Experimental induction of recurrent airway obstruction with inhaled fungal spores, lipopolysaccharide, and silica microspheres in horses

Janet Beeler-Marfisi Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada.

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Mary Ellen Clark Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada.

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Xin Wen Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada.

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William Sears Department of Population Medicine, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada.

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Leslie Huber Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada.

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Cameron Ackerley Department of Pediatric Medicine, Division of Pathology, The Hospital for Sick Children, Toronto, ON M5G 1X8, Canada.

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Laurent Viel Department of Clinical Studies, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada.

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Dorothee Bienzle Department of Pathobiology, Ontario Veterinary College, University of Guelph, Guelph, ON N1G 2W1, Canada.

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Abstract

Objective—To evaluate experimental induction of recurrent airway obstruction (RAO) with inhaled fungal spores, lipopolysaccharide, and silica microspheres in horses.

Animals—7 horses with and 3 horses without a history of RAO.

Procedures—RAO-susceptible horses ranged in age from 17 to approximately 30 years, and control horses ranged in age from 7 to approximately 15 years. Pure mold cultures were derived from repeated culture of hay and identified via gene amplification and sequencing. Pulmonary function testing and bronchoalveolar lavage were performed before and after nebulization with a suspension of spores derived from 3 fungi, lipopolysaccharide, and 1-μm silica microspheres in all horses. This was followed by a 4-month washout period and a further pulmonary function test followed by saline (0.9% NaCl) solution challenge and bronchoalveolar lavage.

ResultsLichtheimia corymbifera, Aspergillus fumigatus, and Eurotium amstelodami were consistently identified in cultures of moldy hay. Nebulization with fungal spores, lipopolysaccharide, and microspheres induced significant increases in pleural pressure in RAO-susceptible but not control horses. Airway neutrophilia developed in both groups of horses with exposure to challenge material but more severely in RAO-susceptible horses.

Conclusions and Clinical Relevance—Results indicated that inhalation of fungal spores in combination with lipopolysaccharide and silica microspheres can induce disease exacerbation in susceptible horses and may thus be a useful model for future standardized studies of RAO in horses.

Abstract

Objective—To evaluate experimental induction of recurrent airway obstruction (RAO) with inhaled fungal spores, lipopolysaccharide, and silica microspheres in horses.

Animals—7 horses with and 3 horses without a history of RAO.

Procedures—RAO-susceptible horses ranged in age from 17 to approximately 30 years, and control horses ranged in age from 7 to approximately 15 years. Pure mold cultures were derived from repeated culture of hay and identified via gene amplification and sequencing. Pulmonary function testing and bronchoalveolar lavage were performed before and after nebulization with a suspension of spores derived from 3 fungi, lipopolysaccharide, and 1-μm silica microspheres in all horses. This was followed by a 4-month washout period and a further pulmonary function test followed by saline (0.9% NaCl) solution challenge and bronchoalveolar lavage.

ResultsLichtheimia corymbifera, Aspergillus fumigatus, and Eurotium amstelodami were consistently identified in cultures of moldy hay. Nebulization with fungal spores, lipopolysaccharide, and microspheres induced significant increases in pleural pressure in RAO-susceptible but not control horses. Airway neutrophilia developed in both groups of horses with exposure to challenge material but more severely in RAO-susceptible horses.

Conclusions and Clinical Relevance—Results indicated that inhalation of fungal spores in combination with lipopolysaccharide and silica microspheres can induce disease exacerbation in susceptible horses and may thus be a useful model for future standardized studies of RAO in horses.

Recurrent airway obstruction is a chronic, inducible, inflammatory lung disease, which occurs in mature horses and has a negative effect on the performance of horses as athletes or companions.1,2 The condition RAO is caused by exposure to environmental organic dusts and particulate matter. It is characterized by reversible small airway obstruction, neutrophilic bronchiolitis, mucus hypersecretion, increased abdominal expiratory effort, and changes in pulmonary function. As such, RAO has similarity with dust-induced asthma in humans.3,4 Excess mucus accumulation and suppurative tracheal inflammation have been identified in approximately 70% of pleasure horses in northern US regions, suggesting that lung inflammation is a common finding among stabled horses, although the exact prevalence of RAO is unknown.2 Clinical signs of RAO are partially reversible with environmental improvement or glucocorticoid and bronchodilator administration.3,5,6 To study the pathogenesis of RAO, it is necessary to exacerbate the disease in susceptible individuals. This is typically accomplished by placing RAO-susceptible horses in an enclosed stall and exposing them to moldy hay or by challenging horses with a nebulized suspension of hay dust, LPS, and mold (hay dust suspension) known to induce inflammation.7–10 Because moldy hay used for challenge is of variable composition and therefore not standardized, comparisons between studies are difficult. Although there has been some success in inducing lung inflammation with hay dust suspension,9 neither the mold nor the inorganic particulate components have been fully characterized or accurately quantified.

The first objective of the study reported here was to identify the fungi present in RAO-inducing moldy hay grown in Ontario during 1 summer season. The second objective was to create a standardized challenge model for experimental induction of RAO with inhaled fungal spores, LPS, and silica microspheres in horses. To determine the effectiveness of the challenge model, changes in 3 PFT variables and changes in the proportions of inflammatory cells in airways were monitored.

Our first hypothesis was that molds contributing to exacerbation of RAO could be found consistently in hay used to induce acute exacerbations of RAO. Our second hypothesis was that challenge with aerosolized fungal spores, LPS, and inorganic particulate matter would exacerbate RAO in susceptible horses but not in unaffected horses.

Materials and Methods

Experimental design—The study was a 3-factor factorial study in a split-plot design. The whole plot factors were history of RAO and challenge with FLS versus sterile saline (0.9% NaCl) solution. The split factor was time (prevs postchallenge), which was split within each horse. Each horse (RAO susceptible or control) was challenged with FLS and saline solution, with a 4-month washout period between challenges. Because this constituted a preliminary study and the horses had been used in previous investigations on RAO, horses were not randomly allocated to treatment or control groups. The challenge suspension was greenish and opaque, precluding masking of personnel to the nature of the nebulized material (FLS or saline solution). The same person assessed the clinical signs of each subject prior to PFT and BAL by use of a standardized scoring sheet (available from the authors upon request).

Fungal preparation—Ten batches of visibly moldy hay historically used to induce RAO in susceptible horses were weighed, and 50 g each was shaken in a sterile container with 10 mL of PBS solution. Three aliquots (0.5 mL) of the resulting suspension were cultured on Sabouraud agar at 20°, 30°, 37°, and 42°C for 24 hours. Plates cultured at 30°C typically contained 5 to 10 fungal colonies after 24 hours. Fungal colonies with unique morphological appearance were repeatedly subcultured via sterile technique until only 1 culture/plate was apparent. The DNA from each of 3 pure cultures identified in each batch of moldy hay was extracted,a and the internal transcribed spacer region was amplified by use of primers ITS 1 and ITS 4 and 0817F and 1196R.11 Resulting amplicons were sequenced, and the National Center for Biotechnology Information database was searched to identify each isolate to the level of genus and species. To prepare the nebulization solution, isolates were cultured and spores were scraped from the surface of the fungal mat with a sterile scalpel blade and counted.b

Challenge material—The challenge suspension (FLS) contained 108 spores from each of the 3 fungal isolates, 10 μL of LPS (10 mg/mL),c and 108 silica spheres (1 μm diameter)d to which sterile PBS solution was added to yield a final volume of 2 mL. Silica microspheres were counted manually in a hemocytometer chamber. One-micrometer silica spheres were chosen because, with nebulization, a hydrophilic, monodisperse particle of this diameter can be expected to reach the alveoli.12 The concentration of spores and microspheres was chosen to approximate particulates identified in hay dust.13 The control mixture was 2 mL of sterile saline solution.

Horses, housing, and handling—The University of Guelph Animal Care Committee, in accordance with the Canadian Council on Animal Care guidelines, approved all procedures under AUP No. R06-067. Seven RAO-susceptible horses (3 mares, 4 geldings) and 3 control horses (1 mare, 2 geldings) were used. All were Standardbreds or Quarter Horses. Susceptible horses ranged in age from 17 to approximately 30 years, and control horses ranged in age from 7 to approximately 15 years. Horses were obtained from the University of Guelph's research herd for use in the study. All but 1 horse had been used in previous research projects on RAO. Research horses received standard vaccinations and anthelminthics, were routinely maintained on pasture with indoor access, and were fed hay. For the trial, horses were housed in separate, enclosed, air-conditioned stalls, bedded on damp wood shavings, fed nondusty extruded feeds, and given free access to water. No hay was fed. Pulmonary function testing, endoscopic examination of the upper and lower portions of the respiratory tract, and BAL were carried out on day 1 and on the day after the last nebulization with FLS or sterile saline solution. Affected horses had RAO for > 4 years, but no abnormalities were detected via the current physical examination. Additionally, affected horses had no evidence of airway edema or inflammation on bronchoscopy, PFT $Ppl ≤ 10 cm of H2O, and BALF neutrophil proportion ≤ 10% and thus were considered to be in a remission state. Control horses had no history of lung disease and normal airway bronchoscopic and pulmonary function findings. Baseline CBCs and serum biochemical evaluations were not performed because these are typically within reference limits in horses with RAO.7 During the study, horses were clinically assessed twice daily. Horses were considered to be in an exacerbated state of RAO if they had ≥ 3 signs of respiratory distress such as nostril flaring, mucoid nasal discharge, cough, tachypnea, increased abdominal expiratory effort, evidence of excessive tracheal mucus heard on auscultation, and wheezes and crackles heard over the lung fields.

PFT—Pulmonary function testing was carried out to determine $Ppl (cm H2O), Cdyn (L/cm H2O), and RL (cm H2O/L/s). These data were derived from integration and analysis of airflow data and corresponding transpulmonary pressure.e Nonsedated horses were restrained in stocks and fitted with a mask that covered the muzzle and was attached to a heated pneumotachograph.f Airflow data were captured and fed through a transducer to integrate the flow signal and derive volume measurements.e An esophageal balloon catheter placed midthorax (10-cm-long latex balloon filled with 3 mL of room air attached to cover the fenestrations in a 2-m polyethylene catheter with a 2.69-mm inner diameter and 3.5-mm outer diameter) and attached to a transducer at the proximal end was used to estimate pleural pressure. Change in pleural pressure was calculated as the difference between atmospheric and estimated pleural pressure. Volume and pressure data were analyzed via respiratory loop analysise to derive values for $Ppl, Cdyn, and RL. Pleural pressure changes > 15 cm of H2O were considered indicative of RAO exacerbation, and pressures < 10 cm of H2O were considered indicative of remission.14 In exacerbated states of RAO, Cdyn is expected to decrease as a result of a loss of lung elastic recoil, and RL is expected to increase as a result of large airway obstruction.15,16

Bronchoscopy and BAL—Horses were restrained in stocks and sedated by administration of romifidineg (10 mg/mL, IV) to effect. Endoscopic examination was performed with a 180-cm-long, 1-cm-external diameter endoscope attached to a light source.h Features consistent with airway inflammation, including mucosal hyperemia and edema, excessive airway mucus, blunting of the carina, and elicitation of cough, were recorded on a score sheet. To minimize discomfort and coughing during airway examination and bronchoalveolar lavage, 60 to 180 mL of warm saline solution containing 2 mg of lidocainei/mL was instilled as needed into the trachea, at the carina, and into the bronchi to be sampled. Prior to challenge, BAL fluid was collected via the diaphragmatic lobar bronchus from the right lung in each horse. After challenge, BAL fluid was collected via the left diaphragmatic lobar bronchus. A fourth- to sixth-generation bronchus, likely corresponding to the middle segmental bronchus, was selected each time. Saline solution was warmed (approx 37°C), and 250 or 500 mL was instilled via the biopsy channel and immediately retrieved into a sterile flask to a minimum volume of 100 mL via vacuum pump.j Fluid was kept on ice until the cell concentration was measuredb and cytocentrifugationk of 150 μL of unfiltered fluid was performed. Both tasks were completed within 4 hours of collection. Cytocentrifuged preparations were stained, and a 400-cell differential count was performed. Neutrophil percentage > 20% was considered consistent with RAO in exacerbation.17,18

Electron microscopy and microprobe spectrometry of BAL macrophages—Four 1-mL aliquots of a BAL fluid sample obtained after FLS nebulization were centrifuged for 10 minutes at 500 × g. Supernatant was removed by pipeting, and cell pellets were fixed for 2 hours in 4% formalin plus 0.1% glutaraldehyde in 0.1M phosphate buffer at ambient temperature. Cells were washed in 1X PBS solution, and a prewarmed (37°C) 20% gelatin solution was mixed with the cell pellets until the gelatin thickened. Tubes were left at 37°C for 15 minutes and then transferred to 4°C for 15 minutes. The solidified material was removed from the tube and cut into 2-mm3 pieces with a scalpel blade. Samples were postfixed in 1% OsO4, dehydrated in acetone, and embedded in epon araldite. Sections (0.5 μm) were cut and stained in uranyl acetate and lead citrate prior to examination in a field emission scanning electron microscope.l Macrophages containing silicon dioxide beads were identified by use of backscatter electron imaging. Energy dispersive x-ray spectrometry was used to confirm the elemental composition of the silicon dioxide microspheres.19

Nebulization with challenge material and saline solution control—Horses received nebulization twice daily with FLS beginning on day 1 immediately after the PFT and BAL by use of a modified maskm attached to a nebulization cup and an air compressorn delivering 20 to 25 psi of air pressure. Twice-daily nebulization with FLS continued until clinical exacerbation of RAO was achieved or 18 nebulizations had been performed. If a horse was in an exacerbated state of RAO, no further challenge material was given and the second PFT, endoscopic examination, and BAL were performed 24 hours later.

Horses were then given a washout period of 4 months at pasture, with supplemental hay feeding, and were subsequently returned for saline solution challenge. Clinical monitoring, PFT, endoscopic examination, and BAL were repeated as in the first part of the study. Sterile saline solution was then nebulized twice daily and continued for 4 days. Four days were allocated for saline solution nebulizations because horses either had responded to FLS by day 4 or they did not achieve clinical exacerbation of RAO.

Statistical analysis—Pulmonary function testing data, including $Ppl, Cdyn, and RL, were evaluated. Proportions of BAL fluid mast cells, macrophages, lymphocytes, and neutrophils before and after challenge with FLS and saline solution were also analyzed. The proportion of eosinophils was not statistically analyzed in either group because eosinophils were rarely present (percentages ranged from 0% to 4.3%) and had a mode of 0 (recorded 26 times of 39 entries) with an arithmetic mean of 0.4%.

A statistical programo was used to analyze data. All terms up to the level of a 3-way interaction were considered; however, if terms were not significant at the 10% level, they were removed from the model. To assess the ANOVA assumptions, comprehensive residual analyses were performed. The assumption of normality was tested by use of Shapiro-Wilk, Kolmogorov-Smirnov, Cramervon Mises, and Anderson-Darling tests.p In addition, residuals were plotted against the predicted values and explanatory variables used in the model to reveal outliers (> 2 SDs greater or less than the mean), bimodal distributions, or the need for data transformations. Differences were considered significant at P ≤ 0.05.

To adequately meet the ANOVA assumptions, a log transformation was applied to all pulmonary function testing data. For $Ppl data, an outlier was identified but not removed. The data were otherwise normally distributed. In the analysis of Cdyn data, 3 outliers were identified. Analyses were carried out with and without these outliers. Because cell data were recorded as percentage values, a logit transform with a bias correction term was applied to data of all cells except macrophages. Macrophage data were not normally distributed and a logit transformation was not corrective. The effects of RAO status, challenge, and time on PFT and BAL results were examined.

Results

Fungi—Fungal growth was most rapid at an incubation temperature of 30°C. Three morphologically distinct and pure fungal cultures were derived from each aliquot of hay after 1 to 3 subcultures. Fungi were identified as Lichtheimia corymbifera, Aspergillus fumigatus, and Eurotium amstelodami, with 100%, 98%, and 89% identity over 678, 520, and 560 bp (BLAST accession Nos. DQ118985, NC_007197.1, and AY373885),q respectively. Cultured on Sabouraud agar at 30°C, L corymbifera colonies were wooly, white, and dark gray on the surface but colorless where touching the culture media. Aspergillus fumigatus cultures were powdery gray-green and pale yellow where touching the culture media, and E amstelodami colonies were a granular dark green, with white-to-yellow fruiting bodies and green-black to pale yellow streaks where touching the culture media. Cytocentrifuge slides of spore suspensions were prepared and Wright stained. Lichtheimia corymbifera spores were 3 to 5 μm, A fumigatus spores were 2 to 3 μm, and E amstelodami spores were 4 to 5 μm in diameter (Figure 1).

Figure 1—
Figure 1—

Photomicrographs of fungal isolates from hay samples used to induce RAO in horses. A—Lichtheimia corymbifera. Wright stain; bar = 20 μm. B—Aspergillus fumigatus. Wright stain; bar = 10 μm. C—Eurotium amstelodami. Wright stain; bar = 20 μm.

Citation: American Journal of Veterinary Research 71, 6; 10.2460/ajvr.71.6.682

Horses—Aside from induction of pulmonary changes, no adverse effects were observed in any horse as a result of FLS challenge. One RAO-affected horse was removed from the study during the washout period for reasons unrelated to the study (severe colic caused by a strangulating lipoma, resulting in euthanasia). All other horses remained healthy and were returned to the research herd.

Response to nebulization challenge—Marked individual variation was seen in the predominant clinical features of RAO in the horses. The changes most consistently identified were increased expiratory effort, nostril flaring, mucoid nasal discharge, increased tracheal mucus, tachypnea, and an expanded lung field.

In RAO-affected horses but not control horses, a significant (P = 0.004) difference from the null hypothesis of no effect was observed. A significant (P = 0.006) postchallenge increase in $Ppl in RAO-affected but not control horses was detected. Saline solution challenge did not induce significant changes in $Ppl in either group. Dynamic compliance did not decrease significantly in either group. Differences in Cdyn after challenge (P = 0.072) or with exposure to saline solution (P = 0.285) were not significant. The RL increased as a combined effect of time and FLS exposure (P = 0.034) in both groups (Figure 2).

Figure 2—
Figure 2—

Results (mean ± SEM) of pulmonary function tests (A, $Ppl; B, Cdyn; C, RL) in 3 healthy control horses and 7 horses with RAO before (solid bars) and after (striped bars) challenge with FLS and saline solution. *Significant (P < 0.05) differences between pre- and postchallenge values.

Citation: American Journal of Veterinary Research 71, 6; 10.2460/ajvr.71.6.682

The BAL fluid cell concentrations ranged from 0.31 to 2.38 × 109 cells/L, but because of variable recovery of fluid volume, differences were not statistically analyzed. Via cytologic analysis, horses in both groups developed neutrophilia as a combined effect of time and exposure to FLS (P = 0.020), but airway neutrophilia in horses with RAO was greater than in control horses. Macrophage proportions were significantly decreased in RAO-affected horses (P = 0.037) and as an effect of time (P = 0.004). The nature of the challenge material did not have a significant effect (P = 0.069; Figure 3). Lymphocyte proportions did not decrease significantly in either group (P = 0.582) or as a combined effect of time and exposure to FLS (P = 0.097). No significant changes in mast cell proportions attributable to group, time, or FLS exposure were detected (P = 0.844, P = 0.191, and P = 0.121, respectively). One RAO-affected horse developed airway neutrophilia after exposure to saline solution.

Figure 3—
Figure 3—

Proportions of BAL fluid macrophages (A), lymphocytes (B), neutrophils (C), and mast cells (D) in the same horses as in Figure 2. See Figure 2 for key.

Citation: American Journal of Veterinary Research 71, 6; 10.2460/ajvr.71.6.682

In cytocentrifuge preparations from horses challenged with FLS, occasional phagocytosed fungal spores and round, refractile, cytoplasmic inclusions were detected in macrophages and neutrophils, consistent in size with 1-μm spheres (Figure 4). Examination of BAL fluid from horses by use of backscatter electron microscopy further identified the cytoplasmic round structures, and energy dispersive x-ray spectrometry revealed that they consisted of oxide and silicate, consistent with the nebulized microspheres.

Figure 4—
Figure 4—

Photomicrograph of BAL fluid macrophages (A) containing a small, round refractile cytoplasmic inclusion (arrow) as well as a neutrophil and lymphocytes (Wright stain; bar = 10 μm). and electron photomicrograph (B) of a silica microsphere in the cytoplasm of a macrophage (bar = 1 μm).

Citation: American Journal of Veterinary Research 71, 6; 10.2460/ajvr.71.6.682

Discussion

Progress in investigating the pathogenesis of RAO has been hampered by a lack of standardized induction of the disease in sensitized horses.20 In the present study, the authors attempted to identify a composite challenge material that induced RAO in susceptible horses. In the challenge suspension, freshly cultured L corymbifera, A fumigatus, and E amstelodami spores were used. Fungal spores (or conidia) differ in polymer composition from hyphae, and only metabolically active, but not dormant, fungal spores have exposed internal B-glucans when germinating.21 Fungal B-glucans are recognized by the innate immune receptor dectin-1, activation of which results in phagocytic and proinflammatory responses through induction of tumor necrosis factor-A and other mediators.21 The size of spores used in the present study was 2 to 5 μm, which would predict that they reached the distal portions of the airways12 and would be phagocytosed by pulmonary alveolar macrophages as was observed on cytocentrifuge preparations. Therefore, nebulization of fresh fungal spores of this size may be more efficacious for inducing inflammation than inhalation of hyphae fragments or spores that have been present for variable lengths of time in hay. The fungal species identified were reasonable candidates as etiologic agents in RAO because A fumigatus can cause direct damage to airway epithelium by serine protease activation,13 and L corymbifera and E amstelodami have been implicated in farmer's lung disease (extrinsic allergic alveolitis) of humans.22 Although additional or different fungi may be involved in causing RAO in Ontario or other geographic regions, inclusion of these 3 particular fungi induced airway inflammation.

Lipopolysaccharide was included in the challenge material because experimentally it induces pulmonary neutrophil influx and asthma in mice23 and has a synergistic effect with particulate matter in the induction of RAO.24 Inorganic particulates play an important role in pulmonary inflammation in humans, and silica microspheres were selected to represent the inorganic particulate fraction of hay dust because silicates from soil could reasonably be expected to form most of the inorganic dust fraction of hay.25–27 The fate of inhaled silica microspheres in the lung has not been determined; however, their in vitro phagocytosis by macrophages induces cell death in a concentration-dependent manner.27 In humans, chronic obstructive pulmonary disease, silicosis, or fibrosis may develop with prolonged exposure to airborne silica.28,29 It is unclear how many of the 108 microspheres in the challenge material actually reached the distal portions of the airways in the present study, but because most phagocytes only contained 1 or 2 microspheres as seen via electron microscopy, the inhaled dose was probably low relative to the surface area of an equine lung. Although persistence of the microspheres in the lung over time was not assessed, prominent cell death was not observed by use of either light or electron microscopy, and all RAO-affected horses recovered from respiratory impairment after the study. Therefore, it was assumed that there were no long-term deleterious effects from this challenge material.

There were several limitations to the challenge material used in this study. Fungal cultures were only derived in a semiquantitative manner from hay washes. It is possible that fungi that contribute to development of RAO but have different growth kinetics or culture requirements were unidentified. Also, horses may vary in their susceptibility to different fungal spores and only 1 fungus, or other isolates, might more optimally induce inflammation in some horses. Further, neither an absolute requirement for LPS nor a specific concentration in hay dust has been reported. Finally, silica particulates were included in the challenge because inorganic dust components were hypothesized to be an essential component, but there are limited data examining such elements in dusty hay. Therefore, although this study aimed to identify a challenge material that can be accurately produced and delivered, future studies should be directed at delineating the role of individual components of this challenge material.

Horses recruited as controls in this study lacked a history of airway disease but developed moderate airway neutrophilia after FLS challenge. These horses were assessed during fall and winter, when they had been fed hay for several months. Although horses were housed without hay or straw exposure immediately prior to the study, clinically silent inflammatory airway disease or low-grade viral infection cannot be ruled out. As assessed by use of pulmonary function testing, $Ppl increased after challenge in RAO-affected horses but not in control horses or with saline solution. The increase was only modest in several RAO-affected horses, which may reflect variable adaptations in respiratory physiology, a relative insensitivity of the PFT method, or only modest efficacy of the challenge protocol. Exacerbated RAO is commonly associated with reduced Cdyn, which was not a consistent finding in horses in this study. Most humans with emphysema have a reproducible decrease in Cdyn, but not all asthmatics have such reduction.15 Individual horses with RAO may vary in how they modify respiratory rate and tidal volume in response to obstruction, which may have contributed to the variable responses observed.16 Furthermore, susceptible horses in this study had long-standing RAO and relatively low Cdyn at baseline, possibly reflecting persistent fibrosis in lung parenchyma. Therefore, assessment of a larger number of horses to more clearly define responding subgroups or an adjustment in challenge frequency should be considered.

Neutrophils in BALF increased in horses with administration of the challenge material, which is consistent with current understanding of the effects of LPS and fungal B-glucans.9,21,23 Although part of the neutrophil influx may have been caused by LPS, nebulization with individual components of the challenge material would be necessary for confirmation. Reduction in lymphocytes and macrophages in BAL fluid was likely only relative because of an influx of neutrophils and an overall increase in leukocytes in the airways. Further, lack of increase in mast cells and eosinophils was consistent with the current definition of RAO.14

ABBREVIATIONS

BAL

Bronchoalveolar lavage

Cdyn

Dynamic compliance

ΔPpl

Change in pleural pressure

FLS

Fungal-lipopolysaccharide-silica microsphere suspension

LPS

Lipopolysaccharide

PFT

Pulmonary function testing

RAO

Recurrent airway obstruction

Rl

Lung resistance

a.

DNeasy Plant kit, Qiagen, Mississauga, ON, Canada.

b.

Z2 Coulter counter, Beckman Coulter, Mississauga, ON, Canada.

c.

Escherichia coli 0111:B4, Sigma-Aldrich, Oakville, ON, Canada.

d.

Polysciences, Warrington, Pa.

e.

Buxco Electronics, Sharon, Conn.

f.

Fleisch No. 4, Gould Electronics, Bilthoven, The Netherlands.

g.

Boehringer-Ingelheim, Burlington, ON, Canada.

h.

Olympus, Markham, ON, Canada.

i.

Bioniche PHARMA, Belleville, ON, Canada.

j.

Medi-Pump, Sheboygan, Wis.

k.

Thermo Shandon, Waltham, Mass.

l.

JSM JEOL 6700F, JEOL USA, Peabody, Mass.

m.

Aeromask, Trudell Medical, London, ON, Canada.

n.

Precision Medical, Northampton, Pa.

o.

Proc MIXED, SAS Institute Inc, Cary, NC.

p.

Proc UNIVARIATE, SAS Institute Inc, Cary, NC.

q.

BLAST, National Center for Biotechnology Information, Bethesda, Md. Available at: blast.ncbi.nlm.nih.gov/Blast.cgi. Accessed Aug 14, 2007.

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