Inflammatory airway disease, as defined by a 2007 consensus statement from the American College of Veterinary Internal Medicine, is a disease of adult horses defined by exercise intolerance, nonseptic airway inflammation, abnormal pulmonary function test results, and excessive tracheal mucus.1 However, a recent study2 has suggested that IAD defined by tracheal inflammation (diagnosed by endoscopic tracheal sampling) and bronchial IAD (diagnosed by cytologic examination of BALF) may have different etiologies, clinical signs, or other distinct features.
Inflammatory processes of the small airways are characterized by an increased percentage of lymphocytes, monocytes, neutrophils, mast cells, eosinophils, or both in BALF,3–10 although the specific criteria for diagnosis of IAD by use of BALF focus on mild neutrophilia, eosinophilia-mastocytosis, or both.10,11 Several studies have suggested that horses with neutrophilic versus eosinophilic or mastocytic inflammation may have 2 or more distinct disease types. Horses with neutrophilic BALF are more likely to be older, have a cough,6 and have abnormally low exercising arterial oxygen tension,12–14 whereas those with pulmonary eosinophilia or mastocytosis are usually racehorses, are younger, and are examined because of poor performance rather than cough and have increased baseline respiratory reactance and resistance as well as hyperreactivity to histamine bronchoprovocation.3,6,15,16 Several authors6,8 have suggested a different etiologic basis for IAD characterized by these different cell types. Asthma, the most common noninfectious IAD of humans, has traditionally been viewed as an allergic response to inhaled triggers such as pollens or other aeroallergens, resulting in a predominantly eosinophilic inflammation.17 Recently, several asthma subtypes are increasingly being associated with neutrophilic rather than eosinophilic inflammation of the airways.18–20 These include occupational asthma, which occurs in workers who are exposed to inhaled particulates ranging from industrial dust and chemicals21–23 to octopus particles and fruit tree residues24,25 and who are sensitized to urban air pollution, especially ozone and diesel fumes.26,27 Additionally, recent data suggest that up to half of all cases of asthma in human patients are characterized as having neutrophils as the predominant inflammatory cell type.19 This occupational asthma pathophysiology may have considerable relevance to equine medicine considering the high loads of dust and endotoxin in the breathing zone of stabled horses.28,29 In horses as well as humans, there is still no clear consensus whether lower airway inflammation results mainly from aeroallergens versus a hypersensitivity to inhaled toxins or whether in fact there are 2 distinct disease processes with different causes, treatments, and outcomes.
Considering the possibility of multiple discrete etiologies or disease processes governing IAD in horses, the objectives of the study reported here were to identify differences in age, career, season of admission, and upper airway endoscopic, cardiologic, and hematologic results between poorly performing racehorses with neutrophilic, eosinophilic-mastocytic, or mixed airway inflammation, compared with horses with noninflammatory BALE We hypothesized that there would be differences in the clinical features of horses examined because of poor performance but without a diagnosis of IAD. We also hypothesized that horses with eosinophilic-mastocytic IAD would be examined clustered in seasons of high concentrations of ambient pollen aeroallergens, whereas horses with neutrophilic IAD will be more likely to be examined in the winter, when they have higher exposure to barn dust. Inflammatory airway disease was diagnosed on the basis of results of cytologic examination of postexercise BALE (vs the presence of tracheal inflammation as diagnosed by endoscopic tracheal sampling).2
Materials and Methods
Data were compiled from a retrospective cohort of racing horses examined because of poor performance at the George D. Widener Hospital for Large Animals at the University of Pennsylvania between 2004 and 2010; inclusion criteria included completion of an HST examination and post-HST BAL. Data tabulated included age, sex, breed, season of admission, career (athletic discipline), HST test results, and results of cytologic examination of postexercise BALE. Horses underwent pre- and intra-HST lameness exam, dynamic upper airway endoscopy, arterial blood gas analysis, blood lactate analysis, telemetric ECG, pre- and post-HST echocardiography, and post-HST BAL, although not every horse had data recorded for every parameter.
The HST protocol used is depicted in detail else-where,30,31 but is briefly described below. After acclimatization to the treadmill, an arterial catheter was placed in the transverse facial artery under physical restraint, and blood gas valuesa were obtained at baseline and every minute during the testing protocol. Horses were then evaluated exercising on an HSTb in a climate-controlled environment for 3 to 4 minutes to a target heart rate of ≥ 200 beats/min, with speeds up to 14.5 m/s and an incline up to 3° for Thoroughbreds and speeds up to 14 m/s at a 0° incline for Standardbreds. This protocol was adjusted as necessary on the basis of the performance and prior conditioning of the horse. Videoendoscopic evaluation of the upper airway was performed at rest and during the HST examination, with images recorded for further analysis. M-mode and 2-D echocardiography were performed before and within 3 minutes after finishing the HST protocol when heart rates still exceeded 100 beats/min. Continuous telemetric base-apex ECG was obtained at rest, throughout the HST, and during the first 5 minutes after recovery (specific data not included).
The BALE was obtained approximately 1 hour after completion of the HST test. Horses were sedated with detomidine hydrochloride (0.005 to 0.01 mg/kg [0.003 to 0.005 mg/lb], IV), and an aseptically prepared 3-m videoendoscopec was passed through the nares and upper airways and wedged in a bronchus of the right lung. Local anesthesia (20 to 30 mL of 0.02% lidocaine infused via the endoscope) was used to reduce coughing prior to infusion of a single 300mL aliquot of warm sterile saline (0.9% NaCl) solution via polyethylene tubing, which was manually aspirated with 60-mL syringes. A pooled sample of BALE was preserved in EDTA, and the cells were isolated by means of cytocentrifugation. Slides were prepared with methanolic Wright-Giemsa and Prussian blue stains, and a differential count was made from a sample of ≥ 200 cells (excluding epithelial cells). Presence or absence of erythrocytes, amount of mucus (none, small amount, or large amount), and amount of hemosiderophages (none, few, or many) were recorded on the basis of interpretation of descriptions on the cytology report as ordinal, categorical variables. Depending on the presence and subtype of lower airway inflammation, the BAL results were used to divide the horses into 4 categories comprised of eosinophilic-mastocytic inflammation, neutrophilic inflammation only, mixed inflammation (neutrophilic and eosinophilic-mastocytic inflammation), or no inflammation (control). These 4 categories, including the control horses, are henceforth referred to as IAD subtypes. The criteria used to define the eosinophilic-mastocytic inflammation group were ≥ 0.5% eosinophils, ≥ 2% mast cells, or both, and ≥ 5% neutrophils for the neutrophilic inflammation group. For the mixed-inflammation group horses, BALE contained ≥ 5% neutrophils as well as ≥ 0.5% eosinophils, ≥ 2% mast cells, or all 3.11 Because the BALE neutrophil counts can be higher after exercise,13,32 a second analysis was also performed that used a more conservative cutoff of ≥ 10% neutrophils.
Season of admission was classified by month of appointment, with winter defined as the months of December through Eebruary, spring as March through May, summer as June through August, and autumn as September through November. Variables analyzed from the HST included maximum heart rate, highest blood lactate concentration, lowest arterial pH, lowest exercising Pao2, and highest exercising Paco2. Because the horses in this study were exercised to a variety of maximal speeds, correction for the expected variation in Pao2 and Paco2 values in mm Hg was calculated via least squares linear regressions formulae (expected Pao2 = 114.2 – [2.7 × running speed in m/s] and expected Paco2 = 35.5 – [0.5 × running speed in m/s] previously generated from clinically normal horses under similar conditions.31,33 The raw values were then compared with the speed-adjusted expected value, and the percentage difference between the raw and the expected value was calculated to quantify abnormalities in gas exchange as a factor of speed. Thus, a negative Δ value suggests that the blood gas value is lower than would be expected in a clinically normal horse. Results of the lameness evaluation, upper airway endoscopy, ECG, and echocardiographic examinations were converted into dichotomous data that specified for each variable whether the horse did or did not have an abnormal result at any testing point. Abnormal results for the lameness examination included any form of gait deficit either in hand before the HST or while undergoing the treadmill exam (generally Standardbreds that had gait abnormalities only while hobbled or at speed; HST was continued at the clinician's discretion and based on the degree of lameness in these cases). Upper airway abnormalities included dynamic collapse of 1 or more pharyngeal walls with or without axial deviation of aryepiglottic folds, dynamic left arytenoid cartilage collapse, intermittent dorsal displacement of the soft palate, and soft palate instability. Echocardiogram abnormalities included valvular insufficiency, chamber size abnormalities, or myocardial dysfunction, whereas ECG findings included heart rate above the expected rate for level of exercise and pathological arrhythmias.
Statistical analysis—Comparisons of IAD subtype (both standard and conservative definitions) with signalment; HST data; cardiac, upper airway, and gait abnormalities; season; and other BALE findings were analyzed. A Shapiro-Wilk test of normality was applied to continuous data, and these assumptions were not met. Therefore, Kruskal-Wallis tests were used to examine the association between the exposure variable of IAD subtype and continuous and ordinal categorical outcome variables. These included all HST and blood gas variables and BALE mucus and hemosiderophages. Simple logistic regression was used to quantify the association between IAD subtype and dichotomous variables including cardiac, upper airway, and gait abnormalities, which were classified as normal or abnormal. Association between the nominal exposure variables of sex, career, and season and IAD subtype (treated as an outcome) were examined via a Eisher exact test. Additionally, the possibility of a directional relationship between BALE neutrophil, eosinophil, and mast cell percentages with HST results (blood gas values, maximum heart rate, minimum pH, and maximum lactate) was explored by means of a Spearman rank correlation coefficient. To reduce the risk of type I error, a level of P ≤ 0.05 was assigned to denote significance. All analyses were performed with a commercial software package.d
Results
Records of 98 horses were obtained for analysis, and horses were subdivided by IAD subtype or control. Seventy-nine (81%) horses met the standard criteria for IAD (≥ 5% neutrophils for neutrophilic inflammation and mixed-inflammation groups and ≥ 0.5% eosinophils or ≥ 2% mast cells or both for eosinophilic-mastocytic and mixed-inflammation groups); 19 horses had noninflammatory BALE and were designated as the control group. There were 17 horses with neutrophilic inflammation only and 22 horses with mixed inflammation, of which 7 had neutrophilic and eosinophilic inflammation, 10 had neutrophilic and mastocytic inflammation, and 5 had neutrophilic, mastocytic, and eosinophilic inflammation. Eorty horses had eosinophilic-mastocytic BALE, of which 4 had eosinophilic inflammation only, 24 had only mastocytic inflammation, and 12 had both. Using the conservative cutoff for neutrophilic inflammation (≥ 10%), 66 horses (67%) met the criteria for IAD. A total of 32 horses were classified as controls, and only 4 horses had ≥ 10% neutrophils without other inflammatory cells (neutrophilic inflammation). Eleven horses that were originally classified as having mixed inflammation were reclassified as having eosinophilic-mastocytic inflammation, and 13 horses with a standard neutrophilic inflammation classification (≥ 5% neutrophils) were reclassified as having no inflammation (control).
Twenty-seven of the 98 (28%) horses were sexually intact males, 29 (30%) were geldings, and 41 (42%) were females, with a median age of 3 years (range, 2 to 8 years). Eifty-three (54%) of the horses were Standardbreds, of which 35 were pacers and 18 trotters. Thoroughbreds comprised 45 (46%) horses, of which 42 trained on the flat, and 3 raced over fences (steeplechase or timber). Thoroughbreds were significantly (P = 0.04) more likely to have mixed inflammation than Standardbreds, and when the conservative cutoff for neutrophils was used, both mixed-inflammation and eosinophilic-mastocytic inflammation groups were significantly (P = 0.009) associated with Thoroughbred breed. Summer and autumn were the most common seasons for horses to be examined because of poor performance (37% and 30% of horses, respectively), with 17% examined in the spring and 16% examined in winter. No significant associations were found between IAD subtype and season, age, sex, lameness, cardiac abnormalities, or upper airway abnormalities (Table 1).
Signalment and comorbidities for 79 horses classified on the basis of IAD subtype (neutrophilic, eosinophilic-mastocytic, or mixed inflammation) and for 19 control horses with normal BALF.
| Variable | Control (n = 19) | Eosinophilic-mastocytic inflammation (n = 40) | Neutrophilic inflammation (n = 17) | Mixed inflammation (n = 22) |
|---|---|---|---|---|
| Median age (range) | 3.0 (2–6) | 3.0 (2–8) | 3.0 (2–7) | 3.0 (2–6) |
| Sex Sexually intact male | 6 (32%) | 10 (25%) | 3 (18%) | 8 (36%) |
| Gelding | 4 (21%) | 14 (35%) | 6 (35%) | 5 (23%) |
| Female | 9 (47%) | 16 (40%) | 8 (47%) | 9 (41%) |
| Breed | ||||
| Standardbred | 13 (68%) | 20 (50%) | 12 (71%) | 8 (36%) |
| Thoroughbred | 6 (32%) | 20 (50%) | 5 (29%) | 14 (64%) |
| Career | ||||
| Thoroughbred—flat racer | 6 (32%) | 19 (48%) | 4 (18%) | 13 (59%) |
| Standardbred—pacer | 9 (47%) | 12 (30%) | 9 (53%) | 5 (23%) |
| Standardbred—trotter | 4 (21%) | 8 (20%) | 3 (18%) | 3 (14%) |
| Thoroughbred—steeplechaser | 0 (0%) | 1 (2%) | 1 (6%) | 1 (4%) |
| Season | ||||
| Spring | 3 (16%) | 8 (20%) | 3 (18%) | 3 (14%) |
| Summer | 8 (42%) | 15 (38%) | 8 (47%) | 5 (23%) |
| Autumn | 5 (26%) | 11 (27%) | 3 (18%) | 10 (45%) |
| Winter | 3 (16%) | 6 (15%) | 3 (18%) | 4 (18%) |
| Arrhythmia | n = 18 | n = 38 | n = 20 | |
| Present | 7 (39%) | 20 (53%) | 11 (65%) | 8 (40%) |
| Absent | 11 (61%) | 18 (47%) | 6 (35%) | 12 (60%) |
| Abnormal echocardiogram | n = 15 | n = 35 | n = 16 | n = 18 |
| Present | 8 (53%) | 20 (57%) | 10 (63%) | 10 (55%) |
| Absent | 7 (47%) | 15 (43%) | 6 (37%) | 8 (45%) |
| Lameness | ||||
| Present | 11 (58%) | 30 (75%) | 10 (59%) | 14 (64%) |
| Absent | 8 (42%) | 10 (25%) | 7 (41%) | 8 (36%) |
| Abnormal upper airway | n = 39 | n = 21 | ||
| Present | 7 (37%) | 26 (67%) | 3 (18%) | 5 (24%) |
| Absent | 13 (63%) | 13 (33%) | 14 (82%) | 16 (76%) |
Values represent absolute numbers of horses (and percentages) within a subtype. Numbers of horses in each subtype are specified at the top of each column, except where noted. Subtypes were assigned by use of the standard cutoff value for neutrophil percentage (≥ 5%).
Most horses had evidence of previous intrapulmonary hemorrhage (Table 2), with many hemosiderophages seen in the BALE of 15 (15%) and occasional hemosiderophages seen in 90 (92%) samples. Recent bleeding demonstrated by the presence of erythrocytes was observed in 30 (31%) postexercise BALE samples. Mucus was seen in the BALE of 32 (33%) horses and was significantly (P = 0.01) associated with IAD subtype, with the mixed-inflammation group significantly (P = 0.016) more likely to have large amounts of mucus than were control horses.
Results of cytologic examination of BALF for 79 horses classified on the basis of IAD subtype (neutrophilic, eosinophilic-mastocytic, or mixed inflammation) and for 19 control horses with normal BALF.
| Variable | Control (n = 19) | Eosinophilic-mastocytic inflammation (n = 40) | Neutrophilic inflammation (n = 17) | Mixed inflammation (n = 22) |
|---|---|---|---|---|
| Median macrophage percentage (range) | 63.0 (49–90) | 54.5 (18–80) | 55.0 (43–80) | 48.5 (34–69) |
| Median lymphocyte percentage (range) | 32.0 (6–47) | 36.5 (14–67) | 36.0 (14–51) | 37.5 (13–55) |
| Median neutrophil percentage (range) | 2.0 (1–4) | 2.0 (0–4) | 6 (5–15) | 9.5 (5–26) |
| Median eosinophil percentage (range) | 0.0 (0–0) | 3.5 (0–12) | 0.0 (0–0) | 1.0 (0–11) |
| Median mast cell percentage (range) | 1.0 (0–1) | 3.0 (0–10) | 1.0 (0–1) | 2 (0–13) |
| Hemosiderophages | ||||
| None | 3 (16%) | 1 (3%) | 1 (6%) | 3 (14%) |
| Few | 13 (68%) | 32 (80%) | 13 (77%) | 17 (77%) |
| Many | 3 (16%) | 7 (17%) | 3 (18%) | 2 (9%) |
| Erythrocytes | ||||
| Absent | 11 (58%) | 25 (63%) | 13 (76%) | 19 (86%) |
| Present | 8 (42%) | 15 (37%) | 4 (24%) | 3 (14%) |
| Mucus | ||||
| None | 14 (74%) | 28 (70%) | 10 (59%) | 14 (64%) |
| Small amount | 3 (16%) | 9 (22%) | 5 (29%) | 2 (9%) |
| Large amount | 2 (10%) | 3 (8%) | 2 (12%) | 6 (27%) |
Values are median and ranges where stated and numbers of horses and percentages otherwise. Subtypes were assigned by use of the standard cutoff value for neutrophil percentage (≥ 5%).
No significant association was noted between IAD subtype and any of the blood gas variables analyzed (Table 3), and no direct relationships were identified between percentage counts of BALE neutrophil, eosinophil, and mast cell and exercising blood gas values, maximum heart rate, minimum pH, and maximum lactate concentration.
Exercise physiology parameters obtained during HST examination for 79 horses classified on the basis of IAD subtype (neutrophilic, eosinophilic-mastocytic, or mixed inflammation) and for 19 control horses with normal BALF.
| Variable | Control (n = 19) | Eosinophilic-mastocytic inflammation (n = 40) | Neutrophilic inflammation (n = 17) | Mixed inflammation (n = 22) |
|---|---|---|---|---|
| Medianraw Pao2 (range) | 76.9 (53.5 to 97.4) | 78.6 (32.7 to 113) | 77.8 (62.1 to 92.5) | 81.0 (34.1 to 118) |
| Median %Δexpected Pao2 (range) | –11.0 (–38.8 to 10.0) | –9.6 (–62.5 to 28.4) | –9.9 (–28.8 to 7.3) | –10.1 (–60.9 to 27.1) |
| Medianraw Paco2 (range) | 40.6 (32.5 to 58.6) | 41.3 (35.0 to 62.9) | 42.0 (34.3 to 52.4) | 41.9 (12.9 to 57.2) |
| Median %Δexpected Paco2 (range) | 0.1 (–20.1 to 44.9) | 3.5 (–14.2 to 55.3) | 4.4 (–14.8 to 28.9) | 4.0 (–68.3 to 44.1) |
| Median minimum pH (range) | 7.40 (7.11 to 7.50) | 7.37 (7.19 to 7.50) | 7.41 (7.27 to 7.46) | 7.41 (7.23 to 7.48) |
| Median maximum lactate (range) | 3.3 (0.7 to 20.2) | 4.1 (0.8 to 12.0) | 2.8 (0.5 to 10.1) | 3.0 (0.7 to 10.0) |
| Mean maximum heart rate (SD) | 212 (13) | 206 (21) | 199 (42) | 203 (20) |
%ΔexpectedPao2 = Percentage difference between the raw and the expected value of Pao2. %Δexpected Paco2 = Percentage difference between the raw and the expected value of Paco2. rawPao2 = Lowest exercising Pao2. rawPaco2 = Highest exercising Paco2.
Numbers of horses within each subtype are specified at the top of each column. No significant differences were noted between values of any subtype. Subtypes were assigned by use of the standard cutoff value for neutrophil percentage (≥ 5%).
Discussion
The present study evaluated horses undergoing HST examination because of poor performance and examined associations between IAD subtype, clinical parameters, and seasonality. We found that Thoroughbreds were more likely to have eosinophilic-mastocytic inflammation, with or without concurrent neutrophilic inflammation, which is consistent with previous data that suggest this pattern of inflammation is most prevalent in young racehorses.1,3,5 However, although Standardbreds typically race at older ages than Thoroughbreds, in the present study, the median age for both groups was 3 years, suggesting that age was not a likely explanation for the differences in IAD subtype between these breeds in the patient population studied. In studies evaluating BAL results in Standardbreds with IAD, increases in eosinophil and mast cell numbers have not generally been reported,8,10,12,34 and most of the reports describing eosinophilic-mastocytic IAD include a high proportion of Thoroughbreds.6,7,9 Although counts of metachromatic cells can vary with BAL technique, slide preparation and staining technique,11,35–38 and number of cells counted per slide, no variation in these factors occurred during the time period of this study. However, the slides were evaluated by several clinical pathologists, and inter-rater variability in technique and interpretation may have affected our results.
Compared with the control group, an increase in mucus in the BALE in the mixed inflammation group was also noted in the present study. Increased tracheal mucus has been associated with poor performance31 and cough,39 but the reported correlation between tracheal mucus and airway neutrophilia is unclear.40–42 We did not identify studies that correlated airway eosinophil or mast cell numbers with lower airway mucus accumulation, although goblet cell hyperplasia has been associated with pulmonary eosinophilia in some murine models.43
Basic pathophysiology would suggest that differing inflammatory cell profiles may well be the result of different etiologies. The classic allergic, or atopic, response of type I hypersensitivity involves a T-helper cell 2 response characterized by IgE-mediated expression of IL-4, IL-5, and IL-13 that are responsible for eosinophil and mast cell recruitment, whereas neutrophilic infiltrates may be consistent with a nonallergic T-helper cell 1 response to airborne particles, viral disease, or other inhaled stimuli8 or simply a nonspecific response to IL-8. Patients with IAD have been shown to have significant increases in proinflammatory cytokines as well as differences in gene expression associated with airway neutrophilia versus mastocytosis.44,45 The closest human model of IAD, asthma, has traditionally been considered an allergic disease resulting in eosinophilic airway inflammation, and decades of investigation have focused on the causes and treatment of atopy.17 Recent evidence suggests that in fact up to 50% of human asthma cases are attributable to neutrophilic inflammation, possibly resulting from nonallergic sensitivity to particulate pollution, inhaled endotoxin, ozone, and other respirable toxins.18–20
In humans with allergic asthma, clinical signs and bronchial hyperreactivity do increase at times of high pollen exposure,46,47 although the causality of that link is increasingly questioned.26 Seasonal asthma also might be caused by nonatopic mechanisms, as ambient concentrations of pollution and aeroallergens often rise contemporaneously,48 and exposure to these toxins is also greater in the spring and summer, when people spend more time outdoors.27 However, the most dramatic seasonal spike in asthma exacerbations occurs in September in children and young adults; this 3- to 4-fold increase over baseline is attributed to infection with rhinoviruses at the beginning of the new school year.49,50 Whether a similar effect is seen in young racehorses at the beginning of the season, when naïve horses congregate under stressful conditions at the racetrack, has yet to be evaluated. Association between respiratory viruses and IAD in horses has been extensively examined with mixed findings51–56; the effect of viruses on airway hyperreactivity or specific BALE inflammatory cell subtype has not been determined.
It is also possible that exposure to inhaled toxins (especially endotoxin in barn dust) or respiratory viruses could result in a seasonal distribution of neutrophilic IAD that might peak in the winter months. As horses typically spend more time within barns during the winter, and barns are also more likely to be closed up and thus have worse ventilation during the cold months, we anticipated that their exposure to inhaled endotoxin may be higher in this season. In support of this theory, healthy racehorses have been shown to develop neutrophilic airway inflammation during the winter season,57 although particle mapping in race barns revealed that mean particle concentrations peaked in the autumn and were lowest in midsummer.58 High levels of inhaled dust have been associated with airway inflammation,29,42 which is seen in both poorly performing5,7,8 and clinically unaffected41,59 horses. Dust concentrations are much higher in horse barns than in pastures, with 8- to 15-fold increases in concentrations of respirable endotoxin, compared with concentrations at pasture,28,29 and exposure to poor ventilation and dust as a risk factor for the development of IAD.51,60 Clinically normal racehorses have been found to have increased percentages of eosinophils in BALE collected in summer, compared with samples collected in winter,57 and a summer seasonal (although strongly geographic) variation of recurrent airway obstruction, summer-pasture associated recurrent airway obstruction,61–63 occurs when high environmental counts of fungal spores and grass pollen are recorded.64 Affected horses have evidence of an allergic cytokine profile, with increased expression of IL-4.65 Nonetheless, the inflammation is exclusively neutrophilic with no evidence of increased percentages of eosinophils or mast cells in BALE.62,63,65,66
Eor the present study, we hypothesized that there would be salient clinical differences between horses with neutrophilic versus eosinophilic-mastocytic airway inflammation with regard to signalment, exercising blood gas values, or comorbidities. It was further postulated that horses examined in seasons of presumed high concentrations of ambient aeroallergens, such as spring and summer months, when pollen counts are high, would have eosinophilic-mastocytic IAD, whereas horses examined in the winter season would have IAD more analogous to human occupational asthma, where triggers are more likely to be inhaled endotoxin or organic dust that may mediate neutrophilic inflammation through nonallergic pathways. In this study, only a small proportion of horses had pure neutrophilic inflammation, and a larger number had inflammation characterized by a mixture of both neutrophilic and eosinophilic-mastocytic inflammation. Therefore, in light of our findings, it seems that these hypotheses may suggest an oversimplification of the etiopathologies and environmental exposures of IAD in horses. As the specific activities undertaken by many of these horses are seasonal (especially racing), the seasonality of admission may relate to the occurrence of perceived poor performance rather than the actual nadir of pulmonary embarrassment. Interestingly, it has not been established that inhaled stable dusts are more likely to contain toxins, and spring and summer months bring a higher concentration of respirable aeroallergens. Along with inhaled endotoxin, stable dust contains a number of potential allergens along with metals and volatile gases that influence lung inflammatory response. Meanwhile, in the spring and summer, racehorses especially are more likely to be in training and thus inhaling particulates from the track as well as environmental pollutants from road traffic in addition to the seasonal pollens and molds that are classically thought of as triggering allergic rhinitis (hay fever) in humans. Because this complex interplay of aeroallergens, pollution, and respiratory viruses is responsible for most increases in human asthma symptoms, it is perhaps unsurprising that this study of horses was unable to identify a seasonal association in the subtype of airway inflammation. Lastly, the diagnostic procedures applied to these clinical cases could not definitively prove that IAD had a causal relationship with the performance limitations; diagnosis may have been unrelated or tangential to the noted exercise intolerance.
The other notable finding in this study was the lack of correlation between IAD cytologic subtypes with abnormal blood gas analysis results at exercise. This finding was conserved even when we compared degree of BALE inflammation or even cytologic evidence of IAD as a whole with blood gas analysis results in a previous study.e The association of neutrophilic airway inflammation and poor performance is well described,7,10,13,40,67,68 but the exact cause (or whether, in fact, the neutrophilic inflammation was simply an epiphenomenon of other causes of exercise intolerance) is unclear. It is hypothesized that airway inflammation leads to reduced gas exchange and that deficits in performance are caused by hypoxemia during exertion.13 Two of the 3 studies12–14 we found that evaluated BALE and exercising blood gases reported a clear association between neutrophilic IAD and hypoxemia. Studies that correlate tracheal airway cytologic examination findings with reduced exercising arterial oxygen tension are also rare and do not consistently support an association between tracheal neutrophilia and hypoxemia during high-speed exercise.31 As a further confounder, there is generally a poor correlation between the presence of BALE inflammation (> 5% neutrophils) and tracheal fluid inflammation (>20% neutrophils),37,69,70 further complicating the evaluation of airway inflammation on gas exchange.
Overall we found only minor differences between the season of admission and clinical parameters of horses with neutrophilic versus eosinophilic-mastocytic pulmonary inflammation. If only subtle variations occur between groups, this study may have been underpowered to observe them, or the treadmill test used may not have resulted in sufficient challenge to identify slight disparities in performance or gas exchange between groups. Additionally, the season of onset of signs may have been quite different from the season of admission, possibly obscuring a link between time of year and IAD subtype. Eurther investigation exploring the cytokine profiles of BALE or even serum may allow a more nuanced evaluation of differences in etiopathophysiology between subtypes. Repeated evaluation of BAL throughout the year may also demonstrate that horses vary both the degree and subtype of inflammation within individual horses, which would also explain the lack of differences in signalment and comorbidity.
ABBREVIATIONS
| BAL | Bronchoalveolar lavage |
| BALF | Bronchoalveolar lavage fluid |
| HST | High-speed treadmill |
| IAD | Inflammatory airway disease |
| IL | Interleukin |
CIBA-GEIGY 288 BG, Norwood, Mass.
Classic 4000 High-Speed Equine Treadmill, Walmanik International, Ereedom, Pa.
GIE-Q Gastroscope, Olympus, Lake Success, NY.
Stata, version 11.0, StataCorp, College Station, Tex.
Davidson E, Harris M, Martin B, et al. Comparison of exercising upper airway obstruction, bronchoalveolar lavage cytology and exercising blood gas analysis in horses with poor performance (abstr), in Proceedings. 28th Annu Symp Vet Comp Respir Soc 2010.
References
1. Couëtil LLHoffman AMHodgson Jet al. Inflammatory airway disease of horses. J Vet Intern Med 2007 21 356–361.
2. Cardwell JMChristley RMGerber Vet al. What's in a name? Inflammatory airway disease in racehorses in training. Equine Vet J 2011 43 756–758.
3. Hare JEViel L. Pulmonary eosinophilia associated with increased airway responsiveness in young racing horses. J Vet Intern Med 1998 12 163–170.
4. Hoffman AM. Inflammatory airway disease: definitions and diagnosis in the performance horse. In: Robinson N, ed. Current veterinary therapy in equine medicine 5th ed. Philadelphia: Saunders, 2003 412–416.
5. Hoffman AM. Bronchoalveolar lavage technique and cytologic diagnosis of small airway inflammatory disease. Equine Vet Educ 1999 11 330–336.
6. Bedenice DMazan MRHoffman AM. Association between cough and cytology of bronchoalveolar lavage fluid and pulmonary function in horses diagnosed with inflammatory airway disease. J Vet Intern Med 2008 22 1022–1028.
7. Eogarty UBuckley T. Bronchoalveolar lavage findings in horses with exercise intolerance. Equine Vet J 1991 23 434–437.
8. Moore BRKrakowka SRobertson JTet al. Cytologic evaluation of bronchoalveolar lavage fluid obtained from Standardbred racehorses with inflammatory airway disease. Am J Vet Res 1995 56 562–567.
9. Hare JEViel LO'Byrne PMet al. Effect of sodium cromoglycate on light racehorses with elevated metachromatic cell numbers on bronchoalveolar lavage and reduced exercise tolerance. J Vet Pharmacol Ther 1994 17 237–244.
10. Richard EAEortier GDPitel PHet al. Sub-clinical diseases affecting performance in Standardbred trotters: diagnostic methods and predictive parameters. Vet J 2010 184 282–289.
11. Hoffman AM. Bronchoalveolar lavage: sampling technique and guidelines for cytologic preparation and interpretation. Vet Clin North Am Equine Pract 2008 24 423–435.
12. Sánchez ACouëtil LLWard MPet al. Effect of airway disease on blood gas exchange in racehorses. J Vet Intern Med 2005 19 87–92.
13. Couëtil LLDenicola DB. Blood gas, plasma lactate and bronchoalveolar lavage cytology analyses in racehorses with respiratory disease Equine Vet J Suppl 1999 77–82.
14. McKane SARose RJEvans DL. Comparison of bronchoalveolar lavage findings and measurements of gas exchange during exercise in horses with poor racing performance. N Z Vet J 1995 43 179–182.
15. Hoffman AMMazan MREllenberg S. Association between bronchoalveolar lavage cytologic features and airway reactivity in horses with a history of exercise intolerance. Am J Vet Res 1998 59 176–181.
16. Richard EAEortier GDDenoix JMet al. Influence of subclinical inflammatory airway disease on equine respiratory function evaluated by impulse oscillometry. Equine Vet J 2009 41 384–389.
17. Pearce NDouwes JBeasley R. Is allergen exposure the major primary cause of asthma? Thorax 2000 55 424–431.
18. Monteseirín J. Neutrophils and asthma. J Investig Allergol Clin Immunol 2009 19 340–354.
19. Douwes JGibson PPekkanen Jet al. Non-eosinophilic asthma: importance and possible mechanisms. Thorax 2002 57 643–648.
20. Jung KSPark HS. Evidence for neutrophil activation in occupational asthma. Respirology 1999 4 303–306.
21. Minov JKaradzinska-Bislimovska JVasilevska Ket al. Occupational asthma in subjects occupationally exposed to herbal and fruit tea dust. Arh Hig Rada Toksikol 2007 58 211–221.
22. Lee SKCho HKCho SHet al. Occupational asthma and rhinitis caused by multiple herbal agents in a pharmacist. Ann Allergy Asthma Immunol 2001 86 469–474.
23. Ereeman ALockey JHawley Pet al. Hypersensitivity pneumonitis in a machinist. Am J Ind Med 1998 34 387–392.
24. Rosado ATejedor MABenito Cet al. Occupational asthma caused by octopus particles. Allergy 2009 64 1101–1102.
25. Irsigler GBVisser PJSpangenberg PA. Asthma and chemical bronchitis in vanadium plant workers. Am J Ind Med 1999 35 366–374.
26. Sears MR. Epidemiology of asthma exacerbations. J Allergy Clin Immunol 2008 122 662–668.
27. D'Amato GLiccardi GD'Amato Met al. Environmental risk factors and allergic bronchial asthma. Clin Exp Allergy 2005 35 1113–1124.
28. Berndt ADerksen EJEdward Robinson N. Endotoxin concentrations within the breathing zone of horses are higher in stables than on pasture. Vet J 2010 183 54–57.
29. McGorum BCEllison JCullen RT. Total and respirable airborne dust endotoxin concentrations in three equine management systems. Equine Vet J 1998 30 430–434.
30. Martin BB JrReef VBParente EJet al. Causes of poor performance of horses during training, racing, or showing: 348 cases (1992–1996). J Am Vet Med Assoc 2000 216 554–558.
31. Durando MMMartin BBDavidson EJet al. Correlations between exercising arterial blood gas values, tracheal wash findings and upper respiratory tract abnormalities in horses presented for poor performance Equine Vet J Suppl 2006 523–528.
32. McKane SACanfield PJRose RJ. Equine bronchoalveolar lavage cytology: survey of Thoroughbred racehorses in training. Aust Vet J 1993 70 401–404.
33. Durando MMMartin BBHammer EJet al. Dynamic upper airway changes and arterial blood gas parameters during treadmill exercise Equine Vet J Suppl 2002 408–412.
34. Couroucé-Malblanc APronost SEortier Get al. Physiological measurements and upper and lower respiratory tract evaluation in Erench Standardbred Trotters during a standardised exercise test on the treadmill Equine Vet J Suppl 2002 402–407.
35. Pickles KPirie RSRhind Set al. Cytological analysis of equine bronchoalveolar lavage fluid. Part 2: comparison of smear and cytocentrifuged preparations. Equine Vet J 2002 34 292–296.
36. Pickles KPirie RSRhind Set al. Cytological analysis of equine bronchoalveolar lavage fluid. Part 1: comparison of sequential and pooled aliquots. Equine Vet J 2002 34 288–291.
37. Hughes KJMalikides NHodgson DRet al. Comparison of tracheal aspirates and bronchoalveolar lavage in racehorses. 1. Evaluation of cytological stains and the percentage of mast cells and eosinophils. Aust Vet J 2003 81 681–684.
38. Sweeney CRHumber KARoby KA. Cytologic findings of tracheobronchial aspirates from 66 Thoroughbred racehorses. Am J Vet Res 1992 53 1172–1175.
39. Christley RMHodgson DRRose RJet al. Coughing in Thoroughbred racehorses: risk factors and tracheal endoscopic and cytological findings. Vet Rec 2001 148 99–104.
40. Holcombe SJRobinson NEDerksen EJet al. Effect of tracheal mucus and tracheal cytology on racing performance in Thoroughbred racehorses. Equine Vet J 2006 38 300–304.
41. Gerber VRobinson NELuethi Set al. Airway inflammation and mucus in two age groups of asymptomatic well-performing sport horses. Equine Vet J 2003 35 491–495.
42. Robinson NEKarmaus WHolcombe SJet al. Airway inflammation in Michigan pleasure horses: prevalence and risk factors. Equine Vet J 2006 38 293–299.
43. Siddiqui SMorris JAvery Net al. Pulmonary eosinophilia correlates with allergen deposition to the lower respiratory tract in a mouse model of asthma. Clin Exp Allergy 2008 38 1381–1390.
44. Lavoie JPCesarini CLavoie-Lamoureux Aet al. Bronchoalveolar lavage fluid cytology and cytokine messenger ribonucleic acid expression of racehorses with exercise intolerance and lower airway inflammation. J Vet Intern Med 2011 25 322–329.
45. Hughes KJNicolson LDa Costa Net al. Evaluation of cytokine mRNA expression in bronchoalveolar lavage cells from horses with inflammatory airway disease. Vet Immunol Immunopathol 2011 140 82–89.
46. Eeo Brito EMur Gimeno PCarnes Jet al. Grass pollen, aeroallergens, and clinical symptoms in Ciudad Real, Spain. J Investig Allergol Clin Immunol 2010 20 295–302.
47. Tilles SABardana EJ Jr. Seasonal variation in bronchial hyperreactivity (BHR) in allergic patients. Clin Rev Allergy Immunol 1997 15 169–185.
48. Eeo Brito EMur Gimeno PMartinez Cet al. Air pollution and seasonal asthma during the pollen season. A cohort study in Puertollano and Ciudad Real (Spain) Allergy 2007 62.
49. Butz AMThompson RETsoukleris MGet al. Seasonal patterns of controller and rescue medication dispensed in underserved children with asthma. J Asthma 2008 45 800–806.
50. Johnston NWJohnston SLDuncan JMet al. The September epidemic of asthma exacerbations in children: a search for etiology. J Allergy Clin Immunol 2005 115 132–138.
51. Burrell MHWood JLWhitwell KEet al. Respiratory disease in Thoroughbred horses in training: the relationships between disease and viruses, bacteria and environment. Vet Rec 1996 139 308–313.
52. Kästner SBHaines DMArcher Jet al. Investigations on the ability of clenbuterol hydrochloride to reduce clinical signs and inflammation associated with equine influenza A infection. Equine Vet J 1999 31 160–168.
53. Christley RMHodgson DRRose RJet al. A case-control study of respiratory disease in Thoroughbred racehorses in Sydney, Australia. Equine Vet J 2001 33 256–264.
54. Wood JLNewton JRChanter Net al. Association between respiratory disease and bacterial and viral infections in British racehorses. J Clin Microbiol 2005 43 120–126.
55. Newton JRWood JLChanter N. A case control study of factors and infections associated with clinically apparent respiratory disease in UK Thoroughbred racehorses. Prev Vet Med 2003 60 107–132.
56. Eortier Gvan Erck EEortier Cet al. Herpesviruses in respiratory liquids of horses: putative implication in airway inflammation and association with cytological features. Vet Microbiol 2009 139 34–41.
57. Riihimäki MRaine AElfman Let al. Markers of respiratory inflammation in horses in relation to seasonal changes in air quality in a conventional racing stable. Can J Vet Res 2008 72 432–439.
58. Millerick-May MLKarmaus WDerksen EJet al. Particle mapping in stables at an American Thoroughbred racetrack. Equine Vet J 2011 43 599–607.
59. Tremblay GMEerland CLapointe JMet al. Effect of stabling on bronchoalveolar cells obtained from normal and COPD horses. Equine Vet J 1993 25 194–197.
60. Clarke AEMadelin TMAllpress RG. The relationship of air hygiene in stables to lower airway disease and pharyngeal lymphoid hyperplasia in two groups of Thoroughbred horses. Equine Vet J 1987 19 524–530.
61. Seahorn TLBeadle RE. Summer pasture-associated obstructive pulmonary disease in horses: 21 cases (1983–1991). J Am Vet Med Assoc 1993 202 779–782.
62. Mair TS. Obstructive pulmonary disease in 18 horses at summer pasture. Vet Rec 1996 138 89–91.
63. Costa LRSeahorn TLMoore RMet al. Correlation of clinical score, intrapleural pressure, cytologic findings of bronchoalveolar fluid, and histopathologic lesions of pulmonary tissue in horses with summer pasture-associated obstructive pulmonary disease. Am J Vet Res 2000 61 167–173.
64. Costa LRJohnson JRBaur MEet al. Temporal clinical exacerbation of summer pasture-associated recurrent airway obstruction and relationship with climate and aeroallergens in horses. Am J Vet Res 2006 67 1635–1642.
65. Beadle REHorohov DWGaunt SD. Interleukin-4 and interferon-gamma gene expression in summer pasture-associated obstructive pulmonary disease affected horses. Equine Vet J 2002 34 389–394.
66. Seahorn TLBeadle REMcGorum BCet al. Quantification of antigen-specific antibody concentrations in tracheal lavage fluid of horses with summer pasture-associated obstructive pulmonary disease. Am J Vet Res 1997 58 1408–1411.
67. Allen KJTremaine WHEranklin SH. Prevalence of inflammatory airway disease in national hunt horses referred for investigation of poor athletic performance Equine Vet J Suppl 2006 529–534.
68. Pirrone EAlbertini MClement MGet al. Respiratory mechanics in Standardbred horses with sub-clinical inflammatory airway disease and poor athletic performance. Vet J 2007 173 144–150.
69. Malikides NHughes KJHodgson DRet al. Comparison of tracheal aspirates and bronchoalveolar lavage in racehorses. 2. Evaluation of the diagnostic significance of neutrophil percentage. Aust Vet J 2003 81 685–687.
70. Derksen EJBrown CMSonea Iet al. Comparison of transtracheal aspirate and bronchoalveolar lavage cytology in 50 horses with chronic lung disease. Equine Vet J 1989 21 23–26.
