglutaminase (gptTGA), antigliadin, deamidated-gliadin-peptides (anti-DGP) and anti-endomysium (EmA)
antibodies compared to the two different control groups.
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
PART 2: MATERIALS AND METHODS
We’ve subjected equine ISBD patients to human coeliac disease diagnostics; to compare the
results two different control groups without ISBD have been tested as well: one group being on a
gluten-rich diet, the other on a gluten-free diet (or at least gluten-poor).
2.1 Animal selection
Patients
Twelve horses with signs of ISBD were referred to the Department of Equine Sciences, Section of
Internal Medicine, Utrecht University in the years 2010 and 2011. The clinical history was taken,
a physical examination was performed and peripheral venous blood was collected for routine
haematological and biochemical examination and further research. The (tentative) diagnosis of
inflammatory small bowel disease was based on the clinical signs, physical examination,
abnormal findings during rectal exploration (thickening of the small intestine), blood chemistry
and a percutaneous ultrasound. Haematocrit and lymphocytes were determined as well as
albumin and total protein. In 7 out of 12 cases an oral glucose tolerance test was performed and
6 out of 12 cases were diagnosed based on histological examination of gastroduodenoscopyassisted biopsies taken from the proximal duodenum. Breed, gender, age, sampling date and
findings by examination are summarized in Table 3. The patient group consists of 5 mares, 6
geldings and 1 stallion, ranging in age from 8 till 18 years (mean 10,8 ± 3,48 SD). Except for 1
Friesian all horses are Dutch Warmbloods. All samples were taken between February 2010 and
January 2011 (Annex 1).
Table 3: Background of the 12 patients suspected to have ISBD
ID
No
Breed
1
2
3
4
KWPN
KWPN
KWPN
Frisian
Sex
Age
(yea
rs)
5
6
7
8
9
10
11
12
gelding
gelding
mare
Clinical
presentation
Ht
Lymphoc
ytes
(L/L)
( x*10^9/L)
Total
protein
( g/L)
Albumin
(g/L)
Percutaneous
ultrasound /
rectal exam (+)
Glucose
tolerance
test
Histopat
hology
18
5
7
Anorexia, fever
0,33
1,1
73
33
+
nd
nd
Weight loss
0,36
1,2
58
34
+
63%
enteritis
Weight loss
0,33
1,1
73
33
+
Normal
Mild LE
Reduced
0,34
5,3
58
34
+
47%
nd
performance,
mare
11 underweight
KWPN
gelding
8 Recurrent colic
0,30
1,3
60
33
+
nd
Mild LPE
KWPN
mare
9 Recurrent colic
0,33
1,4
51
31
5-6mm, +
Normal
nd
KWPN
Weight loss,
0,41
2,4
66
35
+
40%
Broaden
reduced
villi, mild
stallion
14 performance
atrophy
KWPN
gelding
11 Colic
0,34
1,5
63
35
4,5mm, +
nd
nd
KWPN
mare
14 Recurrent colic
0,38
2,0
64
38
+
Lowered
LE
KWPN
gelding
14 Recurrent colic
0,40
1,3
72
36
nd
Lowered
nd
KWPN
gelding
9 Recurrent colic
0,33
1,2
62
38
+
nd
Mild LPE
KWPN
mare
10 Recurrent colic
0,37
2,8
63
37
+
nd
nd
KWPN= Dutch Warmblood horse; LE= Lymphocytic Enteritis; LPE= Lymphocytic-Plasmacytic Enteritis; += distended small intestine are
palpable and/or visible on ultrasound; nd= not determined
In-house: gluten-rich controls
22 “in-house” controls were used for this study. During the experimental period all these
controls were housed at the Department of Equine Sciences, Utrecht University. They were fed
with hay and concentrate according to their nutritional needs. Their mean age is 10,9 ± 4,46 SD
years. The 22 controls consist out of 19 Dutch Warmbloods, 2 Shetland ponies and one
Standardbred. 19/22 are mares, 3/22 are geldings. All samples were taken between February 2010
and January 2011 (Annex 1).
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Sampling
date
160210
040210
020210
160210
040210
180610
080610
060610
150410
200810
101210
060111
14
THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
Wild controls: suspected gluten-free population
A group of 25 Shetland ponies that are housed, year round, in nature reserve park Zeepeduinen
in Zeeland, the Netherlands were used as a gluten-free control group. They are most presumably
living a gluten-poor live, since their diet consists out of wild, non-agricultural grasses and herbs.
All 25 gluten-poor controls are mares and their age ranges from 1-27 years (mean unknown). All
samples were taken the 6 th of October 2010 (Annex 1).
Histology
The duodenal biopsies, taken by endoscopy, from patients after admittance between 2010 and
2011, were used for histopathological evaluation by both a veterinary and a human pathologist.
For comparative histology, between humans and horses, old, paraffin fixed, sections of affected
small bowel were used. These sections were collected, on necropsy, between 1993 and 2004.
These equine patients were diagnosed with eosinophilic enteritis, granulomatous enteritis,
lymphocytic-plasmacytic enteritis or lymphosarcoma. Histopathology rapports are available.
(Annex 7)
2.2. Sampling
Either plasma (using lithium-heparin tubes) or serum (using serum collection tubes) was
collected from peripheral venous blood. Buffy coats were collected using EDTA blood collection
tubes. Both samples were centrifuged after and the plasma/serum respectively buffy coats were
stored at -27°C. The collected biopsies from duodenal mucosa were fixed in 10% formalin and
processed to paraffin wax, cut at 4 mm and stained with haematoxylin and eosin (HE).
2.3 Adaption of human coeliac related ELISA’s for testing equine ISBD patients
Human coeliac disease diagnostics; enzyme-linked immunosorbent assay’s (ELISA) have been
altered for detection of equine IgA antibodies against E. coli, recombinant human tissuetransglutaminase, guinea pig tissue-transglutaminase, gliadin and deamidated gliadin.
Table 4: Antibodies used for ELISA testing.
Marker
Name
Source
Specificity
Format
Goat anti
AAl34P
AbD Serotec
IgA
HRP
horse IgA:HRP
Goat anti
AAl34F
AbD Serotec
IgA
FITC
horse IgA:FITC
HRP= horseradish peroxidise, FITC= fluorescein isothiocyanate isomer 1
Isotype
Polyclonal IgG
Target species
Equine
Polyclonal IgG
Equine
Goat anti horse IgA:HRP testing
To determine whether the goat anti horse IgA:HRP (Table 4.) could be used as a conjugate and at
which dilution, an in-house ELISA for E.coli antibodies was performed. Since E. coli is a common
pathogen all horses are suspected to carry antibodies. The goat anti horse IgA:HRP conjugate
was tested in three dilutions: 1:1000, 1:10.000 and 1:100.000. The serum was diluted to 1:20,
1:40 and 1:80. TMB was initially used as a substrate for the enzyme-substrate reaction. After 15
minutes, when staining had occurred, the reaction was stopped using H 2SO4. The degree of
staining was determined using a spectrophotometer (ELISA reader TECAN Sunrise, 450nm). The
extinctions are thought to reflect the antibody concentration.
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
ELISA: IgA antibodies against E. coli
Because the ELISA’s mentioned above are based on IgA antibodies, all sera were teste d for the
presence of IgA, using an E. coli antibody ELISA, to rule out IgA deficiency. The concentration
antibodies against E. coli is quantitatively determined using 96-wells microtitre plates (Nunc
Maxisorp©), which were coated with E. coli strain EB1. After washing (ELx 405 Auto Plate
washer, Bio-Tek Instruments) the still free binding sites, on the microtitre plates, were filled
using bovine serum albumin (BSA). Next the serum was put on to the plate in an 1:20 dilution. If
the serum contains antibodies against E. coli these will bind to the plate. After washing, the
plates were incubated with the goat anti horse IgA:HRP. After washing again the attached
antibodies were made visible following an enzyme-substrate reaction, using OPD. After 15
minutes the reaction was stopped using H 2SO4. The degree of staining was determined using a
spectrophotometer, at 492 nm. The extinctions are thought to reflect the antibody
concentrations against E. coli. Patient No. 10 is used for a calibration curve and as a positive
standard (per definition containing 100 AU E.coli antibodies/ml).
The exact protocol of this in-house ELISA is retained by the VU medical center.
ELISA: Antibodies against recombinant human and guinea pig tissue-transglutaminase
The most important serological parameter to diagnose human coeliac disease is tissue
transglutaminase antibodies (TGA). The TGA concentration is quantitatively determined based
on the TGA binding to either rh-tTG or to purified guinea pig tTG. 96-wells microtitre plates were
used, which were coated with rh-tTG (source: Diarect) or gp-tTG (source: Sigma) according to
standard operation procedures. After washing the still free binding sites, on the microtitre
plates, were filled using bovine serum albumin (BSA). Next the serum was put onto the plate.
The serum was diluted from 1:20, 1:40, 1:80 till 1:160 in PBS with 1% BSA and 0.05% Tween 20
and pre-incubated at room temperature for 30-60 min to allow potential anti-BSA to bind to the
BSA . If the serum contains TGA these will bind to the rh-tTG. After washing, the plates were
incubated with the goat anti horse IgA:HRP conjugate, in an 1:5000 dilution. After washing again
the attached antibodies were made visible using an enzyme-substrate reaction; reduced from
H2O2 till H2O and when oxidized by HRP, using hydrogen peroxidase, the substrate OPD is turned
into a colored product. After 15 minutes, when staining had occurred, the reaction was stopped
using H2SO4. The degree of staining was determined using a spectrophotometer, at 492 nm. The
extinctions are thought to reflect the concentration of TGA. Patient No. 7 is used as calibration
curve and as a positive standard (per definition containing 100 AU TGA/ml).
The exact protocol of this in-house ELISA is retained by the VU medical center.
ELISA: Antibodies against gliadin
In addition, to determine whether gliadin antibodies were present in the equine plasma an in house ELISA for antibodies against gliadin was executed. 96-wells microtitre plates were used,
which were coated with gliadin (source: Sigma). Next the serum was put onto the plate. The
serum was diluted from 1:20, 1:40, and 1:80 till 1:160. If the serum contains antibodies against
gliadin these will bind to the plate. Bound antibodies are made visible as described abov e for
TGA. Control No. 28 is used for a calibration curve and as positive standard (per definition
containing 100 AU gliadin antibodies/ml).
The exact protocol of this in-house ELISA is retained by the VU medical center.
ELISA: Deamidated gliadin antibodies
After tissue transglutaminase transforms the gliadin (glutamin glutamic acid), the remaining
gliadin is termed “deamidated” gliadin. A commercial ELISA (source: Euro Diagnostica), based on
a set of deamidated gliadin peptides as target, was used for testing antibodies against these
deamidated gliadin peptides.
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
The 'human' ELISA was adapted for measuring specific horse IgA as described for TGA. A 1:50
serum dilution was used. Unfortunately, the kit was limited, so not all horses were tested.
Gluten-rich controls No. 34, 35 and 42 and gluten-free controls 70-75 were not tested.
IFT: Anti-endomysium antibodies
The VUmc also has an IgA-class anti-endomysium immunofluorescence antibody test on hand, by
which an indirect immunofluorescence analysis is performed using unfixed cryostat sections of
monkey oesophagus. Oesophagus is used because the endomysium antibodies are directed
against proteins in between the smooth muscle cells of the muscularis mucosae, such as tissue
transglutaminase. The plasma was put on the 3-wells glasses in a 1:2 dilution. PBS was used as a
negative control. If anti-endomysium antibodies are present these will bind to the endomysium
during incubation. After washing the goat anti horse IgA:FITC conjugate (table 4) was incubated.
The conjugate incubation was tested in three dilutions, 1:20, 1:50 and 1:100. After washing, the
glasses were covered, and viewed under a fluorescence microscope, and scored for staining
pattern and fluorescence intensity of the muscularis mucosae (as -, +/-, +, ++ or +++). Antiendomysium antibodies are ineffective in detecting individuals with silent or subclinical gluten
sensitivity. 21
2.4 Histopathology
A human pathologist performed comparison of histopathological biopsies, from equine ISBD
patients to human coeliac patients. The sections were fixed in 10% formalin, processed to
paraffin wax, cut at 5 µm, stained with HE and examined under a light microscope.
2.5 Statistical analysis
During the study all results were analysed using, the statistical analysis specialised software,
MedCalc. Because the sample sizes are relatively small and it is not clear if these are samples
taken from normally distributed data the Mann-Whitney U-test was chosen to assess the
statistical differences. ρ values < 0.05 were considered significant. Pearson’s correlation was
used to determine the correlation coefficient between two substrates (TMB and OPD) used.
Corrections ELISA testing
The extinctions measured by the spectrophotometer were settled with the extinctions of the
blanks and the positive standards. As mentioned before, patient No. 10 was used as a positive
standard in the antibodies against E. coli ELISA. No.7 is used as a positive standard in both tTG
ELISA’s. Gluten-rich control No. 28 was used as a positive standard in the antibodies against
gliadin ELISA. Patient No. 10 was used as a positive standard in the antibodies against E. coli
ELISA. The extinctions are thought to reflect the concentration of antibodies measured. The
positive standards were used to determine a calibration curve, by diluting them from 1:20 till
1:2560. The extinction of the positive standard per definition containing 100 AU/ml.
Cut offs were not yet determined in this phase of the study.
2.6 Clinical trial/case study: measuring the effects of a gluten-free diet
The owner of patient no. 7, a horse known to suffer from ISBD and with an exceptional high tTGA
concentration, was asked to adjust their horse diet to a gluten-free one (Annex 4). Patient no. 7
was diagnosed with ISBD, based on abnormal findings during rectal examination, a lowered
glucose tolerance test and histopathological examination of a (duodenal) biopsy by a pathologist.
After a dieting period of 6-7 months another plasma sample was collected, from peripheral
venous blood using lithium-heparin tubes. This sample was, again, subject of all ELISA tests.
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
PART 3: RESULTS
3.1 Adaption of human coeliac related ELISA’s for testing equine ISBD patients
Goat anti horse IgA:HRP testing
To determine whether the goat anti horse IgA:HRP (Table 5.) could be used as a conjugate in all
other ELISA’s and at which dilution, an in-house ELISA for E.coli antibodies was performed. This
test was also used to determine which conjugate dilution suited best, 1:1000, 1:10.000 or
1:100.000. Since TMB was initially used as a substrate, extinctions were measured at a
wavelength of 450nm and they are shown in table 5.
Table 5. Extinction overview antibodies against E. coli ELISA at 450nm.
Serum
1:1000 conjugate
1:10.000 conjugate
1:100.000 conjugate
Patient 9
1:20
2,542
2,491
1,142
1,01
0,166
0,161
Patient 9
1:40
2,273
2,195
0,767
0,673
0,121
0,114
Patient 9
1:80
1,841
1,766
0,211
0,432
0,09
0,09
Patient 8
1:20
2,775
2,739
1,779
1,788
0,297
0,305
Patient 8
1:40
2,535
2,53
1,116
1,099
0,177
0,181
Patient 8
1:80
2,192
2,211
0,645
0,614
0,117
0,115
Patient 10
1:20
2,778
2,792
2,161
2,119
0,455
0,467
Patient 10
1:40
2,681
2,669
1,757
1,759
0,317
0,315
Patient 10
1:80
2,549
2,487
1,155
1,136
0,202
0,202
Control 30
1:20
2,676
2,64
1,436
1,257
0,208
0,199
Control 30
1:40
2,504
2,41
0,953
0,868
0,141
0,134
Control 30
1:80
2,079
1,924
0,604
0,508
0,092
0,088
Blanco
0,09
0,066
0,043
0,043
0,039
0,037
Blanco
0,073
0,062
0,042
0,047
0,04
0,041
Blanco
0,058
0,056
0,043
0,04
0,052
0,048
Blanco
0,059
0,056
0,043
0,043
0,049
0,047
The extinctions shown in table 5 confirm that the goat anti horse IgA:HRP conjugate works,
because staining has occurred, resulting in an extinction measured by the spectrophotometer.
Fig. 4 shows the extinction outcome of patient No. 9 for all conjugate and serum dilutions. This
demonstrates that the 1:1000 conjugate dilution has the clearest regression curve.
Fig. 4: Multiple comparison line graph showing the extinctions per dilution.
Outcome extinctions at 3 different dilutions, for
both conjugate as plasma.
Extinction patient no. 9
17
3
2
*1:1000
1
*1:10.000
0
*1:20
*1:40
*1:80
*1:100.000
Plasma dilution
Since the 1:1000 conjugate dilution developed too fast, a 1:5000 conjugate dilution was chosen
for all further experiments.
17 | P a g e
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
Because the substrate TMB has shown too much staining too fast, OPD was chosen as an
alternative substrate. Pearson’s correlation between TMB and OPD is r= 0,999, with a ρ-value of
< 0,0001. So, changing the substrate from TMB to OPD will not influence the overall results.
ELISA: IgA antibodies against E.coli
To exclude IgA deficiency in the study group all horses were tested for IgA antibodies against E.
coli. As shown in Fig. 5 all horses included in this study tested positive for IgA antibodies against
E. coli. As suspected, since no solitary IgA deficiency disorder has been described in horses and
E. coli is such a common pathogen. It also shows that specific IgA is easily and repeatable
detectable in all horses. And that anti-IgA antibodies could be used in further ELISA testing in this
study. The range in antibody concentration is shown in fig. 5. n=12 for group 1, n=22 for group 2
and n=25 for group 3. (See also: annex 2)
Fig. 5: Multiple comparison (dot and box) graph showing the E. coli antibodies in AU/ml per group.
Overview E.coli antibody (IgA) concentrations in arbitrary units (AU) per milliliter per group.
140
P = 0.0015
120
P = 0.5840
P = 0.0001 for
group 1+2 vs. 3
P = 0.0004
100
meidan = 93.85
median = 87.00
80
E. coli
60
[
40
median = 24.10
20
0
1
2
3
group
(1=patients, 2=gluten-rich controls, 3=gluten-free controls)
Using the Mann-Whitney test for independent samples the following results were found:
The ρ-value (P) for group 1 and 2 = 0.5840, since this exceeds the 0.05 = α there is no significant
difference measured between E.coli IgA antibody concentrations in horses with ISBD and horses
without ISDB on a gluten-rich diet. There is however a significant difference between group 1
and 3 and group 2 and 3, with ρ-values being respectively 0.0015 and 0.0004, both being <0.05.
Accordingly, the IgA concentrations against E.coli are significantly higher in horses with ISBD and
in the gluten-rich controls than in the gluten-free, wild controls. This implies that the wild living,
gluten-free horses have lesser antibodies against E. coli then group 1 and 2, with the lowest
concentration being 5.4 AU/ml. In human diagnostics a concentration of 3.0 U/ml is considered
weak positive.
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
ELISA: Antibodies against recombinant human and guinea pig tissue-transglutaminase (TGA)
The TGA concentration is quantitatively determined based on the TGA binding to either rh-tTG or
to purified guinea pig tTG.
The results of the recombinant human tissue transglutaminase antibody ELISA of horses with ISBD
were compared with those of clinically normal horses, both gluten-rich and gluten-free. Results are
shown in fig. 6. n=12 for group 1, n=22 for group 2 and n=25 for group 3. (See also: annex 2)
Fig. 7 shows the staining of a 96-well microtitre plate after the enzyme-substrate reaction with
OPD was stopped using H2SO4.
Fig. 6: Multiple comparison (dot and box) graph showing the rh-tTG antibodies in AU/ml per group.
Overview rh-tTGA concentrations in arbitrary units (AU) per milliliter per group.
100
patient no. 7
P < 0.0001 for
group 1+2 vs. 3
80
60
P = 0.0001
P = 0.2343
40
P = 0.0004
20
median = 12.05
median = 8.40
median = 3.70
0
1
2
3
group
(group 1= patients, group 2= gluten-rich controls, group 3= gluten-free controls
Using the Mann-Whitney test for independent samples the following results were found:
The ρ-value for group 1 and 2 = 0.2343, since this exceeds the 0.05 = α there is no significant
difference measured between the rh-tTGA concentration in horses with ISBD and clinically
healthy horses kept on a gluten-rich diet. There is however a significant difference between
group 1 and 3 and group 2 and 3, with ρ-values being respectively 0.0001 and 0.0004 both being
<0.05. Thus the rh-TGA concentrations are significantly higher in horses with ISBD and in the
gluten-rich controls than in the gluten-free controls.
Remarkable is the, in contrast to other ISBD patients and the gluten-rich controls, repeatable
high recorded rh-TGA concentration of patient no. 7: 100 AU/ml.
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
Fig. 7: ELISA 96-well microtitre plate showing positive staining.
Using a Log transformation, shown in Fig. 8, the differences between groups 1 and 3 and 2 and 3
become even clearer. It shows that ISBD patients do not have an overall higher rh-tTGA
concentration than the clinically healthy controls on a gluten-rich diet. Note the clear separation
in concentration in the gluten-free population, group 3, with some of them having almost no
detectable antibodies at all.
Fig. 8: Multiple comparison (dot and box) graph showing the rh-tTG antibodies in AU/ml per group in Log
transformation.
rh-tTGA concentrations in AU/ml per group (in Log transformation)
100
patient no. 7
10
1
Horizontal reference line.
Value = 2,50
0.1
GR no. 35 +42
0.01
1
2
3
group
(group 1=patients, group 2= gluten-rich controls, group 3= gluten-free controls)
20 | P a g e
21
THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
The results of the guinea pig tissue transglutaminase antibody ELISA from the horses with ISBD
were compared with those of clinically healthy horses, both gluten-rich and gluten-free. n=12 for
group 1, n=22 for group 2 and n=24 for group 3. Gluten-free control no. 61 was not determined.
Results are shown in fig. 9. (See also: annex 2)
Fig. 9: Multiple comparison (dot and box) graph showing the gp-tTG antibodies in AU/ml per group.
Overview gp-tTGA concentrations in arbitrary units (AU) per milliliter per group.
100
Patient no. 7
P = 0.9559 for
group 1+2 vs. 3
GR controls no. 26 & 32
80
P = 0.3305
P = 0.2343
60
P = 0.4748
40
median = 22.20
median = 19.40
20
median = 15.50
0
1
2
3
group
(group 1= patients, group 2= gluten-rich controls, group 3= gluten-free controls)
Using the Mann-Whitney test for independent samples the following results were found:
The ρ-value for group 1 and 2 = 0.2343, since this exceeds the 0.05 = α there is no significant
difference between the gp-tTGA concentration in horses with ISBD and horses without ISDB, kept
on a gluten-rich diet. There was also no significant difference found between group 1 and 3 and
group 2 and 3, with ρ-values being respectively 0.3305 and 0.4748 both being > 0.05.
Summarized: the overall gp-tTGA concentrations are not significantly higher in horses with ISBD
and in the gluten-rich controls than in the gluten-free controls so there is no clear relation
between gp-tTGA and the use of gluten in horses.
Remarkable is the patients median of group 3, m=19.40, being higher than that of group 2,
m=15.50. And patient no. 7 that shows to be strongly positive, as are the gluten-rich controls no.
26 and 32.
21 | P a g e
[GA] in AU/ml
22
THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
ELISA: Antibodies against gliadin
To determine whether significant differences could be found in gliadin antibody concentration
between the three study groups an in-house ELISA for gliadin antibodies was performed. The
results of the antigliadin antibody (GA) ELISA from the horses with ISBD were compared with those of
clinically normal horses, both gluten-rich and gluten-free. n=12 for group 1, n=22 for group 2 and
n=25 for group 3. Results are shown in fig. 10. (See also: annex 2)
Fig. 10: Multiple comparison (dot and box plot) graph showing the GA in AU/ml per group .
Overview GA concentrations in arbitrary units (AU) per milliliter per group.
140
GR no. 32
P = 0.9572 for
group 1+2 vs. 3
120
GR no. 28
100
GF no. 54
P = 0.7827
80
P = 0.7981
P = 0.7731
60
40
median = 24.50
median = 23.75
median = 19.95
20
0
1
2
3
group
(group 1= patients, group 2= gluten-rich controls, group 3= gluten-free controls)
Using the Mann-Whitney test for independent samples the following results were found:
The ρ-values are: ρ = 07731 for 1 and 2, ρ = 0.7827 for 1 and 3 and ρ = 0.7981 for 2 and 3. Since none
of these are < 0.05, no significant between-group differences could be stated. And there is no clear
relation between GA and the use of gluten in horses.
Remarkable however are the 2 clinically healthy gluten-rich controls, no. 28 and 32 that are
strongly positive for GA and also the gluten-free controls no. 54 and two others who have a high
concentration of GA. Patient no. 7 also shows to be strongly positive.
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
ELISA: Deamidated gliadin antibodies
The results of the deamidated gliadin (DGP) antibody ELISA from the horses with ISBD were
compared with those of clinically healthy horses, both gluten-rich and gluten-free. n=12 for group 1,
n=19 for group 2 (gluten-rich controls 34, 35 and 42 were left out) and n=19 for group 3 (glutenfree controls 70-75 were left out). Results are shown in Fig. 11 and 12. (See also: annex 2)
Fig. 11: Multiple comparison (dot and box plot) graph showing the DGP antibody concentration in AU/ml per group .
Overview DGP antibody concentrations in arbitrary units (AU) per milliliter per group.
100
GF no. 59
P = 0.0341 for
group 1+2 vs. 3
GR no. 22 + 24
80
60
P = 0.0244
40
P = 0.4410
P = 0.1403
20
median = 5.60
median = 2.60
median = 1.90
0
1
2
3
group
(group 1= patients, group 2= gluten-rich controls, group 3= gluten-free controls)
Using the Mann-Whitney test for independent samples the following results were found:
The ρ-value for group 1 and 2 = 0.4410, since this exceeds the 0.05 = α there is no significant
difference measured between the DGP antibody concentrations in horses with ISBD and horses
without ISDB, kept on a gluten-rich diet. There has also no significant difference been found
between group 2 and 3, ρ-values= 0.1403 > 0.05. There is however a significant difference
between group 1 and 3, since ρ = 0.0244 < 0.05. Therefore horses with ISBD have a significantly
higher concentration of DGP antibodies than the gluten-free control group.
Striking are the high concentrations of the two horses (No. 22 and 24) in group 2 and the one
(No. 59) in group 3. Patient no. 7 also shows to be strongly positive.
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
Using a Log transformation, seen in Fig. 12, the differences between the groups become clearer.
Fig. 12: Multiple comparison (dot and box plot) graph showing the GA in AU/ml per group, in Log transformation.
DGP antibodies in arbitrary units (AU) per milliliter per group (in Log transformation).
100
10
1
Horizontal reference line
value = 1,0 AU/ml
0.1
1
2
3
group
(group 1= patients, group 2= gluten-rich controls, group 3= gluten-free controls)
IFT: Anti-endomysium antibodies
This test was performed to see if there were any IgA antibodies against endomysium. There were
no anti-endomysium antibodies (EmA) found in patients No. 1,2,7 and 8, when using monkey
oesophageal tissue as a substrate. Also controls 21-24 were found to be negative. Because of the
lack of staining the remaining patients and controls were not tested for EmA in monkey
oesophagus. It is surprising that patient No. 7 does not show any staining since it does have high
rh-TGA. For some horses, however, the smooth muscle cells stained positive, including No. 7.
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
3.2 Comparative histology
A human pathologist performed re-examination of histopathological biopsies, from some of the
equine ISBD patients. The sections were stained with HE and examined under a light microscope.
The histological findings are summarised in table 6.
Table 6: Findings upon histological examination of equines suspected from ISBD.
P. No
2
3
5
7
IEL count*
<30/100
At most a
<30/100
<30/100
minor
increase.
Crypt
hyperplasia
Villous
Somewhat
Mild
atrophy
short.
Lamina
Lamina
PreLamina
Lamina
propria
propria looks
infiltrative
propria looks
propria looks
somewhat
(normal).
somewhat
somewhat
fibrous,
fibrous.
fibrous,
pour amount
pour amount
of cells.
of cells.
8
<30/100
9
<30/100
-
-
Mild?
-
Lamina
propria looks
somewhat
fibrous.
Lamina
propria looks
somewhat
fibrous, only
in the villi.
Deeper
mucosa is
infiltrated
normally.
IEL’s = intraepithelial lymphocytes
The histological findings, in biopsies taken from the proximal duodenum, show no major
abnormalities in the horses with ISBD. None of the horses show a clear IEL increase and only
some show some villous blunting. Most remarkable is the reduced amount of cells in the lamina
propria in almost all patients, compared to that of the human duodenum.
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
3.5 Clinical trial/case study: measuring the effects of a gluten-free diet
The owner of patient no. 7, a horse known to suffer from ISBD and with an exceptional high tTGA
concentration, was asked to adjust their horse’s diet to a gluten-free one (Annex 4). Patient no. 7
was diagnosed with ISBD, based on abnormal findings during rectal examination, a lowered
glucose tolerance test and histopathological examination of a (proximal duodenal) biopsy by a
pathologist. After a dieting period of 6-7 months another plasma sample was collected, from
peripheral venous blood using lithium-heparin tubes. This sample was, again, subject of all ELISA
tests. Results are illustrated in fig. 13 and summarized in table 7.
Fig. 13: The effect of a gluten-free diet on the IgA antibodies against E. coli, rh-tTG, gp-tTG, gliadin and deamidated
gliadin.
In particular the decrease in rh-tTGA from 100 AU/ml to 32,5 AU/ml, after adjustment to a
gluten-free diet, is remarkable. The other parameters show a lesser amount of decrease. As
suspected the IgA antibody concentration against E. coli exposes no changes.
Table 7. IgA antibodies against E. coli, recombinant human tissue-transglutaminase, guinea pig tissuetransglutaminse, gliadin and deamidated gliadin.before and after a gluten-free diet (GFD)
[E.coli antibody] in
AU/ml
[rh-TGA]in AU/ml
[gp-TGA]in AU/ml
[Gliadin antibody]
in AU/ml
[DGP antibody] in
AU/ml
Patient No. 7
100, +++
100
100
61,2
25
Patient No. 7 GFD
100, +++
32,5
95,1
53,1
18.75
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
Other blood parameters measured in patient No. 7 show the following changes:
Table 8. Blood parameters before and after a gluten-free diet (GFD)
ID
No
Breed
Sex
Age
(yea
Clinical
presentation
rs)
7
KWPN
7*
KWPN
stallion
stallion
14
15
Weight loss,
reduced
performance
-
Ht
Lymphoc
ytes
Albumin
( x*10^9/L)
Total
protein
( g/L)
(L/L)
(g/L)
Percutaneous
ultrasound /
rectal exam (+)
Glucose
tolerance
test
0,41
2,4
66
35
+
40%
0,40
-
69
37
ND
ND
Sampling
date
080610
060111
Due to the athletic obligations of the horse a second glucose tolerance test and biopsy have not
yet been performed.
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
PART 4: DISCUSSION
To answer the question if gluten play a role in the aetiology of equine ISBD we’ve subjected
three groups of horses to six different human coeliac disease diagnostic tests; to compare the
results of the ISBD patients, two different control groups without ISBD have been tested: one
group being on a gluten-rich diet, the other on a gluten-free diet.
3.1 animals and sampling
Animal selection
Inflammatory (small) bowel disease represents a poorly characterized group of conditions in the
horse that may present with a dull hair coat, weight loss, colic and at times soft faeces, oedema and
lethargy.22 The ISBD patients used in this study were diagnosed based on their history, clinical
examination and blood work. During rectal examination thickening of the small intestine was
palpated in 9 out of 12 cases and was noted on abdominal ultrasonography in 2 patients. Combining
the results of anthelmintic history and faecal flotation, involvement of parasites was excluded. A
glucose tolerance test was performed in 7 patients and was found to be below standard in 5/7
patients. The results of the glucose tolerance test do not seem to correlate with serum albumin or
the degree of intestinal inflammation in these patients. In only 6 cases a gastroduodenoscopyassisted biopsy of the duodenum was taken. According to a veterinary pathologist all of these
biopsies showed mild enteritis, mostly with infiltration of IEL’s and plasma cells. The clinical
relevance of these findings was taken into question, because none of the biopsies showed signs of
severe enteritis. To make a definitive diagnosis of inflammatory small bowel disease and confirm the
subtype, histopathological examination of affected intestine is necessary. Preferably using full
thickness surgical biopsies. These were however taken in none of the patients, since they were
mainly high performing dressage horses. So diagnoses are more tentative then confirmed. Also, the
age range (8 till 18) and breeds of the included patients are not consistent with what today’s
literature is stating. Most of the horses included in the study are Dutch warmblood horses, being the
main horse breed in the Netherlands, but shouldn’t we look at the 2-4 year old Thoroughbred horses
and Standardbreds, as literature reports these horses as being most susceptible to inflammatory
small bowel disease subtypes eosinophilic and granulomatous enteritis? Only the lymphocyticplasmacytic subtype of ISBD shows no clear age relation.7
The suspected gluten-free population used in this study is housed year round in a nature reserve
park in the Netherlands. They are part of a group consisting of about a hundred Shetland ponies,
ranging in age from 1 till 27 years, all being mares. Their job is to preserve and develop the dune area
with its associated succession stages: open, local dusty dune, pioneer vegetation, dune grasses and
dune valleys, by grazing. As known, the grains such as barley, wheat and ray, which contain gluten,
are part of the grass family. They are however production cereals and it is thought that most wild
grass species don’t contain gluten or contain gluten in far less extent than found in concentrated
feeds. Since the nature reserve park is open to public we cannot, unfortunately, rule out that horses
are fed bread or any other kind of gluten containing feeds.
3.2 Development of a standardized ELISA test
The goat anti horse IgA: horseradish peroxidase testing illustrated that the goat anti horse
IgA:HRP works. All tests were performed using a 1:5000 conjugate dilution, and using OPD as a
substrate for HRP.
ELISA: Antibodies against E. coli
In this study, all horses showed detectable IgA antibodies against E. coli, using a 1:20 serum dilution.
The test was performed to rule out any IgA deficiency, which in humans has a known prevalence of
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
about 1:400 and is even more common in patients with coeliac disease.17, 20 In horses, unlike
selective immunoglobulin M deficiency, no selective immunoglobulin A deficiency has ever been
described.16 Since all horses tested positive this remains this way. So, IgA could be used for further
ELISA testing. The IgA antibody concentrations against E. coli are however significantly higher in
horses with ISBD and in the gluten-rich controls than in the gluten-free, wild controls. Some of them
showing remarkable low antibody titers. For the IgA testing the E. coli strain EB1 was used. EB1 is
known to be a human pathogenic strain of E. coli. It could be hypothesized that since the gluten-free
horses have lesser human contact they are also exposed less to this strain of E. coli resulting in a
lesser serum IgA antibody concentration.
ELISA: Antibodies against recombinant human tissue-transglutaminase (rh-tTG)
The rh-tTGA concentrations were found to be significantly higher in horses with ISBD and in the
gluten-rich control group then in the gluten-free control group. There was no significant difference
found between the patients and the gluten-rich controls. As mentioned before, tTG in man is the
enzyme that, by deamidation, makes gliadin peptides negatively charged and raises the
immunostimulatory effect of gluten. Expression and activity of tTG are raised in the mucosa of
patients with CD. And tTG is thought to be the main target autoantigen. 4 The results in this study
suggest a relation between gluten-uptake and the rh-tTGA concentration in horses but show no clear
relation between clinical signs of ISBD and rh-tTGA concentration. A. Tursi et al. (2003) showed that,
in humans, anti-tTG prevalence and their mean serum value was higher in coeliacs with severe
enteropathy (Marsh IIIb-c lesions) than in those showing slight enteropathy (Marsh I-IIIa). So,
serologic tests without histological evaluation may underestimate the real prevalence of CD. 21 The
correlation between degree of intestinal damage and positivity to rh-tTGA in horses remains
undetermined.
Given that tTG antibodies are antibodies that are directed against the complex of gliadin attached to
tissue transglutaminase all wild controls were expected to be negative for TGA. As shown above
some wild, gluten-free controls do possess some levels of rh-tTGA. This suggests a gluten uptake.
Since the ‘gluten-free’ controls are housed in an ‘open to public’ nature reserve park it is thought
they are in all probability fed bread.
ELISA: Antibodies against guinea pig tissue-transglutaminase (gp-tTG)
No significant relations between group gp-tTGA differences were found during this study. So there is
no clear relation between a gluten containing diet, either with or without clinical signs of ISBD, and
gp-tTGA in horses. Strangely the correlation coefficient r for both TGA tests (rh-tTGA and gp-tTGA) is
0,7005 (p<0,0001), suggesting a linear dependence between both variables. (Annex 3) Patient no. 7,
which shows to be strongly positive in both tests, might illustrate this relation.
ELISA: Antibodies against gliadin
No significant relations between group GA differences were found during this study. Thus there is no
clear relation between a gluten containing diet, either with or without clinical signs of ISBD, and GA
in horses. In human medicine antigliadin antibodies are no longer considered sensitive enough or
specific enough to be used for the detection of coeliac disease. Mainly the sensitivity for the IgA
based test is thought to be poorly and highly variable. Resulting in a higher number of false
negatives. Because of the variable and generally lower sensitivity and specificity associated with the
GA, it is stated that these tests are less suitable for screening purposes.20, 24
ELISA: Antibodies against deamidated gliadin
Unlike the GA concentrations, the DGP antibody concentrations were found to be significantly higher
in horses with ISBD then in the gluten-free control group. Also, when combining the two gluten-rich
groups (both clinically healthy and ISBD horses) a significant difference compared to the gluten-free
group was found. So there seems to be a relation between gluten intake and the DGP antibody
concentration in horses. DGP antibody testing is a relatively new ELISA used in human coeliac disease
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
diagnostics, and although tTG display a higher predictive value then DGP and is still considered the
best CD serological test, DGP antibody testing comes relatively close, showing high sensitivity and
specificity. Volta et al. showed that sensitivities for DGP and tTG antibodies were 87.8% (95% CI:
85.6-89.9) and 93% (95% CI: 91.2-94.5), respectively, and the specificities were 94.1% (95%CI: 92.595.5) and 96.5% (95% CI: 95.2-97.5), respectively. 25
IFT: Anti-endomysium antibodies
In human coeliac disease diagnostics the endomysium IgA antibody IFT is thought to be a highly
specific marker, approaching 100% accuracy. Since it is recognized that tTG is the auto-antigen for
the development of endomysium antibodies it raises the question why staining did not occur in any
of the tested horses, that in some case did show a high rh-tTGA concentration.20 It could be that
equine antibodies don’t recognize the monkey tissue transglutaminase. To rule this out another IFT
needs to be performed, presumably on equine tissue.
3.2 Comparative histology
Definitive diagnosis of the inflammatory small bowel disease subtype necessitates
histopathological examination of the affected intestine. The histological findings in the horses
with ISBD show no major abnormalities. According to a human pathologist, none of the horses
show a clear IEL increase (or any other inflammatory cell infiltrate) and only some show some
villous blunting. The latter may be due to the fact that the biopsies were probably taken from
the duodenal bulb area, just after passing the pylorus, using a 2.2-meter endoscope. Collecting
of intestinal biopsies from the duodenal mucosa is minimally invasive but, intuitively, the
diagnostic usefulness of full thickness surgical biopsies is likely to be greater.
Gastroduodenoscopy-assisted biopsy of the (proximal) duodenum could miss focal small
intestinal infiltrative diseases, such as focal eosinophilic enteritis although lymphocyticplasmacytic enteritis has a more diffuse aspect such that abnormalities would be expected on
the duodenal biopsy specimen. Performing a laparotomy to obtain the intestinal biopsy can be
expensive, may occasionally result in peritonitis or incision infection and may delay introduction
of immunosuppressive therapy until the incision sites are healed in otherwise well performing
horses. 3,22,23 Reports by Packer et al. (2005) and Rotting et al. describe correspondingly the
amount of immune cells in the jejunal mucosa and the mucosal distribution of eosinophilic
granulocytes in the gastrointestinal tract in horses that were euthanized for reasons other that
gastro-intestinal disease. Distribution of immune cells in the villous lamina propria and
intercryptal lamina propria is reported, in mean ± SD cells/9000μm2, for plasma cells (18 ± 10.8,
35 ± 10.2), CD3+ T cells (50 ± 15.2, 50 ± 11.0), CD79a+ B cells (3 ± 3.4, 12 ± 7.6), neutrophils (0 ± 0.3,
0 ± 0.0) and macrophages (5 ± 2.8, 5 ± 3.5). Eosinophils are reported in highest numbers in the
cecum (488 ± 285 cells/mm2) and the ascending and transverse colon, and the lowest are in the
stomach (18 ± 22.4 cells/mm2), the duodenum (100 ± 93 cells/mm2) and the descending colon (83 ±
99 cells/mm2). Plasma cells and B cells have a higher density in the intercryptal than in the villous
regions of lamina propria. The distribution of eosinophils within the mucosal lamina propria also
follows a consistent pattern. In all sections of the gastrointestinal tract most eosinophils are located
in the basilar half of the mucosa.11, 12 Normal distribution of immune cells, other than eosinophils,
in the lamina propria of the horses duodenum remains however unclear.
3.3 Clinical trial/case study: measuring the effects of a gluten-free diet
Since only one patient has been followed shifting from a concentrated, gluten-containing diet to a
gluten-free diet, no statistical significant data are available. Nevertheless, all measured parameters
show a positive decrease, predominantly the serological parameter: rh-tTGA, expect for the IgA
antibodies agains E. coli, which as suspected remained strongly positive. Taking the assumption
that gluten plays a role in the etiology of ISBD, it would seem plausible that plasma rh-tTGA could be
used as a serological marker for gluten intolerance. A challenge study to measure possible relapse
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
after a subsequent gluten intake would be a rational next step. From an ethical and welfare
perspective, this is however unlikely to happen.
PART 5: CONCLUSION
This is the first study to investigate the potential role of gluten in the aetiology of equine
inflammatory small bowel disease. Although some facts in the patient group and gluten-free control
group are questionable and so as researchers we have to be a little sceptic, it is a start in enfolding
the effects of gluten on the equine intestine and its immune system.
IgA deficiency was excluded in all participating horses. The results in this study suggest a relation
between gluten-uptake and the rh-tTGA concentration in horses but show no clear relation between
clinical signs of ISBD and rh-tTGA concentration. Also, there seems to be a relation between gluten
intake and the DGP antibody concentration. The DGP antibody concentrations were found to be
significantly higher in horses with ISBD then in the gluten-free control group. And, in addition, when
combining the two gluten-rich groups, a significant difference compared to the gluten-free group was
found. In human medicine both the rh-tTGA and the DGP antibody ELISA are thought to be highly
sensitive and specific for diagnosing coeliac disease. On the other hand no clear relation was found
between the use of gluten, signs of ISBD and the amount of gp-tTGA and GA. Furthermore, the
endomysium IgA antibody IFT on monkey oesophagus was inconclusive, showing no specific staining.
Thus, on the basis of this data it is not possible to draw any clear conclusions about the role of gluten
in equine inflammatory small bowel disease yet. But, the notion that rh-tTGA and DGP antibody
concentrations are raised in horses fed a gluten rich diet suggest an immunostimulatory effect of
gluten, that could play a role in the aetiology of ISBD. One case, showing both clinically as well as
serological improvement after a gluten-free diet supports this possibility. However, further research
is needed to clarify the specifics.
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
ACKNOWLEDGEMENTS
As mentioned before, this study was performed in joint venture with the VUmc Research Institute for
Cancer and Immunology and the Department of Gastroenterology and Hepatology at the VUmc in
Amsterdam. I would like to thank both departments and within these especially dr. B.M.E. von
Blomberg, Rubi, Martine, Jolien, dr. N.C.T. van Grieken and prof. dr. C.J.J. Mulder for their ideas,
patience, persistence, support and the many practical work hours they put into this research.
This research project was carried out within the study of Veterinary Medicine at the Utrecht
University and was initiated and supervised by dr. J.H. van der Kolk and supported by dr. G.C.M.
Grinwis.
The study could not have been performed without the consent of the horses’ owners and the
cooperation of the veterinary clinics Oisterwijk and Hattem-Wapenveld and the environmental public
organization Staatsbosbeheer.
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REFERENCES
1. Schumacher, J., Edwards, J. F., & Cohen, N. D. (2000). Chronic idiopathic inflammatory bowel
diseases of the horse. J Vet Intern Med , 14 (3), 258-265.
2. Kemper, D. L., Perkins, G. A., Schumacher, J., Edwards, J. F., Valentine, B. A., Divers, T. J. , et
al. (2000). Equine lymphocytic-plasmacytic enterocolitis: a retrospective study of 14 cases.
Equine Vet J Suppl. , 32, 108-112.
3. Archer, D. C., Edwards, B. G., Kelly, D. F., French, N. P., & Proudman, C. J. (2006). Obstruction
of equine small intestine associated with focal idiopathic eosinophilic enteritis: An emerging
disease? The Veterinary Journal , 171, 504-512.
4. Di Sabatino, A., & Corazza, G. R. (2009). Coeliac disease. The Lancet , 373, 1480-1493.
5. Dansen, O., & Van Rutten, J. (2008). The secret of inflammatory small bowel disease. Utrecht
University, Faculty of Veterinary Medicine, Department of Equine Sciences, Internal Medicine,
Utrecht.
6. Roberts, M. C. (2003). Proliferative and inflammatory intestinal diseases associated with
malabsorption and maldigestion. In S. M. Reed, W. M. Bayly, & D. C. Sellon, Equine Internal
Medicine (2 ed.). St. Louis, Missouri, USA: Saunders Elsevier.
7. Kalck, K. A. (2009). Inflammatory bowel disease. Vet Clin North Am Equine Pract , 25, 303-315.
8. Barr, B. S. (2006). Infiltrative intestinal disease. Vet Clin North Am Equine Pract , 22, 1-7.
9. Ettinger, S. J., & Feldman, E. C. The gastrointestinal system. In S. J. Ettinger, & E. C. Feldman,
Textbook of veterinary internal medicine (4 ed., Vol. 2). St. Louis, Missouri, USA: Saunders
Elsevier.
10. Wieser, H. (2007). Chemistry of gluten proteins. Food microbiology , 24, 115-119.
11. Packer, M., Patterson-Kane, J. C., Smith, K. C., & Durham, A. E. (2005). Quantification of
immune cell populations in the lamina propria of equinejejunal biopsy specimens. J Comp Pathol
, 132 (1), 90-95.
12. Rottinger, A. K., Freeman, D. E., Constable, P. D., Eurell, J. A., & Wallig, M. A. (2008).
Mucosal distribution of eosinophilic granulocytes within the gastrointestinal tract of horses. Am J
Vet Res , 69 (7), 874-879.
13. Fogarty, U., Perl, D., Ensley, S., Seawright, A., & Noonan, J. (1998). A cluster of equine
granulomatous enteritis cases: the link with aluminium. Vet Human Toxicol , 40 (5), 297-305.
14. Kagnoff, M. F. (2007). Celiac disease: pathogenesis of a model immunogenetic disease. J Clin
Invest , 117 (1), 41–49.
15. Daminet, S. C. (1996). Gluten-sensitive enteropathy in a family of irish setters. Can Vet J , 37,
745-746.
16. Crisman, M. V., & Kent Scarratt, W. (2008). Immunodificiency disorders in horses. Vet Clin
Equine , 24, 299-310.
17. Cunningham-Rundles, C., Brandeis, W. E., Pudifin, D. J., Day, N. K., & Good, R. A. (1981).
Autoimmunity in selective IgA deficiency: relationship to anti-bovine protein antibodies,
circulating immune complexes and clinical disease. Clin Exp Immunol , 45, 299-304.
18. Greco, L. (1997). From the neolithic revolution to gluten intolerance: benefits and problems
associated with the cultivation of wheat. J Pediatr Gastroenterology Nutr , 24 (5), 14-17.
19. Dickson, B. C., Streutker, C. J., & Chetty, R. (2006). Coeliac disease: an update for
pathologists. J Clin Pathol , 59 (10), 1008-1016.
20. Green, P. H., & Cellier, C. (2007). Celiac Disease. N Engl J Med , 357, 1731-43.
21. Tursi, A., Brandimarte, G., & Giorgetti, G. M. (2003). Prevalence of Antitissue
Transglutaminase Antibodies in Different Degrees of Intestinal Damage in Celiac Disease. J Clin
Gastroenterol , 36 (3), 219-221.
22. Durham, A. E., & Rendle, D. (2010). Inflammatory bowel disease as a cause of colic: diagnosis
and treatment. 49th BEVA Congress (pp. 198-199). Newmarket: Equine Veterinary Journal Ltd.
33 | P a g e
34
THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
23. Divers, T. J., Pelligrini-Masini, A., & McDonough, S. (2006). Diagnosis of inflammatory bowel
disease in a Hackney pony by gastroduodenal endoscopy and biopsy and succesfull treatment
with corticosteroids. Equine vet. Educ. , 18 (6), 284-287.
24. Hill, I. D. (2005). What Are the Sensitivity and Specificity of Serologic Tests for Celiac
Disease? Do Sensitivity and Specificity Vary in Different Populations? Gastroenterology, 128, S25S32.
25. Volta, U., Fabbri, A., Parisi, C., Piscaglia, M., Caio, G., Tovoli, F., et al. (2010). Old and new
serological tests for celiac disease screening. Expert Rev Gastroenterol Hepatol. , 4 (1), 31-35.
26. Brown, I., Mino-Kenudson, M., Deshpande, V., Lauwers, G.Y. (2006) Intraepithelial
lymphocytosis in architecturally preserved proximal small intestinal mucosa: an increasing diagnostic
problem with a wide differential diagnosis. Arch Pathol Lab Med, 130, 1020–1025
ABBREVIATIONS
AGA
AU
BSA
CD
DGP
E. coli
EDTA
ELISA
FACS
FITC
GA
GFD
gp-tTG
gp-tTGA
GRD
HLA
HRP
IEL’s
IFT
Ig
IBD
ISBD
KWPN
LE
LPE
MHC
Nd
OPD
rh-tTG
rh-tTGA
TGA
tTG
anti-gliadin antibodies
arbitrary unit
bovine serum albumin
coeliac disease
deamidated gliadin
Escherichia coli
ethylenediaminetetraacetic acid
enzyme linked immunosorbent assay
fluorescence-activated cell sorting
fluorescein isothiocyanate isomer 1
gliadin antibodies
gluten-free diet
guinea pig tissue transglutaminase
tissue transglutaminase antibodies (guinea pig)
gluten-rich diet
human leukocyte antigen
horseradish peroxidase
intraepithelial lymphocytes
immunofluorescence test
immunoglobulin
inflammatory bowel disease
inflammatory small bowel disease
Dutch Warmblood horse
lymphocytic enteritis
lymphocytic-plasmacytic enteritis
major histocompatibility complex
not determined
o-phenylenediamine dihydrochloride
recombinant human tissue transglutaminase
tissue transglutaminase antibodies (recombinant human)
tissue transglutaminase antibodies
tissue transglutaminase
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THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
ANNEX 1
Overview of the research group, including patients (ISBD), controls (gluten-rich, not known to
have ISBD) and wild (gluten-free) horses.
group
ID No
breed
age
sex
sampling
patient
1 KWPN
18
gelding
160210
patient
2 KWPN
5
gelding
040210
patient
3 KWPN
7
mare
020210
patient
4 Frisian
11
mare
160210
patient
5 KWPN
8
gelding
040210
patient
6 KWPN
9
mare
180610
patient
7 KWPN
14
stallion
080610
patient
8 KWPN
11
gelding
060610
patient
9 KWPN
14
mare
150410
patient
10 KWPN
14
gelding
200810
patient
11 KWPN
9
gelding
101210
patient
12 KWPN
10
mare
060111
Patient* GFD
7* KWPN*
15
Stallion
060111
*All patients were referred to the Department of Equine Sciences, Section of Internal Medicine, Utrecht
University in the years 2010 and 2011.
*GFD= gluten-free diet
group
ID No
breed
age
sex
sampling
control
21 KWPN
11
mare
050310
control
22 KWPN
15
mare
050310
control
23 KWPN
7
mare
050310
control
24 KWPN
14
mare
050310
control
25 KWPN
12
mare
050310
control
26 KWPN
6
mare
050310
control
27 KWPN
8
mare
050310
control
28 KWPN
8
mare
050310
control
29 KWPN
11
mare
050310
control
30 KWPN
8
mare
050310
control
31 KWPN
18
mare
050111
control
32 KWPN
16
mare
050111
control
33 KWPN
12
mare
050111
control
34 KWPN
6
mare
050111
control
35 KWPN
4
mare
050111
control
36 KWPN
18
mare
050111
control
37 KWPN
*
mare
050111
control
38 KWPN
4
mare
050111
control
39 standardbred
*
gelding
050111
control
40 KWPN
11
mare
050111
control
41 shetland pony
12
gelding
050111
control
42 shetland pony
17
gelding
050111
* All gluten-rich controls are housed at the Department of Equine Sciences, Utrecht University for
educational and research purposes.
group
control
ID No
43
breed
KWPN
age
2
sex
stallion
sampling
200410
35 | P a g e
36
THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
group
ID No
breed
age
sex
sampling
wild
51
shetland pony
*
mare
061010
wild
52
shetland pony
*
mare
061010
wild
53
shetland pony
*
mare
061010
wild
54
shetland pony
*
mare
061010
wild
55
shetland pony
*
mare
061010
wild
56
shetland pony
*
mare
061010
wild
57
shetland pony
*
mare
061010
wild
58
shetland pony
*
mare
061010
wild
59
shetland pony
*
mare
061010
wild
60
shetland pony
*
mare
061010
wild
61
shetland pony
*
mare
061010
wild
62
shetland pony
*
mare
061010
wild
63
shetland pony
*
mare
061010
wild
64
shetland pony
*
mare
061010
wild
65
shetland pony
*
mare
061010
wild
66
shetland pony
*
mare
261010
wild
67
shetland pony
*
mare
261010
wild
68
shetland pony
*
mare
261010
wild
69
shetland pony
*
mare
261010
wild
70
shetland pony
*
mare
261010
wild
71
shetland pony
*
mare
261010
wild
72
shetland pony
*
mare
261010
wild
73
shetland pony
*
mare
261010
wild
74
shetland pony
*
mare
261010
wild
75 shetland pony
*
mare
261010
* All “ wild” Shetland ponies are housed in a reserve in Zeeland (in the southwest of the Netherlands) and
belong to Nature Reserves. Their diet consist of grasses and herbs and is considered to be gluten-free.
Other materials used:
A duodenal section in formalin, from control No. 43. Used for comparative histology.
2 x 1ml peripheral venous blood, collected in lithium-heparin tubes (sampling 27-10-’10),
from controls No. 24 & 27. The blood was used for trial FACS analysis.
2x 50ml peripheral venous blood, collected in lithium-heparin tubes (sampling 20-01’11), from controls No. 24 & 27. The blood was used for HLA-DQ2/DQ8 typing.
2x 50ml peripheral venous blood, collected in lithium-heparin tubes (sampling 20-01’11), from patients No. 7* & 11 The blood was used for HLA-DQ2/DQ8 typing.
36 | P a g e
37
THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
ANNEX 2
Results ELISA’s in AU/ml.
Group
1
1
1
1
1
1
1
1
1
1
1
1
4
Group
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
Group
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
ID No.
1
2
3
4
5
6
7
8
9
10
11
12
7*GFD
rhTGA
(AU/ml) 33.9
10.1
5.4
6.8
5.3
8.6
100.0
27.7
14.0
31.6
6.8
16.3
32.5
GA (AU/ml)
34.0
19.8
15.3
26.8
10.5
8.8
61.2
28.0
15.3
46.3
20.7
44.7
53.1
e.coli
(AU/ml)121.8
73.7
53.9
101.7
46.6
33.1
125.9
92.2
36.2
100
106.7
95.5
125.4
e.coli
+/++/+++ +++
++
+
+++
+
+
+++
++
+
+++
+++
++
+++
gpTGA(AU/ml)
42.1
12.5
14.1
21.0
12.3
20.1
100.0
62.8
11.0
30.8
23.4
23.8
95.1
DGP(AU/ml)
12.6
1.3
2.6
2.0
9.2
1.0
25.0
11.9
2.1
11.7
8.0
3.2
ND
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
9.2
21.8
11.4
20.1
6.3
28.0
15.3
21.5
14.0
35.5
5.4
18.9
7.6
3.9
0.03
9.6
7.3
6.9
4.7
6.2
4.9
0.03
30.2
51.8
9.3
53.3
17.5
22.0
17.2
100
22.9
17.9
14.8
135.5
34.3
9.2
13.6
55.8
14.0
29.3
14.1
15.1
22.3
7.4
54.1
92.5
85.4
106.5
96.6
111.2
39.8
41.8
118.4
50.0
72.85
138.9
88.6
41.8
25.0
105.7
65.7
97.6
39.05
90.8
42.1
64.9
+
++
++
+++
++
+++
+
+
+++
+
++
+++
++
+
+
+++
++
++
+
++
+
++
13.6
25.7
13.1
32.5
9.5
77.0
8.2
13.2
13.4
17.0
15.8
74.0
36.9
17.4
9.2
21.1
20.9
20.8
14.7
12.4
15.2
11.1
9.3
88.1
2.1
>100
1.0
1.3
0.8
1.5
1.6
1.4
2.6
10.6
17.1
ND
ND
2.5
3.1
2.7
1.5
4.2
3.4
ND
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
11.5
3.9
0.03
8.4
5.4
0.03
0.03
0.03
3.2
0.03
0.03
0.03
0.03
0.03
0.03
17.2
4.5
4.3
0.03
7.3
10.2
5.3
5.1
6.2
3.7
24.5
24.1
28.8
89.1
21.5
9.9
9.7
24.6
10.1
17.8
10.1
56.4
11.0
28.1
10.2
63.0
29.4
20.4
28.0
26.5
20.0
26.3
24.5
39.3
15.3
109.4
106.9
96.1
117.2
94.8
13.3
9.0
10.6
21.4
9.7
24.1
12.1
14.0
14.4
5.4
90.2
27.0
22.4
9.5
31.4
59.2
34.8
19.2
35.3
26.4
+++
+++
++
+++
++
+
+
+
+
+
+
+
+
+
+
++
+
+
+
+
+
+
+
+
+
22.3
20.2
9.0
21.9
22.5
21.5
33.5
8.6
15.8
8.2
ND
34.1
16.3
7.8
17.2
40.1
14.4
18.6
31.5
37.8
26.7
9.5
17.5
35.8
9.7
2.7
3.4
1.9
1.8
1.9
0.9
3.9
0.7
>100
1.4
0.6
2
0.8
3.3
0.8
10.8
1.5
1.9
1.2
ND
ND
ND
ND
ND
ND
37 | P a g e
38
THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
ANNEX 3
Correlation statistics using Medcalc.
Scatter diagram showing the correlation between the rh-tTGA and the E.coli antibody test.
100
80
60
[
E.coli
40
20
r= 0.4560
P= 0.0003
0
0
20
40
60
[rh-TGA] in AU/ml
80
100
Scatter diagram showing the correlation between the rh-tTGA and the gp-tTGA test.
100
80
60
40
20
r= 0.7005
P<0.0001
0
0
20
40
60
[rh-TGA] in AU/ml
80
100
38 | P a g e
39
THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
Scatter diagram showing the correlation between the rh-tTGA and the GA test.
140
r=0.3822
P=0.0026
120
[GA] in AU/ml
100
80
60
40
20
0
0
20
40
60
[rh-TGA] in AU/ml
80
100
Scatter diagram showing the correlation between the rh-tTGA and the DGP antibody test.
100
r=0.1970
P=0.1704
80
60
40
20
0
0
20
40
60
[rh-TGA] in AU/ml
80
100
39 | P a g e
40
THE POTENTIAL ROLE OF GLUTEN IN EQUINE INFLAMMATORY SMALL BOWEL DISEASE
Scatter diagram showing the correlation between the GA and the DGP antibody test.
100
r=0.1450
P=0.3150
80
60
40
20
0
0
20
40
60
80
[GA] in AU/ml
100
120
140
ANNEX 4
The gluten-free diet of “case study” and patient No. 7.
hour of the day
08.00
sort of feed
Supplements
GF- Concentrate
Roughage
11.30
GF-Concentrate
14.00
16.30
GF-Concentrate
22.00
GF-Concentrate
Roughage
kilograms
30 grams
50 grams
340 grams
960 grams (480 in
rest)
490 grams
3 kilograms
340 grams
960 grams
490 grams
1 kilogram
340 grams
960 grams (480 in
rest)
490 grams
250 grams
3 kilograms
Type of feed
Equistro megabase
Equitop myoplast
Hartog Lucerne
Black crushed oats
Cavalor Strucomix
Hartog compact
grasses
Hartog Lucerne
Black crushed oats
Cavalor Strucomix
Carrots
Hartog Lucerne
Black crushed oats
Cavalor Strucomix
Cavalor Strucomix
Hartog compact
grasses
40 | P a g e