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Gluten-dependent antibodies in horses withinflammatory small bowel disease (ISBD)J.H. van der Kolk a , L.A. van Putten a , C.J. Mulder b , G.C.M. Grinwis c , M. Reijm d ,C.M. Butler e & B.M.E. von Blomberg da Department of Equine Sciences, Medicine Section, Faculty of Veterinary Medicine ,University of Utrecht , Utrecht , the Netherlandsb Department of Gastroenterology and Hepatology , Free University of Amsterdam ,Amsterdam , the Netherlandsc Faculty of Veterinary Medicine, Department of Pathobiology , University of Utrecht ,Utrecht , the Netherlandsd Department of Pathology , Medical Immunology Section, Free University ofAmsterdam , Amsterdam , the Netherlandse Department of Large Animal Medicine and Surgery , St. George's University , Grenada ,West IndiesPublished online: 10 Apr 2012.

To cite this article: J.H. van der Kolk , L.A. van Putten , C.J. Mulder , G.C.M. Grinwis , M. Reijm , C.M. Butler & B.M.E.von Blomberg (2012) Gluten-dependent antibodies in horses with inflammatory small bowel disease (ISBD), VeterinaryQuarterly, 32:1, 3-11, DOI: 10.1080/01652176.2012.675636

To link to this article: http://dx.doi.org/10.1080/01652176.2012.675636

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Veterinary QuarterlyVol. 32, No. 1, March 2012, 3–11

Gluten-dependent antibodies in horses with inflammatory small bowel disease (ISBD)

J.H. van der Kolka*, L.A. van Puttena, C.J. Mulderb, G.C.M. Grinwisc, M. Reijmd, C.M. Butlere

and B.M.E. von Blombergd

aDepartment of Equine Sciences, Medicine Section, Faculty of Veterinary Medicine, University of Utrecht, Utrecht, theNetherlands; bDepartment of Gastroenterology and Hepatology, Free University of Amsterdam, Amsterdam, the Netherlands;

cFaculty of Veterinary Medicine, Department of Pathobiology, University of Utrecht, Utrecht, the Netherlands;dDepartment of Pathology, Medical Immunology Section, Free University of Amsterdam, Amsterdam, the Netherlands;

eDepartment of Large Animal Medicine and Surgery, St. George’s University, Grenada, West Indies

(Received 9 March 2012; final version received 11 March 2012)

Background: Equine inflammatory small bowel disease (ISBD) is an idiopathic pathologic condition seeming toincrease in prevalence.Objective: To investigate the potential role of gluten in equine ISBD.Animals & Methods: Antibodies known to be important in the diagnosis of human coeliac disease (CD): IgAantibodies to human recombinant and guinea pig tissue-transglutaminase (TGA), native gliadin (AGA),deamidated-gliadin-peptides (DGPA), and primate and equine endomysium (EMA) were assessed in bloodsamples from three different groups of horses: ISBD affected (n¼ 12) on a gluten-rich diet and controls either ongluten-rich (n¼ 22) or gluten-poor (n¼ 25) diets. Significant differences ( p< 0.05) between groups were assessedusing the Wilcoxon test.Results: Both ISBD-affected horses and gluten-rich controls had significantly ( p< 0.0004) higher hrTGA titersthan gluten-poor controls. However, ISBD horses did not show significantly increased levels of any of the CDrelated antibodies when compared to gluten-rich controls. Nevertheless, markedly increased antibody levels(TGA, EMA and DGPA) were found in one of the ISBD horses. The introduction of a gluten-free ration in this14-year-old warmblood stallion resulted after 6 months in the reduction of antibody levels and clinical recoveryassociated with improved duodenal histopathology.Conclusion: To the best of our knowledge, this is the first study assessing gluten-related antibodies in horses andresults suggest a potential pathogenic role of gluten in at least some cases of equine ISBD.Clinical importance and impact for human medicine: Given serology and concurrent clinical findings, this studywarrants further investigations into the immunologic basis of possible gluten-sensitive enteropathy in horses andanalogy with human disease.

Keywords: equine; horse; transglutaminase; TGA; TG2; gluten; inflammatory small bowel disease; ISBD;gliadin; endomysium; IgA; E. coli; coeliac disease

1. Introduction

The intestinal immune system is constantly exposed toa vast array of antigens, including those derived from

food, components of the endogenous microbial flora,and pathogenic organisms. Important decisions must

be made about the nature of the antigenic stimulus sothat protective responses are mounted to pathogens

but tolerance to harmless substances is preserved. Ifthis delicate balance is interrupted, a state of chronicuncontrolled inflammation may ensue (German et al.

2003). A syndrome, marked by chronic weight loss andhypoalbuminaemia associated with chronic gastroin-

testinal disorders is well known in Standardbreds withtwo different pathomorphological entities, namely

eosinophilic granulomatosis and granulomatous enter-itis (Lindberg et al. 1985). Lymphocytic–plasmacyticenteritis also belongs to this complex of equine

idiopathic inflammatory small bowel disease (ISBD).Clinical signs include weight loss, recurrent colic, soft

feces, depression, oedema, and a dull hair coat(Schumacher et al. 2000). Lymphocytic–plasmacytic

enteritis is regarded as an uncommon equine intestinal

disease that is difficult to recognize and diagnose ante

mortem and has a poor prognosis (Kemper et al. 2000).

We noticed an increased prevalence of ISBD in equine

elite athletes over the last decade in our veterinary

teaching hospital with more than 70% of these horses

used for dressage (Dansen et al. 2010). By definition,

the exact cause of equine ISBD is unknown urging the

need for studying diet and/or immune function in these

equine elite athletes.Coeliac disease (CD) is an auto-immune inflam-

matory disorder in humans characterized by a partial

or total villous atrophy of the proximal small intestine

occurring after ingestion of gluten in genetically

predisposed patients (Working Group Amsterdam

2001; Tack et al. 2010). The classic form is much

more frequent in children (Tack et al. 2010). The

common factor for all patients with CD is the presence

of a variable combination of gluten-dependent clinical

manifestations, specific auto-antibodies directed to

transglutaminase 2 (TGA) and endomysium (EMA),

*Corresponding author. Email: j.h.vanderKolk@uu.nl

ISSN 0165–2176 print/ISSN 1875–5941 online

� 2012 Taylor & Francis

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HLA-DQ2 and/or DQ8 genes, and different degrees ofenteropathy. Recently, the so-called gluten sensitivity(GS) has received much interest in human medicine,although the limits and possible overlap between GSand CD remain poorly defined (Troncone and Jabri2011). However, in GS antibodies to native gliadin(AGA) (the alcohol-soluble part of gluten) are morepronounced (Volta et al. 2011). For CD diagnostics inchildren, TGA with a sensitivity of almost 100% is nowrecommended as test of preference, while EMA andantibodies against deamidated gliadin can be used forconfirmation or in doubtful cases (Husby et al. 2012).When tissue transglutaminase transforms gliadin byconverting glutamine into glutamic acid, the remaininggliadin is termed ‘‘deamidated’’ gliadin. It should berealized that the clinical demand for both maximalsensitivity and maximal specificity cannot be achievedwith a single test (Rozenberg et al. 2011). Also, in adulthuman patients with abdominal symptoms in primarycare or other unselected populations, IgA TGA and IgAEMA have high sensitivity and specificity for diagnos-ing CD (van der Windt et al. 2010).

The objective of this report was to determine theserological status of elite horses admitted to an equineacademic hospital evaluated for idiopathic chronicallyincreased thickness of the wall of the jejunum regard-ing antibodies known to be important in the diagnosisof human CD.

2. Materials and methods

2.1. Animals

Twelve horses with signs of ISBD referred to theEquine Clinic of Utrecht University were included inthe study. The history was taken, clinical examinationwas performed and peripheral venous blood wascollected for routine haematological and biochemicalexamination as well as further assessment of specificimmunological parameters. The (tentative) diagnosisof ISBD was based on chronic thickening of the wall ofthe jejunum as indicated by the repeated ability topalpate various (non-contractile) loops of the jejunumduring rectal exploration and/or assessment ofincreased thickness of the jejunal wall (above 3.0mm)by transabdominal ultrasound examination. The upperlimit of 3.0mm was based on the statement that theduodenum and jejunum have thin walls that rarelyexceed 3mm in thickness (Reef 1998). Horses sufferingfrom acute (less than 2 weeks duration) diseases andhorses showing diarrhea were excluded from the studyas well as horses with abnormal lymphocyte morphol-ogy in peripheral blood. Horses with a strongyle eggcount above 200 per gram feces were also excludedfrom the study. All affected horses were warmbloodsexcept for one Friesian horse. They performedat the M-level of dressage or above (up to Prix StGeorge/Intermediaire I level and Olympics), as gov-erned by the Federation Equestre Internationale (FEI).The affected horses comprised five mares, six geldings

and one stallion ranging in age from 5–18 years(mean 10.8� 3.5 (SD)). All blood samples were takenbetween February 2010 and January 2011.

For comparison, 22 (gluten-rich) control horsesbelonging to the teaching herd of Utrecht Universitywere used and were fed grass silage ad libitum andconcentrates according to their nutrient requirementsfor maintenance and performance. The total diet of thecontrol horses contained 10% ash, 14.5% crudeprotein, 1.3% crude fat, 20% crude fiber, and 56.2%other carbohydrates. On the average, individualgluten-rich control horses were fed 2 kg of concentratesdaily. The cereals used in the concentrates werespecified by the manufacturer as 25% wheat, 15 %wheat gluten feed, 15% wheat middlings, and 15%maize gluten feed. Water was provided ad libitum. Themean age of the gluten-rich control horses was10.5� 4.3 years ranging from 8 to 16 years and thehorses comprised 19 Dutch warmbloods (weighing633� 60 kg), 2 Shetland ponies, and one Standardbred.The gluten-rich control horses included 21 mares andone gelding. All samples of the gluten-rich controlhorses were taken on 5 March 2010. Furthermore, agroup of 25 Shetland ponies living year-round in anature reserve in the Dutch province of Zeeland, theNetherlands, was used as a gluten-poor control group.They were kept in a gluten-poor (if not gluten-free)environment since their diet consisted exclusively ofwild, non-agricultural grasses, and herbs. All 25 gluten-poor controls were mares ranging in age from 1 to 27years (mean age unknown). All blood samples of thegluten-poor controls were taken on 6 October 2010.

The blood sampling from the gluten-rich controlhorses was approved by the Institutional Animal Careand Use Committee of the University of Utrecht. Thesamples from the other horses were taken on veterinaryindication and approved by the owner.

2.2. Blood analysis

2.2.1. IgA antibodies against Escherichia coli

As CD serology is generally based on IgA antibodies,all horses were tested for the presence of IgA using anE. coli antibody ELISA in order to rule out IgAdeficiency in individual cases. To this end, 96-wellsmicrotiter plates (flat-bottom 96 well plate, NuncMaxisorp�, Bioscience Inc., San Diego, CA, USA)were coated with E. coli strain EB1 and blocked with1% bovine serum albumin (BSA) in PBS/Tween0.05%. Sera were preincubated with the blockingreagent (45min. at room temperature) and tested in a1:20 dilution (120min. at room temperature). Afterwashing, the plates were incubated with goat anti-horse IgA:HRP (AbD Serotec, 1:5000 diluted, 60min.at room temperature) and attached antibodies weredetected by using orthophenylenediamine dihy-drochloride (OPD) as substrate in a routine procedure.After 15 min, the reaction was terminated using H2SO4

and evaluated spectrophotometrically at 492 nm.

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Serum obtained from equine patient number 10 wasused in all experiments as calibration curve andstandard (by definition containing 100 arbitrary units(AU) of E. coli antibodies/mL).

2.2.2. IgA antibodies against human recombinant andguinea pig transglutaminase 2

TGA concentration was determined quantitatively inserum based on binding to either human recombinanttransglutaminase 2 (hrTG2) or purified guinea pigtransglutaminase 2 (gpTG2). 96-Well microtiter plateswere coated with hrTG2 (1mg/well in 0.05M TRIS-HCL pH to 7.5, Diarect, Freiburg, Germany), orgpTG2 (1 mg/well in 0.05M TRIS-HCL pH to 7.5,Sigma, St. Louis, MO, USA) according to standardoperation procedures. After washing, open bindingsites were blocked with 1% BSA and Tween-20 0.05%.The serum was diluted ranging from 1:20, 1:40, 1:80 till1:160 in phosphate buffered saline (PBS) with 1% BSAand 0.05% Tween-20 and pre-incubated at roomtemperature for 30–60min to allow potential anti-BSA to bind. The detection of TGA was performed asdescribed for E. coli antibodies. Serum obtained fromequine patient number 7, being strongly positive, wasused as calibration curve and standard (by definitioncontaining 100 AU of transglutaminase 2 (TG2)/mL).

2.2.3. IgA antibodies against native gliadin

Gliadin antibodies were tested by ELISA. 96-wellsmicrotiter plates were coated with native gliadin(50 mg/well in 0.05M Na-carbonate buffer, pH to 9.6;Sigma, St. Louis, MO, USA) without blocking. Theserum was diluted ranging from 1:20, 1:40, 1:80 till1:160. Bound antibodies were made visible as describedabove. Serum obtained from equine control animalnumber 28, being strongly positive, was used ascalibration curve and standard (by definition contain-ing 100 AU of AGA/mL).

2.2.4. IgA antibodies against deamidated gliadin

When tissue transglutaminase transforms gliadin byconverting glutamine into glutamic acid, the remaininggliadin is termed ‘‘deamidated’’ gliadin. A commercialELISA under development, kindly provided by Euro-Diagnostica AB (Nijmegen, the Netherlands) based ona set of deamidated gliadin peptides (DGPA) as target,was used for testing the presence of antibodies inequine serum directed against these DGPA. The‘‘human’’ ELISA was adapted for measuring specifichorse IgA, as described above. A 1:50 serum dilutionwas used. Unfortunately, the sample size of the kit waslimited, so not all horses could be tested. Gluten-richcontrols 34, 35, and 42 and gluten-poor controls 70–75were not tested. Serum obtained from equine patientnumber 7, being strongly positive, was used ascalibration curve and standard (by definition contain-ing 100 AU of DGPA/mL).

2.2.5. IgA antibodies against endomysium

Endomysium (EMA) were evaluated in an indirectimmunofluorescence analysis (IIFT) using unfixedcryostat sections of monkey (Macaca mulatta) andequine (obtained from a 1-day-old warmblood coltimmediately following euthanasia) esophagus as sub-strate. Esophagus is used because the muscularismucosae of the esophagus has a relatively high contentof TG2. Serum was tested in a 1:2 dilution and PBSwas used as a negative control. After washing, incu-bation was followed with goat anti-horse IgA:FITC-conjugate (AbD Serotec. 1:40). After washing, theglasses were covered, viewed under a fluorescencemicroscope, and scored for staining pattern andfluorescence intensity of the muscularis mucosae byat least two independent observers such as fluorescenceintensity (�, þ/�, þ, þþ or þþþ) of the muscularismucosae by at least two independent observers.

2.3. Statistical analysis

All results were analyzed using specialized software(MedCalc Software, Mariakerke, Belgium and SPPSSoftware, version 16.0, IBM Corporation, New York,USA). Differences between groups were statisticallycompared by means of the Wilcoxon test. p values<0.05 were considered significant.

3. Results

Clinical signs of horses with ISBD included intermittentcolic (n¼ 6), weight loss (n¼ 4), and poor performance(n¼ 2). In 7 out of 12 cases, an oral glucose tolerancetest (according to Benders et al. 2005) was performedwith poor glucose absorption shown in only 2 cases. Inaddition, in 6 out of 12 cases gastro-duodenoscopy-assisted biopsies taken from the proximal duodenumwere available for histopathology revealing predomi-nantly very mild lymphocytic–plasmacytic enteritis.Routine blood analysis in these horses revealednormal mean values for total lymphocyte count(1.8� 1.2 ranging from 1.1 to 5.3G/L), total proteinconcentration (64� 7.0 ranging from 51 to 73 g/L), andalbumin concentration (35� 2.2 ranging from 31 to38 g/L), whereas the mean hematocrit was slightlydecreased (0.35� 0.034 ranging from 0.31 to 0.41L/L)also taken into consideration the fact that the horsesinvolved were elite horses. All horses included in thisstudy tested positive for the presence of IgA antibodiesdirected against E. coli in serum. Remarkably, IgAantibody titers were significantly lower in gluten-poorcontrol horses than in gluten-rich controls ( p¼ 0.0004)or ISBD-affected horses ( p¼ 0.0015).

As shown in Figure 1(a) serum antibody levelsdirected against hrTG2 were also significantly lower ingluten-poor controls as compared to both gluten-richcontrols and ISBD-affected patients ( p¼ 0.0004and p¼ 0.0001, respectively). However, no significantdifferences were found in TGA levels between

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ISBD-affected horses and gluten-rich controls.Remarkably, one ISBD patient (number 7) showed anextremely high hrTGA level. Such high titers were notseen in either control groups. The three groups of horsesdid not differ significantly with respect to serum IgAantibodies directed against gpTG2 (Figure 1b). AgainISBD patient number 7 showed a relatively highgpTGA concentration, but also two gluten-richcontrols had intermediate high levels.

Similarly, differences between groups were notstatistically significant regarding IgA antibodies direc-ted against native gliadin (Figure 1c) and DGPA(Figure 1d), despite incidental clear cut positive horsesoccurring in all groups.

Remarkably, control horse 32 (a 16-year-oldwarmblood mare) showed elevated serum IgA anti-bodies directed against DGPA as well as directedagainst equine oesophageal endomysium (EMA).Blood analysis in this mare revealed serum totalprotein concentrations of 72 g/L (with 37 g/L albumin)on 9 June 2004, 79 g/L (with 44 g/L albumin) on14 February 2005, 60 g/L on 17 February 2010, and63 g/L (with 34 g/L albumin) on 20 December 2010. Aprompted oral glucose tolerance test revealed anincrease of 6% only (normal range 44–174%).

EMA (Figure 2a–h), with the classical reticularstaining pattern of the muscularis mucosae (Figure 2c)were not detectable when using monkey esophagealtissue as substrate (Figure 2a–c). However, the smoothmuscle cells stained positive for some horses, includingpatient number 7. When evaluating the IIFT on equineesophageal tissue, the muscularis mucosae as well asthe arterial walls in the mucosa stained quite positivefor a number of horse sera (Figure 2d). The reticularstaining pattern was however not very clear, since thesmooth muscle cells also stained positive. This was,however, also the case for the human coeliac serum(Figure 2f). Based on the equine esophagus IIFT,EMA (i.e. positive staining of the muscularis mucosae)in ISBD patients tended to be higher than in thegluten-rich control group, but this did not reachsignificance ( p¼ 0.09; Figure 2h).

3.1. Clinical course of patient number 7

A 14-year-old warmblood stallion weighing 589 kg wasadmitted because of poor performance and based onfindings during repeated rectal palpation was includedin the study as patient number 7. Upon admission,

Figure 1. CD-related IgA antibodies in equine ISBD patients and controls.Multiple comparison (box and dot) graph of serum IgA antibodies directed against hrTG2 (Figure 1a), gpTG2 (Figure 1b),native gliadin (Figure 1c), and deamidated gliadin (Figure 1d) in 12 horses with inflammatory small bowel disease (ISBD glut þ),22 gluten-rich controls (CON glut þ), and 25 gluten-poor controls (CON glut�). In the case of DGPA, 19 gluten-rich controlsand 19 gluten-poor controls were tested. IgA antibodies are calibrated with a strong positive serum and expressed as arbitraryunits (AU)/ml. The boxes represent the interquartile range (i.e., 25–75% range) and the horizontal bar in the box represents themedian value. For each box plot, the T-bar extends to 1.5 times the interquartile range, if without a value in that range, to theminimum or maximum values. Statistical analysis was performed by the Wilcoxon test and P values are given in the figures.

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serum total protein measured 66 g/L with 35 g/Lalbumin (see Figure 3 for additional blood valuesand its change over time). An oral glucose tolerancetest performed upon admission indicated moderateglucose absorption (42% increase). Based on its highantibody concentrations (EMA, TGA, DGPA), agluten-free ration was initiated to which the owneradhered strictly. The gluten-free ration was predomi-nantly based on haylage, alfalfa, and black crushedoats containing 126MJ digestable energy and 10.2 kgdry matter. Following feeding of the gluten-free ration,performance was normalized (return to FEIIntermediaire I level). In contrast to the irrelevantE. coli antibodies, which remained strongly positive, allCD-associated antibodies (TGA, EMA, and DGPA)decreased following the gluten-free period of 6 months,though these did not yet normalize completely(Figure 3). In addition, histopathology suggestedincrease in duodenal villous length (Figure 4).

4. Discussion

Serological assessment of antibodies known to beimportant in the diagnosis of human CD disease in

elite horses, evaluated for idiopathic chronicallyincreased thickness of the wall of the jejunum, revealedthat serum IgA antibodies directed against hrTG2 weresignificantly increased in ISBD-affected horses ascompared to gluten-poor controls, but not as com-pared to control horses on a gluten-rich diet. No IgAdeficiency, as based on IgA levels against E. coli, couldbe demonstrated in any of the horses. Regarding AGAand DGPA levels, no statistically significant differ-ences were observed between the groups, thoughincidental clear cut positive horses were seen in allgroups. The classical reticular staining pattern ofhuman EMA on monkey esophageal tissue was notfound in any of the horses. However, on equineesophageal tissue, bright staining of the muscularismucosae and the arterial walls were seen in some of thehorses. Since unequivocal discrimination betweenstaining of the smooth muscle fibers and the endomy-sium was not possible on equine esophagus, theseantibodies were considered to represent EMA in thepresent report. On the whole, EMA in ISBD patientstended to be higher than in the gluten-rich controlswith some individual horses showing strong positiveresponses.

Figure 2. IIFTs for endomysium antibodies (EMA) IgA.EMA was tested on primate (a–c) and equine esophagus (d–h). Cryosections were incubated with serum from equine ISBDpatient 7 (a, d), from a gluten-rich control horse (patient 5; b and e), from a human active CD patient (c and f) and with PBS (g).(h) shows the multiple comparison (box and dot) graph of EMA IgA antibodies directed against equine oesophageal muscularismucosae in 12 horses with inflammatory small bowel disease (ISBD glut þ) and 22 gluten-rich controls (CON glut þ). Antibodyconcentration is reflected by the fluorescence intensity of the muscularis mucosae on a scale from 0–3. The box represents theinterquartile range (i.e., 25–75% range) and the horizontal bar in the box represents the median value. For each box plot, theT-bar extends to 1.5 times the interquartile range, if without a value in that range, to the minimum or maximum values. Notethe high values in patients 7 and 10 as well as in control horse 32 (a 16-year-old warmblood mare). Statistical analysis wasperformed by the Wilcoxon test and p values are given in the figure.

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No significant differences between the ISBD group

and the control horses on a comparable gluten

containing ration were found for any of the CD-

associated antibodies. Remarkably, one ISBD horse

(number 7) showed extremely high IgA levels for all

antibodies known to be important in the diagnosis of

human CD, namely TGA, DGPA as well as EMA. To

evaluate how far these antibodies were related to

gluten consumption, this patient was followed up

during a strict gluten-free diet. The high hrTGA,

DGPA, and EMA levels decreased following a gluten-free ration for 6 months in this 14-year-old warmbloodstallion suffering from ISBD with concurrent increaseof duodenal villous length as visualized histopatholog-ically. Although the majority of oral glucose absorp-tion tests in ISBD horses revealed normal absorption,both the mean hematocrit and serum protein concen-tration were relatively low given the fact that elitehorses were involved. In comparison, poor glucoseabsorption might also be demonstrated in human CD(van Elburg et al. 1995) although results are variable asshown in the current report in horses.

As the ELISA’s used in this study are based on IgAantibodies, all horses were tested for the presence ofIgA using an E. coli antibody ELISA. Although IgAdeficiency has been reported in non-Arabian horses asa very rare disorder (Deem et al. 1979; Freestone et al.1987; MacLeay et al. 1997), all horses included in thisstudy tested positive for the presence of IgA antibodiesdirected against E. coli in serum. As a consequence,IgA deficiency as a confounding factor regardingserology in the current study can be ruled out.Remarkably, mean IgA antibody titer was significantlylower in gluten-poor control horses than in bothgluten-rich and ISBD-affected horses suggesting lowerenvironmental contamination in the nature reserve byE. coli and/or reduced serological response. To theauthors’ best knowledge, no data are available what-soever regarding the relationship between environmen-tal contamination by E. coli and serology in horses.

It should be realized that some of the serologicaltests used were based on human assays modified foruse in horses. Although cross-reactivity between spe-cies are obvious in this study (human coeliac antibodiesreacting to monkey and guinea pig TG2 and equineantibodies reacting to human and guinea pig TG2),species-specific antigen differences might have influ-enced the outcome. Also, the partial reduction inantibodies, together with the improvement in clinicalcourse and blood biochemistry as well as the results ofproximal duodenal histopathology following the initi-ation of a gluten-free ration in equine patient number 7support assay specificity. As a consequence, our

Figure 4. Histopathology of gastro-duodenoscopy-assisted biopsies taken from the proximal duodenum in patient 7 (a 14-year-old warmblood stallion) prior to (left) and following 6 months of a gluten-free diet (right). Arrow depicts a shortened villous inthe initial duodenal biopsy. Note the marked increase in villous length in the control biopsy taken after 6 months of gluten-freefeeding. H&E stain.

Figure 3. Follow up during gluten-free diet in an equinepatient (#7) with ISBD.Change over time in titers of IgA antibodies to E. coli, hrTG2(¼hrTGA), gpTG2 (¼gpTGA), native gliadin (AGA),deamidated-gliadin-peptides (DGPA), and endomysium(EMA) in a 14-year-old warmblood stallion with ISBDprior to and 6 months following a gluten-free diet (GFD)mainly based on haylage, alfalfa, and black crushed oats.

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findings suggest the presence of Gluten-SensitiveEnteropathy (GSE) in at least some horses sufferingfrom ISBD. In addition, control horse 32 (a 16-year-old warmblood mare) retrospectively not only showeda decrease in serum total protein over time, but alsohad elevated DGPA and equine EMA titers. Aprompted oral glucose tolerance test revealed a veryinsufficient glucose absorption (6%), thereby suggest-ing subclinical GSE. Of interest, almost by traditionhorses in some geographic regions were fed oats grainin former times known as a gluten-free cereal. As ourunderstanding of ISBD is far from complete, it isworthwhile to address its potential relationshipwith GSE.

To the authors’ knowledge, GSE has not beenreported in the equine species yet. However, primaryphotosensitization observed in three Appaloosa maresassociated with gluten ingestion has been reported(Yeruham et al. 1999). Dermatitis herpetiformis (DH)is a pruritic papulovesicular skin disorder of unknowncause, characterized by granular IgA deposits in thedermis along the dermoepidermal junction. It isassociated with GSE and increased levels of TGA inman (Karpati 2012). We suggest a DH-like disease dueto GSE in these Appaloosa horses manifested asprimary photosensitization.

As mentioned before, an increased prevalence ofISBD in equine elite athletes over the last decade hasbeen noticed in our equine academic hospital withmore than 70% of these horses being used for dressage(Dansen et al. 2010). Given the fact that the intestinalimmune system is constantly exposed to a vast array ofantigens, including those derived from food (Germanet al. 2003) raises the question about possible alter-ations in the gluten content of equine diets over time.The high prevalence of dressage horses affected withISBD might be explained by differences in feedingmanagement as the equine species responds to differ-ences in the quality of dietary protein (Reitnour andSalsbury 1976). On the other hand, competitioninduced a significant increase in cortisol and ACTHresponses in both jumping and dressage horses beingmost pronounced in the latter (Cayado et al. 2006),thereby possibly negatively influencing the intestinalimmune system.

Based on the current study, IgA antibodies againsthrTG2, DGP, and equine endomysium in horsessuffering from weight loss and/or recurrent colic withchronic increased thickness of the small bowel wall arethe most likely candidates to support the tentativediagnosis of equine GSE besides assessment of theextent of the underlying enteropathy by means ofhistopathology. Last but not the least, clinical remis-sion on a strict gluten-free diet is of importance. Withreference to future research, it is necessary to explorethe potential involvement of HLA-DQ2 and/or DQ8(like) histocompatibility complex genes in horses with atentative diagnosis of GSE.

Villous atrophy of the intestinal mucosa, in com-bination with increased numbers of intraepithelial

lymphocytes and crypt hyperplasia has long beenconsidered the hallmark of CD (Tack et al. 2010). Inequine elite athletes such histopathology is bestperformed based on gastro-duodenoscopy-assistedbiopsies taken from the proximal duodenum.However, sample size is small in the case of gastro-duodenoscopy-assisted biopsies thereby preventingoptimal histopathological evaluation compared to fullthickness biopsies. An additional disadvantage ofgastro-duodenoscopy-assisted biopsies is that no infor-mation can be obtained on sufficient area of the smallintestinal mucosa. The reference values of smallintestine (duodenum and jejunum) wall thickness inThoroughbreds have been reported to range from 0.27to 0.33mm (Bithell et al. 2010). In addition, it might beattractive to study small intestine wall thickness inhorses on a long-term gluten-free diet also.

The question remains if there is a considerable lagtime between improvement in serology and duodenal/jejunal wall thickness and initiation of a gluten-freediet. Preliminary findings derived from follow up ofsome GSE suspected horses (by the first author)suggest that reduction in duodenal/jejunal wall thick-ness is very limited (if not refractory) and takesperhaps years following the initiation of a gluten-freeration similar to findings in human CD serology (Eschet al. 2011) and histopathology (Mallant et al. 2007;Rubio-Tapia et al. 2010). In children with CD, about80% were sero-negative for EMA and anti-TG2 after 2years of the gluten-free diet (Esch et al. 2011). Ofadults with biopsy-proven CD, mucosal recovery attwo years following diagnosis was 34% and at 5 yearswas 66%. However, mucosal recovery was absent in asubstantial portion of adults with CD after a gluten-free diet (Rubio-Tapia et al. 2010).

To date, there are no adequate in vivo models forthe systemic complications of CD; in particular, thereare no genetic knock-out models. However, models areavailable for GSE such as the Irish Setter dog(Daminet 1996) and Balb/c and BDF1 mouse strains(Stazi 2005). In addition, the rhesus macaque model ofGS has been established (Bethune et al. 2008).Exclusion of dietary cereal from birth modified subse-quent expression of the disease in Irish Setter dogs(Hall and Batt 1991c, 1992). In contrast to human CD,susceptibility to canine GSE does not appear to bedetermined by variation within the MHC class II genecluster (Polvi et al. 1997, 1998).

Whether abnormal transport of gluten across thegut epithelium may participate in the pathogenesis ofCD remains debatable. Recent data point to a key rolefor the transcellular pathway and highlights the‘‘Trojan horse’’ role of secretory IgA which canhijack the transferrin receptor and allow the rapidtranslocation of intact gluten peptides into the mucosa(Heyman et al. 2012). In accord, intestinal permeabilitytesting of puppies during controlled oral gluten chal-lenge provides a practical screening test for glutensensitivity in Irish Setter dogs at an early age(Hall and Batt 1991a, b; Garden et al. 1998).

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Microvillar membrane proteins isolated from GSE-affected dogs revealed an essentially normal proteinmap with the exception being an 85 kDa protein(Pemberton et al. 1997).

In conclusion, preliminary findings indicate thatgluten intake might induce clinical pathology in horsesand this study warrants further investigations into theimmunologic basis of possible GSE in horses.Although it cannot be excluded that equine ISBDmust be regarded as a single clinical syndrome, it seemsmore likely that the syndrome is common to severaldifferent enteropathies with different etiologies amongwhich GS might be identified.

Acknowledgements

The support of W. Goesten, DVM, E.P.R. Reijerkerk,DVM, E. Smiet, DVM, D.A. van Doorn, PhD, R.Amirthalingam, and A. Veenhof was greatly appreciated.We also thank Eurodiagnostica, Nijmegen, the Netherlands,for providing us with kits for antibodies to deamidatedgliadin peptides.

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