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.

Prevalence of Clostridium perfringens in faeces and ileal contents from grass sickness affected horses: comparisons with 3 control populations.

REASONS FOR PERFORMING STUDY: While previous studies have demonstrated an association between equine grass sickness (EGS) and the presence of Clostridium botulinum within ileal contents and faeces, no such associations with other intestinal-derived anaerobic bacteria have been extensively investigated. HYPOTHESIS: The prevalence of C. perfringens in the ileal contents and faeces of EGS horses is greater than control horses; the detection of C. perfringens in faeces by ELISA could be diagnostically beneficial in a clinical setting. METHODS: The prevalence of C. perfringens in faeces from EGS horses and healthy grazing control horses was determined by both selective culture and ELISA to permit both validation of the ELISA and inter-group comparisons. Additionally, the prevalence of C. perfringens (ELISA) in ileal contents from EGS horses was compared with that for control horses with nongastrointestinal disease. Finally, the prevalence of C. perfringens (ELISA) in faeces from EGS cases was compared with that from both horses with which they shared pasture at the time of disease onset and non-EGS colic horses. RESULTS: When compared with culture, the ELISA had a sensitivity and specificity of 86 and 98%, respectively. The prevalence of C. perfringens in faeces as determined by both culture and ELISA was significantly higher (P<0.001) for EGS horses (7/9 and 15/37, respectively) than for healthy grazing controls (0/60 and 1/74, respectively). The prevalence of C. perfringens in ileal contents from EGS horses (5/10) was greater than that for horses with nongastrointestinal disease (1/12) at a level that approached significance (P = 0.056). EGS cases had a significantly greater prevalence of C. perfringens in faeces (15/37) than co-grazing horses (1/18) and colic (1/16) horses. The specificity (93%) and PPV (94%) of the detection of C. perfringens by ELISA on faecal samples in relation to disease status (EGS compared with colic horses) was good. Sensitivity (41%) and NPV (39%) were poor. CONCLUSIONS AND POTENTIAL RELEVANCE: The use of a commercial ELISA to detect faecal C. perfringens may be diagnostically beneficial when differentiating EGS cases from colic cases, although further work is required to fully evaluate its potential.

Retrospective evaluation of episodic collapse in the horse in a referred population: 25 cases (1995-2009).

BACKGROUND: Episodic collapse in horses has equine welfare and human safety implications. There are, however, no published case series describing this syndrome. OBJECTIVES: To characterize the cause and outcomes for horses referred for investigation of episodic collapse. ANIMALS: Twenty-five horses referred for investigation of single or multiple episodes of collapse. METHODS: Retrospective study. Clinical records from the Dick Vet Equine Hospital, University of Edinburgh from November 1995 to July 2009 were searched using the following keywords: collapse, collapsing, fall, syncope. Collapse was defined as an incident in which the horse lost postural tone with or without progression to recumbency and with or without loss of consciousness. Long-term follow-up information was obtained by telephone conversation with the owner. RESULTS: A final diagnosis was reached in 11 cases, namely cardiac arrhythmia (4), right-sided heart failure (1), hypoglycemia (2), generalized seizures (2), and sleep disorder (2). A presumptive diagnosis was reached in 8 cases, namely neurocardiogenic syncope (5), exercise-induced pulmonary hemorrhage (2), and generalized seizures (1). No diagnosis was reached in 6 cases despite comprehensive investigations. Three horses were euthanized at presentation. Treatment was attempted in 9 horses with 6 cases having successful outcome before discharge. Follow-up information was available for 14 of 19 horses discharged from the hospital. Only 1 of these horses was observed to collapse after discharge. CONCLUSIONS AND CLINICAL IMPORTANCE: Definitive diagnosis was more likely to be reached in cases with multiple episodes of collapse. Horses in which 1 episode of collapse occurred did not necessarily collapse again.

An update on equine post-operative ileus: Definitions, pathophysiology and management.

Post-operative ileus (POI) is a serious condition which any horse undergoing abdominal surgery is at risk of developing, leading to increased hospitalisation time and resulting costs. Advances in the understanding of the development of equine POI are mainly based on human and rodent literature, where manipulation-induced inflammation has been identified as a trigger, with activation of resident muscularis externa macrophages playing a crucial role in the pathophysiology. Despite many pharmacological trials in all species, there is no single completely successful treatment for POI, highlighting that the condition is multifactorial in cause and requires a multimodal approach to minimise its incidence.

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.

Respirable dust concentrations in equine stables. Part 1: validation of equipment and effect of various management systems.

Traditional methods of measuring airborne dust concentrations (ADC) in animal housing have included the collection of dust onto pre-weighed filters permitting the calculation of mean, not maximum, ADC. However real-time continuous particle monitors are advantageous in identifying short duration elevations in ADC which may be detrimental to equine respiratory health in the face of a relatively low mean ADC. These monitors have not previously been used to measure ADC in equine stables. Comparisons of a filter-based sampler and a real-time continuous particle monitor revealed no significant difference (P=0.079) and good agreement (>or=95% of the points fell within two standard deviations of the mean of the differences and the mean of the differences approximated zero) between the devices, with respect to mean respirable dust concentration (RDC) measurements. Investigations of the influence of various equine management systems on RDC revealed that both mean and maximum breathing zone RDC were significantly reduced (P<0.05) in equine stables by changing the environment from hay feed and straw bedding, to haylage feed and wood shavings bedding (reduction in mean - 0.0867mg/m(3) to 0.0260mg/m(3); reduction in maximum - 4.0758mg/m(3) to 0.2182mg/m(3), respectively).

Respirable dust concentrations in equine stables. Part 2: the benefits of soaking hay and optimising the environment in a neighbouring stable.

The effect of soaking hay to minimise equine breathing zone respirable dust concentration (RDC) is unknown, as is the duration of soaking required. Additionally, the influence of the bedding and forage used in one stable on the mean and maximum RDC in a neighbouring stable within a common airspace is unknown. Consequently, in the management of equine environmental respiratory disease uncertainty remains about the necessity for optimising conditions in neighbouring stables. Investigations using a real-time continuous particle monitor revealed that when feeding hay, horses' mean breathing zone RDC was significantly reduced if the hay was immersed or soaked for a prolonged time, prior to feeding. There was no advantage in soaking for an extended time period. Implementing management changes in one stable (changing from straw bedding and hay feeding to wood shavings bedding and haylage feeding) significantly reduced mean and maximum background RDC in a neighbouring stable within a common airspace.

Comparison of IgG antibody levels to Clostridium botulinum antigens between euthanased and surviving cases of chronic grass sickness.

Serum from 12 horses suffering from chronic grass sickness (CGS) were assayed for IgG antibodies against botulinum neurotoxins C and D (BoNT/C and BoNT/D) and to a surface antigen extract of a neurotoxin negative strain of Clostridium botulinum type C. Collectively, the six surviving CGS cases demonstrated significantly higher initial IgG levels (P=0.05) against surface antigens than the six that were subsequently euthanased. The surviving animals also demonstrated higher initial IgG levels against the BoNT/C but not reaching significance (P=0.06). The two groups demonstrated no difference between IgG levels against BoNT/D. This study supports existing evidence of the involvement of C. botulinum type C in the aetiology of grass sickness.

Preliminary study of mucosal IgA in the equine small intestine: specific IgA in cases of acute grass sickness and controls.

REASONS FOR PERFORMING STUDY: There is much evidence to suggest that group III Clostridium botulinum (types C and D) are involved in the aetiology of equine grass sickness (EGS). Antibodies have been detected previously in the blood and high levels associated with resistance to disease. Specific mucosal antibodies in the gastrointestinal (GI) tract are likely to be important in protection, and this study was performed to ascertain if such antibodies could be detected and if their levels were related to disease state. OBJECTIVES: To develop a method for quantifying IgA antibodies to C. botulinum types C and D in the GI tract of horses and to relate antibody levels to disease status. METHODS: Samples of tissue (n = 25: 6 duodenum, 7 jejunum and 12 ileum) were taken from acute grass sickness (AGS) cases and from control horses (n = 12; 4 samples from each site) at post mortem. They were extracted with the detergent saponin in the presence of protease inhibitors and assayed for total IgA, for specific IgA against botulinum neurotoxins types C and D (BoNT/C or BoNT/D), and against surface antigens of a BoNT/C negative strain of C. botulinum type C (SA) and of Clostridium tetani (TetSA), as a control. Specific IgA was expressed as percentage total IgA. RESULTS: Compared to controls, significantly higher levels of specific IgA against BoNT/C were detected in the jejunum (P = 0.04) and ileum (P = 0.02) of AGS cases. Similarly, higher specific levels against BoNT/D were demonstrated in duodenum (P = 0.01) and jejunum (P = 0.02). Significantly higher levels of IgA against SA were demonstrated only in duodenal samples (P = 0.01). CONCLUSIONS: Levels of IgA antibody to BoNTs in control horses were at near undetectable levels, suggesting no recent exposure to toxins. In AGS cases, significantly higher levels of specific IgA were detected predominantly in jejunum and ileum. POTENTIAL RELEVANCE: If specific IgA is protective then any successful vaccine for EGS should induce a mucosal response.

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.

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.

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 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.

Clinical features and management of equine post operative ileus: Survey of diplomates of the European Colleges of Equine Internal Medicine (ECEIM) and Veterinary Surgeons (ECVS).

REASONS FOR PERFORMING STUDY: There is a need for an improved understanding of equine post operative ileus (POI), in terms of both clinical definition and optimal management. Although the pharmacological strategies that are used to treat POI continue to evolve, little is known about the supplementary strategies used to prevent and manage this condition. OBJECTIVES: To report the current strategies used to diagnose, prevent and manage POI following emergency abdominal surgeries. STUDY DESIGN: Cross-sectional survey. METHODS: An electronic survey invitation was sent by email to 306 European college diplomates (European Colleges of Equine Internal Medicine, ECEIM n = 120, and Veterinary Surgeons, ECVS n = 186). RESULTS: The response rate was 33% (100 of 306). The median reported estimated incidence of POI was 10-20%. The presence of reflux on nasogastric intubation was the main criterion used to define POI. Lesions involving the small intestine were thought to be the leading risk factors for developing POI. Anti-inflammatory drugs, antimicrobial drugs and i.v. fluids were the primary preventative strategies when managing cases at high risk for POI. Flunixin meglumine and lidocaine were the drugs most commonly used to treat horses with POI. Supplementary preventative and treatment strategies for POI included control of endotoxaemia, fluid therapy, early ambulation and judicious timing of post operative feeding. CONCLUSIONS: Appreciation of the potential risk factors associated with the development of POI appeared to have an impact on the choice of management strategies that are implemented. The majority of ECEIM and ECVS Diplomates in the survey used flunixin meglumine and lidocaine, often in combination, to treat horses with POI, which is likely to reflect the contributory role of inflammation in its pathophysiology. Various supplementary strategies were used to prevent and manage POI risk factors intraoperatively and post operatively.

Clinical features and management of equine post operative ileus (POI): Survey of Diplomates of the American Colleges of Veterinary Internal Medicine (ACVIM), Veterinary Surgeons (ACVS) and Veterinary Emergency and Critical Care (ACVECC).

REASONS FOR PERFORMING STUDY: A recent survey of European Colleges (European College of Equine Internal Medicine [ECEIM] and European College of Veterinary Surgeons [ECVS]) revealed the different strategies implemented by, and some of the challenges facing, European clinicians presented with cases of post operative ileus (POI). It was concluded that further comparative analysis of opinions, canvassed from additional colleges of equine veterinary specialism worldwide, would provide valuable additional insight into current POI knowledge on a more global scale. OBJECTIVES: To report and compare the current strategies favoured by American veterinary specialists when managing POI in horses that underwent emergency colic surgery. STUDY DESIGN: Cross-sectional survey. METHODS: Electronic invitations were sent to 814 Large Animal specialists, including 3 colleges: the American College of Veterinary Internal Medicine (ACVIM), American College of Veterinary Surgeons (ACVS) and the American College of Veterinary Emergency and Critical Care (ACVECC). RESULTS: The response rate was 14% (115/814). The majority of respondents (68%) reported an estimated prevalence range of POI of 0-20%. The presence of reflux on nasogastric intubation was the main criterion used to define POI. A lesion involving the small intestine was considered the main risk factor for POI. Anti-inflammatory drugs, intravenous (i.v.) fluids and antimicrobial drugs were the primary strategies used when managing POI. Flunixin meglumine and i.v. lidocaine were the drugs most commonly used in the treatment of horses with POI. Supplementary management strategies targeted mainly the prevention of post operative adhesions, infection and inflammation. CONCLUSIONS: There is a lack of consensus on the clinical definition of POI. Prospective and objective clinical assessment of the effectiveness of the different strategies contained within this and the European survey is necessary in order to identify a standardised approach to the management of equine POI.

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, 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.

Cytokine profiles of peripheral blood and airway CD4 and CD8 T lymphocytes in horses with recurrent airway obstruction.

Equine recurrent airway obstruction (RAO) is thought to result from an aberrant immune response to inhaled antigens, modulated by T lymphocytes via the secretion of pro-inflammatory cytokines. However data relating to the phenotypes of the T lymphocytes present in peripheral blood and bronchoalveolar lavage fluid of RAO horses and their cytokine profiles are contradictory. The aim of this study was to further investigate the cytokine (IL-4, IL-5, IL-13 and INF-gamma) mRNA expression profile in peripheral blood lymphocytes and bronchoalveolar lavage lymphocytes from RAO and control horses, before and at 48 h after horses were exposed to hay/straw. In contrast to previous studies, cytokine expression was quantified in populations of CD4 and CD8 T lymphocytes which were purified using magnetic bead antibody cell separation. Hay/straw exposure induced clinical airway obstruction, airway neutrophilia and airway lymphocytosis in RAO horses, and, induced a mild, but significant, airway neutrophilia in controls. However, hay/straw exposure had no significant effect on peripheral blood lymphocyte or bronchoalveolar lavage lymphocyte cytokine expression in either group. In conclusion, RAO was not associated with alterations in lymphocyte cytokine expression that are consistent with Th1 or Th2 responses, but rather with a general down-regulation in expression of the measured cytokines in peripheral blood lymphocytes and bronchoalveolar lavage lymphocytes.

Effect of composition and different fractions of hay dust suspension on inflammation in lungs of heaves-affected horses: MMP-9 and MMP-2 as indicators of tissue destruction.

REASONS FOR PERFORMING STUDY: Airway matrix metalloproteinases (MMPs) increase following inhalation of organic dust. The relative contribution of dust components to this elevation is unknown. OBJECTIVE: To identify components of organic dust responsible for elevated MMP levels in equine airways. METHODS: Bronchoalveolar lavage (BALF) from 7 heaves-susceptible horses, collected 6 h following inhalation challenges with saline, 2 different hay dust suspensions (HDS-1 and -2) and soluble and particulate fractions of HDS-1, were analysed for MMP-2 and -9 using SDS-page gelatin zymography. RESULTS: HDS-1 challenge increased BALF proMMP-9 and total MMP-9. HDS-1 fractions, or the particulate fraction with added lipopolysaccharide, increased BALF proMMP-9 and total MMP-9 in combination, but not when inhaled separately. HDS-2 inhalation elevated BALF complex forms, proMMP-9, active MMP-9, total MMP-9 and total MMP-2. CONCLUSIONS: The results suggest synergistic action of soluble and particulate organic dust components. The fact that HDS-1 and HDS-2 had different glucan concentrations supports a role for moulds in the activation of MMP-9. POTENTIAL RELEVANCE: Activation and release of MMPs in response to inhaled moulds are involved in the aetiopathogenesis of heaves. Endotoxin contributes to the synergistic action of the dust components, but the overall MMP response to organic dust inhalation in heaves-susceptible horses largely reflects the mould content of the dust. In the future, inhibition of MMP production and release may offer therapeutic means for treatment and prevention of heaves and recommendations for acceptable dust levels can be given.

Dose responses to inhalation of endotoxin, hay dust suspension and Aspergillus fumigatus extract in horses as measured by levels and activation of matrix metalloproteinase-9.

REASONS FOR PERFORMING STUDY: Airway matrix metalloproteinases (MMPs) increase after endotoxin (LPS) exposure, but there are no reports describing dose-dependent increases or activation following exposure. OBJECTIVES: To study matrix metalloproteinase-9 (MMP-9) and -2 (MMP-2) responses in bronchoalveolar lavage fluid (BALF) from heaves-susceptible and control horses following inhalation of hay dust suspension (HDS), LPS and Aspergillus fumigatus extract (AFE). METHODS: Heaves-susceptible (n = 7) and control (n = 6) horses received inhalation challenges with 3 different doses of HDS and LPS. Heaves-susceptible horses (n = 6) also received 3 different doses of AFE and one dose of AFE depleted of endotoxin (AFE-LPS). BALF collected following inhalation challenges was analysed using gelatin zymography. Gelatinolytic bands were identified as complex, pro-MMP-9, active MMP-9, pro-MMP-2 and active MMP-2 based on molecular weights. RESULTS: Each challenge substance induced a dose-dependent elevation in gelatinolytic activity. The dose-dependency was most evident for pro-MMP-9 and total MMP-9 levels in heaves-susceptible horses following LPS challenges. CONCLUSIONS: There is a dose-dependent elevation in MMP-9 in BALF of heaves-susceptible and control horses following inhalation challenge with organic dust and some of its components, elevation being more marked in heaves-susceptible horses. Organic dust components vary in their pro-inflammatory potential. POTENTIAL RELEVANCE: This study supports the role of MMPs in the pathogenesis of heaves and highlights the potential value of protease inhibitors in attenuating the airway inflammatory response to inhaled organic dust.