Pulmonary Eosinophilia Associated with Increased Airway Responsiveness in Young Racing Horses

Horses are known to acquire small airway disease (SAD), an allergen-induced naturally occurring syndrome of reversible obstructive lung disease accompanied by airway hyperresponsiveness and increased inflammatory cell numbers on bronchoalveolar lavage (BAL). This disorder has received scant attention in young racehorses. The purpose of the present report was to examine the effect of BAL eosinophilia in young racehorses on clinical examination, BAL, hematology, airway responsiveness, and on pulmonary function at rest and after a standardized exercise challenge. Five (3 males, 2 females; age 2.6 ± 0.9 years) with a history of respiratory compromise and BAL eosinophil differential count <5% and 6 controls (4 males, 2 females; age 3.5 ± 1.0 years) training and performing to expectation with normal BAL cell differential (eosinophils < 1%) were studied. Respiratory system clinical examination was performed and expressed as a clinical score. Arterial blood gas measurements, CBC, and pulmonary function testing were performed at rest. Pulmonary mechanics measurements were repeated 1 hour and 20 hours after a standardized treadmill exercise challenge. Incremental histamine inhalation challenge was performed and the concentration of histamine effecting a 35% decrease in dynamic compliance (PC35C_Dyn) was determined. Significant differences were noted between and controls with regard to clinical score ( P = .01), blood eosinophils ( P = .04), BAL cell count ( P = .04), BAL macrophage differential ( P = .04), PC35C_Dyn ( P = .008), and tidal volume and respiratory rate at 20 hours following exercise challenge ( P = .05). We conclude that pulmonary eosinophilia and airway hyperresponsiveness are manifest in some young horses without overt airway obstruction at rest. We speculate that these may be early events in the natural progression of heaves.     

Effects of aerosolized albuterol on physiologic responses to exercise in standardbreds.

OBJECTIVE: To examine the effects of an aerosolized beta2-adrenoreceptor agonist, albuterol, on performance during a standardized incremental exercise test in clinically normal horses. ANIMALS: 8 Standardbred pacing mares. PROCEDURE: Clinically normal horses, as judged by use of physical examination, hematologic findings, serum biochemical analysis, and airway endoscopy, were randomly assigned to 2 groups and were given 900 microg of albuterol via a metered-dose inhaler 30 minutes before beginning a standardized incremental exercise test in a crossover design with a 7-day minimum washout. Further examination included measurement of baseline lung mechanics, response to histamine bronchoprovocation, and bronchoalveolar lavage. RESULTS: No significant differences (albuterol vs placebo) were seen for any incremental exercise test variables (ie, maximum oxygen consumption, maximum carbon dioxide consumption, respiratory quotient, treadmill speed at heart rate of 200 beats/min, or number of steps completed during an incremental exercise protocol). Mast cell percentage was significantly (r = -0.84) associated with the concentration of aerosolized histamine that evoked a 100% increase in total respiratory system resistance. No other direct correlations between bronchoalveolar lavage fluid cell types and any indices of exercise capacity or airway reactivity were found. CONCLUSIONS AND CLINICAL RELEVANCE: Although no horse had exercise intolerance, 4 horses had airway hyperreactivity with bronchoalveolar lavage fluid mastocytosis; these horses may have been subclinically affected with inflammatory airway disease. In our study, albuterol did not enhance performance in 8 clinically normal racing-fit Standardbreds.     

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 (PaO₂) 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 PaO₂ provide a good assessment of drug efficacy.     

Comparison of four staining methods for detection of mast cells in equine bronchoalveolar lavage fluid

Mast cells normally are present in equine bronchoalveolar lavage fluid (BALF), but usually represent <2% of all cells in healthy horses. An increased percentage of mast cells has been associated with airway hyperactivity and inflammatory airway diseases, but marked differences are reported between studies in normal and diseased horses. Because an abnormal mast cell count may be of clinical relevance, we compared the ability of a fast Romanowsky method to stain mast cell granules with that of 3 metachromatic stains: automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains. The BALF cells from 24 horses were studied. A differential cell count was performed blindly on 400 cells. The percentages of mast cells obtained were analyzed by means of repeated-measures analysis of variance and Fischer's PLSD test. The Bland and Altman method was used to assess agreement among stains. The mean percentage of mast cells in BALF was significantly lower with the fast Romanowsky than with the automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains. With the fast Romanowsky stain, the metachromatic granules of mast cells were not stained, and their identification was based on morphologic criteria. Toluidine blue staining allowed detection of the highest mean percentage of mast cells, but was inadequate for performing a differential cell count on other cell types. In conclusion, fast Romanosky stain may be inadequate for detection of mast cells in equine BALF, whereas automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains provide metachromatic staining of mast cell granules.     

Pilot study investigating the ability of an herbal composite to alleviate clinical signs of respiratory dysfunction in horses with recurrent airway obstruction

Recurrent airway obstruction (RAO), known previously as chronic obstructive pulmonary disease (COPD), is a debilitating respiratory condition that significantly contributes to lost training days and illness in racehorses. Herbs are becoming increasingly popular for the prophylaxis or treatment of the clinical signs of RAO despite a paucity of research on efficacy and safety. We evaluated the ability of an herbal composite containing garlic, white horehound, boneset, aniseed, fennel, licorice, thyme, and hyssop to reduce the clinical signs of RAO, hypothesizing that the product would safely reduce signs and would improve the inflammatory cell profile within the lungs. The composite was fed to 6 horses with symptomatic RAO for 21 d in a crossover manner. Ventigraphs were used to record respiratory rate and intrapleural pressure; the proportion of inflammatory cells in fluid aspirated from the trachea was determined. Blood biochemical and hematologic screening was conducted to identify possible adverse effects. Treatment with the composite did not result in statistically significant changes in any of the parameters evaluated. A trend to a decrease in respiratory rate (P = 0.1) and an increase in the proportion of macrophages (P = 0.1) was observed in the horses receiving the herbal composite compared with placebo. These data indicate a potential for the herbal composite to safely reduce the elevated respiratory rate in horses with RAO. Future research with a greater number of horses is warranted to further characterize the effect of this product on horses with RAO.     

Stabling is associated with airway inflammation in young Arabian horses

We examined the effect of stabling on upper and lower airway inflammation in 14 yearling Arabian horses that had been at pasture since birth. Horses were divided into 2 groups of 7. One group was stabled for 3 months and the other remained at pasture. The groups were then switched over for another 3 months. The nasopharynx, guttural pouches and trachea were examined endoscopically and bronchoalveolar lavage performed every month. An upper airway inflammation score was devised based on the magnitude of pharyngeal lymphoid hyperplasia and guttural pouch inflammation. During stabling this score remained constant, whereas it decreased during the 3 months at pasture. Stabling was also associated with a higher number and percentage of neutrophils in bronchoalveolar lavage fluid and with a smaller percentage of lymphocytes. There was no correlation between upper airway inflammation score and bronchoalveolar lavage cytology. During a nasal occlusion test, dorsal displacement of the soft palate occurred more times in stabled than in pastured horses, but this was heavily biased by the results from one animal. We conclude that stabling is associated with inflammation of both the upper and lower airway of young horses.     

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.     

Cellular content of secretions obtained by lavage from different levels of the equine respiratory tract

Lavage procedures were used to obtain samples of respiratory secretions from the nasal cavity, trachea, bronchi and bronchoalveolar level of apparently normal conscious horses, and the cellular composition of the lavage fluids was assessed. There was a progressive increase in total cell count of the secretions obtained from the upper to the lower respiratory tracts. Nasal lavage fluid was composed chiefly of epithelial cells, whereas tracheal, bronchial and bronchoalveolar fluids contained higher proportions of macrophages, lymphocytes and neutrophils. Eosinophils and mast cells were identified in small numbers. The results of the differential cell counts were compared with previous published reports, which revealed some major differences between studies. It is suggested that these differences may relate to variations in the collection technique, the cytological interpretation of cell types, the presence of subclinical airway disease, and the degree of exposure to airborne environmental contaminants.     

Clinical signs, evaluation of bronchoalveolar lavage fluid, and assessment of pulmonary function in horses with inflammatory respiratory disease

OBJECTIVE: To evaluate the association among clinical signs, results of cytologic evaluation of bronchoalveolar lavage (BAL) fluid, and measures of pulmonary function in horses with inflammatory respiratory disease. ANIMALS: 9 healthy horses, 5 horses with inflammatory airway disease (IAD), and 9 horses with chronic obstructive pulmonary disease (COPD). PROCEDURES: Clinical examination, lung function tests, and BAL were performed on each horse. RESULTS: Standard lung mechanics of horses with exacerbated COPD differed significantly from those of healthy horses; however, there were few differences among horses with IAD, horses with COPD during remission, and healthy horses. Most variables for forced expiration (FE) in horses with COPD or IAD differed significantly from those for healthy horses. Results of clinical examination had low to moderate sensitivity and predictive values for a diagnosis of COPD (range, 67 to 80%). Results of FE tests had high sensitivity, specificity, and predictive values for a diagnosis of COPD (79 to 100%), and results of standard lung mechanics tests had low sensitivity and predictive values (22 to 69%). Percentage of neutrophils in BAL fluid was highly sensitive (100%) but moderately specific (64%) for a diagnosis of COPD. CONCLUSIONS AND CLINICAL RELEVANCE: Clinical examination is moderately accurate for establishing a diagnosis of COPD. Forced expiration tests can specifically detect early signs of airway obstruction in horses with COPD and IAD that may otherwise be inapparent. Cytologic evaluation of BAL fluid allows early detection of inflammatory respiratory disease, but it is not specific for COPD.     

Purification and biochemical characterization of pulmonary surfactant protein A of horses

OBJECTIVE: To characterize surfactant protein isolated from bronchoalveolar lavage fluids of healthy horses. ANIMALS: 10 Thoroughbreds (5 males, 5 females; 26 to 40 months old) that did not have a history or clinical signs of respiratory tract disease. PROCEDURE: Bronchoalveolar lavage fluid (BALF) was obtained and centrifuged at 33,000 X g. Lipid was removed from precipitated fractions by means of extraction with 1-butanol, and organic solvent-insoluble protein precipitates were dialyzed against Tris buffer. The suspension was centrifuged, and supernatant was placed in a mannose-Sepharose affinity column, with calcium. The bound protein fraction was analyzed by means of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, western immunoblot analysis, and amino acid sequencing. A liposome-aggregation assay was also performed, using purified proteins. RESULTS: Protein isolated by use of mannose-affinity matrices was identified as surfactant protein A (SP-A). It had carbohydrate-binding and phospholipid-aggregation properties characteristic of SP-A isolated from other animal species. The partial primary sequence of the isolated protein had high homology with rat and human SP-A. Furthermore, the equine SP-A reacted with anti-human and anti-rat SP-A specific antibodies. CONCLUSION: Analysis of these findings indicated the existence of SP-A in pulmonary tissues of horses. CLINICAL RELEVANCE: Measurement of SP-A concentrations may be useful for clinicians evaluating pulmonary disease of horses.     

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.     

Evaluation of a risk-screening questionnaire to detect equine lung inflammation: results of a large field study

Reasons for performing study: The diagnosis of equine recurrent airway obstruction (RAO) and inflammatory airway disease (IAD) is based on clinical signs and increased inflammatory cell percentages in the bronchoalveolar lavage (BAL) fluid. Since a BAL is an invasive procedure, a risk‐screening questionnaire (RSQ) would be a valuable screening tool for lung inflammation. Objective: To evaluate the accuracy of a RSQ to detect lower airway inflammation (LAI) in a large population of horses. Methods: A standardised BAL was performed in the field on 167 horses in Alberta, Canada. Horses were separated into 3 categories: 1) BAL normal; 2) BAL mild to moderate LAI (MLAI), and 3) BAL severe LAI (SLAI). The horse owners were asked to complete a RSQ. The RSQ scores were compared to the BAL results to determine the likelihood of a horse having MLAI, SLAI or no LAI. Results: Based on BAL cytology, 28 (17%) horses were normal and 139 (83%) were abnormal, with 110 (66%) showing MLAI and 29 (17%) SLAI. Horses with SLAI and MLAI had a mean RSQ score of 0.95 and 0.70, respectively, compared to 0.60 for normal BAL horses. Horses with SLAI showed more clinical signs than normal and MLAI horses. The sensitivity and negative predictive values of the RSQ for detecting SLAI using a cut‐off score of 0.87, were excellent at 0.90 (95%CI 0.73–0.98) and 0.96 (95%CI 0.82–1.00). Questions on the clinical signs typically found in RAO cases differed significantly between horses with BAL SLAI and those with BAL normal. Conclusions: Prevalence of MLAI was high in this population. Although the RSQ did not allow differentiating normal horses from horses with MLAI, it has a high sensitivity to detect horses with SLAI and is therefore a good screening tool for SLAI.     

Use of successive dynamic videoendoscopic evaluations to identify progression of recurrent laryngeal neuropathy in three horses

CASE DESCRIPTION: 3 racehorses were evaluated because of poor performance or abnormal noise originating from the upper portion of the respiratory tract. CLINICAL FINDINGS: During maximal exercise, initial dynamic videoendoscopy of the upper respiratory tract revealed complete arytenoid cartilage abduction in 2 horses and incomplete but adequate abduction of the left arytenoid cartilage in 1 horse. Subsequent exercising endoscopic evaluation revealed severe dynamic collapse of the left arytenoid cartilage and vocal fold in all 3 horses. TREATMENT AND OUTCOME: 2 horses were treated with prosthetic left laryngoplasty and raced successfully. One horse was retired from racing. CLINICAL RELEVANCE: Idiopathic laryngeal hemiplegia can be a progressive disease. Successive dynamic videoendoscopic upper airway evaluations were used to confirm progression of left laryngeal hemiplegia in these 3 horses. Videoendoscopy of the upper respiratory tract during exercise should be considered as part of the clinical evaluation of horses with signs of upper respiratory tract dysfunction.     

Relationship of horse owner assessed respiratory signs index to characteristics of recurrent airway obstruction in two Warmblood families

Reasons for performing study: The horse owner assessed respiratory signs index (HOARSI‐1–4, healthy, mildly, moderately and severely affected, respectively) is based on owner‐reported clinical history and has been used for the investigation of recurrent airway obstruction (RAO) genetics utilising large sample sizes. Reliable phenotype identification is of paramount importance in genetic studies. Owner reports of respiratory signs have shown good repeatability, but the agreement of HOARSI with an in‐depth examination of the lower respiratory tract has not been investigated. Objectives: To determine the correlation of HOARSI grades 3/4 with the characteristics of RAO and of HOARSI‐2 with the characteristics of inflammatory airway disease. Further, to test whether there are phenotypic differences in the manifestation of lung disease between families. Methods: Seventy‐one direct offspring of 2 RAO‐affected Warmblood stallions (33 from the first family, 38 from the second) were graded as HOARSI‐1–4 and underwent a clinical examination of the respiratory system, arterial blood gas analysis, endoscopic mucus scoring, cytology of tracheobronchial secretion (TBS) and bronchoalveolar lavage fluid (BALF), and clinical assessment of airway reactivity to methacholine chloride. Results: HOARSI‐3/4 animals in clinical exacerbation showed signs consistent with RAO: coughing, nasal discharge, abnormal lung sounds and breathing pattern as well as increased numbers of neutrophils in TBS and BALF, excessive mucus accumulation and airway hyper‐responsiveness to methacholine. HOARSI‐3/4 horses in remission only had increased amounts of tracheal mucus and TBS neutrophil percentages. Clinical phenotypes were not significantly different between the 2 families. Conclusions and clinical relevance: HOARSI reliably identifies RAO‐affected horses in our population.     

Bronchoscopic findings in 48 cats with spontaneous lower respiratory tract disease (2002-2009)

Background: Diagnosis of lower respiratory disease requires collection of airway samples to confirm the etiology of disease. Bronchoscopic evaluation is commonly performed in dogs but less information is available in cats. Hypothesis: The presence and number of bronchoscopic abnormalities visualized during bronchoscopic evaluation of cats with lower respiratory disease will correlate with the type of disease and total and differential cell counts in bronchoalveolar lavage (BAL) fluid. Animals: Forty‐eight cats prospectively evaluated by a single bronchoscopist. Methods: Bronchoscopy was performed during clinical evaluation of cats presenting with cough, respiratory distress, or both. Cats were evaluated for airway hyperemia, stenosis, or collapse, mucus accumulation, bronchiectasis, and epithelial irregularities. Cats were placed into groups of bronchitis/“asthma,” pneumonia, or neoplasia based on BAL findings, histopathology, and response to appropriate medical therapy. Summation of bronchial abnormalities and total and differential cell counts were compared among groups. Results: Endobronchial abnormalities were common in cats with feline bronchitis/asthma, pneumonia, and neoplasia and no differentiating features were found. Excessive mucus accumulation was common (83%), followed by stenosis of bronchial openings and nodular epithelial irregularities (56%), airway hyperemia (54%), airway collapse (48%), and bronchiectasis (27%). Total bronchoscopic score and total cell count did not differ among groups, although differential cell counts were significantly different. A weak correlation (R²= 0.16, P= .006) between age and total bronchoscopic score was noted. Conclusions and Clinical Relevance: Bronchoscopic abnormalities are common in cats with lower respiratory tract disease, and visualization of the airways provides additional nonspecific clinical information in cats.     

Lower respiratory tract disease in Thoroughbred racehorses: analysis of endoscopic data from a UK training yard

REASON FOR PERFORMING STUDY: There has been much research directed at potential causative agents and the epidemiology of lower respiratory tract disease (LRTD), but few reports of the clinical progression and outcome. OBJECTIVES: To define clinical features of LRTD in racehorses, including association with age and average duration of disease, through the analysis of endoscopic data. METHODS: A retrospective analysis of records from a single training yard of endoscopic examinations undertaken over a 2-year period was performed. Horses were subjected to regular endoscopic examination for a variety of reasons, and all horses placed on antibiotic therapy for lower respiratory tract disease were rescoped following treatment. Data analysed included a 0-8 tracheal mucus score based on visible endoscopic mucus and gross tracheal lavage turbidity, as well as age and treatment duration and type. RESULTS: A total of 522 endoscopic examinations undertaken on 123 horses and tracking 169 episodes of lower respiratory tract disease were recorded. Mean duration of disease episode was 15.5 days (median = 11 days, range = 4-61 days). Horses age 2 years were significantly more likely than those age > or=3 years to have at least one episode of respiratory disease (P<0.001). There was a direct association between initial tracheal mucus score and rescope score after treatment. CONCLUSIONS: Lower respiratory tract disease was more common in 2-year-olds than in older horses. Affected horses had endoscopic evidence of increased tracheal mucus accumulation for an average of 15.5 days per episode, a considerably shorter period than that suggested by previous studies. POTENTIAL RELEVANCE: Endoscopic examinations permit longitudinal tracking of lower respiratory tract disease in Thoroughbred racehorses. A prolonged duration of disease, sometimes extending for months, can be expected in a small proportion of cases, some of which appear to be refractory to treatment. There is a clear need for evidence-based analysis of treatment regimes to assist clinicians in decision making when managing disease in both individual and group situations.     

Comparison of tracheal aspirates and bronchoalveolar lavage in racehorses. 1. Evaluation of cytological stains and the percentage of mast cells and eosinophils

OBJECTIVE: To compare a fast Romanowsky cytological stain (Diff-Quik) and Leishman's stain for the detection of mast cells in samples from the lower airways of racehorses, and to compare the proportion of mast cells and eosinophils in the total inflammatory cells in tracheal aspirate (TA) with those in paired bronchoalveolar lavage (BAL) samples. DESIGN: Retrospective case series of 48 young Thoroughbred and Standardbred racehorses. PROCEDURE: Fifty-one paired TA and BAL samples were collected after treadmill exercise from 48 horses with poor racing performance. Two slides were prepared from each sample; one was stained with Diff-Quik stain and the other with Leishman's stain. Differential cell counts of eosinophils and mast cells were recorded from each slide. Comparison of the suitability of the stains for the detection of mast cells, and comparisons of eosinophil and mast cell percentages in TA and BAL samples were analysed using the non-parametric Wilcoxon matched pairs test. RESULTS: Percentages of mast cells were significantly higher in Leishman than in Diff-Quik stained slides in both TA (P = 0.03) and BAL samples (P < 0.0001). Mast cell percentages were significantly higher in BAL than in TA samples using Leishman's stain (P < 0.0001). There was no significant difference in eosinophil percentages between TA and BAL samples (P = 0.07). CONCLUSIONS: Fast Romanowsky type stains (for example Diff-Quik) are not appropriate for the detection of mast cells in samples from the equine lower respiratory tract. Therefore, a metachromatic stain that reliably identifies mast cells (for example Leishman's) should be used if evaluation of mast cells in lower respiratory tract is undertaken. Mast cells are predominantly found in the distal small airways and alveoli sampled with a BAL. In contrast, eosinophils appear to be evenly distributed in the lower respiratory tract. However, high percentages of eosinophils are occasionally found only in TA samples. We recommend that both a TA and BAL be used for the evaluation of eosinophils and mast cells within the equine lower respiratory tract.