The effect of feeding a commercial feedstuff on gastric squamous gastric disease (ESGD) healing and prevention of recurrence.

Feedstuffs are often recommended to mitigate potential damage from acid associated with equine squamous gastric disease (ESGD). In acidic conditions, pectin alters its structure to one like mucus and binds the stomach mucosa, whilst alfalfa has a strong intrinsic acid buffering capacity. The study aimed to determine whether feeding a commercial beet pulp/alfalfa/oat fibre mix aids ESGD healing and/or prevention of recurrence. Ten adult horses with naturally occurring ESGD were included. All animals were treated with omeprazole as per the attending veterinarian's recommendation and randomly allocated to also be fed a commercial beet pulp/alfalfa/oat fibre mix (1Kg/horse divided into 2 meals/day; n=5) or no additional feed (n=5) for one month. Gastroscopy was then repeated to assess response to therapy. If the ESGD had healed, omeprazole therapy was discontinued, and the commercial feed given to all horses for a further month. Gastroscopy was repeated to determine ESGD recurrence. The mean (±SD) age of the horses was 11.6 (±3.8) years; 4 mares and 6 geldings; various breeds were represented; and the median (range) initial ESGD grade was 2 (2-4). ESGD had healed (grade 0/4) in all animals after one month. After a further month, ESGD had recurred in significantly (p=0.04) more animals that did not receive the commercial feed initially (3/5; 60%; mean [range] ESGD grade 3 [0,4]) compared to those that did (0/5; 0%; mean [range] ESGD grade 0 [0,0]). Thus, the commercial beet pulp/alfalfa/oat fibre mix aided prevention of ESGD recurrence when fed during the healing and prevention phases.

Short Communication: Identification of equine corticotropin-like intermediate lobe peptide (CLIP) binding to an adrenocortipcotrophic hormone (ACTH) assay capture antibody.

A chemiluminescent immunoassay is commonly employed to measure adrenocorticotrophic hormone (ACTH) concentrations to assist pituitary pars intermedia dysfunction diagnosis. In a previous study, seasonally-dependent assay cross-reactivity to endogenous equine corticotropin-like intermediate lobe peptide (CLIP, ACTH 18-39) was suspected. The present study aimed to demonstrate binding of endogenous equine CLIP to the capture antibody of the ACTH chemiluminescent immunoassay. Liquid chromatography - mass spectrometry (LCMS) methods were optimised to identify selected ions from synthetic human ACTH, α-melanocyte stimulating hormone (α-MSH, ACTH 1-17) and CLIP. Synthetic ACTH and CLIP bound to the capture antibody of the chemiluminescent ACTH assay, but α-MSH did not. Equine endogenous CLIP was detected by LCMS in pony plasma taken in the autumn and could be eluted from the capture antibody of the ACTH chemiluminescent immunoassay. Further research is required to enable quantification of CLIP. Equine CLIP may alter measured ACTH concentrations in vivo.

Validity and application of immunoturbidimetric and enzyme-linked immunosorbent assays for the measurement of adiponectin concentration in ponies.

BACKGROUND: Circulating adiponectin concentrations were lower in ponies with a history of endocrinopathic laminitis and in nonlaminitic ponies that subsequently developed laminitis. The assays used in these studies have been discontinued or are no longer valid. OBJECTIVES: (1) to determine the validity of immunoturbidimetric (IT) and enzyme linkedimmunosorbent (ELISA) assays for equine total and high molecular weight (HMW) [adiponectin] measurement and (2) to investigate the association between [adiponectin] measured using these assays and endocrinopathic laminitis. STUDY DESIGN: Method validation and cohort study. METHODS: Accuracy and precision of IT and ELISA assays for measuring total (TAC) and HMW (HMWAC) [adiponectin] were determined. Using the IT assay, the effects of anti-coagulant and storage temperature were assessed, TAC was measured in previously laminitic (PL) and never laminitic (NL) ponies (n = 6/group). Comparison with a previously validated radioimmunoassay was made in NL ponies (n = 223). Association between TAC and subsequent laminitis development in NL ponies was investigated using univariable logistic regression and ROC curve analysis. RESULTS: The IT assay was precise and demonstrated good agreement with the previously validated radioimmunoassay. TAC was significantly (P<0.01) lower in PL (mean ± s.d. 8.9 ± 2.9 µg/mL) compared to NL (24.2 ± 11.8 µg/mL) ponies and in NL ponies that developed laminitis within 12 months (median 4.8 µg/mL; IQR 2.65-13.4 µg/mL) compared to those that remained nonlaminitic (19.9 µg/mL; 9.95-31.5 µg/mL). TAC was significantly (P = 0.01) associated with laminitis occurrence within 12 months. Use of the area under the ROC curve to distinguish animals that did and did not develop laminitis showed good accuracy (0.76). None of the ELISA methods validated satisfactorily. MAIN LIMITATIONS: Laminitis risk is based on data from ponies in one region. CONCLUSIONS: The IT method is suitable for measurement of equine TAC. TAC is lower in ponies with previous or future laminitis. The ELISA methods are not suitable for measurement of equine HMWAC or TAC.

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.

Effect of varying the dose of corn syrup on the insulin and glucose response to the oral sugar test.

BACKGROUND: The oral sugar test (OST) is used to identify equine insulin dysregulation (ID); however only a dose of 0.15 mL/kg bwt corn syrup has been evaluated. OBJECTIVES: To determine the effect of varying the dose of corn syrup on insulin and glucose response to the OST and the test's ability to distinguish between ponies with a history of laminitis (PL) and without laminitis (NL). STUDY DESIGN: Randomised crossover experiment. METHODS: After an overnight fast, in a three-way randomised crossover study with a 7-day washout, 0.15, 0.3 or 0.45 mL/kg bwt corn syrup was administered orally to eight ponies (5 PL and 3 NL) and blood obtained between 0 and 120 min. Serum [insulin] and [glucose] were measured using previously validated radioimmunoassay and colorimetric assays respectively. The repeatability of and the effect of continued pasture access on the dose that best distinguished PL and NL ponies were then assessed. The effect of dose, laminitis history and fasting on serum [insulin] and [glucose] responses were assessed using mixed-effects models. RESULTS: The serum [insulin] following 0.15 mL/kg bwt were not significantly different from 0.3 mL/kg bwt at any time point, while serum [insulin] following 0.45 mL/kg bwt significantly (P<0.01) differed from 0.15 and 0.3 mL/kg bwt at all time points apart from 0 min. The serum [insulin] concentration significantly (P<0.01) differed between NL (mean 86 [95% CI 59, 113] µiu/mL) and PL (146 [95% CI 124, 167] µiu/mL) only following 0.45 mL/kg bwt at 60 min. Repeatability of serum [insulin] at 60 min following 0.45 mL/kg bwt dose under fasted conditions was 0.51. Using AUC insulin improved repeatability to 0.83. There was no significant difference between the fasted and at pasture results. MAIN LIMITATIONS: The OST was performed in small numbers of ponies on limited occasions. CONCLUSIONS: A dose of 0.45 mL/kg bwt corn syrup may be preferable to differentiate PL and NL ponies.

Plasma adrenocorticotropic hormone (ACTH) concentrations in ponies measured by two different assays suggests seasonal cross-reactivity or interference.

BACKGROUND: Analysis of plasma adrenocorticotropic hormone concentration [ACTH] aids diagnosis of pituitary pars intermedia dysfunction (PPID). Comparisons of the validated chemiluminescent-immunoassay (CI) and immunofluorescent (IF) assays are limited. OBJECTIVES: To compare the results of [ACTH] analysis by CI and IF methods of samples collected in autumn and spring and assess cross-reactivity. STUDY DESIGN: Method comparison. METHODS: Plasma from nonlaminitic ponies was analysed concurrently using the IF and CI methods in autumn and the following spring. Diagnostic thresholds for the IF method were derived using ROC curves and Youden indices to correspond with CI thresholds. Assay specificity was assessed using commercially available ACTH fragments and degradation products of endogenous ACTH. RESULTS: CI and IF methods yielded different results (P<0.001); mean differences (CI-IF), (95% confidence intervals): Autumn (n=99) 38.6 (30.6-46.5) pg/ml, Spring (n=88) 5.1 (3.9-6.3) pg/ml. The association between CI and IF results differed in autumn and spring, consistent with seasonally dependent cross-reactivity or interference. Good (κ=0.66-0.74) agreement was obtained for binary interpretation in spring between IF and CI using thresholds of >24 and >29 pg/ml respectively and in autumn between IF and CI using thresholds of >27 and >47 pg/ml respectively or >33 and >77 pg/ml respectively. Of 88 ponies with both spring and autumn samples, 56 (64%) exceeded a published autumn CI threshold (>47 pg/ml), of which 39 (70%) were below the equivalent threshold (<29 pg/ml) the following spring without treatment. The CI assay showed apparent increases in [ACTH] following addition of CLIP (ACTH 18-39). Degradation of ACTH during storage affected the assays differently. MAIN LIMITATIONS: Limited numbers of PPID cases were included. Immunoreactivity of commercially available peptides may differ from their endogenous equivalents. CONCLUSIONS: The methods yielded different absolute values but the agreement for binary classification was good. An altered pituitary secretome in autumn that affects apparent [ACTH] values is likely. The Summary is available in Spanish - see Supporting information.

The effect of mare obesity and endocrine function on foal birthweight in Thoroughbreds.

BACKGROUND: Birthweight of Thoroughbred foals has increased in recent years. It is unknown whether this is associated with increased broodmare obesity or endocrine dysfunction. OBJECTIVES: To determine insulin, leptin and triglyceride concentrations in Thoroughbred mares throughout gestation and investigate their association with obesity and foal birthweight. STUDY DESIGN: Cohort study. METHODS: A total of 66 mares were included from 40 days post-breeding. Body condition score (BCS), weight and blood samples were obtained every 60 days throughout gestation. Serum/plasma insulin, leptin and triglyceride concentrations and foal birthweight were recorded. Associations between hormone/triglyceride concentration with BCS, stage of gestation and birthweight were analysed using a linear mixed effects model. RESULTS: Serum insulin concentrations were greater at 1-60 days (4.31 µiu/mL) compared with 241-300 days (3.13 µiu/mL) and 61-120 days (5.33 µiu/mL) compared with 181-240, 241-300 and 301-360 days (3.78, 3.13, 3.37 µiu/mL) gestation (P<0.05). There was no significant hyperinsulinaemia and no association of insulin concentration with BCS. Leptin concentration was greater at 181-240 days (2.28 µg/L, P<0.0001) compared with all other time points and correlated with BCS (P<0.0003). Triglyceride concentration was greater at 241-300 days (0.245 mmol, P<0.02) compared with earlier time points, but was not associated with BCS. Foal birthweight was weakly positively correlated with BCS (r = 0.13, P<0.001) and inversely correlated with leptin concentrations at 61-120 and 241-300 days gestation (r = -0.64, P<0.05). MAIN LIMITATIONS: Reduction in sample size over the study and tight clustering of BCS. CONCLUSIONS: Mare BCS correlated with foal birthweight; obese mares had heavier foals. Significant hyperinsulinaemia was not identified in this population. Increased leptin concentration in early and late gestation was associated with decreased foal birthweight. Further work is required to establish whether leptin concentration in late gestation could predict foal birthweight.

Prospective cohort study evaluating risk factors for the development of pasture-associated laminitis in the United Kingdom.

BACKGROUND: Certain individuals appear to be predisposed to recurrent pasture-associated laminitis. Previous studies have predominantly investigated risk factors only after disease occurrence. OBJECTIVES: To investigate risk factors for pasture-associated laminitis prior to disease occurrence. STUDY DESIGN: Prospective cohort study. METHODS: Non-laminitic ponies aged ≥7 years were recruited. Body condition score (BCS), height, weight, crest height and thickness were measured and an overnight dexamethasone suppression test performed. Plasma or serum adiponectin, leptin, triglyceride, basal insulin, insulin post-dexamethasone, insulin-like growth factor 1 (IGF-1), IGF binding protein 1 (IGFBP-1), IGFBP-3, C-reactive protein, von Willebrand's factor, soluble E-selectin and P-selectin concentrations were assayed. Follow-up data were obtained from owners annually for 3 years to ascertain occurrences of veterinarian-diagnosed pasture-associated laminitis. Data were analysed by multivariate logistic regression. Receiver operating characteristic (ROC) curves analysis was performed for significant risk factors and cut-off values determined. RESULTS: A total of 446 animals with a median (interquartile range) age of 15 (10-20) years were recruited. Of these, 50.4% were mares and 49.6% were geldings. The most common breeds were Welsh (36.4%), Shetland (17.0%) and cob (9.4%). Overall, 72.2% of animals were overweight/obese (BCS 7-9/9), 27.3% were of ideal weight (BCS 4-6/9) and 0.5% were underweight (BCS 1-3/9). After 1, 2 and 3 years, respectively, 18 (4.0%), 30 (6.7%) and 44 (9.9%) animals were reported to have had laminitis. Plasma adiponectin, and serum basal (insulin) and (insulin) post-dexamethasone levels were significantly (P ≤ 0.05) associated with laminitis occurrence cumulatively after 1, 2 and 3 years. Use of the area under the ROC curves to distinguish animals that did and did not develop laminitis showed good (basal [insulin] after 1 year), fair (all others) or poor ([insulin] post-dexamethasone) levels of accuracy. MAIN LIMITATIONS: Animals were evaluated at a single time point and biomarkers were assayed using single assays. CONCLUSIONS: Risk factors for future laminitis prior to disease occurrence include low plasma adiponectin and high serum basal insulin or insulin post-dexamethasone concentrations.

Use of the oral sugar test in ponies when performed with or without prior fasting.

BACKGROUND: It is recommended that the oral sugar test (OST) for insulin dysregulation (ID) be performed after an overnight fast, but fasting is impractical in ponies kept solely at pasture. There are few data on OST repeatability and reliability in ponies. OBJECTIVES: To report 1) whether OST results obtained in the morning after an overnight fast or without fasting in the afternoon (FASTING/FED) can be used interchangeably, 2) time of highest insulin concentration Tmax [insulin], repeatability and reliability of insulin response to the OST when FASTING or FED and 3) dichotomous agreement (ID/normal) within a small sample when FASTING or FED. STUDY DESIGN: Method comparison study. METHODS: Oral sugar tests were performed on four occasions in 10 adult native British ponies, twice FASTING and twice FED. Insulin concentrations were measured by radioimmunoassay at 0-120 min (T0,30,60,75,90,120 ). Differences between FASTING and FED results were assessed using mixed effects models. Indices of repeatability and reliability were calculated; dichotomous agreement was reported using kappa statistics. RESULTS: Serum [insulin] was significantly (P≤0.05) higher at T60 -T90 with prior fasting (estimated differences [95% confidence intervals]): T60 : 23.5 µiu/ml (8.7-38.4 µiu/ml), T75 : 27.1 µiu/ml (12.3-41.8 µiu/ml), T90 : 15.1 (0.36-29.9 µiu/ml). Most frequently, Tmax [ins] occurred at T30 . At any single time point, within-subject coefficients of variation were: FASTING: 40% and FED: 31%. The 95% limits for repeatability were FASTING: 29-340%, FED: 41-240%. Test reliabilities were FASTING: 0.70 and FED: 0.67. For dichotomous interpretation similar results (kappa = 0.7) were obtained using cut-offs of [Insulin] >60 µiu/ml at T60 or T90 for FASTING and [Insulin] >51 µiu/ml at T30 or T60 for FED samples. MAIN LIMITATIONS: Oral sugar tests were performed on a small number of animals on one pasture during one season (spring). CONCLUSIONS: Clinicians should beware of interpreting changes in absolute OST results owing to poor repeatability. When stabling is unavailable, OSTs of ponies at pasture may yield similar dichotomous results without prior fasting.

Comparison of the in-feed glucose test and the oral sugar test.

REASONS FOR PERFORMING STUDY: The in-feed oral glucose test (OGT) and oral sugar test (OST) are advocated as field tests of insulin sensitivity in horses and ponies but have not been directly compared. OBJECTIVES: To compare the insulin response to OGT and OST in 8 ponies and 5 horses of unknown insulin sensitivity. STUDY DESIGN: Experimental, randomised crossover study. METHODS: Animals were fasted for 8 h overnight before and throughout testing. They were fed 1 g/kg bwt glucose powder with chaff (OGT) or 0.15 ml/kg bwt corn syrup (Karo™ Light Syrup; OST) was administered per os in a randomised crossover study with 48 h between tests. Blood samples were obtained at 0, 30, 60, 75, 90, 120 and 180 min. The maximal insulin concentration (Cmaxi ), time to maximal insulin concentration (Tmaxi ) and area under the curve of insulin concentration over time (AUCi ) for the tests were compared using Student's paired t test. The effect of individual subject, horse or pony and test were analysed using a linear mixed model. RESULTS: The OGT Cmaxi (mean ± s.d.; 154 ± 116 µiu/ml), Tmaxi (136 ± 52 min) and AUCi (15,308 ± 9886 µiu/ml/min) were significantly (P<0.05) greater compared with the OST Cmaxi (72 ± 55 µiu/ml), Tmaxi (63 ± 25 min) and AUCi (5980 ± 4151 µiu/ml/min). The Cmaxi , Tmaxi and AUCi varied significantly between individual subjects. The Tmaxi was significantly different between horses and ponies during OGT and OST. Using previously defined criteria of insulin dysregulation, OGT identified 7/13 animals as insulin resistant, whereas OST identified 5/13 animals as insulin resistant. CONCLUSIONS: The OGT and OST showed agreement in identification of insulin dysregulation in 85% of equine subjects. Results of the OGT and OST are not comparable in all cases. Further work is required to establish which test more accurately diagnoses insulin dysregulation in horses and ponies.

Clinical Research Abstracts of the British Equine Veterinary Association Congress 2015.

REASONS FOR PERFORMING STUDY: The birthweight of Thoroughbred foals has increased in recent years. It is unknown whether this is associated with increased broodmare obesity or endocrine dysfunction. OBJECTIVES: To determine insulin, leptin and triglyceride concentrations in obese and non-obese Thoroughbred mares throughout gestation and to investigate their association with foal birthweight. STUDY DESIGN: Prospective, cohort study. METHODS: Fifty-seven pregnant Thoroughbred mares were included in the study. From 40 days post-breeding, body condition score (BCS), weight and venous blood samples were obtained every 60 days throughout gestation. Feed was withheld for 6 h before blood sampling. Serum/plasma insulin, leptin and triglyceride concentrations were measured using validated/standard methods. Foal birthweight was recorded. Association of hormone or triglyceride concentration with time, BCS and birthweight were analysed using a linear mixed effects model. A Pearson correlation co-efficient was calculated between hormone or triglyceride concentration, BCS and birthweight. RESULTS: Serum insulin concentrations were significantly greater (P<0.05) at 0-59 days compared with 240-299 days and at 60-119 days compared with 180-359 days gestation. 55% of mares had BCS ≥ 7. There was 1.5% incidence of fasting hyperinsulinaemia throughout gestation and no association of fasting insulin concentration with BCS. Leptin concentration was significantly (P<0.0001) greater at 180-239 days compared with all other time points and was significantly (r = 0.29, P<0.0003) correlated with BCS. Triglyceride concentration was significantly (P<0.02) greater at 240-299 days compared with earlier time points but was not associated with BCS. Foal birthweight was significantly positively correlated with BCS (r = 0.13, P<0.001) and inversely correlated with leptin concentration at 60-119 days and 240-299 days gestation (r = -0.64, P<0.05). CONCLUSIONS: Mare BCS correlated with foal birthweight such that obese mares had heavier foals. Significant fasting hyperinsulinaemia was not identified in this population. Increased leptin concentration in early and late gestation was associated with decreased foal birthweight and may be useful to predict foal birthweight. Ethical animal research: Informed client consent was obtained for all animals used in the study. SOURCE OF FUNDING: Private donor. Competing interests: None declared.

Plasma fructosamine concentrations in horses with pituitary pars intermedia dysfunction with and without laminitis.

REASONS FOR PERFORMING STUDY: Plasma fructosamine concentration ([fructosamine]) is believed to reflect medium term, average blood glucose concentration and in a previous study was higher in horses with active laminitis than in normal horses. Pituitary pars intermedia dysfunction (PPID) is associated with hyperglycaemia and laminitis. OBJECTIVES: To test the hypotheses that: [fructosamine] is higher in PPID cases than normal animals; furthermore, that within cases of PPID [fructosamine] is higher in those with active laminitis than nonlaminitic cases and in cases that have been affected by active laminitis in the preceding year than those that have not. STUDY DESIGN: Observational, case-control/cross-sectional study. METHODS: [Fructosamine] was measured in cases of PPID (n = 46) and normal animals (n = 139). A normal range was calculated; values were compared between the 2 groups and within the PPID group, between cases with and without active laminitis and between cases that had and had not been affected by active laminitis in the preceding year. RESULTS: In normal animals mean [fructosamine] was 248.7 µmol/l; the normal range (mean ± 2 s.d.) was 195.5-301.9 µmol/l. Plasma [fructosamine] was not higher in PPID cases than in normal animals. In PPID cases, [fructosamine] was significantly (P = 0.006) higher in cases with active laminitis (mean ± s.d. 261.2 ± 39.2 µmol/l) compared with those without active laminitis (234.5 ± 32.9 µmol/), but [fructosamine] was not higher in cases that had been affected by active laminitis in the preceding year than those that had not. CONCLUSIONS: In horses affected by PPID, [fructosamine] is higher in cases with active laminitis than in cases without active laminitis. POTENTIAL RELEVANCE: Clinical use of [fructosamine] is limited due to overlap with the normal range. Further studies into glucose and protein metabolism in cases of PPID are warranted.

The effect of exercise on plasma concentrations of inflammatory markers in normal and previously laminitic ponies.

REASONS FOR PERFORMING STUDY: The mechanisms underlying predisposition to pasture-associated laminitis remain unclear; chronic inflammation is implicated, and this may be exacerbated by physical inactivity. OBJECTIVES: To determine whether exercise affects the inflammatory profile of normal and previously laminitic ponies. STUDY DESIGN: Prospective case-control study. METHODS: The short (1 day) and longer term (14 days) effects of low intensity (10 min walking and 5 min trotting) exercise on plasma inflammatory marker concentrations in normal (NL) and previously laminitic (PL) nonobese ponies (n = 6/group) was determined. Plasma concentrations of TNF-α, serum amyloid A (SAA), haptoglobin, insulin, adiponectin and fibrinogen were assayed by validated/standard methods. Data were analysed using a linear mixed effects model. RESULTS: Before exercise, plasma [adiponectin] was significantly (P = 0.0001) lower in PL (mean ± s.d. 2.4 ± 0.1 ng/l) than in NL (4.03 ± 0.2 ng/l), but exercise had no effect. Previous laminitis and exercise had no effect on plasma [TNF-α] or [fibrinogen]. Serum amyloid A concentrations in all ponies were significantly (P = 0.00001) reduced after longer term exercise compared to Day 1 values. Plasma [haptoglobin] was significantly (P = 0.00001) higher in PL compared to NL on Day 1. This difference was no longer apparent after longer term exercise, such that [haptoglobin] in PL had decreased to concentrations similar to NL. Following short-term exercise, all ponies had an initial decrease in serum [insulin] immediately after exercise, followed by an increase peaking 10 min after exercise cessation, before returning to pre-exercise values. On Day 14 these fluctuations were significantly (P = 0.001) reduced in all ponies. CONCLUSIONS: Fourteen days of low intensity exercise significantly decreased [SAA] in all ponies and plasma [haptoglobin] in PL such that it was no longer increased compared to NL. Regular low intensity exercise appears to have an anti-inflammatory effect, which is possibly greater in PL and so may be beneficial in reducing this putative risk factor in pasture-associated laminitis.

Seasonal and annual influence on insulin and cortisol results from overnight dexamethasone suppression tests in normal ponies and ponies predisposed to laminitis.

REASONS FOR PERFORMING STUDY: A simple, accurate test for identifying individual animals at increased risk of laminitis would aid prevention. Laminitis-prone ponies have a greater serum insulin response to dexamethasone administration than normal ponies in the summer, but the response during different seasons is unknown. OBJECTIVE: To test the hypothesis that previously laminitic ponies have a greater insulin response to dexamethasone than normal ponies, which is present during all seasons. STUDY DESIGN: Prospective longitudinal study. METHODS: Overnight dexamethasone suppression tests were performed on 7 normal ponies and 5 previously laminitic ponies in spring 2009 and 2010, summer 2008 and 2010, autumn 2009 and winter 2008, while the ponies were at pasture. In spring 2010, a dexamethasone suppression test was performed after the ponies had been fed only hay for 3 weeks. Serum cortisol and insulin concentrations pre- and post dexamethasone were measured. Linear mixed models were used to analyse the data. RESULTS: Insulin concentrations pre- and post dexamethasone were significantly higher in previously laminitic ponies than in normal ponies during spring 2009 and summer 2008, but there was no difference between groups in spring 2010, summer 2010, autumn 2009 or winter 2008. Insulin concentration varied significantly with season. Diet had no apparent effect on insulin concentration pre- or post dexamethasone in spring 2010. Cortisol concentrations post dexamethasone were significantly higher in previously laminitic ponies than in normal ponies in autumn 2009, with concentrations above the reference range (<25 nmol/l) in both groups in summer 2008 and autumn 2009. Individual ponies had insufficient cortisol suppression in all seasons. CONCLUSIONS: There were significant differences between groups in insulin and cortisol concentrations post dexamethasone during some seasons, but this was not present in all years. Wide interindividual variation in response limits the usefulness of a dexamethasone suppression test for predicting the susceptibility of an individual animal to laminitis. POTENTIAL RELEVANCE: Abnormal insulin and cortisol responses to dexamethasone must be interpreted in the light of the individual animal, seasonal and annual variation reported here.

Seasonal variation in maintenance of phenylephrine-induced tone in isolated equine digital arteries under hypoxic or hyperoxic conditions in vitro.

Digital vasoconstriction, ischaemia and hypoxia may predispose to acute laminitis. Laminitis incidence varies seasonally, peaking in spring and summer. Direct seasonal influences on equine digital artery (EDA) contractility have not been investigated. This study assessed seasonal variation in maintenance of phenylephrine (PHE)-induced tone in isolated EDAs under hypoxic (95% nitrogen) and hyperoxic (95% oxygen) conditions. The objective was to measure change in arterial tone over time after constriction to a plateau with PHE. Tone was measured at plateau and over time and percentage change calculated. Hyperoxic EDAs maintained PHE-induced tone over 1 h with no seasonal variation. Hypoxic EDAs relaxed in fall (median [inter-quartile range] 59% [44-77%] decrease from plateau; P=0.008), contracted in spring (65% [20-192%] increase from plateau; P=0.03) and did not significantly change tone in winter (18% [0-28%] decrease; P=0.13). Continued contraction under hypoxic conditions in spring may contribute to digital vasoconstriction.

Plasma concentrations of inflammatory markers in previously laminitic ponies.

REASONS FOR PERFORMING STUDY: The mechanisms underlying individual animal predisposition to pasture-associated laminitis remain unclear; however, chronic inflammation is implicated. OBJECTIVES: To identify differences in the inflammatory profile of a group of previously laminitic ponies compared with control animals at pasture in late spring and winter. METHODS: Previously laminitic (PL; n = 38 and 42) and nonlaminitic control ponies (NL; n = 41 and 39) were sampled in late spring and winter. Body condition score, height, weight and crest height and thickness were measured. Plasma concentrations of tumour necrosis factor-α, serum amyloid A, haptoglobin, insulin, adiponectin, triglyceride, fibrinogen, interleukin-17, interleukin-4 and interferon-γ were assayed by validated/standard methods. Factors independently associated with each cytokine were determined by multivariate analysis. RESULTS: Plasma [adiponectin] was significantly influenced by laminitis status, being lower in PL (median [interquartile range] 2.1[1.4-3.2] µg/l) than in NL ponies (3.4 [2.6-4.1] µg/l; P<0.0001). No other cytokines or inflammatory markers were associated with laminitis status. Plasma fibrinogen and serum amyloid A concentrations were significantly (P = 0.04 and P = 0.01) higher in geldings (3.5 [3.0-4.0] g/l; 2.2 [0.5-3.6] mg/l) than in mares (3.0 [3.0-4.0] g/l; 1.5 [0.4-2.1] mg/l) and significantly (P = 0.04 and P<0.001) higher in winter (3.5 [3.0-4.0] g/l; 2.5 [0.9-3.6] mg/l) than in late spring (3.0 [3.0-3.5] g/l; 1.1 [0.3-1.9] mg/l). Serum haptoglobin concentration showed the same significant (P<0.001) seasonal difference (winter 2.1 [1.6-2.6 g/l; late spring 1.8 1.4-2.4 g/l) and was significantly (P = 0.01) inversely associated with weight. Serum interleukin-4 concentration was significantly (P<0.0001) higher in winter (2.0 [1.2-3.0] ng/l) than in late spring (0.0 [0.0-0.0] ng/l). Serum insulin concentration was significantly (P = 0.02) influenced by season (winter 31.7 [9.6-43.5] miu/l; late spring 84.0 [7.0-131.0] miu/l). Plasma triglyceride concentration was significantly (P = 0.02) higher in PL (0.5 [0.3-0.7] mmol/l) than in NL ponies (0.4 [0.2-0.5] mmol/l). CONCLUSIONS: There were significant effects of season, gender and bodyweight on a number of proinflammatory mediators or markers of inflammation. The only marker influenced by laminitis status was adiponectin, and concentrations of this anti-inflammatory marker were lower in previously laminitic animals. POTENTIAL RELEVANCE: Recurrent laminitis may be associated with reduced anti-inflammatory capacity rather than a proinflammatory state.

Effect of feeding glucose, fructose, and inulin on blood glucose and insulin concentrations in normal ponies and those predisposed to laminitis.

Identification of ponies (Equus caballus) at increased risk of pasture-associated laminitis would aid in the prevention of the disease. Insulin resistance has been associated with laminitis and could be used to identify susceptible individuals. Insulin resistance may be diagnosed by feeding supplementary water-soluble carbohydrate (WSC) and measuring blood glucose and insulin concentrations. The aim of this study was to assess the glycemic and insulinemic responses of 7 normal (NP) and 5 previously laminitic (PLP), mixed breed, native UK ponies fed glucose, fructose, and inulin [1 g/(kg·d) for 3 d] or no supplementary WSC (control) in spring and fall after a 7-d adaptation to a pasture or hay diet. Blood samples were taken for 12 h after feeding on each day, and baseline and peak concentrations and area under the curve (AUC) for glucose and insulin were recorded. Linear mixed models were used for statistical analysis. Differences between PLP and NP groups were most marked after glucose feeding with differences in peak glucose (P = 0.02) and peak insulin (P = 0.016) concentrations. Season and diet adaptation also affected results. Peak concentrations of glucose and insulin occurred 2 to 4 h after WSC feeding. Peak insulin concentration was greater and more variable in fall, particularly in PLP adapted to fall pasture. Baseline glucose and insulin concentrations varied between individuals and with season and diet adaptation but were not greater in PLP than NP. Insulin AUC was greater in PLP than NP after feeding both glucose and fructose (P = 0.017), but there were no differences between PLP and NP in glucose AUC. Glycemic and insulinemic changes were less (P ≤ 0.05) after feeding fructose than glucose, although differences between PLP and NP were still evident. Minimal changes in glucose and insulin concentrations occurred after inulin feeding. Measurement of peak insulin 2 h after feeding of a single dose of glucose (1 g/kg) may be a simple and practical way to aid identification of laminitis-prone ponies before the onset of clinical disease, particularly when ponies are adapted to eating fall pasture.

Antibiotic resistance in faecal bacteria isolated from horses receiving virginiamycin for the prevention of pasture-associated laminitis.

Enterococcus faecium, a major cause of potentially life-threatening hospital-acquired human infections, can be resistant to several antimicrobials, such that streptogramin quinupristin-dalfopristin (Q/D) is one of the few antibiotics still effective. Consequently use of the streptogramin virginiamycin as an animal growth promoter was banned in the EU in 1999 as some believed this contributed to the emergence of Q/D resistant E. faecium. Virginiamycin is advocated for preventing equine pasture-associated laminitis, but its effect on equine faecal bacterial Q/D resistance has not been determined. Faecal samples were obtained from horses receiving virginiamycin, horses co-grazing and horses not exposed to virginiamycin. Streptogramin resistant E. faecium were cultured from 70% (21/30) of animals treated with virginiamycin, 75% (18/24) of co-grazing animals and 69% (11/16) of animals not exposed. ermB and vatD genes were detected using real time PCR in 63% and 66% of animals treated with virginiamycin, 75% and 71% of co-grazing animals and 63% and 69% of animals not exposed. Antimicrobial resistance genes were present only in samples which had cultured Q/D resistant E. faecium. There was no significant difference between groups with respect to antimicrobial resistance. The gene load of vatD was significantly (p=0.04) greater in unexposed animals compared to those treated with virginiamycin. The use of virginiamycin to prevent pasture-associated laminitis does not appear to be related to an increased Q/D resistance frequency. However, in view of the high frequency of resistance within all groups, the horse is a reservoir of Q/D resistant genes and clones that potentially could be transferred transiently to humans.