Sharanne Raidal

Flow cytometric determination of oxidative burst activity of equine peripheral blood and bronchoalveolar lavage-derived leucocytes

Flow cytometric techniques were developed for the evaluation of oxidative burst activity in equine peripheral blood neutrophils and lymphocytes, as well as bronchoalveolar lavage derived pulmonary alveolar macrophages and lymphocytes. The oxidation of dichlorofluorescin was measured by the increased fluorescence of cells stimulated with phorbol myristate acetate or a variety of other stimulants. Flow cytometry was a suitable method for the evaluation of the intracellular oxidation in all cell populations evaluated. Analysis was rapid and cell separation before analysis was not required. Heterogenous cell populations with differing responsiveness to phorbol myristate acetate stimulated oxidative burst were identified in peripheral blood neutrophil and alveolar macrophage populations. The current study characterizes flow cytometric techniques for the evaluation of oxidative burst activity in equine peripheral blood and bronchoalveolar lavage-derived leucocytes.

Effects of training on resting peripheral blood and BAL-derived leucocyte function in horses

In this study, the effects of prolonged, high intensity training on aspects of peripheral blood and bronchoalveolar lavage (BAL)-derived leucocyte function were evaluated in 8 horses. All horses undertook a 7 week endurance training programme, followed by 5 weeks of high intensity training (HIT). Thereafter, horses were divided into control (C) and overtraining (OT) groups. The frequency and intensity of training were increased more substantially for horses in the OT group. Training was terminated in week 32 when horses in the OT group demonstrated a significant performance reduction. Peripheral blood and BAL samples were collected from 4 horses in C and OT groups in training weeks 7, 11, 14, 18, 22, 28 and 32. Flow cytometric techniques were used to assess phagocytosis by peripheral blood neutrophils and pulmonary alveolar macrophages (PAM), and oxidative burst activity of neutrophils, PAM, peripheral blood and BAL-derived lymphocytes. Peripheral blood neutrophil phagocytosis (internalisation) increased during the initial HIT period and decreased from week 16 when the training workload was increased for both groups. The oxidative burst activity of peripheral blood neutrophils and lymphocytes similarly increased and then decreased in response to training. The oxidative burst activity of PAM was reduced towards the end of the overtraining phase of the programme. Pulmonary alveolar macrophage phagocytosis and oxidative burst activity of BAL-derived lymphocytes demonstrated no change throughout the course of the study. There was no difference in results obtained from C or OT group horses, suggesting that protracted HIT, rather than overtraining, was associated with impaired cell function. The detrimental effects observed in peripheral blood neitrophil and PAM function may indicate impaired nonspecific immunity which may adversely affect the health and performance of horses undergoing protracted periods of intense training.

Pharmacokinetics and Safety of Single and Multiple Oral Doses of Meloxicam in Adult Horses

BACKGROUND Safety of meloxicam, a potent NSAID with selective COX-2 inhibition, has not been evaluated in horses. OBJECTIVES To evaluate pharmacokinetics and safety of single and repeated oral doses of meloxicam in adult horses. ANIMALS Forty-nine healthy, university-owned adult lightbreed horses. METHODS Study conducted in 2 parts. Part I addressed pharmacokinetics of single oral dose meloxicam (0.6 mg/kg) in 16 horses. Part II, 33 horses were randomly assigned to 5 treatment groups to assess prolonged administration (0.6 mg/kg PO q24h for 6 weeks, n = 7) or higher doses (1.8 mg/kg, n = 7, or 3.0 mg/kg PO q24h, n = 7) of meloxicam for 2 weeks, compared with control horses (placebo, n = 7, or phenylbutazone, 4.4 mg/kg q12h on day 1, 2.2 mg/kg q12h for 4 days, then 2.2 mg/kg q24h for 9 days, n = 5). RESULTS Maximum plasma concentration following a single oral dose of meloxicam was 915.1 ± 116.9 ng/mL and elimination half-life 10.2 ± 3.0 hours. Meloxicam (0.6 mg/kg, q24h, PO for 6 weeks) yielded plasma concentrations between 100 and 1000 ng/mL and was well tolerated by healthy adult horses. Administration of 3-5 times the recommended dose of meloxicam was associated with decreased total serum protein and albumin concentrations, gastrointestinal damage, renal damage, or bone marrow dyscrasia. PBZ administration was associated with the development right dorsal colitis, gastric ulceration, and protein losing enteropathy in 2 horses. CONCLUSIONS AND CLINICAL IMPORTANCE Administration meloxicam at 0.6 mg/kg q24h was well tolerated for 6 weeks, without drug accumulation in plasma. Higher doses were associated with dose-dependent adverse effects typical of class of drugs.

Effects of meloxicam and phenylbutazone on equine gastric mucosal permeability.

BACKGROUND Newer NSAIDs that more selectively target the induced isoform of the cyclooxygenase enzyme (COX2) activity might reduce adverse effects while preserving therapeutic benefits of these drugs. OBJECTIVES To compare the effect of oral administration of multiple dose rates of meloxicam and phenylbutazone (PBZ) on gastric mucosal integrity in horses. ANIMALS Twenty-five light breed horses. METHODS In vivo toxicity study. Horses were randomly assigned to 5 treatment groups, receiving placebo, PBZ (4.4 mg/kg PO q12h day 1, 2.2 mg/kg PO q12h for 4 days, 2.2 mg/kg PO q24h for 9 days), or 3 dose rates of meloxicam (0.6 mg/kg q24h, 1.8 mg/kg q24h, 3.0 mg/kg q24h) for 14 days. Sucrose permeability testing was performed on Day 0 (before treatment) and on Day 13. All personnel involved with data collection or analysis were blinded to treatment. RESULTS Administration of PBZ at the above dose rate significantly increased gastric permeability to sucrose, evidenced by increased peak serum sucrose concentrations (280-1,580 pg/μL, P = .001) after treatment. Similar changes were not evident after administration of meloxicam at any dose rate tested, or in control horses (P > .05). Treatment was not associated with significant differences in ulceration of the squamous or glandular mucosa. Peak sucrose concentrations were not correlated with serum total protein or albumin concentrations (R(2) = -0.07, P = .61, R(2) = -0.08, P = .58, respectively). CONCLUSION AND CLINICAL IMPORTANCE These results suggest that PBZ was associated with greater compromise to gastric mucosal integrity than meloxicam.

Pharmacokinetics of potassium bromide in adult horses.

OBJECTIVE To determine the pharmacokinetics of potassium bromide (KBr) in horses after a single and multiple oral doses. ANIMALS Twelve adult Standardbred and Thoroughbred mares. PROCEDURE Horses were randomly assigned into two treatment groups. In Part 1 of the study, horses were given a single oral dose of 120 mg/kg KBr. Part 2 of the study evaluated a loading dose of 120 mg/kg KBr daily by stomach tube for 5 days, followed by 40 mg/kg daily in feed for 7 days. Serum concentrations of bromide were determined by colorimetric spectrophotometry following drug administration to permit determination of concentration versus time curves from which pharmacokinetic parameters could be calculated. Treated horses were monitored twice daily by clinical examination. Serum concentrations of sodium, potassium and chloride ions and partial pressures of venous blood gases were determined. RESULTS Maximum mean serum bromide concentration following a single dose of KBr (120 mg/kg) was 284 +/- 15 microg/mL and the mean elimination half-life was 75 +/- 14 h. Repeated administration of a loading dose of KBr (120 mg/kg once daily for 5 days) gave a maximum serum bromide concentration of 1098 +/- 105 microg/mL. The administration of lower, maintenance doses of KBr (40 mg/kg once daily) was associated with decreased serum bromide concentrations, which plateaued at approximately 700 microg/mL. Administration of KBr was associated with significant but transient changes in serum potassium and sodium concentrations, and possible changes in base excess and plasma bicarbonate concentrations. High serum concentrations of bromide were associated with an apparent increase in serum chloride concentrations, when measured on an ion specific electrode. CONCLUSIONS AND CLINICAL RELEVANCE A loading dose of 120 mg/kg daily over 5 days and maintenance doses of approximately 90-100 mg/kg of KBr administered once daily are predicted to result in serum bromide concentrations consistent with therapeutic efficacy for the management of seizures in other species. The clinical efficacy of this agent as an anticonvulsant medication and/or calmative in horses warrants further investigation.

Health Problems and Risk Factors Associated with Long Haul Transport of Horses in Australia

Simple Summary Records from road transport of horses from Perth to Sydney over a two year period were analysed to explore the incidence of transport related issues and identify risk factors. Transportation resulted in health problems in 2.8% of the transported horses, and in fatalities in 0.24%. Journey duration and season were risk factors for the development of transport related health problems, while breed, sex and age did not predict disease or injury risk. Overall, this study provides statistics to inform policy development for the equine transport industry and enhance management of the transported horse. Abstract Equine transportation is associated with a variety of serious health disorders causing economic losses. However; statistics on horse transport are limited and epidemiological data on transport related diseases are available only for horses transported to abattoirs for slaughter. This study analysed reports of transport related health problems identified by drivers and horse owners for 180 journeys of an Australian horse transport company transporting horses between Perth and Sydney (~4000 km) in 2013–2015. Records showed that 97.2% (1604/1650) of the horses arrived at their destination with no clinical signs of disease or injury. Based on the veterinary reports of the affected horses; the most common issues were respiratory problems (27%); gastrointestinal problems (27%); pyrexia (19%); traumatic injuries (15%); and death (12%). Journey duration and season had a significant effect on the distribution of transport related issues (p < 0.05); with a marked increase of the proportion of the most severe problems (i.e., gastrointestinal; respiratory problems and death) in spring and after 20 h in transit. Although not statistically significant; elevated disease rate predictions were seen for stallions/colts; horses aged over 10 years; and Thoroughbreds. Overall; the data demonstrate that long haul transportation is a risk for horse health and welfare and requires appropriate management to minimize transport stress.

Survey of horse transportation in Australia: issues and practices

OBJECTIVE To survey amateur and professional participants on equine transportation management, practices and outcomes in Australia. METHODS An online survey targeting people who organised horse movements at least monthly was made available to a broad cross-section of amateur and professional equine associations. Respondents were invited to provide demographic details and information relating to their routine transportation management practices and their experiences of issues relating to the transportation of horses. RESULTS Of 797 usable responses involving approximately 17,000 horses and 313,000 individual horse transport events, transport-related behavioural problems were reported by 38% of respondents, particularly at loading. Transport-related health problems had been experienced during or after transportation by horses in the care of 67% of respondents. The most common problems reported were traumatic injuries (45.0%), diarrhoea (20.0%), muscular problems (13.0%), respiratory problems (12.3%), overheating (10.5%) and colic (10.3%). In the 2 years reviewed in the survey, 9.4% of participants reported at least one case of transport-associated pneumonia and 35 horses had died, most commonly from fractures, colic or pneumonia. Although respondents identifying as amateurs transported horses less frequently and over shorter distances, the incidence of transport-related problems was similar between amateurs and professionals. Respondents reported specific precautions before, during and after transportation, although management was often not compliant with the Australian Code of horse transportation. CONCLUSIONS Responses indicated that there remains a substantial risk of adverse welfare and health outcomes for horses transported in Australia and management practices reported may not be compliant with current recommendations for transportation.

The influence of spasmolytic agents on heart rate variability and gastrointestinal motility in normal horses.

The effects of hyoscine-N-butylbromide (hyoscine) and propantheline-bromide (propantheline) on heart rate (HR), HR variability (HRV) and gastrointestinal tract (GIT) contractions in the normal horse were determined. Five adult horses had ECG recordings for 180 min after treatment with propantheline (100mg), hyoscine (120 mg) or saline. Both propantheline and hyoscine reduced GIT sounds, with propantheline having a longer duration of effect (≥120 min). Both drugs elevated HR relative to the control baseline period (P<0.05), with the effects of propantheline again being of longer duration. HRV analysis indicated that propantheline suppressed Total Power (P<0.05), and both the high frequency (HF) and low frequency (LF) components of the power spectral analysis for up to 60-90 min post treatment. Hyoscine had no effect on HRV Total Power but reduced the HF component for 30 min after drug injection. Time domain variables correlated with Total Power and HF data (P<0.01). The marked effect of these compounds on parasympathetic control of cardiac and GIT function in normal horses should be taken into consideration when evaluating a clinical response to these agents.

Immunological, clinical, haematological and oxidative responses to long distance transportation in horses

Horses are transported frequently and often over long distances. Transportation may represent a physiological stressor with consequential health and welfare implications. This study reports the effects of a long distance journey on immunological, clinical, haematological, inflammatory and oxidative parameters in an Experimental Group (EG) of ten horses, comparing them with six horses of similar age and breed used as a non-transported Control Group (CG). Clinical examination and blood sampling were performed twice on all horses: immediately after unloading for the EG, and at rest on the same day for the CG (day 1); at rest on the same day one week later for both groups (day 7). On day 1 EG horses showed increased heart and respiratory rates (P<0.01), rectal temperature (P<0.05), capillary refilling time (P<0.01), neutrophil numbers (P<0.01), serum albumin (P<0.01), plasma total antioxidant status (P<0.01), and a lower rate of mitogen induced proliferation of lymphocytes (P<0.05), in comparison with CG. On day 7 only an increase in total serum protein (P<0.05) and serum globulins (P<0.001) was seen in the EG. No difference in serum cortisol concentration was found. Long distance transportation induced an acute phase response impairing the cell-mediated immune response. Clinical examinations, including assessing CRT and body weight loss, and the monitoring of redox balance may be useful in evaluating the impact of extensive transport events on horses. A better understanding of the link between transportation stress, the immune system and the acute phase response is likely to inform strategies for enhancing the welfare of transported horses.

Determination of sucrose in equine serum using liquid chromatography-mass spectrometry (LC/MS)

Mucosal integrity may be objectively assessed by determination of the absorption of exogenous substances such as sucrose. Gas chromatography-mass spectrometry (GC/MS) and liquid chromatography-mass spectrometry (LC/MS) have been reported for the accurate quantification of low concentrations of sucrose in serum. LC/MS offered the advantage of high sensitivity and mass selectivity without the need for extensive sample derivatization required for GC/MS methods. However, the high polarity and non-volatile nature of the sucrose molecule renders LC/MS techniques challenging. Previously published reports lacked sufficient detail to permit replication of methodology. Problems encountered with existing protocols included poor peak resolution and weak fragmentation of the parent molecule. This communication describes a LC/MS protocol developed to provide improved resolution and product detection.