Comparison of the antioxidant status in tracheal and bronchoalveolar epithelial lining fluids in recurrent airway obstruction

REASONS FOR PERFORMING STUDY: Following a period of airway inflammation the clearance of inflammatory cells along the mucociliary escalator may impose a considerable oxidant load on the trachea. OBJECTIVES: To determine the degree of oxidative stress in tracheal epithelial lining fluid (ELF) in comparison to that present in peripheral airways after an acute exposure to organic dust. METHODS: Tracheal wash fluid and bronchoalveolar lavage fluid (BALF) were collected for cytology and antioxidant analyses from 6 recurrent airway obstruction (RAO)-affected horses and 6 healthy control horses before and after stabling on straw bedding for 24 h. RESULTS: In RAO-affected horses, organic dust exposure resulted in a significant decrease in ascorbic acid concentration in tracheal ELF (P<0.0001), which was greater than the decrease in bronchoalveolar ELF (P = 0.0003). The percentage decrease in tracheal ELF ascorbic acid correlated with the percentage decrease in bronchoalveolar ELF ascorbic acid (r = 0.76; P = 0.004) following exposure. CONCLUSIONS: Acute organic dust exposure results in significant antioxidant depletion in the trachea, which may reflect inflammation and oxidative processes in peripheral airways. POTENTIAL RELEVANCE: Further work is required to evaluate the role of ascorbic acid depletion in the pathogenesis of RAO.     

Antioxidant and inflammatory responses of healthy horses and horses affected by recurrent airway obstruction to inhaled ozone

REASONS FOR PERFORMING STUDY: Inhaled ozone can induce oxidative injury and airway inflammation. Horses affected by recurrent airway obstruction (RAO) have a decreased pulmonary antioxidant capacity, which may render them more susceptible to oxidative challenge. It is currently unknown whether RAO-affected horses are more susceptible to oxidative stress than those unaffected by RAO. OBJECTIVES: To determine whether ozone exposure induces greater oxidative stress and airway inflammation in RAO-affected horses in remission than in healthy horses. METHODS: Seven healthy control horses and 7 RAO-affected horses were exposed to 0.8 ppm ozone for 2 h at rest. RESULTS: At baseline, bronchoalveolar lavage fluid (BALF) ascorbic acid concentrations were lower in RAO-affected horses than healthy controls. Ozone appeared to preferentially oxidise glutathione rather than ascorbic acid 6 h after exposure. Individual healthy and RAO-affected horses demonstrated oxidation of BALF glutathione after ozone exposure. Overall, RAO-affected horses did not demonstrate increased oxidative stress following ozone exposure, compared with healthy horses. Ozone did not induce significant airway inflammation in either group. CONCLUSIONS: RAO-affected horses in remission are not more sensitive to ozone despite a decreased pulmonary antioxidant capacity. Sensitivity to ozone appears to be independent of initial pulmonary antioxidant status. POTENTIAL RELEVANCE: Horses with high susceptibility to oxidative stress may benefit from antioxidant supplementation.     

Coughing,mucus accumulation,airway obstruction,and airway inflammation in control horses and horses affected with recurrent airway obstruction

OBJECTIVE: To investigate relationships between cough frequency and mucus accumulation, airway obstruction, and airway inflammation and to determine effects of dexamethasone on coughing and mucus score. ANIMALS: 13 horses with recurrent airway obstruction (RAO) and 6 control horses. PROCEDURE: 6 RAO-affected and 6 control horses were stabled for 3 days. Coughing was counted for 4 hours before and on each day horses were stabled. Before and on day 3 of stabling, tracheal mucus accumulation was scored, airway obstruction was assessed via maximal change in pleural pressure (deltaPpl(max)), and airway inflammation was evaluated by use of cytologic examination of bronchoalveolar lavage fluid (BALF). Effects of dexamethasone (0.1 mg/kg, IV, q 24 h for 7 days) were determined in 12 RAO-affected horses. RESULTS: To assess frequency, coughing had to be counted for 1 hour. In RAO-affected horses, stabling was associated with increases in cough frequency, mucus score, and deltaPpl(max). Control horses coughed transiently when first stabled. In RAO-affected horses, coughing was correlated with deltaPpl(max), mucus score, and airway inflammation and was a sensitive and specific indicator of deltaPpl(max) > 6 cm H2O, mucus score > 1.0, and > 100 neutrophils/microL and > 20% neutrophils in BALF Dexamethasone reduced cough frequency, mucus score, and deltaPpl(max), but BALF neutrophil count remained increased. CONCLUSIONS AND CLINICAL RELEVANCE: Because of its sporadic nature, coughing cannot be assessed accurately by counting during brief periods. In RAO-affected horses, coughing is an indicator of airway inflammation and obstruction. Corticosteroid treatment reduces cough frequency concurrently with reductions in deltaPpl(max) and mucus accumulation in RAO-affected horses.     

Assessment of leukotriene B4 production in leukocytes from horses with recurrent airway obstruction

OBJECTIVE: To determine the ex vivo leukotriene (LT) biosynthesis in peripheral blood neutrophils (PBNs) and inflammatory cells in bronchoalveolar lavage fluid (BALF) obtained from horses affected with recurrent airway obstruction (RAO). ANIMALS: 6 RAO-affected and 6 control horses. PROCEDURES: Before and 6, 24, and 48 hours after stabling, disease severity was determined subjectively by clinical and mucus scores and measurement of the maximal change in pleural pressure (deltaPpl(max)); PBNs were isolated and BALF samples were examined cytologically. The PBN and BALF cells were activated with a calcium ionophore in the presence of arachidonic acid, and production of LTC4 and LTB4 was measured per 10(6) cells. RESULTS: Clinical and mucus scores and deltaPpl(max) increased during stabling in RAO-affected horses, but not in control horses. In neutrophils and BALF cells from both groups, production of LTB4 exceeded that of LTC4. At all times, LTB4 production by PBNs was less in RAO-affected horses than it was in control horses. Before stabling, LTB4 production by cells in BALF was low in RAO-affected horses, but increased considerably after 6 hours of stabling. This increase coincided with the migration of neutrophils into the airways. In control horses, production of LTB4 did not change during stabling. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested increased production of LTB4 in airways of RAO-affected horses, compared with control horses, that may contribute to the infiltration of neutrophils into the lungs and the sustained inflammation associated with RAO.     

Evaluation of leukotriene biosynthetic capacity in lung tissues from horses with recurrent airway obstruction

OBJECTIVE: To evaluate leukotriene (LT) biosynthetic capacity in lung tissue from healthy horses and horses with recurrent airway obstruction (RAO). SAMPLE POPULATION: Lung parenchyma and airway specimens from 8 RAO-affected and 5 healthy horses. PROCEDURE: Horses were stabled for > or = 72 hours. Blood was drawn before euthanasia, after which lung specimens were collected. Tissue strips from small airways and parenchyma were incubated in organ baths with the precursor LTA4 or stimulated with calcium ionophore A23187 or the tripeptide N-formyl-Met-Leu-Phe (fMLP), with or without exogenous arachidonic acid, in the presence of isolated blood neutrophils. RESULTS: Stabling induced typical clinical signs of airway obstruction in RAO-affected horses but not control horses. When lung parenchyma or airway specimens from both groups of horses were incubated with calcium ionophore, with or without arachidonic acid, they did not form LT. In contrast, addition of LTA4 to both tissues resulted in conversion to LTB4, although concentrations of LTC4 were negligible in airways and parenchymal strips from healthy and RAO-affected horses. Incubation of airway and parenchymal strips with suspensions of autologous neutrophils did not influence formation of LT stimulated by calcium ionophore or fMLP, with or without exogenous arachidonic acid. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that lung parenchyma and airway tissues themselves are not of substantial importance for LT formation in the lungs, although these tissues possessed some LTA4 hydrolase activity, enabling LTB4 formation. It may be speculated that LTB4 originates primarily from neutrophils and may play a role in the inflammatory events of RAO.     

IgE-bearing cells in bronchoalveolar lavage fluid and allergen-specific IgE levels in sera from RAO-affected horses

Recurrent airway obstruction (RAO) is a common condition in stabled horses characterized by small airway inflammation, airway neutrophilia and obstruction following exposure of susceptible horses to mouldy hay and straw and is thus regarded as a hypersensitivity reaction to mould spores. However, the role of immunoglobulin E antibodies (IgE) in the pathogenesis of RAO is unclear. We hypothesized that the number of cells with receptor-bound IgE in bronchoalveolar lavage fluid (BALF) and IgE levels in serum would be higher in RAO-affected than in healthy horses living in the same environment. Therefore, IgE-positive (+) cells were identified by immunocytochemistry on cytospins from BALF and counted. IgE levels against the mould extracts Aspergillus fumigatus (Asp. f.) and Alternaria alternata (Alt. a.) and the recombinant mould allergen Aspergillus fumigatus 8 (rAsp f 8) were measured by enzyme-linked immunosorbent assay (ELISA) in the sera of seven RAO-affected and 22 clinically healthy mature horses housed in the same conventional stable environment. After correcting for the number of neutrophils, there were no significant differences in IgE+ cells on cytospins from BALF between both groups of horses (5% versus 7%, P > 0.1). Serum IgE levels against the mould extracts were significantly higher in RAO-affected than in clinically healthy horses [median = 119 versus 66 relative ELISA units (REU), P < 0.05]. Furthermore, significantly more RAO-affected than healthy horses had detectable serum IgE against the recombinant allergen rAsp f 8 (4/7 and 3/22, respectively, P < 0.05). Age had no significant effect on BALF cell ratios or on specific serum IgE levels. These results show that high IgE levels against mould antigens are associated with RAO under controlled environmental conditions but ranges of mould-specific serum IgE levels overlapped too much between diseased and clinically healthy animals to be of any diagnostic value. Further studies are needed to assess whether IgE-mediated reactions contribute to the pathogenesis of RAO.     

Effect of hay dust extract and cyathostomin antigen stimulation on cytokine expression by PBMC in horses with recurrent airway obstruction

Equine recurrent airway obstruction (RAO) is an inflammatory, obstructive airway disease induced by exposure of susceptible horses to inhaled organic dust particles. The immunological process underlying RAO is still unclear. Previous studies have shown that RAO is linked to the Interleukin-4 receptor (IL-4R) gene in one Warmblood family (F1), but not in another (F2). It has also been shown that in F1, but not in F2, RAO is associated with resistance against parasites, suggesting that this association may have an immuno-genetic basis. Therefore, we hypothesized that the T helper (h)1/Th2/regulatory (Treg) cytokine profiles of RAO-associated antigen- and parasite-antigen-stimulated peripheral blood mononuclear cells (PBMC) differ between RAO-affected and healthy horses depending on their genetic background. In our study, PBMC from 17 RAO-affected and 14 healthy control horses of F1 and F2 were stimulated for 24 h with antigens relevant to RAO [hay dust extract (HDE), Aspergillus fumigatus extract (AFE) and lipopolysaccharids (LPS)]; cyathostomin extract (CE) and recombinant cyathostomin antigen (RCA) or with concanavalin A (ConA). Total mRNA levels of IL-4, IL-4R, IL-13, interferon (INF)-γ and IL-10 were examined by qRT-PCR. Stimulation with either HDE or RCA resulted in significant differences in IL-4R mRNA levels between RAO-affected and control horses in F1, but not in F2. For IL-10 mRNA expression, a significant difference between RAO-affected and control horses in F1 but not in F2 was observed only following stimulation with HDE. In contrast to HDE, stimulation with CE resulted in a significant difference of IL-10 mRNA expression level between RAO-affected horses of F2 and healthy horses of F1. No significant differences were detected upon stimulation with any of the other challenge agents. These findings indicate that the immunological response, specifically IL-4R expression, in response to hay dust and cyathostomin antigens, differs between RAO-affected and healthy horses depending on their genetic background. This study shows that analysis of PBMC reveals systemic changes associated with RAO and helps to elucidate immunological pathways involved in this disease.     

Neurokinin receptors in recurrent airway obstruction: A comparative study of affected and unaffected horses

The purpose of the study was to compare in vitro airway responses to neurokinin A & B (NKA and NKB) and expression of NK-2 receptors in airways of horses affected and unaffected with recurrent airway obstruction (RAO). Neurokinin-A, an inflammatory mediator belonging to the tachykinin family of neuropeptides, causes bronchoconstriction by binding to NK-2 receptors. Neurokinin-B is a lesser-known neuropeptide that acts on NK-3 receptors. Horses were placed into RAO-affected and RAO-unaffected groups based on their history, clinical scoring, and pulmonary function testing. Lung tissue from each lobe was collected for immunohistochemical staining for NK-2 receptors. Cumulative concentration-response relationships were determined on bronchial rings (4-mm wide) collected and prepared from the right diaphragmatic lung lobe to graded concentrations (half log molar concentrations 10⁻⁷M to 10⁻⁴M) of NKA and NKB. The results showed that NKA caused significantly greater contraction than NKB in both groups. In RAO-affected horses, both agents produced significantly greater bronchial contractions than those in the RAO-unaffected horses. Immunohistochemical staining showed that the overall NK-2 receptor distribution was significantly increased in bronchial epithelium and smooth muscles of bronchi and pulmonary vessels of RAO-affected than RAO-unaffected horses. The findings indicate that NK-2 receptors are up-regulated in RAO, suggesting that NK-2 receptor antagonists may have some therapeutic value in controlling the progression of airway hyperreactivity in horses affected with RAO.     

Proteomic analysis in canine leishmaniasis

In horses, Recurrent Airway Obstruction (RAO) is an allergic disease that involves IgE mediated Type I Hypersensitivity responses. The development of this type of allergy involves a series of events that begins with reaginic antibodies, mainly IgE and some IgG subclasses. These reaginic antibodies bind with high affinity, via the Fc portion, to FcεRI receptors on the membrane of mast cells and basophils. Once bound, environmental allergens cross-link the antibodies, which results in mast cell degranulation leading to the production of histamine and other chemical mediators that act together to induce airway inflammation. RAO-affected horses present with coughing, respiratory distress, airway obstruction and poor performance. The aspect of the RAO has been extensively studied, yet the precise sequence of events is still not well understood. Therefore, this study proposes a bioassay for reaginic antibody detection from horse serum of RAO-affected individuals, in order to determine the etiology of disease, which mediate immediate type reactions. The technique involves measuring in vitro calcium mobilization in RBL-2H3 cells following incubation with horse serum from affected or unaffected horses and one of the RAO antigens (Aspergillus fumigatus). The results presented here demonstrate that 30% of RAO-affected horses react positively in this in vitro bioassay, whereas unaffected horses do not. This bioassay may facilitate further research on RAO and other allergic diseases in horses.     

Mast cells and IgE-bearing cells in lungs of RAO-affected horses

Recurrent airway obstruction (RAO) is a common condition in stabled horses characterised by small airway inflammation and obstruction following exposure of susceptible horses to mouldy hay and straw. The aim of the present study was to investigate whether lung tissue from horses with RAO contains higher numbers of IgE-protein positive (+) cells and mast cells compared to controls after mouldy hay challenge. Furthermore, mast cell subtypes in lung tissue were investigated. IgE+ cells were detected in most lung tissue samples but no significant differences between RAO-affected and control horses were found. In the wall of the bronchi and bronchioli of both RAO-affected and control horses, mainly chymase+ mast cells (MC(C)) were present (85% in the bronchial wall and 77% in the wall of the bronchioli), while 73% of the mast cells (MC) around blood vessels were tryptase+ mast cells (MC(T)). No double stained MCs were detected. RAO-affected horses had significantly more MC(C) than controls in the wall of the bronchi (median=7.6 and 1.7 cell/mm(2), respectively, P< or =0.05). They also showed a tendency for more MC(C) in the wall of the bronchioli than controls (median=21 and 2.9 cells/mm(2), respectively, P=0.07) but there were no differences in MC(T) numbers. The data suggest an involvement of MC(C) in the pathogenesis of RAO. Independently of the clinical diagnosis, there was a significant relationship between high MC(C) numbers in the bronchial wall and lung fibrosis, suggesting that these MC(C) may be involved in tissue remodelling. Furthermore, high MC(C) numbers were also associated with increased infiltration with lymphocytes and neutrophils.     

Indices of oxidative stress in blood and pulmonary epithelium lining fluid in horses suffering from recurrent airway obstruction.

To test the hypothesis that reactive oxygen species could be associated to the lower airway disorders occurring in horses suffering from recurrent airway obstruction (RAO), indices of oxidative stress were studied in blood and pulmonary epithelium lining fluid in 5 RAO horses either in clinical remission or 24 h after the onset of a crisis of bronchospasm and in 5 healthy horses. Venous blood and bronchoalveolar lavage fluid (BALF) samples were collected and analysed for reduced glutathione (GSH), oxidised glutathione (GSSG), total glutathione (TGSH), glutathione redox ratio (GRR) in blood haemolysate and pulmonary epithelium lining fluid (PELF). The haemolysate concentrations of GSH, GSSG, TGSH and GRR were similar in the 3 groups. The PELF glutathione status was significantly different in the RAO horses in acute crisis compared to healthy horses, indicating the occurrence of an oxidative stress. When RAO horses were in crisis their GSH and TGSH remained unchanged but their GSSG and GRR were significantly increased compared to the remission. These results support the hypothesis that oxidative stress is associated with lower airway disorders occurring in horses suffering from RAO.     

Randomized, controlled study of inhaled fluticasone propionate, oral administration of prednisone, and environmental management of horses with recurrent airway obstruction

OBJECTIVE: To determine whether administration of glucocorticoids provides additional benefits to environmental management of horses with recurrent airway obstruction (RAO). ANIMALS: 28 horses with RAO. PROCEDURE: Horses were classified as having mild, moderate, or severe RAO. Within each category, horses were randomly assigned to receive inhaled fluticasone propionate, inhaled control substance, or oral administration of prednisone. During the 4-week study, horses were maintained outdoors and fed a pelleted feed. Clinical scores, pulmonary function, results of cytologic examination of bronchoalveolar lavage fluid (BALF), and adrenal gland function were determined before and 2 and 4 weeks after initiation of treatment. RESULTS: Clinical score and pulmonary function of all RAO-affected horses improved during the treatment period. After 4 weeks, clinical scores and pulmonary function of horses treated with a glucocorticoid were not different from those for the control treatment. In horses with severe RAO, treatment with fluticasone for 2 weeks resulted in significantly greater improvement in pulmonary function, compared with pulmonary function after treatment with prednisone or the control substance. Treatment with a glucocorticoid for 4 weeks and a low-dust environment did not have any effect on cellular content of BALF Treatment with prednisone for 2 weeks resulted in a significant decrease in serum cortisol concentration, compared with concentrations after administration of fluticasone or the control substance. CONCLUSIONS AND CLINICAL RELEVANCE: Environmental management is the most important factor in the treatment of horses with RAO. Early treatment with inhaled fluticasone can help accelerate recovery of horses with severe RAO.     

Effects of in vitro exposure to hay dust on expression of interleukin-17, -23, -8, and -1beta and chemokine (C-X-C motif) ligand 2 by pulmonary mononuclear cells isolated from horses chronically affected with recurrent airway disease

OBJECTIVE: To examine effects of in vitro exposure to solutions of hay dust, lipopolysaccharide (LPS), or beta-glucan on cytokine expression in pulmonary mononuclear cells isolated from healthy horses and horses with recurrent airway obstruction (RAO). ANIMALS: 8 RAO-affected and 7 control horses (experiment 1) and 6 of the RAO-affected and 5 of the control horses (experiment 2). PROCEDURES: Bronchoalveolar lavage cells were isolated from horses that had been stabled and fed dusty hay for 14 days. Pulmonary mononuclear cells were incubated for 24 (experiment 1) or 6 (experiment 2) hours with PBS solution or solutions of hay dust, beta-glucan, or LPS. Gene expression of interleukin (IL)-17, IL-23(p19 and p40 subunits), IL-8, IL-1beta, and chemokine (C-X-C motif) ligand 2 (CXCL2) was measured with a kinetic PCR assay. RESULTS: Treatment with the highest concentration of hay dust solution for 6 or 24 hours increased expression of IL-23(p19 and p40), IL-8, and IL-1beta in cells from both groups of horses and increased early expression of IL-17 and CXCL2 in RAO-affected horses. Lipopolysaccharide upregulated early expression of IL-23(p40) and IL-8 in cells from both groups of horses but only late expression of these cytokines in cells from RAO-affected horses. Treatment with beta-glucan failed to increase cytokine expression at 6 or 24 hours. CONCLUSIONS AND CLINICAL RELEVANCE: Cells from RAO-affected horses were not more responsive to the ligands tested than were cells from control horses, which suggests a minimal role of mononuclear cells in propagation of airway neutrophilia in horses with chronic RAO.     

Expression of thymic stromal lymphopoietin in equine recurrent airway obstruction

Thymic stromal lymphopoietin (TSLP) is a cytokine involved in lymphocyte development. In humans and mice, TSLP drives the differentiation of T helper 2 (Th2) cells and the development of allergic inflammation. The equine TSLP gene has been previously identified and characterized, but its role in the pathogenesis of equine allergic diseases is not known. Our objective was to assess the expression of TSLP in bronchoalveolar lavage (BAL) cells and in primary bronchial epithelial cells (BEC) isolated from horses with recurrent airway obstruction (RAO). RNA was isolated from BAL cells sampled from clinical cases of RAO (n=8) and from control horses (n=12). Furthermore, BAL samples were taken from an additional group of 8 RAO-susceptible and 8 control horses when on pasture (remission) and after 30 days of exposure to moldy hay (exacerbation). In order to study epithelial cells as a potential source of TSLP, cultures of primary bronchial epithelial cells (BEC) were established from 6 RAO-affected and 6 healthy horses and stimulated in vitro with hay dust solution (HDS). Expression of TSLP mRNA was assessed by quantitative real-time RT-PCR (qPCR). Clinical RAO-cases had higher TSLP expression in BAL than control horses (p<0.05). In an experimental group of horses there was no difference between healthy and susceptible horses in remission, whereas after 30-day experimental exposure to moldy hay, all susceptible horses upregulated TSLP expression in BAL (p=0.008, average 6.36-fold increase), whereas in healthy horses there was no significant increase in TSLP expression. BEC generated both from healthy and RAO-affected horses strongly upregulated TSLP expression after 6 h stimulation with HDS, which identifies epithelial cells as potential sources of TSLP in RAO. Finding of increased TSLP expression by BAL cells of RAO-affected horses is in agreement with the contribution of Th2-driven allergic inflammation in the pathogenesis of RAO.     

Rapid, multiwell colorimetric assay for measuring neutrophil chemoattractant activity in bronchoalveolar lavage fluid of horses with recurrent airway obstruction.

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

Comparison of results for intradermal testing between clinically normal horses and horses affected with recurrent airway obstruction

OBJECTIVE: To evaluate differences in response to ID injection of histamine, phytohemagglutinin (PHA), and Aspergillus organisms between clinically normal horses and horses with recurrent airway obstruction (RAO). ANIMALS: 5 healthy adult horses and 5 adult horses with RAO. PROCEDURE: Intradermal testing (IDT) was performed on the neck with 2 positive control substances (histamine and PHA) and a mixture comprising 5 Aspergillus species. Four concentrations of each test substance plus a negative control substance were used. Equal volumes (0.1 mL) of each test substance were prepared to yield 15 syringes ([4 concentrations of each test substance plus 1 negative control substance] times 3 test substances) for each side of each horse (ie, 30 syringes/horse). Intradermal injections were administered; diameter of wheals was recorded 0.5, 4, and 24 hours after injection. RESULTS: Hypersensitive responses to ID injection of histamine were detected 0.5 hours after injection, and a delay in wheal formation after ID injection of Aspergillus mixture 24 hours after injection was detected in RAO-affected horses but was not observed in clinically normal horses. No differences were detected between the 2 groups after ID injection of PHA. CONCLUSIONS AND CLINICAL RELEVANCE: RAO-affected horses are hypersensitive to histamine, suggesting that RAO is associated with a heightened vascular response to histamine. Higher concentrations of Aspergillus mixture may be needed to detect horses that are sensitive to this group of antigens. Wheal reactions to Aspergillus may be a delayed response, suggesting that IDT results should be evaluated 0.5, 4, and 24 hours after ID injection.     

Tracheobronchial mucus viscoelasticity during environmental challenge in horses with recurrent airway obstruction

The goal of this study was to compare the rheological properties of mucus from horses with recurrent airway obstruction (RAO) to that from healthy controls during environmental challenge by stabling in stalls with straw as bedding and hay as feed. We determined viscoelasticity (log G* dyn/cm2, at 10 radian/s) and calculated mucociliary clearability index (MCI) and cough clearability index (CCI), which are derivative parameters of G* and the ratio of viscosity and elasticity measured at 1 and 100 radian/s, respectively. We also investigated the solids content of mucus, and cytology of bronchoalveolar lavage fluid (BALF). Samples were obtained before (0 h) and 6, 24 and 48 h after environmental challenge. The central findings were rheological changes in airway mucus, which occurred over time in RAO-affected animals, but not in controls. Mucus rheology was similar in both groups at 0 and 6 h. In RAO-affected horses, mucus viscoelasticity, as measured by log G*, increased from 2.49 +/- 0.18 dyn/cm2 (mean +/- s.e.) at 0 h to 3.05 +/- 0.13 dyn/cm2 at 24 h after environmental challenge, and was accompanied by significant decreases in MCI and CCI. Percent solids of mucus did not differ significantly between the 2 groups, nor over time. Rheological values did not correlate with BALF cytology. We conclude that viscoelastic properties of tracheal mucus samples from RAO horses in remission do not differ from those of normal horses. However, environmental challenge causes clinical signs of small airway disease and a concurrent increase in mucus viscoelasticity only in RAO horses. Therefore, we infer that unfavourable changes in mucus rheology may contribute to stasis and accumulation of mucus in RAO horses in exacerbation, but not in clinical remission.     

Lung function and airway cytologic profiles in horses with recurrent airway obstruction maintained in low-dust environments

BACKGROUND: The effects of long-term environmental management on airway obstruction and inflammation in horses with recurrent airway obstruction (RAO) are unknown. HYPOTHESIS: Horses with RAO maintained in low-dust environments have persistent airway obstruction and neutrophilic inflammation. ANIMALS: Study horses were treated for RAO and then maintained in low-dust environments with no medical management. Horses were classified into 3 groups by years after diagnosis: 1 year (time 1, n = 9), 2-3 years (time 2, n = 7), and 5-6 years (time 3, n = 8). The comparison groups were age-matched healthy horses. METHODS: In this cross-sectional study, a clinical examination was performed, and the clinical score was calculated. Standard lung function, forced expiratory maneuvers, and the cytology of bronchoalveolar lavage fluid (BALF) were evaluated. RESULTS: The clinical scores of the RAO horses were higher than those of the non-RAO horses at time 2 (P = .018). Standard lung function data were not different between the groups at any time point. The forced expiratory flow between 75-95% of exhaled vital capacity was lower in RAO horses than in non-RAO horses at all time points (P < .02), indicating persistent peripheral airway obstruction. Cytologic evaluation of BALF revealed no difference in total nucleated cell numbers or differential cell counts between RAO and non-RAO horses at any time point. CONCLUSIONS AND CLINICAL IMPORTANCE: The peripheral airway obstruction detected in horses with RAO maintained in low-dust environments likely is due to irreversible airway remodeling but is not associated with cytologic evidence of airway inflammation.