Histological assessment of ß-amyloid precursor protein immunolabelled rectal biopsies aids diagnosis of equine grass sickness.

BACKGROUND: An accurate, minimally invasive, ante-mortem diagnostic test for equine grass sickness (EGS) is currently lacking. Although histological examination of haematoxylin and eosin-stained rectal biopsies for chromatolytic neurons is insensitive as a diagnostic test for EGS, we hypothesised that its diagnostic accuracy could be improved by immunolabelling for ß-amyloid precursor protein (ß-APP), which has increased expression in cranial cervical ganglia (CCG) neuronal perikarya in EGS. OBJECTIVES: To develop a grading scheme for assessing the distribution and intensity of ß-APP immunoreactivity within individual rectal submucosal neurons and subsequently to determine the value of the distribution of different grades of neurons in EGS diagnosis. STUDY DESIGN: Retrospective case-control diagnostic accuracy study. METHODS: Initially, a standardised grading scheme was developed and ß-APP immunoreactivity in individual neuronal perikarya and axons was compared in sections of CCG and ileum from EGS and control horses. The grading scheme was then refined before being blindly applied to submucosal neurons in rectal biopsies derived from 21 EGS and 23 control horses. RESULTS: ß-APP immunoreactivity was increased in neuronal perikarya and axons in sections of CCG, ileum and rectum from EGS horses compared with controls. For rectal biopsies, a mean immunoreactivity grade exceeding 1.1 was 100% specific and sensitive for EGS, and the presence of at least one neuron with diffuse labelling of the entire cytoplasm (grade 3) was 95% sensitive and 100% specific for EGS. MAIN LIMITATIONS: Although the diagnostic criteria facilitated the discrimination of the EGS and control biopsies evaluated in this study, further prospective validation using a larger sample set is required. CONCLUSIONS: Histological assessment of ß-APP immunolabelled rectal biopsies is more sensitive than conventional histological examination in EGS diagnosis. Further validation is required before this technique can be advocated for use in clinical decision making.

Neurodegeneration in equine grass sickness is not attributable to niacin deficiency.

BACKGROUND: The aetiology of equine grass sickness (EGS) is currently unknown. We hypothesised that an acute deficiency of niacin (vitamin B3), which plays a key role in neural homeostasis, may contribute to neurodegeneration in EGS. Niacin deficiency can potentially result from ingestion of niacin antagonists produced by pasture mycotoxigenic fungi. OBJECTIVES: To compare the niacin status of EGS and control grazing horses. A secondary objective was to compare blood concentrations of vitamins B1, B2 and B6 in EGS and control grazing horses to determine if the status of these vitamins was altered in EGS. STUDY DESIGN: Case-control study. METHODS: Indices of niacin status, namely the erythrocyte nicotinamide adenine dinucleotide:nicotinamide adenine dinucleotide phosphate ratio (NAD:NADP ratio) and erythrocyte concentrations of NAD and NADP, were compared in blood collected from EGS and healthy control grazing horses. Blood concentrations of vitamins B1, B2 and B6 were also compared. RESULTS: There was no significant intergroup difference in the NAD:NADP ratio, the main index of functional niacin status (control group: median 2.1, interquartile range [IQR] 1.8-2.6; EGS group: median 2.1, IQR 1.9-2.6). EGS horses had significantly higher (median value increased by 25%) concentrations of NADP. There were no intergroup differences in blood concentrations of vitamins B1, B2 and B6. MAIN LIMITATIONS: The interpretation of data was limited by the lack of previously defined equine reference ranges for many of the analytes. Sample size was low. CONCLUSIONS: Niacin deficiency does not contribute to EGS neurodegeneration.

Factors affecting the efficiency of aerosolized salbutamol delivery via a metered dose inhaler and equine spacer device.

Despite frequent use of metered dose inhalers (MDIs) and spacers in equine practice, limited information exists on the efficiency of aerosol delivery using such devices. We determined the particle size distribution within an MDI-generated salbutamol aerosol delivered via an equine spacer using 'best practice' delivery technique and assessed the effect of variations in MDI use technique (shaking prior to each actuation, rapid repetitive actuations, and MDI angulation) on aerosol delivery efficiency. Under optimal conditions, only 53(±18) µg salbutamol per 100 µg actuation was delivered beyond the spacer. Although this aerosol had a high [89.6% (±2.4)] fine particle (<5 µm) fraction, and a low mass median aerodynamic diameter [2.52 (±0.29) µm], and particle size variability [geometric SD - 1.66 (±0.16) µm], within all particle size fractions, there was a high coefficient of variance (31-79%) of the percentage salbutamol delivered between experimental runs, thus impeding any effort to predict drug delivery to the patient during equine inhalation therapy. Despite observable trends and with the exception of minor statistically significant changes in the least abundant particle sizes, none of the deviations from a 'best practice' delivery technique significantly altered the relative salbutamol delivery beyond the spacer, a finding which has potential relevance with regard to maintaining user compliance.

Comparative transcriptome analysis of equine alveolar macrophages.

REASONS FOR PERFORMING STUDY: Alveolar macrophages (AMs) are the first line of defence against pathogens in the lungs of all mammalian species and thus may constitute appropriate therapeutic target cells in the treatment and prevention of opportunistic airway infections. Therefore, acquiring a better understanding of equine macrophage biology is of paramount importance in addressing this issue in relation to the horse. OBJECTIVES: To compare the transcriptome of equine AMs with that of equine peritoneal macrophages (PMs) and to investigate the effect of lipopolysaccharide (LPS) on equine AM. STUDY DESIGN: Gene expression study of equine AMs. METHODS: Cells from both bronchoalveolar and peritoneal lavage fluid were isolated from systemically healthy horses that had been submitted to euthanasia. Cells were cryopreserved. RNA was extracted and comparative microarray analyses were performed in AMs and PMs, and in AMs treated and untreated with LPS. Comparisons with published data derived from human AM studies were made, with particular focus on LPS-induced inflammatory status. RESULTS: The comparison between AMs and PMs revealed the differential basal expression of 451 genes. Gene expression analysis revealed an alternative (M2) macrophage polarisation profile in AMs and a hybrid macrophage activation profile in PMs, a phenomenon potentially attributable to a degree of induced endotoxin tolerance. The gene expression profile of equine AMs following LPS stimulation revealed significant changes in the expression of 240 genes, including well-known upregulated inflammatory genes. This LPS-induced gene expression profile of equine AMs more closely resembles that of human rather than murine macrophages. CONCLUSIONS: This study improves current understanding of equine macrophage biology. These data suggest that the horse may represent a suitable animal model for the study of human macrophage-associated lung inflammation and data derived from human macrophage studies may have significant relevance to the horse.

Neuronal chromatolysis in the subgemmal plexus of gustatory papillae in horses with grass sickness.

REASONS FOR PERFORMING STUDY: Diagnosis of equine grass sickness (EGS) can be challenging. We hypothesised that subgemmal plexus neurons are chromatolytic in EGS. If correct, histopathological examination of gustatory papillae biopsies could aid premortem diagnosis of EGS, and EGS could represent a spontaneous model of subgemmal neuronal chromatolysis to facilitate study of the pathology of structures involved in taste. OBJECTIVE: To compare subgemmal plexi and gustatory papillae in EGS and control horses. STUDY DESIGN: Observational study. METHODS: Conventional histology and immunohistochemistry were used to compare subgemmal plexi and gustatory papillae in post mortem samples from 10 EGS and 13 control horses. RESULTS: Chromatolytic neurons were present in all 57 EGS sections which had identifiable neurons, and in only one of 57 control sections. Blinded examination of all haematoxylin-eosin stained sections from each horse for chromatolysis facilitated accurate differentiation of EGS and control horses, with a sensitivity of 100% (95% confidence interval 93.7-100) and specificity of 98.2% (90.6-100) for diagnosing EGS; however, the presence of chromatolytic neurons in one control section indicated that multiple sections per horse must be analysed to achieve diagnostic accuracy. Equine grass sickness was not associated with alterations in taste bud density or morphology, proportion of taste buds with neurofilament immunopositive intragemmal axons or proportion of taste buds containing cells undergoing apoptosis, suggesting taste buds had adequate neurotrophic support at the time of sampling. Horses with EGS had no detectable alteration in lingual gland morphology, but had increased proportions of apoptotic lingual serous gland cells. CONCLUSIONS: While identification of chromatolytic subgemmal neurons in post mortem samples correctly differentiated EGS and control horses, further study is required to evaluate this technique for premortem EGS diagnosis. Equine grass sickness represents a spontaneous model of subgemmal neuronal chromatolysis that facilitates study of the pathology of structures involved in taste.

Equine grass sickness, but not botulism, causes autonomic and enteric neurodegeneration and increases soluble N-ethylmaleimide-sensitive factor attachment receptor protein expression within neuronal perikarya.

REASONS FOR PERFORMING STUDY: Equine grass sickness (EGS) is of unknown aetiology. Despite some evidence suggesting that it represents a toxico-infection with Clostridium botulinum types C and/or D, the effect of EGS on the functional targets of botulinum neurotoxins, namely the soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins, is unknown. Further, while it is commonly stated that, unlike EGS, equine botulism is not associated with autonomic and enteric neurodegeneration, this has not been definitively assessed. OBJECTIVES: To determine: 1) whether botulism causes autonomic and enteric neurodegeneration; and 2) the effect of EGS on the expression of SNARE proteins within cranial cervical ganglion (CCG) and enteric neuronal perikarya. STUDY DESIGN: Descriptive study. METHODS: Light microscopy was used to compare the morphology of neurons in haematoxylin-eosin stained sections of CCG and ileum from 6 EGS horses, 5 botulism horses and 6 control horses. Immunohistochemistry was used to compare the expression of synaptosomal-associated protein-25, synaptobrevin (Syb) and syntaxin within CCG neurons, and of Syb in enteric neurons, from horses with EGS, horses with botulism and control horses. The concentrations of these SNARE proteins in extracts of CCG from EGS and control horses were compared using quantitative fluorescent western blotting. RESULTS: EGS, but not botulism, was associated with autonomic and enteric neurodegeneration and with increased immunoreactivity for SNARE proteins within neuronal perikarya. Quantitative fluorescent western blotting confirmed increased concentrations of synaptosomal-associated protein-25, Syb and syntaxin within CCG extracts from EGS vs. control horses, with the increases in the latter 2 proteins being statistically significant. CONCLUSIONS: The occurrence of autonomic and enteric neurodegeneration, and increased expression of SNARE proteins within neuronal perikarya, in EGS but not botulism, suggests that EGS may not be caused by botulinum neurotoxins. Further investigation of the aetiology of EGS is therefore warranted.

Bodyweight change aids prediction of survival in chronic equine grass sickness.

REASONS FOR PERFORMING STUDY: Objective criteria for predicting survival of chronic grass sickness cases are currently lacking. OBJECTIVES: To determine whether the rate and/or magnitude of bodyweight change during hospitalisation of chronic grass sickness cases can provide an objective predictor of survival to discharge from hospital. Clinicians' recorded indication(s) for euthanasia were also reviewed. STUDY DESIGN: Single centre retrospective observational study. METHODS: Case records of all horses admitted for management of chronic grass sickness to The Dick Vet Equine Hospital between 1998 and 2013 were analysed. Case background, survival to hospital discharge, indication(s) for euthanasia, disease duration at admission and bodyweight changes during the hospitalisation period were analysed, and data for survivors and nonsurvivors compared. Percentage weight change was calculated for 7 day intervals up to 28 days (0-7, 7-14, 14-21, 21-28 days) and for entire periods from the first weight recorded (0-7, 0-14, 0-21, 0-28 days). These results were used to estimate survival probability conditional on weight change. RESULTS: The study sample comprised 213 horses, with 114 survivors (53.5%) and 99 (46.5%) nonsurvivors. Compared with nonsurvivors, survivors had significantly lower median maximum bodyweight loss as a percentage of first weight (survivors 5.9%, interquartile range 1.8-13.5; nonsurvivors 12.7%, 6.4-17.3). Throughout all time periods analysed, survivors had significantly lower median bodyweight loss than nonsurvivors, but no specific time period was more predictive of survival. Highest percentages of total bodyweight loss for individual horses were comparable for survivors (36%) and nonsurvivors (37%). Survival prediction curves reporting percentage survival rates for all time periods analysed provided data to aid prediction of chronic grass sickness survival. CONCLUSIONS: Overall, nonsurvivors had greater bodyweight loss than survivors. Rapidity and magnitude of bodyweight loss were equally predictive of outcome. Percentage survival prediction curves provide objective data to aid discussion of prognosis, but greater predictive specificity with associated sensitivity is required for clinical decision making in individual cases.

Equine grass sickness in Scotland: A case-control study of environmental geochemical risk factors.

REASONS FOR PERFORMING STUDY: We hypothesised that the apparent geographical distribution of equine grass sickness (EGS) is partly attributable to suboptimal levels of soil macro- and trace elements in fields where EGS occurs. If proven, altering levels of particular elements could be used to reduce the risk of EGS. OBJECTIVES: To determine whether the geographical distribution of EGS cases in eastern Scotland is associated with the presence or absence of particular environmental chemical elements. STUDY DESIGN: Retrospective time-matched case-control study. METHODS: This study used data for 455 geo-referenced EGS cases and 910 time-matched controls in eastern Scotland, and geo-referenced environmental geochemical data from the British Geological Survey Geochemical Baseline Survey of the Environment stream sediment (G-BASE) and the James Hutton Institute, National Soil Inventory of Scotland (NSIS) datasets. RESULTS: Multivariable statistical analyses identified clusters of three main elements associated with cases from (i) the G-BASE dataset - higher environmental Ti and lower Zn, and (ii) the NSIS dataset - higher environmental Ti and lower Cr. There was also some evidence from univariable analyses for lower Al, Cd, Cu, Ni and Pb and higher Ca, K, Mo, Na and Se environmental concentrations being associated with a case. Results were complicated by a high degree of correlation between most geochemical elements. CONCLUSIONS: The work presented here would appear to reflect soil- not horse-level risk factors for EGS, but due to the complexity of the correlations between elements, further work is required to determine whether these associations reflect causality, and consequently whether interventions to alter concentrations of particular elements in soil, or in grazing horses, could potentially reduce the risk of EGS. The effect of chemical elements on the growth of those soil microorganisms implicated in EGS aetiology also warrants further study.

Equine grass sickness in Scotland: a case-control study of signalment- and meteorology-related risk factors.

REASONS FOR PERFORMING STUDY: Equine grass sickness (EGS) remains a frequently fatal disease of equids in Britain. Since previous investigations of signalment- and meteorology-related risk factors for EGS have yielded some conflicting data, further investigation is warranted. OBJECTIVES: To identify signalment- and meteorology-related risk factors for EGS in Scotland. STUDY DESIGN: Retrospective time-matched case-control study. METHODS: This study was undertaken using data for 455 EGS cases and 910 time-matched controls that were referred to the Royal (Dick) School of Veterinary Studies, and average UK Meteorological Office weather station meteorological values from the month of admission of the animal, from the 3, 6 and 12 months prior to admission, and for the entire 1990-2006 period. RESULTS AND CONCLUSION: Signalment-related risk factors associated with an increased risk of EGS were native Scottish pure breeds compared with crossbreeds (odds ratio [OR] = 3.56, 95% confidence interval [CI] 2.43-5.43) and animals living on premises located further north within the study region (OR = 1.08, 95% CI 1.06-1.10). There was a decreased risk of EGS in animals aged 11-20 years compared with animals 2-10 years (OR = 0.32, 95% CI 0.22-0.45), non-native Scottish pure breeds compared with crossbreeds (OR = 0.71, 95% CI 0.54-0.94), and stallions compared with mares (OR = 0.43, 95% CI 0.22-0.86). Meteorology-related risk factors associated with an increased risk of EGS were (if Ordnance Survey northing is excluded) more sun hours (OR>1.43) and more frost days (OR>1.13), while there was a decreased risk of EGS with higher average maximum temperature (OR<0.83). POTENTIAL RELEVANCE: The signalment-related risk factors will help owners identify high-risk animals, thereby allowing them to prioritise management strategies. The identification of meteorological risk factors may assist studies on the aetiology of EGS.

An investigation of anthelmintic efficacy against strongyles on equine yards in Scotland.

REASONS FOR PERFORMING STUDY: Cyathostomins comprise 50 helminth species, considered the most problematic equine endoparasites. Three classes of anthelmintic are currently licensed for their control, namely the benzimidazoles (fenbendazole), tetrahydropyrimidines (pyrantel) and macrocyclic lactones (ivermectin and moxidectin). Anthelmintic resistance in cyathostomins is common. With no new classes expected in the near future, it is essential to determine the efficacy of the available anthelmintics to inform future control programmes. OBJECTIVES: To determine the efficacy of all 3 anthelmintic classes against strongyles in equids on livery yards in east and central Scotland. STUDY DESIGN: Anthelmintic efficacy testing using the faecal egg count reduction test (FECRT). METHODS: FECRTs were performed on equids with initial strongyle faecal egg counts (FECs) of ≥50 eggs per gram. Efficacy was determined by comparing pretreatment (Day 0) and 14 days post treatment (Day 14) FECs. The following chemicals were tested: fenbendazole, pyrantel, ivermectin and moxidectin. Group arithmetic mean FECR of >90% for fenbendazole and pyrantel, and >95% for ivermectin and moxidectin, represented efficacy, whereas lower mean FECR indicated potential resistance. RESULTS: A total of 447 FECRTs were performed on 15 yards, as follows (the numbers in parentheses represent the number of yards each anthelmintic was tested on): 55 equids (7 yards) fenbendazole, 111 (8 yards) pyrantel, 163 (13 yards) ivermectin and 118 (10 yards) moxidectin. Fenbendazole resistance was documented on all yards (range of mean FECR, 15.8-83.4%), whereas pyrantel (90.4-99.6%), ivermectin (99.5-100%) and moxidectin (99.4-100%) treatment had acceptable efficacy. CONCLUSIONS: Reduced efficacy of fenbendazole was widespread, whereas >90% efficacy was found after pyrantel, and >95% efficacy after ivermectin and moxidectin. Overall, efficacies were higher than reported previously in Europe and the USA, potentially reflecting differences in management and anthelmintic use on the yards surveyed. POTENTIAL RELEVANCE: The use of fenbendazole for strongyle control in Scotland should be questioned. Targeted use of pyrantel should be encouraged to reduce reliance on macrocyclic lactones. Further work to correlate management practices with the presence of anthelmintic resistance is warranted.

A questionnaire study of equine gastrointestinal parasite control in Scotland.

REASONS FOR PERFORMING STUDY: Anthelmintic resistance in equine gastrointestinal nematodes is a threat to equine health and welfare. Detailed knowledge of anthelmintic use and parasite control methods is a prerequisite to identification of potential risk factors for resistance. OBJECTIVES: To identify parasite control practices employed by equine owners in Scotland and investigate management factors associated with anthelmintic resistance. STUDY DESIGN: Questionnaire study of equine parasite control in Scotland. METHODS: Questionnaires were available electronically, distributed at a conference and mailed to clients. Key areas explored included general background, grazing management, anthelmintic treatment practices and use of diagnostic tests. RESULTS: A total of 193 responses detailing information on parasite control programmes of 993 equids were analysed. Moxidectin (MOX) and ivermectin or related combination products were the most commonly administered anthelmintics in the preceding 12 months. Treatments licensed for use against cyathostomin encysted larvae and tapeworms were administered by 80% and 90% of respondents, respectively. This was often achieved through indiscriminate use of MOX and MOX-praziquantel products. Faecal egg count (FEC) analysis had been performed on 62% of yards and regular use of FECs reduced annual anthelmintic treatment frequency. Veterinarians had the greatest influence on control practices. While 40% of respondents believed that they practised targeted dosing, this was not associated with delaying treatment beyond the egg reappearance period of the anthelmintic used. CONCLUSIONS: Responses indicated increasing veterinary involvement and use of FECs. The majority of respondents administered anthelmintics licensed against cyathostomin encysted larvae and tapeworms. However, responses suggested that owners did not understand the definition of 'targeted' dosing regimens. POTENTIAL RELEVANCE: The high frequency of MOX use represents a potential risk factor for macrocyclic lactone resistance. As veterinarians were the most influential factor in anthelmintic choice, awareness of macrocyclic lactone resistance and potential risk factors for its development and spread should be incorporated into client advice.

In equine grass sickness, serum amyloid A and fibrinogen are elevated, and can aid differential diagnosis from non-inflammatory causes of colic.

Equine grass sickness (EGS) is a debilitating and often fatal neurodegenerative disease. A presumptive diagnosis of EGS may be made on the basis of clinical signs and subjective ancillary tests, but a definitive antemortem diagnosis can only be made following histopathological examination of intestinal biopsies. It has previously been reported that horses with EGS may show clinical and clinicopathological signs of systemic inflammation. The objective of this study was to (a) quantify acute inflammatory markers in blood samples collected from acute, subacute and chronic EGS cases, and (b) compare them with (i) clinically normal horses co-grazing with acute EGS cases (co-grazers), (ii) horses with other causes of colic and (iii) healthy horses. Serum amyloid A (SAA), serum activin A and plasma fibrinogen were quantified. There were marked increases in SAA and fibrinogen in EGS cases compared with healthy horses, co-grazers and non-inflammatory colic cases. The concentrations of SAA and fibrinogen in EGS cases were not significantly different from inflammatory colic cases. When concentrations of SAA, fibrinogen and activin A in each EGS subgroup were compared, no significant differences were detected. Activin A concentrations were significantly elevated in EGS cases and co-grazing horses; this could reflect the presence of subclinical disease in some horses that do not develop clinical signs of EGS, and suggests widespread exposure to the aetiological agent. When faced with sparse antemortem diagnostic techniques, identification of marked increases in acute phase protein concentrations may help to differentiate EGS from other causes of abdominal pain, such as intestinal obstructions; however, there could be diagnostic difficulty in differentiating other inflammatory abdominal conditions, such as peritonitis or enteritis.

Plasma neurofilament pNF-H concentration is not increased in acute equine grass sickness.

Although a presumptive diagnosis of acute grass sickness (AGS) can be made on the basis of clinical signs, a definitive ante mortem diagnosis currently requires histological examination of enteric ganglia. Development of an accurate noninvasive ante mortem diagnostic test is therefore warranted. The objective of this study was to determine whether quantification of the plasma concentrations of the heavily phosphorylated form of major neurofilament subunit NF-H (pNF-H), which mirror the degree of axonal degeneration in some human and animal neurodegenerative disorders, could distinguish AGS-affected and control horses. The pNF-H was quantified in plasma from 20 AGS cases and 20 control horses using a commercial enzyme-linked immunosorbent assay kit. Five AGS and 4 control samples had detectable pNF-H concentrations (>0.0759 ng/ml). There was no significant intergroup difference in pNF-H concentrations. It was concluded that plasma pNF-H is not a useful biomarker for the diagnosis of AGS.

Bronchodilator activity of the selective muscarinic antagonist revatropate in horses with heaves.

Bronchodilators are frequently used to attenuate airway obstruction in equine heaves (or recurrent airway obstruction). This study evaluated the selective (M(3) and M(1)) muscarinic antagonist revatropate, which offers potential advantages over non-specific antimuscarinic agents such as ipratropium. Protocol 1 assessed the response to inhaled revatropate (1, 2 and 7 mg) using a blinded, negative (inhaled saline) and positive (inhaled ipratropium bromide; 0.3, 0.7 and 2mg) controlled, dose escalation study, with six heaves horses. The lowest doses of revatropate and ipratropium induced a rapid (within 1h) and significant improvement in airway function. The highest doses of both drugs had no significant effect on gastrointestinal sound score or iris function, but resulted in tacky mucous membranes and reduced gastrointestinal sound score in some horses. In Protocol 2, a cross-over design comparing the duration of action of inhaled revatropate (1mg), ipratropium (0.3mg) and saline, some indices of airway function were improved for between 5 and 6h after revatropate administration, and for between 6 and 24h after ipratropium administration. Inhaled revatropate and ipratropium had similar effects on airway function, with no significant difference between their efficacies. Importantly, however, only revatropate significantly improved clinical scores of breathing effort, improving combined clinical score at the 1h time point and abdominal score at the 1-3h time points. No significant adverse events were observed in Protocol 2, although some horses had reduced gastrointestinal sound scores. Inhaled revatropate is therefore a safe and effective bronchodilator for treating airway obstruction in heaves.

Short-term incubation of equine laminar veins with cortisol and insulin alters contractility in vitro: possible implications for the pathogenesis of equine laminitis.

This study investigated the effects of cortisol and insulin, hormones that affect both glycaemic status and vascular function, on the in vitro contractility of isolated healthy equine small laminar veins. Small veins (150-500 µm) draining the digital laminae from healthy horses or ponies were investigated by wire myography. Concentration response curves were constructed for noradrenaline (NA), phenylephrine (PE), endothelin-1 (ET-1) and 5-hydroxytryptamine (5-HT) in the presence of either cortisol (10(-6 ) m) or insulin (1000 µIU/mL). Cortisol significantly increased the maximum contractility of laminar veins to the vasoconstrictors NA and 5-HT but decreased the maximal contraction to ET-1. Insulin decreased the contractility of vessels to PE and ET-1. It is possible that short-term cortisol excess could enhance venoconstrictor responses to 5-HT and NA in laminar veins in vivo, thereby predisposing to laminitis. Additionally, a reduction in the ability of insulin to counteract alpha-adrenoreceptor and ET-1-mediated contraction, likely to occur in subjects with insulin resistance, may further exacerbate venoconstriction in animals prone to laminitis. These mechanisms may also predispose horses with disorders such as equine Cushing's disease and equine metabolic syndrome to laminitis.

An update on cyathostomins: anthelmintic resistance and diagnostic tools.

Cyathostomins represent a potential cause of equine morbidity and have become the main focus of endoparasite control in managed horses. All grazing horses are at risk of infection with cyathostomins; therefore, the application of appropriate management measures is essential. Anthelmintics currently comprise the main method of control for equine nematodes and the ready availability of these products in some countries has resulted in their use becoming dissociated from veterinary involvement. This is concerning given the levels of anthelmintic resistance that have been recorded in cyathostomin populations. It is important that veterinarians re-establish control over the implementation of parasite control programmes, a major objective of which should be the preservation of anthelmintic efficacy. This article details the principles of cyathostomin control in horses with particular reference to anthelmintic resistance, and the use and interpretation of diagnostic tests for detecting cyathostomins and identifying anthelmintic resistance.

A nationwide surveillance scheme for equine grass sickness in Great Britain: results for the period 2000-2009.

REASONS FOR PERFORMING STUDY: Equine grass sickness (EGS) remains a frequently fatal disease of equids in Great Britain (GB). The first nationwide surveillance scheme for EGS was developed to obtain information on the occurrence of EGS and to help facilitate future proposed intervention studies, such as vaccine trials. OBJECTIVES: To collect both retrospective and prospective data on cases of EGS occurring in GB since 2000 and to assess potential risk factors for categories of EGS and survival after chronic EGS. METHODS: Prospective data were collected using a nationwide surveillance scheme for EGS that was developed and initiated in 2007. Data were collected by means of postal and online questionnaires documenting both retrospective premises-level and prospective case-level information. Data on outcomes of EGS category and survival after chronic grass sickness (CGS) were analysed using univariable logistic regression. RESULTS: Descriptive data for 1410 EGS cases occurring in GB in the decade between the beginning of 2000 and the end of 2009 are presented. Univariable logistic regression analyses identified that CGS cases in Scotland were significantly more likely to survive than those occurring elsewhere in GB. There was no relationship between case details and the category of EGS or the outcome among CGS cases. CONCLUSIONS: EGS affected equids throughout England, Scotland and Wales. Although an overall average of 141 cases were reported to the nationwide surveillance scheme annually, this number of cases was not consistent between years. Around 50% of CGS cases survived, although regional differences in survival rates existed, probably due to variation in expertise of care and/or disease severity. POTENTIAL RELEVANCE: This study provides information on the spatiotemporal occurrence of EGS in GB during the last decade. Data from the nationwide surveillance scheme can be used in developing protocols for future intervention studies such as Clostridium botulinum vaccine trials.

Effects of equine grass sickness on sympathetic neurons in prevertebral and paravertebral ganglia.

Acute equine grass sickness (EGS) is a fatal disease of horses that is thought to be due to ingestion of a neurotoxic agent causing extensive damage to autonomic neurons. The aim of this study was to compare the effects of EGS on neurons in two sympathetic ganglia, the paravertebral cranial cervical ganglion (CCG) and the prevertebral coeliac/cranial mesenteric ganglion (CG/CMG). Specimens from horses with EGS and controls were obtained post mortem and processed using single and double immunofluorescence labelling for PGP 9.5 and HuC/HuD (pan-neuronal markers), TUNEL and caspase 3 (markers for apoptosis), vasoactive intestinal polypeptide (VIP) and galanin (markers of the cell body response to injury following axotomy or exposure to sympathetic neurotoxins) and tyrosine hydroxylase (TH, marker for noradrenaline synthesis). In control horses, all neurons contained PGP 9.5 and HuC/HuD. There was a significant loss of PGP 9.5 and HuC/HuD expression in samples from horses with EGS that occurred to a greater extent in the CG/CMG than the CCG. The number of caspase 3-positive neurons increased significantly in both ganglia, but TUNEL staining of sympathetic neurons was only significantly increased in the CG/CMG in EGS. No VIP was observed in any ganglia; however, there was a significant increase in galanin-positive neurons in both ganglia in EGS. In the CCG, there was a significant shift towards increased fluorescence intensity for TH, possibly indicating an initial accumulation of TH within the cell body. In contrast, TH fluorescence intensity was significantly reduced in the CG/CMG in EGS correlating with the greater loss of neurons. These results demonstrate that EGS can induce a cell body response that is similar to the response of sympathetic neurons to a chemical neurotoxin. EGS also causes loss of sympathetic neurons, some of which occurs via apoptosis. Changes were more marked in the CG/CMG than the CCG indicating that the prevertebral ganglia were affected earlier than the paravertebral ganglia in the pathological process and had undergone greater neurodegeneration.

Sudden death in racing Thoroughbred horses: an international multicentre study of post mortem findings.

REASONS FOR PERFORMING STUDY: To improve the understanding of exercise related sudden death in Thoroughbred racehorses. OBJECTIVES: To describe the post mortem findings in cases of sudden death associated with exercise in 268 Thoroughbred racehorses. METHODS: Gross and histological post mortem findings of 268 cases of sudden death were collated and reviewed. Cases originated from 6 racing jurisdictions around the world. Sudden death was defined as acute collapse and death in a closely observed and previously apparently healthy Thoroughbred racehorse, during, or within one hour after, exercise. Cause of death as determined by the attending pathologist was categorised as definitive, presumptive or unexplained and compared between the different populations. Cardiopulmonary lesions recorded at post mortem examination were compared between different populations. RESULTS: Pathologists recorded a definitive cause of death in 53% (143/268) of cases. Major definitive causes of sudden death included cardiac failure, apparent pulmonary failure, pulmonary haemorrhage, haemorrhage associated with pelvic fractures or with idiopathic blood vessel rupture, and spinal cord injury. A presumptive cause of death was made in 25% (67/268) of cases and death remained unexplained in 22% (58/268) of cases. There were several statistically significant inter-population differences in the cause of death and in reporting of cardiopulmonary lesions. CONCLUSIONS: Sudden death can be attributed to a variety of causes. Causes of sudden death and the lesions found in cases of exercise-related sudden death are similar in different racing jurisdictions. However, the lesions are often not specific for the cause of death and determination of the cause of death is therefore affected by interpretation by the individual pathologist.