Bronchoalveolar Lavage Fluid Of Horses Research Articles

Aspergillus fumigatus binding IgA and IgG1 are increased in bronchoalveolar lavage fluid of horses with neutrophilic asthma.

Equine asthma (EA) is a common lower airway disease in horses, but whether its pathogenesis is allergic is ambiguous. Extrinsic stimuli like hay dust induce acute exacerbation of clinical signs and sustained local neutrophilic inflammation in susceptible horses. Aspergillus fumigatus is an EA stimulus, but it is unclear if it merely acts as an IgE-provoking allergen. We aimed to comprehensively analyze immunoglobulin (Ig) isotypes in EA, elucidating their binding to different A. fumigatus antigens, and their quantities systemically in serum and locally in bronchoalveolar lavage fluid (BALF). Serum and BALF from healthy horses (HE, n = 18) and horses with mild-moderate asthma (MEA, n = 20) or severe asthma (SEA, n = 24) were compared. Ig isotype (IgG1, IgG3/5, IgG4/7, IgG6, IgA, and IgE) binding to nine antigens (A.fumigatus lysate, and recombinant Asp f 1, Asp f 7, Asp f 8, dipeptidyl-peptidase 5, class II aldolase/adducin domain protein, glucoamylase, beta-hexosaminidase, and peptide hydrolase) was compared by enzyme-linked immunosorbent assays. Total Ig isotype contents were determined by bead-based assays. MEA and SEA differed from HE but hardly from each other. Compared to HE, asthmatic horses showed increased anti-A. fumigatus binding of IgG (BALF and serum) and IgA (BALF). Serum and BALF IgE binding and total IgE contents were similar between HE and EA. Single antigens, as well as A. fumigatus lysate, yielded similar Ig binding patterns. Serum and BALF IgG1 binding to all antigens was increased in SEA and to several antigens in MEA. Serum IgG4/7 binding to two antigens was increased in SEA. BALF IgA binding to all antigens was increased in SEA and MEA. Total BALF IgG1 and IgG4/7 contents were increased in SEA, and serum IgG4/7 content was increased in MEA compared to HE. Yet, total isotype contents differentiated EA and HE less clearly than antigen-binding Ig. A. fumigatus immunogenicity was confirmed without identification of single dominant antigens here. A. fumigatus provoked elevated BALF IgG1 and IgA binding, and these isotypes appear relevant for neutrophilic EA, which does not support allergy. BALF Ig isotype differentiation beyond IgE is crucial for a comprehensive analysis of immune responses to fungi in EA pathogenesis.

The Relationship between Lung Inflammation and Aerobic Threshold in Standardbred Racehorses with Mild-Moderate Equine Asthma.

Simple SummaryHorses can be affected by equine asthma, a disease that shares several similarities with human asthma. Young racehorses can be affected by a mild form of equine asthma, which can have a negative impact on racing performance. In this study, we evaluated the relation between the increase in inflammatory cells in the bronchoalveolar lavage fluid of horses affected by poor performance due to equine asthma and the speed at which the horse blood lactate reaches the value of 4 mmol/L, which is a parameter of athletic capacity. With this aim, we evaluated the results of a treadmill test with blood lactate analysis of 30 horses that had a bronchoalveolar lavage fluid cytology consistent with a mild form of equine asthma. The results showed a significant relation between the increase in the differential count of neutrophils in bronchoalveolar lavage and the decrease in the speed at 4 mmol/L of lactate. This confirm the negative impact of neutrophilic lung inflammation in the presence of equine asthma on athletic capacity and, consequently, on racing performance.Mild–moderate equine asthma (MEA) is a common disease of young racehorses characterized by inflammation of the lower airways diagnosed by cytological examination of bronchoalveolar lavage fluid (BALF), and it is considered an important cause of poor performance. The most common parameter of athletic capacity associated with horse performance is the speed at 4 mmol/L of lactate (VLA4). The aim of the present work was to evaluate the relation between the different BALF inflammatory cells and VLA4 in racehorses affected by MEA. A population of Standardbred racehorses that underwent an accurate protocol for poor performance evaluation was collected for a retrospective study. Horses with any other alteration potentially influencing performance were excluded, thus considering only subjects with BALF cytology consistent with MEA. A sample of 30 horses (3.4 ± 1.0 years old) was selected. The relationship between BALF inflammatory cell differential count and VLA4 was evaluated by means of linear regression. Statistical analysis showed a significant relation (p = 0.015, r2 = 0.19) between the increase in the differential count of neutrophils in BALF and the decrease in VLA4. The results obtained suggested that the accumulation of neutrophils in the airways of MEA horses may have a direct impact on athletic capacity, possibly due to impaired alveolar blood-gas exchanges during strenuous exercise.

Effect of injected dexamethasone on relative cytokine mRNA expression in bronchoalveolar lavage fluid in horses with mild asthma

BackgroundMild equine asthma is a common inflammatory airway disease of the horse. The primary treatment of mild equine asthma is corticosteroids. The purpose of this study was to investigate the effects of injected dexamethasone on relative IL-1β, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p35, IL-17, IL-23, IFN-γ, Eotaxin-2 and TNF-α mRNA expression in bronchoalveolar lavage (BAL) fluid in healthy Thoroughbred horses (n = 6), and those with mild equine asthma (n = 7).ResultsHorses with mild equine asthma had a significantly greater bronchoalveolar lavage mast cell percentage than healthy horses both before and after treatment. Mild equine asthma was associated with a 4.95-fold up-regulation of IL-17 (p = 0.026) and a 2.54-fold down-regulation of IL-10 (p = 0.049) compared to healthy horses. TNF-α was down-regulated in response to dexamethasone treatment in both healthy horses (3.03-fold, p = 0.023) and those with mild equine asthma (1.75-fold, p = 0.023). IL-5 was also down-regulated in horses with mild asthma (2.17-fold, p = 0.048).ConclusionsHorses with mild equine asthma have a lower concentration of IL-10 in BAL fluid than healthy controls which concurs with human asthmatics. The marked up-regulation of IL-17 in horses with mild asthma suggests these horses had a true tendency of “allergic” airway inflammation in response to environmental allergens. Dexamethasone administration exerted anti-inflammatory effects associated with down-regulation of TNF-α in all horses, and decreased levels of IL-5 mRNA expression in horses with mild equine asthma. The inhibition of the Th-2 response, without any alterations to the airway cytology, indicates that maintained exposure to environmental allergens perpetuates airway inflammation.

Reliability of cytological evaluation of mast cells from bronchoalveolar lavage fluid in horses: Intraobserver agreement and mast cell identification

SummaryEndoscopy, including bronchoalveolar lavage (BAL) fluid cytology, are invaluable tools for equine lower airway diagnostics. Objectives of the study were to evaluate the reliability of two staining methods, May–Grünwald–Giemsa (MGG) and toluidine blue (TB), for the evaluation of mast cells in cytospin smears from BAL fluid samples and the reliability of a 500‐cell differential cell count using both staining methods. Endoscopies and BAL fluid sampling were performed and BAL cytology was evaluated from cytospin smears. BAL cytology slides from 78 horses were evaluated. Three experienced observers evaluated the BAL cytology cytospin smears for the mast cell comparison study. The intra‐ and interobserver agreements were calculated using intraclass correlation coefficients (ICC). BAL cytology mast cell counts were significantly higher with the TB staining method compared with the MGG staining method. Observer agreements showed below acceptable agreement between observers for mast cell counts for both staining methods, even with fixed coordinates. Based on results from this study recommendation for BAL cytology staining would be to use both the MGG and the TB staining method, relying on the TB for the enumeration of mast cells.

The effect of single pretreatment with salbutamol on recovery of bronchoalveolar lavage fluid in horses with suspected or confirmed severe equine asthma.

BackgroundBronchoalveolar lavage (BAL) is a method for the recovery of respiratory secretion from the lower airways.ObjectivesTo investigate if the administration of a single dose of a bronchodilatator in horses with a suspected or confirmed severe equine asthma could improve recovery of bronchoalveolar lavage fluid (BALF).AnimalsTwenty‐eight horses with severe equine asthma.MethodsHorses were divided into 2 groups: group “treated” was given salbutamol before endoscopic examination and BALF collection, whereas group “not treated” was not given. BAL was performed with BAL‐catheter by instilling 350 mL of sterile saline. Amount of recovered fluid was recorded. Statistical analysis was performed with a two‐tailed Student's t test.ResultsThe average fluid recovery in the horses treated with salbutamol was 52% ± 15% (mean +/− SD), compared with 38% ± 13% for the group of horses not treated with salbutamol (P = 0.013).Conclusions and Clinical ImportanceClinicians should consider administration of salbutamol before performing BAL on horses with asthma.

Secretoglobin and Transferrin Expression in Bronchoalveolar Lavage Fluid of Horses with Chronic Respiratory Disease

BackgroundLower expression of secretoglobin and transferrin has been found in the bronchoalveolar lavage fluid (BALF) of a small number of horses with experimentally induced signs of recurrent airway obstruction (RAO) compared to healthy controls.Hypothesis/ObjectivesSecretoglobin and transferrin BALF expression will be similarly decreased in horses with naturally occurring clinical signs of RAO and in horses with experimentally induced clinical signs of RAO as compared to healthy controls and intermediate in horses with inflammatory airway disease (IAD).AnimalsRecurrent airway obstruction‐affected and control horses were subjected to an experimental hay exposure trial to induce signs of RAO. Client‐owned horses with a presumptive diagnosis of RAO and controls from the same stable environments were recruited.MethodsPulmonary function and BALF were evaluated from control and RAO‐affected research horses during an experimental hay exposure trial (n = 5 in each group) and from client‐owned horses (RAO‐affected horses, n = 17; IAD‐affected horses, n = 19; healthy controls, n = 5). The concentrations of secretoglobin and transferrin in BALF were assessed using Western blots.ResultsNaturally occurring and experimentally induced RAO horses had similar decreases in BALF transferrin expression, but secretoglobin expression was most decreased in naturally occurring RAO. Secretoglobin and transferrin expression were both lower in BALF of RAO‐affected horses than in IAD‐affected and control horses.Conclusions and Clinical ImportanceSecretoglobin and transferrin expression is decreased in BALF of RAO‐affected horses after both experimental and natural exposure. Secretoglobin and transferrin likely play clinically relevant roles in the pathophysiology of RAO, and may thus be used as biomarkers of the disease.

Effect of inhaled hydrosoluble curcumin on inflammatory markers in broncho-alveolar lavage fluid of horses with LPS-induced lung neutrophilia

Background: Horses commonly suffer from chronic respiratory disease and are also used in large animal models of spontaneous or induced airway inflammation. The anti-inflammatory properties of curcumin are largely described but its low bioavailability precludes its clinical use. NDS27, a lysin salt of curcumin incorporated in beta-cyclodextrine, has high bioavailability and can be administered by inhalation. The aim of this study was to investigate the effects of inhaled NDS27 on inflammatory cytokines and proteins in the broncho-alveolar lavage fluid using a model of neutrophilic airway inflammation.
 Methods: Airway neutrophilia was induced in eight horses by inhalation of lipopolysaccharides (LPS). Horses were treated with either inhalation of NDS27 or with placebo in a randomized cross-over design. Broncho-alveolar lavages were performed 6 hours after stimulation with LPS. Percentage of neutrophils, concentrations of IL-1β, TNF-α, IL-6, Club cell secretory protein, myeloperoxidase (MPO) and elastase (ELT) concentrations were determined.
 Results: LPS stimulation induced significant increases in neutrophil counts and concentrations of IL-6 (70.2 ± 66.0 pg/ml), TNF-α (43.9 ± 31.2 pg/ml), MPO (580.9 ± 327.0 ng/ml) and ELT (27.6 ± 16.7 ng/ml). Treatment with NDS27 significantly prevented the increase in active and total MPO (216.4 ± 118.1 ng/ml) and ELT (5.9 ± 3.2 ng/ml) while there was a trend towards reduced IL-6 concentration.
 Conclusions: Results show that, although not reducing neutrophil recruitment, NDS27 largely abolishes LPS-induced neutrophil degranulation. Reduced levels of ELT and MPO, as well as reduced MPO activity may have beneficial effects via a reduced production of reactive oxygen species implicated in chronic inflammation and airway remodeling.

Effect of inhaled hydrosoluble curcumin on inflammatory markers in broncho-alveolar lavage fluid of horses with LPS-induced lung neutrophilia.

BackgroundHorses commonly suffer from chronic respiratory disease and are also used in large animal models of spontaneous or induced airway inflammation. The anti-inflammatory properties of curcumin are largely described but its low bioavailability precludes its clinical use. NDS27, a lysin salt of curcumin incorporated in beta-cyclodextrine, has high bioavailability and can be administered by inhalation. The aim of this study was to investigate the effects of inhaled NDS27 on inflammatory cytokines and proteins in the broncho-alveolar lavage fluid using a model of neutrophilic airway inflammation.MethodsAirway neutrophilia was induced in eight horses by inhalation of lipopolysaccharides (LPS). Horses were treated with either inhalation of NDS27 or with placebo in a randomized cross-over design. Broncho-alveolar lavages were performed 6 hours after stimulation with LPS. Percentage of neutrophils, concentrations of IL-1β, TNF-α, IL-6, Club cell secretory protein, myeloperoxidase (MPO) and elastase (ELT) concentrations were determined.ResultsLPS stimulation induced significant increases in neutrophil counts and concentrations of IL-6 (70.2 ± 66.0 pg/ml), TNF-α (43.9 ± 31.2 pg/ml), MPO (580.9 ± 327.0 ng/ml) and ELT (27.6 ± 16.7 ng/ml). Treatment with NDS27 significantly prevented the increase in active and total MPO (216.4 ± 118.1 ng/ml) and ELT (5.9 ± 3.2 ng/ml) while there was a trend towards reduced IL-6 concentration.ConclusionsResults show that, although not reducing neutrophil recruitment, NDS27 largely abolishes LPS-induced neutrophil degranulation. Reduced levels of ELT and MPO, as well as reduced MPO activity may have beneficial effects via a reduced production of reactive oxygen species implicated in chronic inflammation and airway remodeling.

Increased adenosine concentration in bronchoalveolar lavage fluid of horses with lower airway inflammation

Several reports have suggested a role for adenosine in the pathogenesis of chronic airway conditions and this has led to new therapeutic strategies to limit airway inflammation. In this study, detectable levels of adenosine in bronchoalveolar lavage (BAL) samples from 11 horses with non-infectious lower-airway inflammation and 14 healthy controls are reported, with significantly higher values in horses with airway inflammation. Although these increased levels did not correlate with changes in neutrophil percentage in BAL, a positive association between adenosine levels and signs of lower airway inflammation (clinical score) was observed. These novel findings support the hypothesis that adenosine may contribute to bronchoconstriction and also act as a pro-inflammatory mediator in the bronchoalveolar milieu of horses with airway inflammation. Further investigation of this axis could lead to new approaches for the treatment of highly prevalent lower airway inflammatory conditions in the horse.

Cytology Results of Bronchoalveolar Lavage Fluid in Horses with Recurrent Airway Obstruction

Bronchoalveolar lavage is essential in evaluating airway’s status and response to treatment in horses with recurrent airway obstruction. In this study we investigated the changes of bronchoalveolar lavage (BAL) fluid in horses with recurrent airway obstruction undergoing treatment with corticosteroids. Cytologic examination revealed decreased neutrophils percentage in BAL fluid obtained from horses with recurrent airway obstruction at 21 days of treatment after receiving dexamethasone, compared with initial results before treatment. Cytologic examination of BAL fluid provided important data regarding the local inflammatory status of the airways before and after treatment.

Evaluation of variations in bronchoalveolar lavage fluid in horses with recurrent airway obstruction

To determine variations in cytologic counts of bronchoalveolar lavage (BAL) fluid attributable to month of collection, first and second aliquots, and left and right lung sites in horses with recurrent airway obstruction (RAO). 5 horses with RAO and 5 healthy horses without respiratory tract disease. Horses were housed in a stable for 5 months prior to and throughout the study. Bronchoalveolar lavage fluid was collected from the right and left lung of each horse 3 times at monthly intervals (February, March, and April). Each BAL fluid collection was performed by use of 2 incremental instillations of 250 mL of isotonic saline (0.9% NaCl) solution in the same bronchial site. Analysis of BAL fluid included volume of BAL fluid recovered, a CBC, and differential cytologic counts. Volume of BAL fluid recovered and cytologic counts did not differ in horses with RAO across time or between right and left lungs, except for the number of mast cells. Horses with RAO had significantly lower volumes of BAL fluid recovered, significantly lower percentages of macrophages and lymphocytes, and significantly higher percentages of neutrophils than did healthy horses. Despite individual variation, all horses with RAO had > 25% neutrophils throughout the study period. Despite variation among horses, BAL fluid cytologic counts were repeatable over short and long periods and samples can be used for longitudinal studies as a diagnostic tool of pulmonary inflammation in horses with RAO.

Evaluation of cytokine mRNA expression in bronchoalveolar lavage cells from horses with inflammatory airway disease

Inflammatory airway disease (IAD) is a common disorder of performance horses and is associated with poor performance and accumulation of mucus and inflammatory cells in lower airway secretions. Horses with IAD frequently have increased relative counts of neutrophils in bronchoalveolar lavage fluid (BALF); less commonly relative counts of eosinophils and/or mast cells may be increased. The aetiopathogenesis of IAD is unknown and may involve innate and/or acquired immune responses to various factors including respirable dust constituents, micro-organisms, noxious gases and unconditioned air. The molecular pathways and role of the immune system in the pathogenesis of IAD remain poorly defined and it is unknown whether polarised T cell responses occur in the disease, as have been reported to occur in equine recurrent airway obstruction and asthma in humans. Elucidating cytokine responses that develop in horses with IAD may allow a greater understanding of the possible aetiopathological pathway(s) involved and could contribute to development of novel treatments. We compared the mRNA expression of tumour necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin (IL)-1β, IL-2, IL-4, IL-8, IL-13, IL-17 and IL-23 in cell pellets extracted from BALF of horses with IAD ( n = 21) and horses free of respiratory tract disease ( n = 17). Horses with IAD had significantly increased levels of TNF-α, IL-1β and IL-23 mRNA; no significant differences in the other cytokine mRNAs were detected. The results of this study indicate that IAD of horses is associated with increased mRNA expression of pro-inflammatory cytokines in BALF cells, which may reflect stimulation of the innate immune responses to inhaled antigens. There was no evidence of a polarised T-cell cytokine response suggesting hypersensitivity responses may not be involved in the aetiopathogenesis of IAD.

Rapid, Multiwell Colorimetric Assay for Measuring Neutrophil Chemoattractant Activity in Bronchoalveolar Lavage Fluid of Horses with Recurrent Airway Obstruction

The criteria used to diagnose recurrent airway obstruction (RAO) in affected horses include demonstration of reversible lower airway obstruction and greater than 25% neutrophils in bronchoalveolar lavage fluid (BALF). Additional objective laboratory tests are needed to improve diagnostic accuracy and to monitor response to treatment. The goal of this study was to determine if neutrophil chemoattractant activity of BALF could be measured by using a previously described, rapid, multiwell colorimetric assay for chemotaxis. In this assay, neutrophils that have migrated through a membrane filter are collected into the bottom well of a disposable chemotaxis-cell migration chamber. The number of viable cells collected in the bottom well is quantified by measurement of the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenol tetrazolium bromide (MTT), which is reduced by dehydrogenase in mitochondria of live cells. The number of migrating cells corresponds to the amount of MTT reduced, which is measured with an enzyme-linked immunosorbent assay plate reader. Fourteen adult horses were enrolled in this study, 7 of which had owner histories consistent with RAO. Each horse was sedated, a bronchoalveolar lavage tube was passed, and saline was infused and immediately aspirated. An aliquot of BALF was used for differential cell count, and BALF supernatant was harvested to assess neutrophil chemoattractant activity. Normal control horses and RAO-affected horses were distinguished according to clinical signs and percent neutrophils in BALF. Neutrophil chemoattractant activity of BALF was significantly greater in RAO-affected horses (P = 0.001) compared with control horses. This assay may be useful in future studies for monitoring response to therapy in RAOaffected horses.

Comparison of TGF-beta 1 concentrations in bronchoalveolar fluid of horses affected with heaves and of normal controls

Airway remodeling may play an important role in heaves pathophysiology. Transforming growth factor-beta 1 (TGF-beta1) is a potent profibrotic cytokine, which might contribute to airway wall thickening and fibrosis of bronchiolar and alveolar submucosa. An ELISA designed for the measurement of human TGF-beta1 was used to measured total TGF-beta1 released in bronchoalveolar lavage fluid (BALF) of normal horses and of those affected with heaves in remission. The specificity of the assay for TGF-beta1 of the horse was confirmed using recombinant equine TGF-beta1. The influence of hay exposure on TGF-beta1 release in the airways was also examined by stabling horses in a dusty environment. TGF-beta1 was found in the BALF of all horses. However, no significant difference between basal concentration of TGF-beta1 in BALF of control horses versus that of horses affected with heaves was found. Furthermore, no differences were identified in these populations 1 and 9 days after allergen challenge. In conclusion, these data indicate that TGF-beta1 is released in BALF fluid of horses in biologically active concentrations. Other studies are necessary for a better definition of the role of this cytokine within the lung, as our study does not establish a causal relationship between TGF-beta1 and the pathophysiology of heaves in the horse.

Effects of pentoxifylline on pulmonary function and results of cytologic examination of bronchoalveolar lavage fluid in horses with recurrent airway obstruction.

To determine the effects of pentoxifylline (PTX) administration on lung function and results of cytologic examination of bronchoalveolar lavage fluid in horses affected by recurrent airway obstruction (RAO). 10 RAO-affected horses. 6 horses were orally administered PTX (16 g) mixed with corn syrup, and 4 horses were administered corn syrup alone, twice daily for 14 days. Pulmonary function was evaluated before administration (day 0) and on days 8 and 15. Bronchoalveolar lavage (BAL) was performed on days 0 and 15. Reversibility of airway obstruction was assessed by measuring pulmonary function before and after administration of atropine (0.02 mg/kg, IV). Serum concentration of PTX was measured in 4 horses 30 minutes and 2 and 4 hours after administration of PTX on days 1, 2, 3, 7 and 14. Administration of PTX to BAO-affected horses resulted in a decrease in elastance value on day 8 and on elastance and resistance (RL) values on days 8 and 15. Results for cytologic examination of BAL fluid obtained on day 15 did not differ significantly, compared with values for day 0. Values of RL decreased in all horses following administration of atropine. When mixed in corn syrup and administered orally, PTX was poorly absorbed in horses, and there was noticeable variation in serum PTX concentrations over time and among horses. Based on these results, it can be concluded that administration of PTX at high doses improved respiratory function of RAO-affected horses maintained in an unfavorable environment.

Surfactant proteins in bronchoalveolar lavage fluid of horses: assay technique and changes following road transport

An enzyme-linked immunosorbent assay (ELISA) was developed for equine surfactant proteins SP-A and SP-D in bronchoalveolar lavage fluid (BALF). Anti-equine SP-A or SP-D monoclonal antibodies (mAb) were produced by hybridoma...

Interleukin-8 concentration and neutrophil chemotactic activity in bronchoalveolar lavage fluid of horses with chronic obstructive pulmonary disease following exposure to hay.

To analyze effects of hay dust exposure on interleukin-8 (IL-8) concentration, percentage of neutrophils, and neutrophil chemotactic activity in bronchoalveolar lavage fluid (BALF) of horses with chronic obstructive pulmonary disease (COPD). 16 healthy horses and 29 horses with COPD. IL-8 concentration, percentage of neutrophils, and neutrophil chemotactic activity in BALF were measured. Values were analyzed with respect to hay dust exposure. These variables were also measured in 5 asymptomatic horses with COPD after the induction of clinical signs by changing feed from silage to hay. L-8 concentrations and chemotactic activity in BALF were greater in horses with COPD, compared with healthy horses, and greater in horses with COPD exposed to hay dust, compared with nonexposed affected horses. An increase in IL-8 concentration accompanied by an increase in percentage of neutrophils in BALF and development of clinical signs of COPD were induced in asymptomatic horses with COPD by changing feed from silage to hay. Exposure of horses with COPD to hay dust components resulted in an increase in IL-8 secretion at the bronchoalveolar surface. This chemokine may play a role in the pathogenesis of COPD, because it causes neutrophil accumulation in the bronchoalveolar space. Our results underscore the importance of eliminating dust sources for the treatment and prevention of COPD in horses.

Blood gas, plasma lactate and bronchoalveolar lavage cytology analyses in racehorses with respiratory disease.

The purpose of this study was to evaluate the relationship between bronchoalveolar lavage (BAL) cytology, arterial blood gases and plasma lactate concentration during a standardised treadmill test (STT) in racehorses with small-airway inflammation (SAI), or exercise-induced pulmonary haemorrhage (EIPH). Sixteen Thoroughbred and 20 Standardbred actively racing racehorses, were divided into a control group (n = 10), EIPH group (n = 13) and SAI group (n = 13). Each STT consisted of a 2 min trot at 4 m/s followed by 5 x 1 min, incremental speed steps (6, 8, 10, 11 and 12 m/s) at 10% incline for Thoroughbred and 5% for Standardbred horses, followed by a 15 min recovery period. Blood was collected via a transverse facial artery catheter at the end of each step. Total nucleated cell count of bronchoalveolar lavage fluid (BALF) collected from horses with EIPH was significantly higher than controls. Neutrophil count and percentage in BALF collected pre-STT from horses with SAI were significantly higher than controls. Horses with EIPH or SAI exhibited a more severe exercise-induced arterial hypoxaemia than control horses during the first 4 steps of the STT (P < 0.001). Poorly performing horses were more severely hypercapnic than control horses during the STT recovery period (P < 0.05). Five and 15 min post exercise, horses with SAI and EIPH had higher blood lactate and lower blood bicarbonate concentrations than control horses (P < 0.05). The results of this study suggest that lung ventilation-perfusion mismatch may be more severe in horses with respiratory disease. BALF of horses with SAI and EIPH was characterised by neutrophilic and lymphocytic inflammation, respectively. Very few parameters were significantly different between groups of good and poor performers.

Effect of exercise and COPD crisis on isoprostane concentration in plasma and bronchoalveolar lavage fluid in horses.

To test whether isoprostanes could be used as markers of oxidative stress in horses, their concentration was determined in plasma and in pulmonary epithelial lining fluid (PELF) in 3 models of oxidative stress: (1) strenuous exercise, (2) acute COPD crisis and (3) exercise combined with COPD crisis. Four horses were investigated twice, once in crisis and once in remission. The animals underwent a standardised treadmill exercise test. Isoprostane assessment was performed in plasma and bronchoalveolar lavage fluid 24 h before and 1 h after exercise and in plasma also immediately after exercise. Exercise in remission induced a significant increase of isoprostanes in plasma and in PELF. In horses in crisis, the isoprostane concentrations did not increase in plasma, while they did increase in PELF. Lastly, exercise in crisis increased plasma levels of isoprostanes, but did not change PELF isoprostanes. In conclusion, 1) isoprostanes are increased by systemic oxidative stress induced by strenuous exercise in COPD horses in remission either in PELF or in plasma; 2) only PELF and not plasma isoprostanes are increased by pulmonary oxidative stress induced by COPD crisis and 3) unexpectedly, exercise in crisis increased plasma but not PELF isoprostanes.

Phenotypic analysis of bronchoalveolar lavage fluid lymphocytes in horses with chronic pulmonary disease.

A panel of specific antibodies against CD3, CD4, CD5, CD8, MHC I and II was used in single and two colour flow cytometry to define T cell subpopulations in bronchoalveolar lavage fluid of horses affected with chronic obstructive pulmonary disease and of healthy controls. According to the results of the clinical examination including bronchoscopy and cytology of the tracheal aspirate the horses were divided into four groups (healthy, subclinically to mildly affected; moderately affected, and severely affected). All groups of horses had a similar percentage of CD3+ cells in the BALF. Compared to controls, severely affected horses had a significantly increased number of CD4+ cells in the BALF, but a similar percentage of CD4+ cells whereas mildly and moderately affected horses had a decreased percentage. The percentage and number of CD8+ cells and the percentage of CD8+/MHCII+ cells in the BALF was found to be higher than normal and varied according to the disease state. This novel finding raises the possibility that not only the CD4+ cells but also the CD8+ cells are involved in the pathogenesis of COPD. The percentage and the number of CD8+ cells in BALF might be of diagnostic value to detect subclinical to mild cases of COPD.