Healthy Ponies Research Articles

Associations between feeding and glucagon-like peptide-2 in healthy ponies.

Gastrointestinal peptides, such as glucagon-like peptide-2 (GLP-2), could play a direct role in the development of equine hyperinsulinaemia. To describe the secretory pattern of endogenous GLP-2 over 24 h in healthy ponies and determine whether oral administration of a synthetic GLP-2 peptide increases blood glucose or insulin responses to feeding. A cohort study followed by a randomised, controlled, cross-over study. In the cohort study, blood samples were collected every 2 h for 24 h in seven healthy ponies and plasma [GLP-2] was measured. In the cross-over study, 75 μg/kg bodyweight of synthetic GLP-2, or carrier only, was orally administered to 10 ponies twice daily for 10 days. The area under the curve (AUC0-3h ) of post-prandial blood glucose and insulin were determined before and after each treatment. Endogenous [GLP-2] ranged from <0.55 to 1.95 ± 0.29 [CI 0.27] ng/mL with similar peak concentrations in response to meals containing 88-180 g of non-structural carbohydrate, that were ~4-fold higher (P < 0.001) than the overnight nadir. After GLP-2 treatment peak plasma [GLP-2] increased from 1.1 [0.63-1.37] ng/mL to 1.54 [1.1-2.31] ng/mL (28.6%; P = 0.002), and AUC0-3h was larger (P = 0.01) than before treatment. The peptide decreased (7%; P = 0.003) peak blood glucose responses to feeding from 5.33 ± 0.45 mmol/L to 5.0 ± 0.21 mmol/L, but not AUC0-3h (P = 0.07). There was no effect on insulin secretion. The study only included healthy ponies and administration of a single dose of GLP-2. The diurnal pattern of GLP-2 secretion in ponies was similar to other species with no apparent effect of daylight. Although GLP-2 treatment did not increase post-prandial glucose or insulin responses to eating, studies using alternative dosing strategies for GLP-2 are required.

Investigating the effects of experimentally induced insulin dysregulation on adiponectin concentrations in metabolically healthy ponies

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Influence of endocrine disease on l-lactate concentrations in blood of ponies.

BackgroundBlood l‐lactate concentrations are higher in people with developing or established diabetes mellitus and insulin resistance.ObjectivesTo investigate whether blood l‐lactate concentrations are positively correlated with measures of insulin dysregulation (ID) or increased autumnal ACTH concentrations in ponies.AnimalsSystemically healthy client‐owned ponies (n = 101).MethodsProspective case‐control study. Blood samples were obtained from 101 clinically healthy ponies. Breed, weight, height, and subjective and objective measures of body condition were recorded. Blood l‐lactate, glucose, triglyceride, total adiponectin, and ACTH concentrations were measured and an oral sugar test (OST) was carried out. Correlations between blood l‐lactate and variables of endocrine health were determined.ResultsUsing a seasonal cutoff point of ACTH concentrations ≥47 pg/mL, 55 ponies had increased autumnal ACTH concentrations and 45 did not. Using a basal insulin concentration of >50 μiU/mL, 42 ponies were diagnosed with ID and 58 were not. Using a 60 minutes after OST cutoff point of >45 μiU/mL, 57 ponies had ID and 37 did not. Blood l‐lactate concentrations were significantly lower in obese (average body condition score ≥ 7/9) compared to nonobese ponies (0.6 mmol/L; range, 0.0‐1.9 mmol/L vs 0.8 mmol/L; range, 0.3‐2.7 mmol/L; P = .01). No other significant correlations were detected. No differences were detected between ponies with and without increased autumnal ACTH concentrations (0.7 mmol/L; range, 0.0‐2.7 mmol/L vs 0.7 mmol/L; range, 0.3‐1.8 mmol/L; P = .84) and with and without ID (0.7 mmol/L; range, 0.3‐2.7 mmol/L vs 0.8 mmol/L; range, 0.0‐1.6 mmol/L; P = .63).Conclusions and Clinical ImportanceResults do not support an effect of endocrine status on l‐lactate concentrations in blood of ponies.

Volumetric measurements of paranasal sinuses and examination of sinonasal communication in healthy Shetland ponies: anatomical and morphometric characteristics using computed tomography

BackgroundDespite clinical importance and frequent occurrence of sinus disease, little is known about the size of paranasal sinuses and their communication in ponies and small horses. To examine the shape and volume of the paranasal sinuses and evaluate the sinonasal communication, three-dimensional (3D) reconstructions of computed tomography (CT) datasets of 12 healthy adult Shetland ponies were performed and analysed. Linear measurements of head length and width were taken. Using semi-automatic segmentation, 3D-models of all sinus compartments were created. Volumetric measurement of the seven sinus compartments were conducted and statistical analysis was performed. Sinus volumes were compared between the left and right sinuses and the relation to age and head size was evaluated.ResultsStructure and shape of the paranasal sinus system in Shetland ponies was similar to that of large horses. All seven sinus compartments on each side of the head were identified (rostral maxillary sinus, ventral conchal sinus, caudal maxillary sinus, dorsal conchal sinus, middle conchal sinus, frontal sinus, sphenopalatine sinus). The existence of a bilateral cranial and a caudal system formed by a maxillary septum was visible in all 12 individuals. The volumetric sizes of the left and right sinuses did not differ significantly (p > 0.05). A positive correlation between the size of the paranasal sinuses and the head length was shown. A relation between sinus volumes and age could not be proved in adult ponies aged > six years. Communication between single sinus compartments was identified. Furthermore, communication with the nasal cavity over the nasomaxillary aperture (Apertura nasomaxillaris) and a common sinonasal channel (Canalis sinunasalis communis) as well as its splitting up into a rostral and a caudolateral channel could be seen. Examination of the sinonasal communication was challenging and only a descriptive evaluation was possible.ConclusionsOur findings concerning the size, shape and volumetric dimensions of Shetland pony CT images could help improve CT interpretation of abnormal clinical cases as well as aiding clinicians to develop and select appropriate instruments for medical inspection and treatments.

Variations in haematological and biochemical parameters in healthy ponies

BackgroundBreed specific reference ranges for selected blood parameters are recommended for proper interpretation of blood tests, but there are only few reports dealing with ponies. The purpose of this study was to investigate if blood parameters differ among ponies’ classes and to check if general normal values for equine species are applicable to ponies.ResultsAll, except total protein concentration, biochemical parameter significantly (p < 0.05) differed among ponies’ classes. The most pronounced difference was noted in blood lactate concentrations, higher (p < 0.001) in the smallest ponies (class A). In all groups of ponies muscle enzymes (aspartate aminotransferase and creatine kinase) and urea were high when compared to normal values for equine species, but triglycerides and creatinine were low. Blood lactate concentration was high in comparison with normal values for horses only in class A ponies’.ConclusionsIn healthy ponies, blood lactate concentration significantly differs between height classes. Normal values for equine species should not be directly applied to interpret the lactate, triglycerides, aspartate aminotransferase and creatine kinase values in ponies.

A single bout of high-intensity exercise modulates the expression of vitamin D receptor and vitamin D-metabolising enzymes in horse skeletal muscle.

The expressions of vitamin D receptor (VDR) and vitamin D-metabolising enzymes (CYP27B1 and CYP24A1) in skeletal muscle have been reported. However, the regulation of this vitamin D system in horse skeletal muscle after high-intensity exercise has not yet been elucidated. To investigate the effect of high-intensity exercise on the expression of vitamin D system-related proteins in horse skeletal muscle and its associations with skeletal muscle stem cell (SMSC) activity and serum 25(OH)D level. Longitudinal study. Six healthy ponies (5 geldings, 1 mare; age 6.3±2.2years) were studied. Serum and muscle samples were taken from the jugular vein and gluteus medius respectively. Samples were collected at pre-exercise, post-exercise, 1 and 3weeks after a single bout of high-intensity exercise. Protein expression levels of VDR, CYP27B1, CYP24A1, OxPhos and Pax7 (SMSC marker) were determined using immunohistochemical analysis. Oxidative capacity and intramuscular glycogen content were evaluated using histochemical analysis. Blood biochemistry was analysed for lactate concentration and creatine kinase (CK), and 25(OH)D activity. High-intensity exercise significantly upregulated Pax7 and VDR protein expression, which correlated with significantly increased blood lactate and serum CK levels immediately post-exercise. Serum 25(OH)D2 level correlated with CYP27B1 protein expression in skeletal muscle, and it reduced significantly immediately post-exercise and at 1 and 3weeks post-exercise. However, CYP24A1 protein expression was unchanged throughout study periods. The healthy ponies could not represent a fit population of racehorses and eventers. The rapid increase in Pax7 and VDR protein expression along with serum CK level after high-intensity exercise demonstrated an association between SMSC activity and activation of the vitamin D system in response to muscle injury in horses. Moreover, a decrease in CYP27B1 protein expression, correlated with a reduction in serum 25(OH)D2 , may indicate a compromised vitamin D metabolism after high-intensity exercise.

The effect of diet change and insulin dysregulation on the faecal microbiome of ponies.

The equine microbiome can change in response to dietary alteration and may play a role in insulin dysregulation. The aim of this study was to determine the effect of adding pasture to a hay diet on the faecal bacterial microbiome of both healthy and insulin-dysregulated ponies. Faecal samples were collected from 16 ponies before and after dietary change to enable bacterial 16S rRNA sequencing of the V3-V4 region. The dominant phyla in all samples were the Firmicutes and Bacteroidetes. The evenness of the bacterial populations decreased after grazing pasture, and when a pony was moderately insulin dysregulated (P=0.001). Evenness scores negatively correlated with post-prandial glucagon-like peptide-1 concentration after a hay-only diet (r²=-0.7, P=0.001). A change in diet explained 3% of faecal microbiome variability. We conclude that metabolically healthy ponies have greater microbial stability when challenged with a subtle dietary change, compared with moderately insulin-dysregulated ponies.

An investigation of the equine epidermal growth factor system during hyperinsulinemic laminitis.

Equine laminitis is a disease of the digital epidermal lamellae typified by epidermal cell proliferation and structural collapse. Most commonly the disease is caused by hyperinsulinemia, although the pathogenesis is incompletely understood. Insulin can activate the epidermal growth factor (EGF) system in other species and the present study tested the hypothesis that upregulation of EGF receptor (EGFR) signalling is a key factor in laminitis pathophysiology. First, we examined lamellar tissue from healthy Standardbred horses and those with induced hyperinsulinemia and laminitis for EGFR distribution and quantity using immunostaining and gene expression, respectively. Phosphorylation of EGFR was also quantified. Next, plasma EGF concentrations were compared in healthy and insulin-infused horses, and in healthy and insulin-dysregulated ponies before and after feeding. The EGFR were localised to the secondary epidermal lamellae, with stronger staining in parabasal, rather than basal, cells. No change in EGFR gene expression occurred with laminitis, although the receptor showed some phosphorylation. No difference was seen in EGF concentrations in horses, but in insulin-dysregulated ponies mean, post-prandial EGF concentrations were almost three times higher than in healthy ponies (274 ± 90 vs. 97.4 ± 20.9 pg/mL, P = 0.05). Although the EGFR does not appear to play a major pathogenic role in hyperinsulinemic laminitis, the significance of increased EGF in insulin-dysregulated ponies deserves further investigation.

Insulin and incretin responses to grazing in insulin-dysregulated and healthy ponies.

BackgroundSupraphysiological insulin and incretin responses to a cereal‐based diet have been described in horses and ponies with insulin dysregulation (ID). However, the hormonal responses to grazing have not yet been described.ObjectivesTo determine if there is a difference in the insulin and incretin responses to grazing pasture between insulin‐dysregulated and healthy ponies.AnimalsA cohort of 16 ponies comprising 5 with normal insulin regulation (NIR), 6 with moderate ID (MID), and 5 with severe ID (SID).MethodsIn this case‐control study, an oral glucose test (OGT) was used to determine the insulin responsiveness of each pony to PO carbohydrate before grazing pasture (4 hours) for 3 consecutive days. Serial blood samples collected during grazing were analyzed for glucose, insulin, glucose‐dependent insulinotropic peptide (GIP) and active glucagon‐like peptide‐1 (aGLP‐1), and compared among pony groups and day of pasture access.ResultsThe area under the insulin curve when grazing increased with ID severity (P < .03). The median (range) maximal insulin concentration was greater in the MID (72.5 [129] μIU/mL) and SID (255 [338.5] μIU/mL) groups, compared to the NIR (11.7 [24.9] μIU/mL) group (P < .03) and occurred within 2‐4 hours of grazing. Postprandial OGT insulin concentration was positively correlated with 2 hours post‐grazing insulin across all 3 grazing days (P ≤ .03). The aGLP‐1 and GIP concentrations increased in response to grazing but did not differ among groups.Conclusions and Clinical ImportanceGrazing pasture provoked an increased insulin and incretin response in insulin‐dysregulated ponies within 4 hours of grazing. The pasture and OGT insulin concentrations were correlated.

Cell specific microvesicles vary with season and disease predisposition in healthy and previously laminitic ponies

Microvesicles are small (up to 1 μm) vesicles found in plasma and other bodily fluids. They are recognised as part of the normal system of inter-cellular communication but altered numbers are also used as biomarkers of disease. Microvesicles have not been studied in detail in the horse but may be relevant to diseases such as laminitis. Identification of equine cell specific microvesicles was performed by developing a panel of cross reactive antibodies to use in flow cytometry to detect microvesicles of platelet, leucocyte and endothelial origin in plasma from healthy ponies and those predisposed to laminitis. The total number and proportion of microvesicles from the different cell types varied with season and there were more annexin V positive endothelial MV in non laminitic ponies compared to previously laminitic ponies. Development of this antibody panel and the technique for measuring microvesicles in the horse opens a new field for further investigation of these important structures in equine health and disease.

Glucagon-like peptide-2: A potential role in equine insulin dysregulation.

Equine insulin dysregulation (ID) is a common and poorly understood disorder that increases the risk of laminitis. Recent data show that the condition may be associated with alteration of the enteroinsular axis and enhanced glucose bioavailability. Upregulation of glucagon-like peptide-2 (GLP-2), an intestinotrophic peptide, leads to enhanced nutrient uptake and metabolic dysfunction in other species. The study aimed to 1) determine whether GLP-2 is differentially expressed in insulin-dysregulated ponies, compared with healthy ponies, and 2) confirm intestinal expression of the GLP-2 receptor in horses (eGLP-2R). Cohort study. Fasting and post-prandial GLP-2 concentrations were measured in archived plasma samples obtained from 25 mixed-breed ponies during two feeding studies. Measurements were undertaken with an ELISA that was validated for equine use as part of the current study. Ponies were designated as healthy or insulin-dysregulated based on an oral glucose test, and the results were compared between groups. The gene expression of the eGLP-2R was determined with polymerase chain reaction. Basal, fasted plasma GLP-2 concentrations were higher in ponies with ID, compared with healthy ponies. Grazing increased GLP-2 in healthy, but not in insulin-dysregulated, ponies. The eGLP-2R gene was expressed in the small intestine and pancreas. The study was performed with a relatively small sample size. The specificity of the GLP-2 assay could not be determined due to the lack of equine-specific assay standards. This study has demonstrated that GLP-2 may be important in the pathogenesis of equine ID and suggests that the eGLP-2R may be a novel therapeutic target for the treatment of equine ID.

Impact of season, age and gender on some clinical, haematological and serum parameters in Shetland ponies in east province, Saudi Arabia

The Shetland ponies, which originate from harshest environments in the world islands in the north of the Atlantic Ocean, are now most popular pony breed distributed in almost every corner of the world. Reference ranges of physiological, biochemical and haematological values, which are widely used in veterinary clinics, may differ according to season, age, sex, type of feeding, and environmental circumstances of the area. Reference ranges of haematological and biochemical values of Shetland ponies are rare. The present study was therefore undertaken to evaluate the impact of season, age and sex on some haematological and biochemical values and cortisol levels in healthy ponies in Saudi Arabia. The study was conducted between December 2016 to June 2017 on ponies in and around Al-Hasa, Saudi Arabia. Twenty-three clinically healthy ponies males and females of different ages were included for haematological and biochemical analysis in this study. For each animal, blood samples were collected in summer and winter. Four physiological, fourteen haematological, sixteen biochemical parameters as well as serum cortisol levels were analysed.Heart rate, respiratory rate, pulse and rectal temperature were increased in summer in comparison to winter. Values of heart rate proved significant at P < 0.05. The results of blood haematology and biochemistry of the ponies revealed that there was no significant variation between summer and winter in most of the haematological and biochemical parameters, while there was a slight significant difference in leucocyte counts, monocytes, MCH, MPVK+, platelets and AST activity. There were significant differences in serum cortisol concentration regarding season and age but not regarding gender.

Effects of stomach loading on lung aeration, Fshunt, and oxygenation in adult healthy ponies in dorsal recumbency

Effects of stomach loading on lung aeration, Fshunt, and oxygenation in adult healthy ponies in dorsal recumbency

A comparison of the effects of a 4% modified fluid gelatin and a 6% hydroxyethyl starch on haemodilution, colloid osmotic pressure, haemostasis and renal parameters in healthy ponies.

Adverse effects on renal health and haemostasis have been documented in human patients administered hydroxyethyl starches (HESs). Gelatins may represent useful substitutes should similar adverse effects be identified in horses. To compare the effects of a 4% modified fluid gelatin (MFG) with a 6% (130/0.4) HES on haemodilution, colloid osmotic pressure (COP), haemostasis and renal parameters in healthy ponies. Randomised crossover experiment. Three treatments (Treatment A: 10 ml/kg bwt HES; Treatment B: 10 ml/kg bwt MFG; Treatment C: 20 ml/kg bwt MFG) were administered to 6 healthy ponies with a 1 week washout period between treatments. Haematocrit, platelet count, total serum protein, COP, thromboelastography (TEG), prothrombin time (PT), activated partial thromboplastin time (aPTT) and fibrinogen concentration were measured at baseline and at multiple time points up to 24 h post-infusion. Serum creatinine concentration, urine specific gravity (USG), urine protein:creatinine ratio (UPC), urine γ-glutamyltransferase:creatinine ratio (UGC) and urine sediment examination (USE) were performed before and at 24 h after each treatment, as well as at 1 week after the final treatment. All treatments resulted in significant haemodilution and increases in COP. Treatment C had a significantly greater effect on haematocrit than the other treatments. The platelet count decreased with all treatments and was significantly lower following Treatment C compared with Treatment B. No clinically relevant differences were observed in any of the TEG parameters within or between treatments. No significant differences in PT, aPTT or fibrinogen concentration were observed among treatments. Serum creatinine concentration, UPC and UGC did not change significantly between pre- and post-study measurements. USG and USE remained within normal limits. Modified fluid gelatin could be considered as an alternative to HES for volume expansion and oncotic support. Neither MFG nor HES were associated with clinically significant adverse effects on haemostasis or renal parameters.

Cryopreservation protocol for equine platelet-rich plasma

In this preliminary study, a new equine platelet-rich plasma (PRP) cryopreservation protocol was evaluated. PRP was obtained by a double centrifugation technique of whole blood collected from 8 adult healthy ponies. A fresh sample of PRP was analyzed for total platelet count, mean platelet volume (MPV), and platelet morphology. Upon morphological evaluation, 200 platelets were counted using a differential interference contrast microscope with a 40x phase objective and classified as activated (with pseudopodia), inactivated (normal discoid shape), or uncertain state (spherical shape, without pseudopodia). Two other PRP samples, one containing DMSO as a cryoprotectant and the other without DMSO, were stored in a mechanical freezer at –80ºC. After 14 days, the frozen samples were thawed and submitted to the same analysis as described above. The fresh PRP showed a platelet count of 830 (±95.3) x103 uL-1, an MPV of 5.2 (±0.07) fL, and composed of 4% activated platelets. There was no significant difference in platelet count, MPV, and activated platelets between fresh and 6% DMSO frozen PRP samples (617.9±65.5x103uL-1; 5.3±0.06fL; 9.5%) (p &amp;gt; 0.05). On the other hand, samples frozen without DMSO showed a significantly lower platelet count (519.6±66.1x103uL-1), higher MPV (5.7±0.08fL), and more activated platelets (13.9%) than the other groups (p &amp;lt; 0.05). The 6% DMSO was able to preserve platelet morphology in PRP stored at –80oC for 14 days, but studies on platelet function of thawed PRP are still needed.

Recombinant Culicoides obsoletus complex allergens stimulate antigen-specific T cells of insect bite hypersensitive Shetland ponies in vitro.

Ponies may suffer from Insect bite hypersensitivity (IBH), an allergic IgE-mediated pruritic skin disorder, induced by allergens from biting midges of the Culicoides spp. To determine whether recombinant Culicoides obsoletus allergens are able to activate T cells of ponies exposed to C. obsoletus and whether these allergen-specific responses differ between IBH-affected and healthy ponies. Ten IBH-affected Shetland ponies and 10 age-matched healthy controls taken from the same stables, to ensure similar exposure to midges. Peripheral blood mononuclear cells (PBMC) were cultured with two different pools of recombinant C. obsoletus complex allergens to expand the allergen-specific T cells. These PBMC cultures were subsequently co-cultured with mature dendritic cells (DCs) loaded with the same antigens. Induction of Th1, Th2 and regulatory T (Treg) cells in these DC/PBMC co-cultures was assessed by analysis of IFN-γ, IL-4, IL-10 and FoxP3 expression levels using quantitative RT-PCR and phenotyping by flow cytometry. Recombinant C. obsoletus allergens increased IFN-γ mRNA expression levels, percentages of IFN-γ expressing (Th1) cells and CD25(high) FoxP3(+) IL-10(+) Tregs compared to unstimulated DC/PBMC co-cultures. Stimulation of IL-4 expressing Th2 cells by the recombinant allergens was far less pronounced. The DC/PBMC co-cultures did not reveal significant differences between healthy and IBH-affected ponies for any of the analysed parameters, except for higher IL-4 mRNA levels in IBH affected ponies after stimulation with one of the two allergen pools. The recombinant C. obsoletus complex allergens can stimulate antigen-specific Th1 and IL10 producing Treg cells and are therefore promising candidates for the immunotherapy of IBH.

A Comparison of A 4% Modified Fluid Gelatin and A 6% Hydroxyethyl Starch on Haemodilution, Colloid Osmotic Pressure, Haemostasis and Renal Parameters in Healthy Ponies

Reasons for performing studyAdverse effects on renal health and haemostasis have been documented in human patients administered hydroxyethyl starches (HES). Gelatins could provide useful substitutes for HES should similar adverse effects be identified in horses.ObjectivesTo compare the effects of a 4% modified fluid gelatin (MFG) with a 130/0.4 6% tetrastarch (TES) on haemodilution, colloid osmotic pressure (COP), haemostasis and renal parameters in healthy ponies.Study designRandomised crossover.MethodsThree treatments (A = 10 ml/kg bwt TES, B = 10 ml/kg bwt MFG and C = 20 ml/kg bwt MFG) were administered to 6 healthy ponies with a one‐week washout period. Packed cell volume (PCV), total serum protein (TSP), COP, platelet count, fibrinogen, prothrombin time (PT), activated partial thromboplastin time (aPTT) and thromboelastography (TEG) were measured at baseline and at multiple time points up to 24 h post infusion. Serum creatinine, urine specific gravity (USG), urine protein:creatinine (UPC), urine GGT:creatinine (UGC) and urine sediment examination (USE) were performed before and 24 h after each treatment, and one week after the final treatment.ResultsAll treatments caused significant haemodilution and increases in COP with treatment C having a significantly greater effect on PCV than other treatments. The platelet count decreased with all treatments and was significantly lower for treatment C compared with treatment B. No significant differences were observed in any TEG parameter within or between treatments. No significant differences in PT, aPTT or fibrinogen were observed between treatments. Serum creatinine, UGC and UPC did not change significantly pre‐ and post study. USG and USE remained within normal limits.Conclusions4% MFG could be considered as an alternative to 130/0.4 6% TES for volume expansion and oncotic support. Neither MFG nor TES were associated with clinically significant adverse effects on haemostasis or renal parameters.Ethical animal research: This research was approved by the University of Pretoria's Animal Ethics Committee. The animals used in the study were part of a research herd belonging to The University of Pretoria. Sources of funding: The Department of Companion Animal Clinical Studies, Faculty of Veterinary Science, University of Pretoria, The Abe Bailey Trust Fund, The South African Veterinary Foundation and The South African Equine Veterinary Association. Competing interests: None declared.

The activity and inhibition of poly(ADP-ribose) polymerase-1 in equine peripheral blood mononuclear cells in vitro.

To evaluate the poly (ADP-ribose) polymerase-1 (PARP1) enzyme and its inhibition in horses and explore its potential as a novel therapeutic target for equine intestinal ischemia-reperfusion injury by (1) identifying poly (ADP-ribose) (PAR) as an indication of PARP1 activation in equine cells using available immunoblot analytical techniques, (2) inducing PARP1 activation in an in vitro oxidative DNA damage model, (3) and demonstrating the inhibition of PARP1 in equine cells using commercially available PARP1 inhibitors. Experimental study. Blood samples were collected from systemically healthy ponies (n = 3) and horses (n = 3). (1) Equine peripheral blood mononuclear cells were exposed to 3 different concentrations of hydrogen peroxide (H2 O2 ) and were lysed at specific time points. PARP1 activity was then assessed by using immunoblot analyses to determine PAR levels. (2) Equine peripheral blood mononuclear cells were preincubated with defined concentrations of PARP1 inhibitors prior to H2 O2 -mediated PARP1 stimulation. PAR levels reflecting PARP1 activity were determined using immunoblot analyses. Commercially available anti-PAR antibodies were used successfully to identify equine PAR. There was a significant increase in PAR accumulation following treatment with H2 O2 . All of the tested PARP inhibitors significantly reduced PAR accumulation to or below basal levels following treatment with H2 O2 . This proof of principle study demonstrated that PAR, an indicator of PARP1 activity, can be identified in the equine species using immunoblot techniques, that equine PARP1 can be activated by H2 O2 -induced DNA damage, and that this activation can be inhibited by PARP1 enzyme inhibitors. The data suggest that the PARP1 pathway plays a role in the equine cellular response to oxidative DNA damage and supports its potential as a novel therapeutic target. Further research documenting an increase in PAR levels in vivo and the efficacy of PARP1 inhibitors in an equine intestinal ischemia-reperfusion model is needed.

Allergen-Specific Cytokine Polarization Protects Shetland Ponies against Culicoides obsoletus-Induced Insect Bite Hypersensitivity.

The immunological mechanisms explaining development of an allergy in some individuals and not in others remain incompletely understood. Insect bite hypersensitivity (IBH) is a common, seasonal, IgE-mediated, pruritic skin disorder that affects considerable proportions of horses of different breeds, which is caused by bites of the insect Culicoides obsoletus (C. obsoletus). We investigated the allergen-specific immune status of individual horses that had either been diagnosed to be healthy or to suffer of IBH. Following intradermal allergen injection, skin biopsies were taken of IBH-affected and healthy ponies and cytokine expression was determined by RT-PCR. In addition, allergen-specific antibody titers were measured and cytokine expression of in vitro stimulated, allergen-specific CD4 T-cells was determined. 24 hrs after allergen injection, a significant increase in mRNA expression of the type-2 cytokine IL-4 was observed in the skin of IBH-affected Shetland ponies. In the skin of healthy ponies, however, an increase in IFNγ mRNA expression was found. Analysis of allergen-specific antibody titers revealed that all animals produced allergen-specific antibodies, and allergen-specific stimulation of CD4 T-cells revealed a significant higher percentage of IFNγ-expressing CD4 T-cells in healthy ponies compared to IBH-affected ponies. These data indicate that horses not affected by IBH, in contrast to the so far established dogma, are not immunologically ignorant but have a Th1-skewed allergen-specific immune response that appears to protect against IBH-associated symptoms. To our knowledge this is the first demonstration of a natural situation, in which an allergen-specific immune skewing is protective in an allergic disorder.

A commercially available immunoglobulin E-based test for food allergy gives inconsistent results in healthy ponies

Commercial immunoglobulin E (IgE)-based tests are available for diagnosis of food allergies and are commonly used in equine practice. However, these tests have been proven unreliable as a screening method in man and other species, but not critically evaluated in equids. Therefore, a commercially available IgE-based test for horses was evaluated. To evaluate the consistency of the results obtained with a commercially available IgE-based test for food allergy diagnosis in ponies (Phase I) and to subject ponies to a provocation trial with the presumed allergens (Phase II). Allergen screening followed by experimental food provocation trials in healthy ponies. Blood samples of 17 healthy Shetland ponies were taken at 2 different time points, sent blinded to a commercial laboratory for screening of common food allergens and the results were evaluated for consistency (Phase I). Ponies that were positive for food allergens were consecutively challenged orally with each allergen separately for 14 days (Phase II). A washout period of one week was applied in ponies with multiple positive results. Clinical parameters and serum amyloid A were monitored during the provocation trial. Only 7/17 ponies were negative on the IgE-based test at the 2 time points, 3 had positive results twice but only one tested positive twice for the same food allergen. No abnormalities were noted during the provocation trials. This study demonstrated that this IgE-based test is not a reliable screening tool for food allergy in healthy equids.