Exercise induced pulmonary hemorrhage in horses: American College of Veterinary Internal Medicine consensus statement.

BACKGROUND: Published studies of exercise-induced pulmonary hemorrhage (EIPH), when assessed individually, often provide equivocal or conflicting results. Systematic reviews aggregate evidence from individual studies to provide a global assessment of the quality of evidence and to inform recommendations. OBJECTIVES: Evaluate evidence to determine: if EIPH adversely affects the health, welfare or both of horses; if EIPH affects the athletic capacity of horses; the efficacy of prophylactic interventions for EIPH; and if furosemide affects the athletic capacity of horses. ANIMALS: None. MATERIALS AND METHODS: Systematic review. A panel of 7 experts was formed to assess evidence in the peer reviewed literature addressing each of the 4 objectives. Methodology followed that of the Grading of Recommendations, Assessment, Development, and Evaluation (GRADE). Publications were assessed for quality of evidence by working groups of the panel, and a summary of findings was presented in tables. Recommendations were based on quality of evidence and were determined by a vote of the panel. RESULTS: Much of the evidence was of low to very low quality. Experimental studies frequently lacked adequate statistical power. There was moderate to high quality evidence that EIPH is progressive, is associated with lung lesions, that it adversely affects racing performance, that severe EIPH (Grade 4) is associated with a shorter career duration, that furosemide is efficacious in decreasing the incidence and severity of EIPH, and that administration of furosemide is associated with superior race performance. CONCLUSIONS AND CLINICAL SIGNIFICANCE: Strong recommendation that EIPH be considered a disease and a weak recommendation for use of furosemide in management of racehorses with EIPH.

Airborne particulates (PM10) and tracheal mucus: A case-control study at an American Thoroughbred racetrack.

REASONS FOR PERFORMING STUDY: Particle mapping within stalls has demonstrated a relationship between particulate concentrations and visible accumulations of tracheal mucus. However, measurement of breathing zone particulate concentrations, which is the most accurate way to measure exposure, has not been performed to determine the relationship between exposures and accumulations of tracheal mucus sufficient to affect performance. OBJECTIVES: To compare breathing zone particulate exposures between horses with tracheal mucus scores (MS) previously demonstrated to affect performance (MS≥2) and those without (MS = 0) visible tracheal mucus. STUDY DESIGN: Case-control study. METHODS: We endoscopically examined 649 Thoroughbred racehorses over 7 months and selected 113 age-matched cases and controls based on the presence (MS≥2) or absence (MS = 0) of tracheal mucus, respectively. Inflammatory cell numbers in tracheal lavage were also determined. Breathing zone monitors recorded particulate exposure during 3 time periods (mid-day, evening and overnight). Total recording time averaged 17.5 h/horse. RESULTS: The overall prevalence of MS≥2 in the observed stables was 23%. Breathing zone particular matter (PM10) concentrations were very similar to previously reported ambient concentrations. During the evening and overnight, cases had significantly higher breathing zone PM10 concentrations and neutrophil counts than controls. Inflammatory cell counts were associated with average PM10 concentrations throughout the day. CONCLUSIONS: Breathing zone particulate concentrations are associated with MS≥2 and inflammatory cell numbers. If breathing zone particulate exposures are predominantly influenced by ambient PM concentrations, racing stable management practices to reduce particle exposures should greatly reduce the prevalence of tracheal mucus.

Ultrasound-guided biopsy of the cricoarytenoideus lateralis muscle: technique and safety in horses.

REASONS FOR PERFORMING STUDY: Current diagnosis of recurrent laryngeal neuropathy (RLN) depends upon disease recognition in the clinically affected horse. Biopsy of the intrinsic laryngeal muscles may provide a method to identify the changes in fibre-type composition that occur in RLN before clinical signs become apparent. OBJECTIVE: To develop an ultrasound-guided biopsy technique of the left cricoarytenoideus lateralis muscle (CALM) and evaluate its efficacy and safety in vivo. STUDY DESIGN: A longitudinal descriptive study. METHODS: Six standing horses underwent ultrasound-guided biopsy of the left CALM. Frozen muscle cores were obtained with a breast biopsy tool. Serial endoscopic, ultrasonographic and physical examinations before and for 8 weeks after the biopsy were assessed for iatrogenic trauma. Histologies of representative muscle core cross-sections were analysed for the total number of muscle fibres obtained with each biopsy. RESULTS: There were no immediate complications of the procedure and the left CALM was harvested in all instances. Biopsy samples had an average weight of 0.043 g (range = 0.023-0.077 g) and contained 3418 fibres in cross-section (range = 711-7143). Laryngeal endoscopic grade did not change significantly between prebiopsy and the end of the 8 week follow-up. The left CALM had significantly greater echogenicity than the right throughout the study (P<0.001), but there was no difference between the prebiopsy CALM echogenicity and that at completion of the study. CONCLUSIONS: Ultrasound-guided biopsy of the left CALM is safe and well tolerated, providing a minimally invasive method to obtain muscle from healthy horses. This new technique may be applicable in research and clinical settings.

Plasma cortisol concentration increases within 6 hours of stabling in RAO-affected horses.

REASONS FOR PERFORMING STUDY: In many inflammatory diseases plasma cortisol concentration (CORT) increases at the onset of acute inflammation, but the situation in recurrent airway obstruction (RAO) of horses is unknown. STUDY DESIGN: Split-plot repeated measures design with one grouping factor (disease) and two repeated factors (day and 3-hour intervals). OBJECTIVE: To test the hypothesis that CORT increases as acute exacerbations of RAO develop. METHODS: Four RAO-susceptible and 4 control horses were placed in a low dust environment (LDEnv) for 2 days followed by 2 days in a high dust environment (HDEnv). Exacerbations of RAO were indicated by increases in maximal change in pleural pressure (ΔPplmax) and decreases in breathing frequency variability (BFV), which was continuously measured by respiratory inductance plethysmography. Plasma samples for determination of CORT were collected every 6 h. RESULTS: In control horses, ΔPplmax and BFV were unaffected by the HDEnv, whereas in RAO-affected horses ΔPplmax increased and BFV decreased significantly. In the LDEnv, there was a circadian variation in CORT in both control and RAO-affected horses. In HDEnv, CORT was unaffected in control horses, but increased significantly in RAO-affected horses between 6 and 12 h after entering the HDEnv. CONCLUSIONS: Plasma cortisol concentration increases concurrently with the development of acute exacerbations of RAO.

Local airborne particulate concentration is associated with visible tracheal mucus in Thoroughbred racehorses.

REASON FOR PERFORMING STUDY: Accumulations of tracheal mucus assessed by endoscopic examination are associated with poor performance in racehorses. The air quality in horses' stalls may contribute to this visible tracheal mucus. OBJECTIVES: To determine whether the concentration and number of airborne particulates in stalls are associated with visible accumulations of tracheal mucus and with the number of inflammatory cells in tracheal aspirates. METHODS: We studied 107 racehorses from 3 stables, in 3 different months, and measured airborne particulate matter 3 times daily in each of the stalls. On each monthly visit, horse airways were examined endoscopically and assigned a mucus score, and tracheal lavage was performed. Bivariate procedures, general estimating equations and linear mixed models were applied to estimate the association between PM and the presence of accumulations of mucus and number of inflammatory cells. RESULTS: Stable, stall, month and PM were all significantly associated with the presence of accumulations of tracheal mucus, which had an overall prevalence of 67%. The odds of horses having visible accumulation of mucus were increased when horses occupied enclosed stables or stalls with higher particulate concentrations, and when concentrations of larger particles (≤ 10 µm in diameter) were elevated. Sixty-eight percent of tracheal wash samples contained more than 20% neutrophils. Increased numbers of neutrophils were associated with the concentration of smaller particles (≤ 2.5 µm in diameter). POTENTIAL RELEVANCE: Careful consideration of stable construction and management practices focused on maintaining the lowest possible dust concentrations throughout the day should reduce the prevalence of visible accumulations of tracheal mucus, potentially improving racing performance.

The effect of intravenous lidocaine infusion on bronchoalveolar lavage cytology in equine recurrent airway obstruction.

BACKGROUND: Lidocaine decreases neutrophilic inflammation in models of acute lung injury and decreases inflammation in asthmatic patients. Neutrophilic bronchiolitis develops in recurrent airway obstruction (RAO), but it remains unknown if lidocaine infusion decreases neutrophil migration into the airways. HYPOTHESIS: Lidocaine decreases neutrophilic inflammation as measured in BALF in RAO-affected horses. ANIMALS: Six RAO-susceptible horses in remission. METHODS: In a randomized cross-over design, horses received lactated Ringer's solution (LRS) IV or lidocaine hydrochloride IV with a minimum of 4 weeks at pasture between treatments. Treatments were delivered as continuous infusions beginning 4 hours before and for 68 hours during exposure to hay and straw challenge. Clinical score (CS, grade 0-8), maximal change in pleural pressure (∆Ppl(max) ), and bronchoalveolar lavage fluid (BALF) cytology were measured at baseline and the end of challenge (day 4). Plasma lidocaine concentrations were monitored daily. RESULTS: At baseline, there were no significant differences in variables between treatments. Plasma lidocaine concentration was consistently > 1100 ng/mL. After challenge, CS increased significantly [baseline: 2/8 (2-3), [median (interquartile range)]; day 4: 4/8 (4-5) P = .0006] as did ∆Ppl(max) [baseline: 3.6 (2.63-4.95) cmH(2) 0; day 4: 9.62 (6.5-16) P = .0036], but there was no difference between treatments. Percentage of neutrophils was not different between treatments, but lidocaine infusion significantly increased BALF total cells [baseline: LRS 2.18 ± 0.82 × 10(5) cells/mL (mean ± SD), lidocaine 1.6 ± 0.3 × 10(5) , day 4: LRS 2.0 ± 0.88 × 10(5) , lidocaine 4.4 ± 2 × 10(5) (P = .0045)]. CONCLUSIONS AND CLINICAL IMPORTANCE: Lidocaine does not decrease neutrophilic inflammation in RAO.

Owner assessment in judging the efficacy of airway disease treatment.

REASONS FOR PERFORMING STUDY: Efficacy of medications for recurrent airway obstruction is typically tested using clinical, cytological and lung function examinations of severely affected animals. These trials are technically challenging and may not adequately reflect the spectrum of disease and owner complaints encountered in clinical practice. OBJECTIVE: To determine if owners of horses with chronic airway disease are better able to detect drug efficacy than a veterinarian who clinically examines horses infrequently. METHOD: In a double-blinded randomised controlled trial, owners and a veterinarian compared the efficacy of dexamethasone (0.1 mg/kg bwt per os, q. 24 h, for 3 weeks; n = 9) to placebo (n = 8) in horses with chronic airway disease. Before and after treatment, owners scored performance, breathing effort, coughing and nasal discharge using a visual analogue scale (VAS). The clinician recorded vital parameters, respiratory distress, auscultation findings, cough and nasal discharge, airway mucus score, bronchoalveolar lavage fluid (BALF) cytology and arterial blood gases. RESULTS: The VAS score improved significantly in dexamethasone- but not placebo-treated horses. In contrast, the clinician failed to differentiate between dexamethasone- and placebo-treated animals based on clinical observations, BALF cytology or endoscopic mucus score. Respiratory rate (RR) and arterial oxygen pressure (PaO(2)) improved with dexamethasone but not placebo. CONCLUSIONS AND CLINICAL RELEVANCE: In the design of clinical trials of airway disease treatments, more emphasis should be placed on owner-assessed VAS than on clinical, cytological and endoscopic observations made during brief examinations by a veterinarian. Quantifiable indicators reflecting lung function such as RR and PaO(2) provide a good assessment of drug efficacy.

Particle mapping in stables at an American Thoroughbred racetrack.

REASONS FOR PERFORMING STUDY: Airway inflammation and mucus in the trachea are common in racehorses. Fine airborne particles can initiate and coarse particles can worsen airway inflammation in man and in animal models of airway disease. The regional and seasonal distribution of particles of different sizes has never been investigated in American racing stables. OBJECTIVES: To determine the regional and seasonal concentration and number of airborne particles of different sizes in racing stables. METHODS: Direct reading instruments were used to determine the mass concentration and numbers of particles 3 times daily (early morning, midday and late afternoon) in July, September and November, in 3 different racing stables. RESULTS: Average particle concentrations were lowest in July and highest in September and November. Early morning concentrations were significantly higher than those measured throughout the rest of the day. The completely enclosed stable with little natural ventilation, had significantly higher particulate concentrations than the open-sided stable. With regard to numbers of particles, those 2-5 µm were greatest in July and least in November; those 0.5-1.0 µm were greatest in September and least in November. Location of stall within stable also affected concentrations and numbers. CONCLUSIONS: The concentration and number of particles in sizes known to reach the lower airways varies with stable design/management, time of day, season of year and location of the stall within the stable. POTENTIAL RELEVANCE: Particle mapping is a useful tool in the identification of stables, season, and location of stalls within stables where horses may be at greater risk of exposure to offending particulates.

Repeated blood instillation into the airway of the horse does not cause pulmonary fibrosis.

REASONS FOR PERFORMING THE STUDY: Exercise-induced pulmonary haemorrhage (EIPH) occurs in nearly all strenuously exercising horses. Recent studies have attempted to identify the role of free blood within the airspaces, in the lung fibrosis that develops within the lungs of EIPH horses. HYPOTHESIS: Repeated exposure of the equine lung to autologous blood results in lung fibrosis similar to that observed in spontaneous EIPH. METHODS: Forty ml of autologous blood from the jugular vein was instilled into preselected lung regions of 6 horses one, 2, 3, 4 or 5 times at 2 week intervals, with 40 ml of saline instilled into the contralateral lung serving as a control. The time interval between instillation of the first blood and euthanasia ranged from 2-10 weeks. The lung from each instillation site was harvested, and the histopathology was scored from each region based upon the presence and abundance of blood, haemosiderin and interstitial collagen. Consequently, at the time of euthanasia, the time since instillation of the first blood ranged from 2-10 weeks. RESULTS: Beyond retention of blood, and the accumulation of haemosiderin, there was no visible increase in perivascular and interstitial collagen within the blood-instilled lung sites. In a small number of regions, there were foci of bronchiolitis obliterans organising pneumonia with collagen accumulation within these foci, but no collagen accumulation with the characteristic perivascular and interstitial histological distribution seen in EIPH. CONCLUSIONS: Free blood within the airways of horses does not result in a qualitative increase in the amount of interstitial collagen within 8-10 weeks, and is therefore an unlikely aetiological factor in the lung collagen accumulation that occurs in EIPH. POTENTIAL RELEVANCE: This study emphasises the efficiency of the equine lung in clearing blood from the airspaces. Further, it suggests that the aetiopathogenesis of EIPH is not driven by events within the airspace lumen, but rather emanates from within the vasculature and lung interstitium.

Regional distribution of collagen and haemosiderin in the lungs of horses with exercise-induced pulmonary haemorrhage.

REASONS FOR PERFORMING STUDY: Regional veno-occlusive remodelling of pulmonary veins in EIPH-affected horses, suggests that pulmonary veins may be central to pathogenesis. The current study quantified site-specific changes in vein walls, collagen and haemosiderin accumulation, and pleural vascular profiles in the lungs of horses suffering EIPH. HYPOTHESIS: In the caudodorsal lung regions of EIPH-affected horses, there is veno-occlusive remodelling with haemosiderosis, angiogenesis and fibrosis of the interstitium, interlobular septa and pleura. METHODS: Morphometric methods were used to analyse the distribution and accumulation of pulmonary collagen and haemosiderin, and to count pleural vascular profiles in the lungs of 5 EIPH-affected and 2 control horses. RESULTS: Vein wall thickness was greatest in the dorsocaudal lung and significantly correlated with haemosiderin accumulation. Increased venous, interstitial, pleural and septal collagen; lung haemosiderin; and pleural vascular profiles occurred together and changes were most pronounced in the dorsocaudal lung. Further, haemosiderin accumulation colocalised with decreased pulmonary vein lumen size. Vein wall thickening, haemosiderin accumulation and histological score were highly correlated and these changes occurred only in the caudodorsal part of the lung. CONCLUSION: The colocalisation of these changes suggests that regional (caudodorsal) venous remodelling plays an important role in the pathogenesis of EIPH. POTENTIAL RELEVANCE: The results support the hypothesis that repeated bouts of venous hypertension during strenuous exercise cause regional vein wall remodelling and collagen accumulation, venous occlusion and pulmonary capillary hypertension. Subjected to these high pressures, there is capillary stress failure, bleeding, haemosiderin accumulation and, subsequently, lung fibrosis.

Fluticasone propionate aerosol is more effective for prevention than treatment of recurrent airway obstruction.

BACKGROUND: Efficacy of inhaled fluticasone propionate (FP) for management of recurrent airway obstruction (RAO) has only been evaluated after several weeks' treatment. OBJECTIVES: To compare efficacy of (1) 3-day treatments with FP to dexamethasone (DEX) for management of RAO; and (2) FP and DEX to no treatment in prevention of acute RAO exacerbations. ANIMALS: Nine RAO affected horses. METHODS: Crossover studies in RAO-affected horses compared (a) 3-day treatment of RAO exacerbation with FP (3 and 6 mg q12h) and DEX (0.1 mg/kg q24h) and (b) FP (6 mg q12h) and DEX (0.1 mg/kg q24h) to no treatment for prevention of acute exacerbations of RAO. Treatment efficacy and unwanted effects were judged from maximal change in pleural pressure (DeltaPpl(max)), serum cortisol (COR), bronchoalveolar lavage (BAL) cytology, and subjective scores for respiratory distress and lameness. RESULTS: In treatment trial, DEX and FP (6 mg) significantly decreased DeltaPpl(max) by 48 and 72 hours, respectively; FP (3 mg) had no significant effect. DEX decreased COR more than did FP. In prevention trial, both DEX and FP (6 mg) prevented the increase in DeltaPpl(max) that occurred in untreated horses. Both treatments decreased COR to the same degree. FP and DEX had no effects on bronchoalveolar lavage fluid (BALF) cytology and there was no evidence of laminitis. CONCLUSIONS AND CLINICAL IMPORTANCE: FP (6 mg q12h) is as effective as DEX for prevention of acute exacerbations of RAO and lower doses should be evaluated. High-dose FP is not as effective as DEX for treatment of RAO exacerbations.

Effect of magnitude and direction of force on laryngeal abduction: implications for the nerve-muscle pedicle graft technique.

REASONS FOR PERFORMING STUDY: The nerve-muscle pedicle graft technique is a treatment for recurrent laryngeal neuropathy (RLN), but the optimal placement of the pedicles within the cricoarytenoideus dorsalis (CAD) muscle is unknown. HYPOTHESIS: The magnitude and direction of force placed on the muscular process of the left arytenoid cartilage affects the magnitude of laryngeal abduction. METHODS: Five larynges were harvested from cadavers. Using increments of 0.98 N, a dead-weight force generator applied a force of 0-14.7 N for 1 min each to the left muscular process at 0, 10, 20, 30, 40, 50, 60 and 70 degrees angles. The rima glottis was photographed digitally 1 min after each force had been applied. Distances between biomarkers (Lines 1-4) and right to left angle quotient (RLQ) were used to assess the degree of left arytenoid abduction. RESULTS: Increasing force from 0-14.7 N progressively and significantly increased the length of all lines and RLQ, indicating abduction. Furthermore, there was a significant interaction between force and angles. Applying forces of 7.84 N or greater (Lines 2-4 and RLQ) or 11.76 N or greater (Line 1) at angles 0, 10, 20 and 30 degrees resulted in significantly greater abduction than applying the same forces at 40, 50, 60 and 70 degrees. Angles of 0-30 degrees correspond with the direction of pull exerted by the lateral compartment of the CAD muscle. CONCLUSION: In RLN, nerve-muscle pedicle grafts should be placed preferentially in the lateral rather than in the medial compartment of the CAD muscle. POTENTIAL RELEVANCE: The information presented can be used to assist surgeons in the planning and application of the nerve-muscle pedicle graft procedure.

Expression of toll-like receptor 2 mRNA in bronchial epithelial cells is not induced in RAO-affected horses.

REASONS FOR PERFORMING STUDY: Airway inflammation in recurrent airway obstruction (RAO) is triggered by housing affected horses in stables.It has been suggested that RAO is an allergic condition, but innate immune mechanisms are also involved. Fungal products activate innate immune mechanisms through toll-like receptor 2 (TLR2). In human airway epithelium, TLR2 activation leads to interleukin (IL)-8 production. This pathway is negatively regulated by the zinc finger protein A20. This study was performed to enhance understanding of innate immune mechanisms in RAO. HYPOTHESIS: TLR2 and IL-8 mRNA are elevated in RAO during stabling compared with controls. A20 mRNA is negatively associated with the numbers of airway inflammatory cells. OBJECTIVES: To determine TLR2, IL-8 and A20 mRNA expression in lungs of stabled and pastured RAO-affected and control horses. METHODS: Airway obstruction and inflammatory cell counts in bronchoalveolar lavage were measured, and TLR2, IL-8 and A20 mRNA expression quantified by qRT-PCR in 6 RAO-affected and 6 control horses, during and after exposure to hay and straw. RESULTS: Airway obstruction and neutrophils were increased in RAO-affected horses during stabling. While stabling increased IL-8, TLR2 and A20 mRNA were unaffected. TLR2 and A20 were significantly correlated (r = 0.83) and A20 mRNA was negatively associated with inflammatory cells. POTENTIAL RELEVANCE: Stabling does not lead to an increase in TLR2 expression. Other molecules or processes in the TLR2 cascade might be important in fungal-induced airway inflammation. Equine epithelial-derived A20 may be involved in modulation of airway inflammation.

Effect of ventriculectomy versus ventriculocordectomy on upper airway noise in draught horses with recurrent laryngeal neuropathy.

REASONS FOR PERFORMING STUDY: Little is known about the efficacy of bilateral ventriculectomy (VE) or bilateral ventriculocordectomy (VCE) in draught horses. OBJECTIVES: To compare the effect of VE and VCE on upper airway noise in draught horses with recurrent laryngeal neuropathy (RLN) by use of quantitative sound analysis techniques. HYPOTHESIS: In competitive draught horses with grade 4 RLN, VE and VCE reduce upper airway noise during exercise, but VCE is more effective. METHODS: Thirty competitive hitch or pulling draught horses with grade 4 RLN were evaluated for upper airway sound during exercise. Respiratory rate (RR), inspiratory (Ti) and expiratory time (Te), the ratio between Ti and Te (Ti/Te), inspiratory (Sli) and expiratory sound levels (Sle), the ratio between Sli and Sle (Sli/Sle), and peak sound intensity of the second formant (F2) were calculated. Eleven horses were treated with VE and 19 with VCE. After 90 days of voice and physical rest and 30 days of work, the horses returned for post operative upper airway sound evaluation and resting videoendoscopy. RESULTS: VE significantly reduced Ti/Te, Sli, Sli/Sle and the sound intensity of F2. Respiratory rate, Ti, Te and Sle were unaffected by VE. VCE significantly reduced Ti/Te, Ti, Te, Sli, Sli/Sle and the sound intensity of F2, while RR and Sle were unaffected. The reduction in sound intensity of F2 following VCE was significantly greater than following VE. After VE and VCE, 7/11 (64%) and 15/18 (83%) owners, respectively, concluded that the surgery improved upper airway sound in their horses sufficiently for successful competition. CONCLUSIONS: VE and VCE significantly reduce upper airway noise and indices of airway obstruction in draught horses with RLN, but VCE is more effective than VE. The procedures have few post operative complications. POTENTIAL RELEVANCE: VCE is recommended as the preferred treatment for RLN in draught horses. Further studies are required to evaluate the longevity of the procedure's results.

Differential association of MUC5AC and CLCA1 expression in small cartilaginous airways of RAO-affected and control horses.

REASONS FOR PERFORMING STUDY: Airway mucus accumulation is associated with indoor irritant and allergen exposure in horses with recurrent airway obstruction (RAO). Epidermal growth factor receptor (EGFR) and a chloride channel (calcium activated, family member 1; CLCA1) are key signalling molecules involved in mucin gene expression. OBJECTIVES: We hypothesised that exposure to irritants and aeroallergens would lead to increased expression of the mucin gene eqMUC5AC and increased stored mucosubstance in the airways of RAO-affected horses, associated with increased neutrophils and CLCA1 and EGFR mRNA levels. METHODS: We performed quantitative RT-PCR of eqMUC5AC, CLCA1 and EGFR; volume density measurements of intraepithelial mucosubstances; and cytological differentiation of intraluminal inflammatory cells in small cartilaginous airways from cranial left and right and caudal left and right lung lobes of 5 clinically healthy and 5 RAO-affected horses that had been exposed to indoor stable environment for 5 days before euthanasia. RESULTS: Neutrophils were increased in RAO-affected horses compared to clinically healthy controls. EqMUC5AC mRNA levels were positively correlated with both CLCA1 and EGFR mRNA levels in RAO-affected horses but only with CLCA1 in controls. The relationship between eqMUC5AC and CLCA1 differed in the 2 groups of horses with RAO-affected animals overexpressing CLCA1 in relation to eqMUC5AC. CONCLUSIONS: These data implicate CLCA1 as a signalling molecule in the expression of eqMUC5AC in horses but also suggest differential regulation by CLCA1 and EGFR between horses with RAO and those with milder degrees of airway inflammation.

In vitro allergy tests compared to intradermal testing in horses with recurrent airway obstruction.

Recurrent airway obstruction (RAO) is a common condition in stabled horses characterised by small airway inflammation, airway neutrophilia and obstruction following exposure of susceptible horses to mouldy hay and straw and is thus regarded as a hypersensitivity reaction to mould spores. However, the role of IgE-mediated reactions in RAO remains unclear. The aim of the study was to investigate with a serological IgE ELISA test (Allercept), an in vitro sulfidoleukotriene (sLT) release assay (CAST) and with intradermal testing (IDT) whether serum IgE and IgE-mediated reactions against various mould, mite and pollen extracts are associated with RAO. IDT reactions were evaluated at different times in order to detect IgE-mediated immediate type reactions (type I hypersensitivity reactions, 0.5-1 h), immune complex-mediated late type reactions (type III reactions, 4-10 h) and cell-mediated delayed type reactions (type IV hypersensitivity reactions 24-48 h). In the serological test, overall the control horses displayed more positive reactions than the RAO-affected horses but the difference was not significant. Comparison of the measured IgE levels showed that the RAO-affected horses had slightly higher IgE levels against Aspergillus fumigatus than controls (35 and 16 AU, respectively, p<0.05), but all values were below the cut off (150 AU) of the test. In the sLT release assay, seven positive reactions were observed in the RAO-affected horses and four in the controls but this difference was not significant. A significantly higher proportion of late type IDT reactions was observed in RAO-affected horses compared to controls (25 of 238 possible reactions versus 12 of 238 possible reactions, respectively, p<0.05). Interestingly, four RAO-affected but none of the control horses reacted with the recombinant mould allergen A. fumigatus 8 (rAsp f 8, p<0.05), but only late phase and delayed type reactions were observed. In all three tests the majority of the positive reactions was observed with the mite extracts (64%, 74% and 88% of all positive reactions, respectively) but none of the tests showed a significant difference between RAO-affected and control animals. Our findings do not support that IgE-mediated reactions are important in the pathogenesis of RAO. Further studies are needed to investigate whether sensitisation to mite allergens is of clinical relevance in the horse and to understand the role of immune reactions against rAsp f 8.

Plasma and urinary concentrations of trimetoquinol by LC-MS-MS following intravenous and intra-tracheal administration to horses with heaves.

Trimetoquinol (TMQ) is a very potent and fast acting bronchodilator in horses with heaves. This study assessed the plasma and urinary concentrations of TMQ in horses with heaves following administration via the intravenous (IV, 0.2 microg/kg) and intra-tracheal (IT, 2 microg/kg) routes. TMQ was administered to six horses affected with heaves (RAO - Recurrent Airway Obstruction, used interchangeably) by the above routes and plasma and urine samples collected and stored at -20 degrees C until analyzed. Solid Phase Extraction (SPE) of TMQ was followed by highly sensitive ESI(+)-LC-MS-MS (ElectroSpray Ionization, positive mode - Liquid Chromatography - Mass Spectrometry - Mass Spectrometry); with a Limit of Detection (LOD) estimated at 1 pg/mL. Following IV administration, TMQ plasma levels peaked at 1 min at 707 pg/mL, and at 9 min at 306 pg/mL following IT administration. Our results show that TMQ plasma concentrations decline rapidly following IV administration, which is consistent with the fast onset and short duration of TMQ effect that was observed in our previous studies. On the other hand, IT administration showed a very unique plasma concentration pattern. From a regulatory standpoint, the current available TMQ ELISA kit was also used in an attempt to detect TMQ from the plasma and urine samples. We report that the ELISA kit was unable to detect TMQ from any of the samples generated in these studies.

Regional pulmonary veno-occlusion: a newly identified lesion of equine exercise-induced pulmonary hemorrhage.

Exercise-induced pulmonary hemorrhage (EIPH) is common in horses following intense exertion, occurring in up to 75% of racing Thoroughbreds and Standardbreds. In spite of this, the pathogenesis of EIPH is poorly understood. In 7 racing Thoroughbred horses with EIPH, 6 sections were collected from the left and right lung, representing the cranial, middle, and caudal region of the dorsal and ventral lung (84 sites total). Grossly, both right and left lungs had numerous dark brown to blue-black foci along the caudodorsal visceral pleura. Tissue sections were stained with hematoxylin-eosin, Masson's trichrome, and Prussian blue. Verhoeff Van Gieson and immunohistochemistry for alpha-smooth muscle actin were used to assess the pulmonary vasculature. Histologic scores (HS = 0-3) were assigned to each region/slide for the presence and severity of 5 findings: interstitial fibrosis, hemosiderin accumulation, pleural/interlobular septal thickness, arterial and venous wall thickness, and evidence of angiogenesis (maximum cumulative HS = 15). Thirty-nine of the 84 (46%) sections were histologically normal (HS = 0); 33/84 (39%) were mildly to moderately affected, with small amounts of hemosiderin and fibrosis (HS = 1-9) while 12/84 (14%), primarily from the dorsocaudal lung, had severe vascular remodeling, fibrosis, and hemosiderin accumulation (HS = 10-15). In the latter, veno-occlusive remodeling of the intralobular veins colocalized with hemosiderosis, fibrosis, hypertrophy of vessels within the pleura, and interlobular septa and bronchial neovascularization. We propose that regional veno-occlusive remodeling, especially within the caudodorsal lung fields, contributes to the pathogenesis of EIPH, with the venous remodeling leading to regional vascular congestion and hemorrhage, hemosiderin accumulation, fibrosis, and bronchial angiogenesis.