N. J. Menzies-Gow

Hypertension and insulin resistance in a mixed-breed population of ponies predisposed to laminitis

OBJECTIVE To determine the metabolic phenotype of a group of laminitis-prone ponies when at pasture in summer, compared with when at pasture in winter. ANIMALS 40 ponies of various breeds predisposed to recurrent pasture-associated laminitis and 40 unaffected control ponies. PROCEDURES Body condition score and size of the crest of the neck were assessed, blood samples obtained, and blood pressure measured by use of an indirect oscillometric technique, while ponies were kept on winter pasture (last week of November or beginning of December) and again on summer pasture (June). Serum insulin concentration and plasma glucose, triglyceride, uric acid, and ACTH concentrations were measured. Insulin sensitivity was calculated with proxies derived from basal serum insulin and plasma glucose concentrations. RESULTS No significant differences were apparent between ponies predisposed to laminitis and control ponies during winter. However, in June, laminitis-prone ponies had increased serum insulin concentration and plasma triglyceride and uric acid concentrations, compared with control ponies. Also, laminitis-prone ponies were relatively insulin resistant, compared with control ponies. Mean blood pressure was significantly higher during summer in laminitis-prone ponies (median [interquartile range], 89.6 mm Hg [78.3 to 96.9 mm Hg]), compared with control ponies (76.8 mm Hg [69.4 to 85.2 mm Hg]). CONCLUSIONS AND CLINICAL RELEVANCE Summer pastures appear to induce metabolic responses in some ponies, leading to expression of the prelaminitic phenotype, which includes hypertension as well as insulin resistance. Signs of this metabolic syndrome may not be apparent in affected ponies during periods of grazing winter pasture. Understanding this syndrome may enable improved countermeasures to be devised to prevent laminitis.

Endotoxin-induced digital vasoconstriction in horses: associated changes in plasma concentrations of vasoconstrictor mediators

REASONS FOR PERFORMING STUDY Lipopolysaccharide (LPS) infusion reduces digital perfusion, but the mediators responsible remain undetermined. OBJECTIVES To identify vasoconstrictor mediators released following LPS infusion and relate their appearance in plasma to digital blood flow alterations. METHODS Blood flow in the lateral digital vessels of 6 Thoroughbred horses, following a 30 min infusion of LPS (E. coli 055:B5; 30 ng/kg), was measured using Doppler ultrasonography. Concomitant measurements of hoof wall and coronary band surface temperatures (HWST and CBST) were made. Serial blood samples were collected and plasma LPS, tumour necrosis factor alpha (TNFalpha), 5-HT, thromboxane B2 (TxB2) and endothelin measured. RESULTS Plasma LPS concentrations reached a maximum of 13.2 pg/ml during the infusion, followed by an increase in plasma TNFalpha concentration. Digital arterial and venous blood flow decreased by 43 and 63%, respectively; HWST and CBST similarly decreased. Systemic blood pressure remained unaltered. Plasma concentrations of TxB2 and 5-HT increased, coinciding with the onset of digital hypoperfusion. Plasma endothelin concentrations remained unchanged. CONCLUSIONS The temporal relationship between the onset of digital hypoperfusion and increases in plasma 5-HT and TxB2 concentrations is consistent with these platelet-derived mediators being associated with LPS-induced laminitis. POTENTIAL RELEVANCE These experimental data support the use of anti-platelet therapy in the prevention of laminitis associated with endotoxaemic conditions.

The effects of vasoactive amines found in the equine hindgut on digital blood flow in the normal horse

REASONS FOR PERFORMING STUDY Disturbances of digital blood flow are thought to be fundamental to the pathophysiology of acute laminitis. However, factors linking the initiating events in the equine hindgut with these disturbances in the foot remain to be determined. HYPOTHESIS Amine compounds, formed by bacteria in the equine hindgut, have digital vasoconstrictor effects in vivo. METHODS Tryptamine (1.6 microg/kg/min) and phenylethylamine (2.13 microg/kg/min) were infused i.v. into standing nonsedated horses. Digital blood flow was measured by Doppler ultrasound and foot surface temperature was monitored. Plasma 5-hydroxytryptamine (5-HT) concentrations were measured by HPLC. RESULTS Tryptamine and phenylethylamine infusions had no effect on systemic arterial blood pressure or heart rate, but caused significant decreases in digital arterial blood flow (mean +/- s.e. 29.2 +/- 8.5 and 18.4 +/- 6.8%, respectively). Both amines also caused decreases in dorsal hoof wall temperature (0.6 +/- 0.1 and 0.5 +/- 0.1 degrees C for tryptamine and phenylethylamine, respectively) and concomitant increases in plasma 5-HT concentration. CONCLUSIONS Tryptamine and phenylethylamine caused reduction of digital blood flow, effects which may have been mediated, in part, via displacement of 5-HT from platelets. POTENTIAL RELEVANCE Amine compounds occurring in the equine hindgut, if released into the circulation following carbohydrate overload, could contribute to selective digital vasoconstriction. Further work in ponies and horses, with naturally occurring laminitis, is necessary to determine whether amines represent a therapeutic target in this disease.

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.

Epidemiological study of pasture-associated laminitis and concurrent risk factors in the South of England

A retrospective study of laminitis was carried out to identify risk factors associated with this disease on an East Anglian farm with approximately 1000 animals living in an area of 1000 acres. Medical records between January 1997 and May 2000 and between April 2005 and March 2008 were reviewed, and the age, sex, weight (kg), height (inches [in] and hands [H]) and weight-to-height ratio (kg/in) was recorded. The prevalence, incidence and seasonality of laminitis were determined and their relationship to the monthly temperature, rainfall and hours of sunshine was evaluated. Averaged over the six years, the highest prevalence (2.6 per cent) and incidence (16 cases/1000 animals) of laminitis occurred in May. The findings of a multivariate analysis revealed that females (P=0.007, odds ratio [OR] 1.46, 95 per cent confidence interval [CI] 1.1053 to 1.9646) and light animals (P≤0.001, OR=0.995, 95 per cent CI =0.9932 to 0.9963) had the greatest risk of developing laminitis. A positive association was found between hours of sunshine and incidence (P=0.007, relative risk [RR] 1.009, 95 per cent CI 1.001 to 1.012) and prevalence (P=0.002, RR 1.008, 95 per cent CI 1.003 to 1.012) of laminitis. The data suggest that there is a relationship between season, sex of the animal and the development of laminitis.

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.

Conjunctival bacterial and fungal flora in healthy horses in the UK.

OBJECTIVES To describe the bacterial and fungal flora of the normal conjunctiva of horses in the UK; to determine the effect of horse age, sex, geographic location, and housing on this flora; and to determine the most appropriate antimicrobial drug(s) for prophylactic treatment of corneal ulcers. ANIMAL STUDIED A total of 60 adult healthy horses were studied. PROCEDURE Swabs of the conjunctiva were obtained from 60 horses housed in two locations within the UK. Specimens were cultured for aerobic bacteria and fungi, and sensitivity against six ophthalmic antimicrobials assessed. The effect of age, sex, location and housing on the frequency of microbial isolation was evaluated. RESULTS Fifty-four bacterial isolates, representing thirteen genera of bacteria were cultured from 31 (52%) horses. The most frequently isolated bacterial species was Acinetobacter sp (17/32 horses). The majority of isolates (28/54: 52%) were gram-positive. Three genera of fungi (Mucor, Absidia and Aspergillus spp) were isolated from eight (13%) horses. There was no significant effect of geographic location, sex, age or housing on frequency of microbial isolation. Horses from which gram-negative bacteria were isolated were significantly older than horses from which gram-positive bacteria were isolated. High efficacy (greater than 90% of isolates sensitive in vitro) was displayed by chloramphenicol, gentamicin and tetracycline. CONCLUSIONS The microbial species isolated are comparable with studies performed in other countries, although the frequency of Acinetobacter isolation was higher which may reflect a geographic difference. The topical antimicrobials gentamicin and chloramphenicol are appropriate first line antimicrobials for empirical treatment of corneal ulcers in the UK.

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 Willebrands 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.

Severity and outcome of equine pasture-associated laminitis managed in first opinion practice in the UK

Data from 107 cases of pasture-associated laminitis were obtained from first opinion practices to study factors associated with severity, survival and return to ridden exercise. There were 43 mares and 64 geldings, with a median age of 11 years. Of the 107 animals, 33 were small ponies, 45 were large ponies/cobs, 17 were small horses and 12 were large horses. Ninety-seven animals were categorised as having laminitis as defined by Cripps and Eustace 1999): 76 had mild (Obel grade 1 or 2) laminitis and 31 had severe (Obel grade 3 or 4) laminitis. Forty-three animals had previously had laminitis, and were significantly less likely (P=0.02) to have severe laminitis than those that had not. Eighty-nine animals were overweight, and there was a trend (P=0.09) towards severe laminitis cases having a higher body mass index. Eight weeks after disease onset, 102 animals were alive. Lower bodyweight, optimal body condition, mild laminitis and category of acute/chronic founder as defined by Cripps and Eustace (1999) were significantly associated with survival. There was a trend (P=0.06) towards treatment with acepromazine being associated with survival. Of the 81 animals that were used for riding, 48 were being ridden again; this was 2.6 times more likely in animals without previous laminitis. The clinical outcome was judged by a panel of three veterinarians as ‘good’ in 77 of 107 of cases. Clinical outcome was significantly associated (P=0.03) with horse type: the outcome was ‘bad’ in none of the small horses, compared with 15 of 45 large ponies/cobs, 11 of 33 small ponies and three of 12 large horses.

Roles of thromboxane A2 and 5-hydroxytryptamine in endotoxin-induced digital vasoconstriction in horses

OBJECTIVE To evaluate the roles of 5-hydroxytryptamine (5-HT), thromboxane A2 (TxA2), and platelet-activating factor (PAF) in endotoxin-induced digital hypoperfusion in horses. ANIMALS 6 healthy adult Thoroughbreds. PROCEDURES Horses were treated with IV administration of saline (0.9% NaCl) solution (control treatment) or the 5-HT 1B/D selective antagonist, GR55562 (0.3 mg/kg), prior to tryptamine infusion (1.6 microg/kg/min for 30 minutes) to establish an effective GR55562 dose. In a crossover study, horses were treated with IV administration of saline solution (control treatment), aspirin (4 mg/kg, 2 hours or 4 days before lipopolysaccharide [LPS] infusion), GR55562 (0.3 mg/kg), the PAF antagonist WEB2086 (3 mg/kg), or aspirin plus GR55562 prior to LPS infusion (30 ng/kg for 30 minutes). Digital blood flow was measured by use of Doppler ultrasonography. Concomitant measurements of hoof wall and coronary band surface temperatures were made. Serial blood samples were collected and plasma 5-HT and TxA2 concentrations determined. RESULTS GR55562 abolished tryptamine-induced digital hypoperfusion. Neither WEB2086 nor GR55562 affected LPS-induced alterations in digital perfusion or plasma mediator concentrations. Aspirin given 2 hours before LPS administration abolished the increase in plasma TxA2 concentration and significantly attenuated LPS-induced digital hypoperfusion. Aspirin given 4 days before LPS significantly attenuated the increase in plasma TxA2 concentration and digital hypothermia. Aspirin plus GR55562 had a greater effect on LPS-induced digital hypothermia than aspirin alone. CONCLUSIONS AND CLINICAL RELEVANCE Thromboxane A2 and 5-HT played a role in mediating LPS-induced digital hypoperfusion in horses. Platelet-activating factor appeared unimportant in mediating LPS-induced 5-HT or TxA2 release or digital hypoperfusion.