Journal of Equine Veterinary Science

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Short Communication

Preliminary Notes on Equine Tissue Transglutaminase Serology and A Case of Equine Gluten-Sensitive Enteropathy and Dermatitis in an 11-Year-Old Dutch Warmblood Horse

Rick van Proosdij

^a^,^*

, Chris Mulder

^b

, Martine Reijm

^c

, Hetty Bontkes

^c

, Mary von Blomberg

^c

, Han van der Kolk

^d^,^e

aDe Klomp Dierenartsen, De Klomp, the Netherlands

bDepartment of Gastroenterology and Hepatology, Amsterdam University Medical Center, Amsterdam, the Netherlands

cLaboratory of Medical Immunology, Amsterdam UMC, Vrije Universiteit Amsterdam, Department of Pathology and Department of Clinical Chemistry, Amsterdam Infection and Immunity Institute, Amsterdam, the Netherlands

dEuregio Laboratory Services, Maastricht, the Netherlands

eSwiss Institute for Equine Medicine (ISME), Vetsuisse Faculty, Department of Clinical Veterinary Medicine, University of Bern and Agroscope, Bern, Switzerland

a r t i c l e i n f o

Article history:

Received 14 November 2019 Received in revised form 17 March 2020 Accepted 20 March 2020 Available online 2 April 2020

Keywords:

Transglutaminase TGA

Gluten

Inﬂammatory small bowel disease ISBD

a b s t r a c t

It has been suggested that gluten may play a role in equine inﬂammatory small bowel disease (ISBD).

Previous work showed an association between equine gluten-sensitive enteropathy and IgA antibodies to tissue transglutaminase (TGA) in serum. The purpose of this study is to investigate the prevalence of IgA antibodies to TGA in a group of healthy nonegluten-free sport ponies and to present a case of tentative gluten-sensitive enteropathy and dermatitis in a horse. Blood samples were obtained from 40 healthy jumping ponies. The ponies comprised 12 mares, 8 stallions, and 20 geldings with an average age of 9.0± 3.8 years (±SD; range 3e19 years). Sera were tested for IgA antibodies against human recombinant TGA.

Significance (P<.05) of the correlation between TGA titer and age in these ponies was assessed using Pearson test (two tailed). In addition, to further illustrate tentative equine gluten-sensitive enteropathy and dermatitis, the clinical course in an 11-year-old Dutch Warmblood sport horse gelding has been described. The average TGA titer was 21.4±13.6 AU/mL (range 2e65 AU/mL). There was a significant (P¼ .013) correlation (r¼0.389) between age and TGA titer in ponies. One of the 40 ponies (2.5%) showed an elevated TGA titer. An elevated TGA titer decreased after a gluten-free ration for 3 months in an 11-year- old Warmblood gelding with a tentative diagnosis of ISBD associated with full remission of the generalized skin reaction. To our best knowledge, this is thefirst study assessing TGA antibodies in sera from healthy nonegluten-free ponies and showing a correlation with age. The presented case could be thefirst one of a horse with a tentative diagnosis of gluten-sensitive enteropathy combined with dermatitis.

Given the reportedﬁndings, this study warrants further investigations into gluten-sensitive enteropathy and dermatitis in individual horses affected with ISBD.

1. Introduction

The intestinal immune system is constantly exposed to a vast array of antigens, including those derived from food, components of the endogenous microbialﬂora, and pathogenic organisms. It is important that the immune system discriminates between patho- gens and harmless (food derived) substances. If this delicate bal- ance is interrupted, a state of chronic uncontrolled inﬂammation may ensue [1]. A syndrome marked by chronic weight loss and hypoalbuminemia associated with chronic gastrointestinal Animal welfare/ethical statement:Blood collection for TGA in the ponies was per-

formed during the blood collection for training and/or performance evaluation. The case was examined and treated without any delay. No blood collection or other test was specially performed for this case report.

Conﬂict of interest statement:The last author (JHvdK) is director of a commercial laboratory which also performs equine IgA antibodies to TGA.

*Corresponding author at: Rick van Proosdij, De Klomp Dierenartsen, De Klomp, the Netherlands.

E-mail address:rick@deklompdierenartsen.nl(R. van Proosdij).

Contents lists available atScienceDirect

Journal of Equine Veterinary Science

j o u r n a l h o m e p a g e : w w w . j - e v s . c o m

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disorders is well known in Standardbreds with two different pathomorphological entities, namely eosinophilic granulomatosis and granulomatous enteritis [2]. Lymphocytic-plasmacytic enteritis also belongs to this complex of equine idiopathic inﬂammatory small bowel disease (ISBD). Clinical signs include weight loss, recurrent colic, soft feces, depression, edema, and a dull hair coat [3]. By deﬁnition, the exact cause of (equine) ISBD is unknown urging the need for studying diet and/or immune function in these patients. Previously, it has been indicated that gluten sensitivity might be among the different etiologies associated with equine ISBD [4].

Celiac disease (CD) is an autoimmune inflammatory 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 [5,6]. The classic form is much more frequent in children [6]. The common factor for all patients with CD is the presence of a variable combination of gluten- dependent clinical manifestations, specific autoantibodies directed to tissue transglutaminase (TGA) and endomysium, HLA- DQ2, and/or DQ8 and different degrees of enteropathy. Recently, so-called non-celiac gluten sensitivity (NCGS) has received much interest in human medicine, although the limits and possible overlap between NCGS and CD remain poorly defined. Both NCGS and CD have, as diagnostic criterium, the presence of IgG antibodies to native gliadin. Autoantibodies to TGA are not present in patients with NCGS. Patients with NCGS have an abnormal intestinal permeability, and patients with CD have villous atrophy [7].

The aim of the present study was to investigate the prevalence of IgA antibodies to TGA in a group of healthy nonegluten-free sport ponies and to further illustrate equine gluten-sensitive enteropathy and dermatitis with the clinical course in an 11-year-old Dutch Warmblood sport horse gelding.

2. Materials and Methods 2.1. Animals

Forty healthy jumping ponies comprising 12 mares, 8 stallions, and 20 geldings with an average age of 9.0 ± 3.8 years (range 3e19 years) were used. The ponies performed at the basis to international-level jumping (as governed by the Federation Equestre Internationale). A clinical examination was performed, and peripheral venous blood was collected for routine hemato- logical and biochemical examination as part of the regular veterinary health check as performed by the team veterinarian. All ponies were deemed clinically healthy based on the veterinary health check. In addition, to further illustrate equine gluten-sensitive enteropathy and dermatitis, the clinical course in an 11-year-old Dutch Warmblood sport horse gelding has been described. Sera from these equines were tested for IgA antibodies to tissue TGA.

The samples from the other horses were taken on veterinary indication and approved by the owners.

2.2. Serology

TGA titers were determined quantitatively in serum based on binding to recombinant human tissue transglutaminase (rh-tTG).

Ninety-sixewell microtiter plates were coated with rh-tTG (1m^g/

well in 0.05 M TRIS-HCL, pH 7.5, Diarect, Freiburg, Germany) as per standard operation procedures. After washing, open binding sites were blocked with 1% bovine serum albumin (BSA) and 0.05%

Tween-20. The serum was diluted ranging from 1:100 to 1:200 in phosphate-buffered saline with 1% BSA and 0.05% Tween-20 and preincubated at room temperature for 30e60 minutes to allow potential anti-BSA to bind to the BSA. TGA antibodies were detected

using orthophenylenediamine dihydrochloride as substrate and Goat Anti Horse IgA:HRP (Bio Rad, Hercules, California, US) as conjugate in a routine procedure. After 15 minutes, the reaction was terminated using H2SO4and evaluated spectrophotometrically at 492 nm. Serum previously obtained from an equine patient was used as calibration curve and standard (by deﬁnition containing 100 TGA AU/mL). This method was previously validated by van der Kolk et al [4].

2.3. Statistical Analysis

Signiﬁcance (P<.05) of the correlation between TGA titer and age in these ponies was assessed using Pearson’s test (two tailed) as the one-sample Kolmogorov-Smirnov test showed normality in data. The Mann-WhitneyUtest was used to compare TGA titers between these ponies and Warmblood horses described in a previous study [4].

3. Results

The average (n¼40) TGA titer was 21.4±13.6 AU/mL (range 2e65 AU/mL) in the sport ponies. The result showed a normal distribution. There was a signiﬁcant (P¼.0013) correlation (r¼ 0.389) between age and TGA titer (seeFig. 1). One of the 40 ponies (2.5%) showed an elevated TGA titer (95% conﬁdence interval). All the ponies of this study were lost in follow-up. There is no extra information about their clinical status or development of TGA titer over the years after initial testing.

3.1. Clinical Course of an 11-Year-Old Warmblood Gelding

An 11-year-old Dutch Warmblood sport horse gelding was examined because of skin problems since two weeks. The horse had not suffered from any skin problems previously and was regularly dewormed with ivermectin. Examination of the skin revealed papula and urticaria covering almost the complete body with the highest density of urticaria found on the neck region (seeFigs. 2A and 2B). The urticaria was associated with pruritis. The tentative diagnose was a hypersensitive reaction, and as a consequence, dexamethasone was administered IV (at a dosage of 0.06 mg/kg BW). Within 2 weeks after administration, symptoms recurred and worsened. The administration of dexamethasone IV had been repeated several times.

Fig. 1.TGA titer versus age in a group of 40 sport ponies. TGA, transglutaminase.

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The following year, owing to arthritis and arthrosis of the cervical facet joints C4-C5 left side (minor arthritic changes, grade 3a), C6-C7 left side (major arthritic changes, grade 5a), and right side (minor changes, grade 3a) (grading scale by Down and Henso [8]), treatment was initiated at a dose of 20 mg triamcinolone (Deport;

Ceva Sante Animale B.V., Naaldwijk, the Netherlands) per facet joint in the left facet joint C4-C5 and left facet joint C6-C7. This treatment was repeated every three months during a year. During this period, no dermal problems were observed.

Afterﬁnishing the triamcinolone treatments, a wide array of clinical symptoms developed. Clinically most signiﬁcant was chronic weight loss with a body condition score of 2/9 (according to

Henneke et al [9]), despite normal appetite and adequate nutrient intake in relation to its workload. Furthermore, the recurrent skin problems were categorized as papular with urticaria. In addition, clinical examination showed increased muscle tone in the caudo- ventral abdominal wall after gentle palpation reﬂecting abdominal discomfort. Muscle tremors of the triceps were observed. Abdom- inal ultrasound revealed thickening of the walls of the small intestine (>5 mm) on multiple sites (one of these images is shown in Fig. 3).

Remarkable ﬁndings regarding blood analysis were a low normal total protein concentration (55 g/L; reference range:

55e75 g/L), hematocrit (Ht) (31%; reference range: 35%e50%), Fig. 2.Gluten-sensitive dermatitis in an 11-year-old Dutch Warmblood gelding (A and B) before and (C and D) after a month on a gluten-free diet.

Fig. 3.Ultrasound image of the small intestines of an 11-year-old Dutch Warmblood gelding suspected of gluten-sensitive enteropathy showing wall thickening (solid arrow).

Measurement is of two adherent small intestinal walls.

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leukocytes (5.7 G/L; reference: 7e10 G/L), and vitamin E (1.4m^mol/

L; reference range>4.0mmol/L) (SeeTable 1column“6-2-2015”).

An oral glucose absorption test (using 1 g dextrose/kg BW) after a 12-hour period of food deprivation showed a normal glucose response with 202% increase. In addition, antibodies to rh-tTG were tested revealing 45 AU/mL. Based on the clinical ﬁndings and serology, the tentative diagnosis reached was gluten-sensitive enteropathy and dermatitis (seeTable 1).

In line, the horse was put on a box bedding of wood shavings and was fed hay and a commercially available (presumably)

“gluten-free”concentrate (PAVO, Boxmeer, the Netherlands). Via this concentrate, the horse was given 600 IU vitamin E and 0.9 mg selenium a day. Next to this, the horse was given 2100 IU vitamin E and 0.5 mg selenium by a commercially available product daily (Vetoquinol, Lure, France). These management interventions did result in some clinical improvement: there was full remission of the skin abnormalities and muscle tremors were not observed anymore (Figs. 2C and 2D). However, total recovery was not achieved after application of this management protocol. The horse was still too skinny (body condition score: 3/9) and the abdomen remained too sensitive on palpation. Besides, the skin abnormalities recurred.

TGA antibodies were tested again four weeks after initial diet change resulting in 70 AU/mL with negative controls (horses without symptoms of ISBD associated with low TGA titers) showing 11 and 14, whereas positive controls (horses with ISBD and elevated TGA titers) tested 48, 97, and 254 AU/mL. As a consequence, the presumed gluten-free concentrate was tested for its concentration of gluten. Feed analysis revealed a concentration of 83 mg/kg concentrate feed similar to 8.3 mg gluten/kg food per day.

Subsequently, the horse was put on a strict diet without concentrate or other sources of gluten. In accord, the skin problems disappeared within a few weeks. Afterﬁve months without clinical symptoms, TGA was 28 AU/mL with negative controls 17, 19, 23, and 35, whereas positive controls tested 121 and 290 AU/mL. The blood parameters like the concentration of total protein (57 g/L), Ht (36%), and vitamin E (4.3mmol/L) were all within their reference ranges after full clinical recovery (seeTable 1column“8-9-2016”).

The clinical course in response to changes in ration and the normalization of blood parameters are in line with a tentative diagnosis of gluten-sensitive enteropathy and dermatitis. The TGA test results supported the diagnosis with an elevation of TGA after (accidental) stimulation with gluten and a normal TGA concentration after a gluten-free diet.

4. Discussion

Little is known about so-called CD in horses. In a previous study, tissue TGA antibody was found a promising value for assessing a gluten-dependent immune response with gluten-free controls

(healthy ponies on a gluten-free diet) revealing values lower than 18 AU/mL and nonegluten-free controls (healthy horses on a commercial nonegluten-free ration) lower than 36 AU/mL [4].

Other antibodies associated with CD, such as antibodies to native gliadin, deamidated gliadin peptides, and primate and equine endomysium were not tested in the equine patient discussed previously. This study shows an age-dependent TGA titer in clinical healthy ponies. In contrast, in 18 nonegluten-free healthy Warm- blood horses on a commercial nonegluten-free ration, there was no significant (r¼0.016;P¼.950) correlation between rh-TGA titer and age. Of note, the 40 nonegluten-free healthy ponies had a mean± SD titer of 21.4 ± 13.6 AU/mL, which was significantly different (P<.05 in Mann-WhitneyUtest) from 18 nonegluten- free control Warmblood horses with 13.4±9.3 AU/mL in a previous study from our group [4] performed in the same laboratory using identical methodology. The TGA titers in the 40 control ponies were normally distributed, and we calculated the cutoff value for ponies as an average value of±2*SD equal to 48.6 AU/mL. In line, given that the TGA titers in the 18 nonegluten-free control horses were normally distributed, the cutoff value for Warmblood horses can be calculated as an average value of±2*SD equal to 32.0 AU/mL. Of note, following assessment of TGA titers, in ponies, age indepen- dence should be considered. The pony with the elevated TGA titer of 65 AU/mL could be suffering of subclinical gluten-sensitive enteropathy. Owing to lack of follow-up, this cannot be confirmed.

The equine patient presented here had multiple general symptoms also seen in humans with CD, failure to thrive, muscle wasting, anorexia, joint disease, and anemia. In addition to intestinal pathology, this horse also showed signs of CD-associated dermal pathology. Unfortunately, we had no permission by the owner to take skin or intestinal biopsies. In human patients, dermal pathology due to CD is well documented [10]. However, to the authors’knowledge, dermal pathology associated with CD is poorly documented in veterinary medicine. The muscle tremors found in this case could be caused by CD owing to neural pathology. Neural pathology caused by CD is both described in human medicine [11]

and in canine medicine [12]. No cases of CD-associated neural pathology in horses have been described yet, but the diagnosis is also very difﬁcult to make.

This horse developed clinical signs on a commercially available concentrate with a gluten concentration of 8.3 mg per kilogram food as daily intake. Interestingly, this concentration is well lower than the threshold value for a human gluten-free diet. Human diet can be called“gluten-free”if it contains less than 20 mg gluten per kg (or 20 parts per million) food. Although the claim“gluten-free” is legal, humans with CD are prone to development of clinical problems based on a so-called“gluten-free”diet [13]. One should also keep in mind that the total daily intake of food by a horse is several times more than that of humans. By that the absolute amount of gluten in the previously described gluten-free diet is a plural of that in human diets containing less than 20 mg gluten per kg food.

An elevated TGA titer decreased after a gluten-free ration for 5 months in an 11-year-old Warmblood gelding with ISBD, with associated full remission of the generalized skin reaction. This decrease most likely was caused by the gluten-free ration when using 32.0 AU/mL as a cutoff point. Furthermore, it seems as there is no effect of age on TGA titer in nonegluten-free Warmblood horses.

Unfortunately, owing to lack of intestinal or skin biopsies, a conclusive diagnosis cannot be made.

To the authors’experience based on a few cases, small bowel wall thickness does not reverse after a gluten-free ration fed to a gluten-sensitive horse, and as a consequence, the absorption capacity in such horses might never reverse to normal. In the current case, total protein as well as the Ht and leukocyte count tends to Table 1

Blood analysis.

19-8-2014 6-2-2015 10-11-2015 8-9-2016

TP gr/L 60 55 54 57

Alb gr/L (%) 35.5 (59.3) 33.9 (61.6) 32.1 (59.6) 26.4 (46.3) Alfa gr/L (%) 7.9 (13.2) 4.0 (7.4) 7.6 (14) 12.2 (21.4) Beta gr/L (%) 5.8 (9.6) 7.0 (12.8) 3.8 (7.1) 8.1 (14.2) Gamma gr/L (%) 10.7 (17.9) 10.0 (18.2) 10.4 (19.3) 10.3 (18.1)

Ht L/L 0.31 0.32 34 0.36

WBC giga/L 5.4 5.7 5.8 6.1

19-8-2014, examination was before symptoms of ISBD. 6-2-2015,ﬁrst examination with symptoms of ISBD; 10-11-2015, examination after 6 months of gluten-free diet and with no symptoms of ISBD; 8-9-2016 examination after 16 months of gluten- free diet and no symptoms of ISBD. TP, total protein; Alb, albumin; Ht, hematocrit; WBC, white blood cells.

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increase, but it turns out to be a slow process. The persistently low albumin might reﬂect the chronically decreased absorption capacity.

The increased thickness of the wall of the small bowel both on rectal palpation and ultrasonographically combined with the persistent leukopenia is the basis of our clinical tentative diagnosis of chronic ISBD. Of course, this clinical tentative diagnosis usually is conﬁrmed at histopathology on a duodenal biopsy, which was refused by the owner.

Chronic ISBD in horses is well described. Hence, we suggest evaluating so-called CD in horses as well and measure TGA antibodies to exclude gluten-induced inflammatory diseases in horses especially skin-induced dermatitis as identified in a horse in this report. However, in these cases, histopathology revealing duodenal villous atrophy further strengthens the diagnosis also considering the fact that duodenal and/or jejunal villous atrophy is a very rare finding in adult horses.

Acknowledgments

The authors thank Drs Tamarinde Laan and Jan Greve for their practical contribution.

Disclosure: The last author is director of a commercial laboratory which also performs equine IgA antibodies to TGA

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