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.     

Pulmonary epithelial lining fluid and plasma ascorbic acid concentrations in horses affected by recurrent airway obstruction

OBJECTIVE: To determine the pulmonary epithelial lining fluid (ELF) concentrations and degree of oxidation of ascorbic acid in horses affected by recurrent airway obstruction (RAO) in the presence and absence of neutrophilic airway inflammation. ANIMALS: 6 RAO-affected horses and 8 healthy control horses. PROCEDURE: Nonenzymatic antioxidant concentrations were determined in RBC, plasma, and ELF samples of control horses and RAO-affected horses in the presence and absence of airway inflammation. RESULTS: ELF ascorbic acid concentration was decreased in RAO-affected horses with airway inflammation (median, 0.06 mmol/L; 25th and 75th percentiles, 0.0 and 0.4 mmol/L), compared with RAO-affected horses without airway inflammation (1.0 mmol/L; 0.7 and 1.5 mmol/L) and control horses (2.2 mmol/L; 1.4 and 2.2 mmol/L). Epithelial lining fluid ascorbic acid remained significantly lower in RAO-affected horses without airway inflammation than in control horses. Moreover, the ELF ascorbic acid redox ratio (ie, ratio of the concentrations of dehydroascorbate to total ascorbic acid) was higher in RAO-affected horses with airway inflammation (median, 0.85; 25th and 75th percentiles, 0.25 and 1.00), compared with RAO-affected horses without airway inflammation (0.04; 0.02 and 0.22). The number of neutrophils in bronchoalveolar lavage fluid was inversely related to the ELF ascorbic acid concentration (r = -0.81) and positively correlated with the ascorbic acid redox ratio (r = 0.65). CONCLUSIONS AND CLINICAL RELEVANCE: Neutrophilic inflammation in horses affected by RAO is associated with a reduction in the ELF ascorbic acid pool. Nutritional supplementation with ascorbic acid derivatives in horses affected by RAO is an area for further investigation.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Mucin genes in horse airways: MUC5AC,but not MUC2, may play a role in recurrent airway obstruction

REASONS FOR PERFORMING STUDY: Increased mucin gene expression may be an important cause of mucus accumulation observed in recurrent airway obstruction (RAO)-affected horses. To date, however, no mucin gene sequences are available for the horse. OBJECTIVES: To identify equine homologues of gel-forming mucins and investigate their expression at different airway generations of healthy and RAO-affected horses. METHODS: Two equine homologues were identified by cloning and sequencing fragments of equine (eq)MUC5AC and eqMUC2. RESULTS: Semiquantitative RT-PCR on RNA from airways (generations 1, 5, 10, 15; small airways and parenchyma), stomach (glandular), and colon revealed that eqMUC5AC is expressed in equine stomach and in all of the airway samples. In contrast, eqMUC2 steady-state mRNA levels were detected in colon and very faintly in stomach, but not in airway tissue. EqMUC5AC expression was also compared to that of ZO-1, a tight junction protein, and eqMUC5AC/ZO-1 ratios were higher in RAO-affected compared to control horses at all airway generations. CONCLUSIONS: That eqMUC5AC is expressed in horse airways, but any expression of MUC2 is undetectable and unlikely to be of physiological consequence. POTENTIAL RELEVANCE: EqMUC5AC up-regulation may be a primary mechanism responsible for mucus hypersecretion and accumulation in RAO.     

Environment and prednisone interactions in the treatment of recurrent airway obstruction (heaves)

Recurrent airway obstruction (RAO) or heaves is a manifestation of a hypersensitivity to dust, moulds, and spores in the environment of a susceptible horse. Although in the majority of RAO-affected horses, clinical remission can be achieved by keeping horses at pasture to reduce their allergen exposure, this often is not practicable. For this reason, we investigated if changing the environment of a single stall in a 4 stall stable was sufficient to improve lung function and reduce inflammation in RAO-affected horses. In addition, we determined if addition of oral prednisone provided additional benefit. Twelve RAO-susceptible horses were stabled, fed hay, and bedded on straw until they developed airway obstruction. At this point, bedding was changed to wood shavings and they were fed a pelleted diet for 2 weeks. Lung function was measured and bronchoalveolar lavage was performed before and 3, 7, and 14 days after environmental modification. In a crossover design, horses were treated for the 14 days with prednisone tablets (2.2 mg/kg bwt, q. 24 h). Horses then returned to pasture for 30 days. Airway obstruction was greatest before environmental modification. Significant improvement in lung function occurred within 3 days of the change in environment and continued to Day 7. Airway function was best after 30 days at pasture. The clinical response achieved by environmental modification was not significantly improved by addition of oral prednisone. The total number of cells, total neutrophils, and percent neutrophils was greatest before environmental modification. In the absence of prednisone, total and percent neutrophils did not decrease until Day 14 and total cell number until 30 days at pasture. In the presence of prednisone, total cells and total and percent neutrophils decreased by Day 3 and again at pasture. The fact that lung function can be improved within 3 days by environmental management alone emphasises the need for allergen reduction as the cornerstone of treatment of RAO. Although prednisone induced a more rapid reduction in airway inflammation, this was not associated with a more rapid improvement in airway function.     

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.     

Lower shedding of strongylid eggs by Warmblood horses with recurrent airway obstruction compared to unrelated healthy horses

An association between equine recurrent airway obstruction (RAO) and increased resistance to intestinal parasites has been demonstrated in descendants of an RAO-affected stallion. It was hypothesised that members of another high-incidence RAO family (F) and unrelated RAO-affected Warmblood horses (UA) would shed fewer strongylid eggs than unrelated RAO-unaffected pasture mates (PM) under the same environmental conditions. Faecal worm egg counts were performed on faecal samples (63 F, 86 UA, 149 PM) and classified into three categories: 0, 1-100 and >100 eggs per gram. While results for F did not differ from PM, UA were 2.5-times less likely to shed strongylid eggs than PM. RAO-affected Warmblood horses may be more resistant to strongylid nematodes than unrelated unaffected pasture mates and a family history of RAO does not necessarily confer protection against helminth infections.     

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.     

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.     

Detection of reaginic antibodies against Faenia rectivirgula from the serum of horses affected with Recurrent Airway Obstruction by an in vitro bioassay

Reaginic antibodies, mainly of the IgE and some IgG subclasses, play an important role in the induction of type I immediate hypersensitivity reactions. These antibodies bind through their Fc fragment to high affinity receptors (FcεRI) present in the membrane of mast cells and basophils. Previously, several studies have investigated the role of reaginic antibodies in the pathogenesis of RAO. However, whereas immunological aspects of RAO have been extensively studied, the precise sequence of events is still not well understood and role of IgE in this disease still remains controversial. Therefore, in this study a bioassay was developed for reaginic antibody determination in serum from RAO-affected horses in order to determine the etiology of disease. The technique involves measuring in vitro calcium mobilization in RBL-2H3 cells following incubation with horse serum from RAO-affected or unaffected horses and one of the RAO antigens (Faenia rectivirgula). Results demonstrated that 15% of samples from the RAO-affected horses reacted positively in this in vitro bioassay, whereas the samples from unaffected horses did not. This bioassay indicates that reaginic antibodies could be involved in the immunological mechanism leading to RAO; and this technique may facilitate future research in other allergic diseases in horses.     

Dietary (n-3) fatty acids from menhaden fish oil alter plasma fatty acids and leukotriene B synthesis in healthy horses

The study objective was to determine the effect of feeding corn oil or fish oil to horses on plasma fatty acid profiles and leukotriene B (LTB) synthesis by stimulated peripheral blood neutrophils. Two groups of horses (n = 5) were randomly assigned to diets supplemented with either 3.0% (by weight) corn oil or fish oil for a period of 14 weeks. The ratio of (n-6) to (n-3) fatty acids in oil supplements was 68.1:1 for corn oil and 0.12:1 for fish oil. Production of LTB4 and LTB, by peripheral blood neutrophils stimulated with calcium ionophore A23187 and plasma cholesterol, triacylglycerol, and alpha-tocopherol concentrations were measured. At 12 weeks, horses fed fish oil had increased plasma concentrations of eicosapentaenoic acid (27-fold; 8.5 versus 0.3 g/100 g fatty acids; P < .0001), docosahexaenoic acid (34-fold; 5.1 versus 0.1 g/100 g fatty acids; P < .0001), and arachidonic acid (8.3-fold; 4.1 versus 0.5 g/100 g fatty acids; P < .0001) compared with horses fed corn oil. Neutrophils from horses fed fish oil produced 78-fold (P = .01) more LTB5 and 9.5-fold (P = .003) more LTB4 compared with predietary levels, and 17.6-fold (P = .01) and 3.3-fold (P = .02), respectively, more than horses fed corn oil, and the ratio of LTB5 to LTB4 concentrations was 4.0-fold (P = .002) higher in horses fed fish oil. This study suggests that dietary polyunsaturated fatty acids modulate the leukotriene inflammatory response of horses. If the ratio of LTB5 to LTB4 concentrations is important in determining how inflammatory processes are mediated, then fish oil supplementation may have value in treatment of equine inflammatory diseases.