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OBJECTIVE To evaluate the effect of 2 bronchoalveolar lavage (BAL) sampling techniques and the use of N-butylscopolammonium bromide (NBB) on the quantity and quality of BAL fluid (BALF) samples obtained from horses with the summer pasture endophenotype of equine asthma. ANIMALS 8 horses with the summer pasture endophenotype of equine asthma. PROCEDURES BAL was performed bilaterally (right and left lung sites) with a flexible videoendoscope passed through the left or right nasal passage. During lavage of the first lung site, a BALF sample was collected by means of either gentle syringe aspiration or mechanical suction with a pressure-regulated wall-mounted suction pump. The endoscope was then maneuvered into the contralateral lung site, and lavage was performed with the alternate fluid retrieval technique. For each horse, BAL was performed bilaterally once with and once without premedication with NBB (21-day interval). The BALF samples retrieved were evaluated for volume, total cell count, differential cell count, RBC count, and total protein concentration. RESULTS Use of syringe aspiration significantly increased total BALF volume (mean volume increase, 40 mL [approx 7.5% yield]) and decreased total RBC count (mean decrease, 142 cells/μL), compared with use of mechanical suction. The BALF nucleated cell count and differential cell count did not differ between BAL procedures. Use of NBB had no effect on BALF retrieval. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that retrieval of BALF by syringe aspiration may increase yield and reduce barotrauma in horses at increased risk of bronchoconstriction and bronchiolar collapse. Further studies to determine the usefulness of NBB and other bronchodilators during BAL procedures in horses are warranted.

Bronchial hyper-responsiveness (BHR) describes a lung abnormality in which airways are easily triggered to constrict in response to normally harmless inhaled stimuli, and is a key element of human asthma pathophysiology. BHR contributes to equine respiratory diseases including inflammatory airway disease and recurrent airway obstruction. Collectively these diseases account for over 80% of poor performance in equine athletes, and at least 10% of veterinary admissions. BHR is also a contributing factor in ‘exercise induced pulmonary hemorrhage’. Increased sensitivity to airway constriction that characterizes BHR is a documented sequel to viral respiratory infections in several species, including horses and humans. Five respiratory viruses known to circulate extensively in equine populations place the horse at risk for BHR. Despite adverse effects of BHR on equine health, there remains a gap in our fundamental understanding of how gene products coordinate in the lung to cause BHR. Leveraging the equine genome sequence, we employ systems biology including proteomics and RNA sequencing to model the complex biology of BHR in the lungs of horses with pasture asthma. Using a self controlled experimental design, gene products that segregate with seasonal asthma exacerbation in diseased horses are being identified and their relevant physiology identified to address the need for better recognition and management of BHR in equine disease.

Lauren A Bright,1 Wellesley Dittmar,1 Bindu Nanduri,2 Fiona M McCarthy,3 Nisma Mujahid,2 Lais RR Costa,2 Shane C Burgess,3 Cyprianna E Swiderski11Department of Clinical Sciences, College of Veterinary Medicine, Mississippi State University, Starkville, MS 39762, USA; 2Department of Basic Sciences, College of Veterinary Medicine, Mississippi State University, Starkville, MS 39762, USA; 3School of Animal Comparative and Biomedical Sciences, College of Agriculture and Life Sciences, University of Arizona, Tucson, AZ 85721, USABackground: Pasture-associated severe equine asthma is a warm season, environmentally-induced respiratory disease characterized by reversible airway obstruction, persistent and non-specific airway hyper-responsiveness, and chronic neutrophilic airway inflammation. During seasonal exacerbation, signs vary from mild to life-threatening episodes of wheezing, coughing, and chronic debilitating labored breathing.Purpose: In human asthma, neutrophilic airway inflammation is associated with more severe and steroid-refractory asthma phenotypes, highlighting a need to decipher the mechanistic basis of this disease characteristic. We hypothesize that the collective biological activities of proteins in bronchoalveolar lavage fluid (BALF) of horses with pasture-associated severe asthma predict changes in neutrophil functions that contribute to airway neutrophilic inflammation.Methods: Using shotgun proteomics, we identified 1,003 unique proteins in cell-free BALF from six horses experiencing asthma exacerbation and six control herdmates. Contributions of each protein to ten neutrophil functions were modeled using manual biocuration to determine each protein’s net effect on the respective neutrophil functions.Results: A total of 417 proteins were unique to asthmatic horses, 472 proteins were unique to control horses (p

Severe equine asthma, formerly recurrent airway obstruction (RAO), is the horse counterpart of human asthma, affecting horses maintained indoors in continental climates. Equine pasture asthma, formerly summer pasture RAO, is clinically similar but affects grazing horses during hot, humid conditions in the southeastern United States and United Kingdom. To advance translational relevance of equine pasture asthma to human asthma, histologic features of airway remodeling in human asthma were scored in lung lobes from 15 pasture asthma-affected and 9 control horses of mixed breeds. All noncartilaginous airways were scored using a standardized grading rubric (0–3) in hematoxylin and eosin (HE) and Movat’s pentachrome-stained sections; 15 airways were chosen randomly from each lobe for analysis. Logistic regression identified disease, age, and lobe effects on probability of histologic outcomes. Airway smooth muscle (odds ratio [OR] = 2.5, P < .001), goblet cell hyperplasia/metaplasia (OR = 37.6, P < .0001), peribronchiolar elastic system fibers (OR = 4.2, P < .001), peribronchiolar fibrosis (OR = 3.8, P = .01), airway occlusion by mucus/inflammation (OR = 4.2, P = .04), and airway adventitial inflammation (OR = 3.0, P = .01) were significantly greater in diseased airways. A novel complex tissue disorganization, designated terminal bronchiolar remodeling, was overrepresented in diseased airways (OR = 3.7, P < .0001). Distribution of terminal bronchiolar remodeling corresponded to putative sites of air trapping in human asthma, at secondary pulmonary lobules. Age (>15 years) was an independent risk factor for increased peribronchiolar fibrosis, elastic system fibers, and terminal bronchiolar remodeling. Remodeling differed significantly between lung lobes, congruent with nonhomogeneous remodeling in human asthma. Equine pasture asthma recapitulates airway remodeling in human asthma in a manner not achieved in induced animal asthma models, endorsing its translational relevance for human asthma investigation.

Acupuncture is part of an Eastern system of medicine used for diagnosing, treating, and preventing disease. It has been used for the treatment of reproductive disorders in both human beings and domestic species. We hypothesized that acupuncture could be used to induce cyclicity in anestrous mares. In experiment 1, six anestrous mares were treated with acupuncture at Bai Hui, governing vessel (GV)-2, bladder (BL)-22, BL-23, BL-31, and BL-33 twice a week for 3 weeks, then once a week for an additional 8 weeks, whereas six anestrous mares served as controls. In experiment 2, six anestrous mares received acupuncture stimulation at Bai Hui, GV-1, BL-22, BL-23, and BL-28, with electroacupuncture (10 minutes at 50 Hz) at BL-22 and BL-28 bilaterally. In addition, vitamin B12 (3 mL, 1,000 mcg/mL) was injected in the vaginal fornix at the 3, 6, and 9 o’clock positions around the cervix. Six anestrous mares served as controls. Examinations and treatments were carried out twice a week until ovulation. In both experiments, ovarian activity was examined in all mares by transrectal ultrasonography and blood sample was obtained twice a week to determine progesterone concentrations. The time to first ovulation for experiment 1 was 14.0 ± 0.7 and 13.5 ± 1.0 weeks (mean ± SD) for the control and acupuncture groups, respectively ( P = .60). In experiment 2, the time to ovulation was 9.0 ± 0.6 and 10 ± 0.3 weeks (mean ± SD) for the control and acupuncture groups, respectively ( P = .19). No significant difference was found between the treated and control mares in either study.

Objective—To immunohistochemically determine the expression of endothelin (ET) receptors in bronchial smooth muscle and epithelium of healthy horses and horses affected by summer pasture-associated obstructive pulmonary disease (SPAOPD). Sample Population—Tissue specimens obtained from 8 healthy and 8 SPAOPD-affected horses. Procedure—Horses were examined and assigned to healthy and SPAOPD groups. Horses were then euthanatized, and tissue specimens containing bronchi of approximately 4 to 8 mm in diameter were immediately collected from all lung lobes, fixed in zinc-formalin solution for 12 hours, and embedded in paraffin. Polyclonal primary antibodies against ET-A or ET-B receptors at a dilution of 1:200 and biotinylated IgG secondary antibodies were applied to tissue sections, followed by the addition of an avidin-biotin immunoperoxidase complex. Photographs of the stained slides were digitally recorded and analyzed by use of image analysis software to determine the intensity of staining. Two-way ANOVA was used for statistical analysis. Results—The left diaphragmatic lung lobe of SPAOPD-affected horses had a significantly greater area of bronchial smooth muscle that immunostained for ET-A, compared with that for healthy horses. All lung lobes of SPAOPD-affected horses, except for the right diaphragmatic lobe, had significantly greater staining for ET-B receptors in bronchial smooth muscle, compared with results for healthy horses. Conclusions and Clinical Relevance—This study revealed overexpression of ET-A and, in particular, ETB receptors in the bronchial smooth muscle of SPAOPD-affected horses, which suggested upregulation of these receptors. These findings improve our understanding of the role of ET-1 in the pathogenesis of SPAOPD.

Objective—To determine concentrations of nitric oxide (NO) in plasma and bronchoalveolar lavage fluid (BALF) and localize nitric oxide synthesis in the lungs of horses with summer pasture-associated obstructive pulmonary disease (SPAOPD).Animals—7 adult horses with SPAOPD and 6 clinically normal adult horses.Procedure—Severity of SPAOPD was determined by use of clinical scores, change in intrapleural pressure (ΔPpl) during tidal breathing, cytologic analysis of BALF, and histologic evaluation of lung specimens obtained during necropsy. Nitric oxide concentrations in plasma, BALF, and epithelial lining fluid (ELF) were determined by use of a chemiluminescent method. Inducible nitric oxide synthase (iNOS) and nitrotyrosine (NT) were localized in formalin-fixed lung specimens by use of immunohistochemical staining, and nicotinamide adenine dinucleotide phosphate diaphorase (NADPHd) activity was localized in cryopreserved specimens by use of histochemical staining.Results—Plasma concentration of NO in affected horses was slightly but not significantly greater than concentration in nonaffected horses. Nitric oxide concentrations in BALF or ELF did not differ between groups. Immunoreactivity of iNOS in bronchial epithelial cells of 3 of 5 lung lobes was greater in horses with SPAOPD, compared with nonaffected horses. However, staining for NT and NADPHd activity did not differ between groups.Conclusions and Clinical Relevance—Expression of iNOS was greater in bronchial epithelial cells of horses with SPAOPD, compared with nonaffected horses, suggesting that NO may play a role in amplifying the inflammatory process in the airways of horses with this disease. (Am J Vet Res2001;62:1381–1386)

Objective—To correlate clinical score, intrapleural pressure, cytologic findings of bronchoalveolar lavage fluid (BALF), and histologic lesions of pulmonary tissue in horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD). Animals—8 adult horses affected with SPAOPD and 6 adult horses without evidence of respiratory tract disease. Procedure—Clinical score, change in intrapleural pressure (ΔPpl) during tidal breathing, results of cytologic examination and bacteriologic culture of BALF, and results of histologic examination of pulmonary parenchyma were evaluated. Results—Clinical scores for SPAOPD-affected horses (median, 5.75; range, 4.0 to 7.5) were significantly greater, compared with clinically normal horses (median, 2.0; range, 2.0 to 3.0). Cytologic examination of BALF from SPAOPD-affected horses revealed predominantly nondegenerate neutrophils. Histologic lesions were identified throughout pulmonary tissue and included severe accumulation of mucus and neutrophils within the small airways, metaplasia of bronchiolar goblet cells, and mild peribronchial infiltrate. Histologic examination of specimens collected via percutaneous biopsy was predictive of disease and corresponded to findings at postmortem examination. Clinical score and δPpl were highly correlated with mucus accumulation in the airways of affected horses. Peribronchial inflammatory infiltrate correlated with percentage of neutrophils in BALF of affected horses. Conclusions and Clinical Relevance—Clinical scoring and ΔPpl provided valid estimates of disease severity. Findings from cytologic examination of BALF of SPAOPD-affected horses varied, although, in most instances, it was diagnostically useful. Severe mucus accumulation in the airways was the most remarkable histopathologic finding in SPAOPDaffected horses. Examination of biopsy specimens collected from pulmonary parenchyma was consistently useful in diagnosing SPAOPD. (Am J Vet Res 2000;61:167–173)

Background: Summer pasture-associated recurrent airway obstruction (SPA-RAO), a seasonal airway obstructive disease of horses, is characterized by clinical exacerbation after exposure to pasture during warm months of the year. Endothelin (ET)-1, potent bronchoconstrictor, mitogen, secretagogue, and proinflammatory mediator, has been implicated in the pathogenesis of asthma and equine heaves. Hypothesis: Immunoreactive ET-1 concentrations increase during clinical exacerbation and return to basal values during periods of disease remission. Animals: Twelve horses, 6 affected with SPA-RAO and 6 nonaffected. Methods: Prospective, observational study. Bronchoalveolar lavage fluid (BALF), arterial and venous plasma samples, and clinical variables were obtained from affected horses during clinical exacerbation and remission. Samples and data of nonaffected horses were collected during the summer and winter on dates similar to affected horses. Immunoreactive ET-1 was determined using a commercial ELISA. Results: The median and range ET-1 concentrations (pg/ml) in arterial (1.3, 0.7–1.8) and venous (1.3, 1.2–1.7) plasma and in BALF (0.3, 0.2–0.4), and pulmonary epithelial lining fluid (PELF) (25.5, 21–50) were greater in affected horses during clinical exacerbation compared with remission (P < .01). The concentrations of immunoreactive ET-1 were greater in affected horses during clinical exacerbation compared with nonaffected horses (P < .05). Conclusions and Clinical Importance: During clinical exacerbation of SPA-RAO, ET-1 is increased in circulation and pulmonary secretions. Intervention with ET receptor antagonists should provide further information on the role of ET-1 in SPA-RAO.

Background:Lactoferrin is a colostral glycoprotein with antimicrobial properties. Hypotheses: (1) Serum lactoferrin and immunoglobulin G (IgG) concentrations are correlated and increase in healthy foals after ingestion of colostrum; (2) compared to healthy foals, ill foals will have lower lactoferrin concentrations that correlate with their IgG concentration, neutrophil count, the diagnosis of sepsis, and survival; and (3) plasma concentrations of lactoferrin will be less than serum concentrations. Animals:Healthy foals (n = 16), mature horses (n = 10), and ill foals 1–4 days old (n = 111) that were examined for suspected sepsis were used for blood collection. Colostrum was obtained from 10 healthy mares unrelated to the foals. Methods:Blood was obtained from the healthy foals at birth and 1–3 days of age and from the ill foals at admission. Serum IgG was quantified by single radial immunodiffusion (SRID). Lactoferrin concentrations in colostrum and blood were determined by an enzyme-linked immunosorbant assay. The sepsis score, blood culture results, neutrophil counts, and survival were obtained on ill foals. Results:The mean colostral lactoferrin concentration was 21.7 μg/mL. Compared to values at birth, serum IgG (18 ± 2 versus 2,921 ± 245 mg/dL, SEM) and lactoferrin (249 ± 39 versus 445 ± 63 ng/mL, SEM) concentrations were significantly greater in healthy foals 1–3 days old. Serum lactoferrin concentration in 1–3-day-old healthy foals was not different from mature horses or ill foals. IgG and lactoferrin concentrations were significantly correlated only in healthy foals. Serum lactoferrin concentrations were significantly lower in ill neutropenic foals. The serum IgG concentration was significantly lower in ill foals as compared to healthy foals. Only serum IgG was significantly less in ill foals with a positive sepsis score and in nonsurvivors. Plasma lactoferrin concentrations were lower than serum concentrations, although values were significantly correlated. Clinical Importance: Although both serum IgG and lactoferrin concentrations increase in healthy foals after ingestion of colostrum, only serum IgG is significantly correlated with the sepsis score and outcome.

Objective—To describe the seasonal pattern of clinical exacerbation of summer pasture-associated recurrent airway obstruction (SPA-RAO) in relation to climate and aeroallergens in horses. Animals—19 horses with SPA-RAO and 10 nonaffected horses. Procedures—Daily examinations were performed on all horses while they were kept on pasture for 3 years. Onset and progression of clinical exacerbation based on a clinical score of respiratory effort were evaluated in relation to changes in maximum temperature, minimum temperature, mean temperature, maximum dew-point temperature, minimum dew-point temperature, and delta dew-point temperature. Seasonal pattern of clinical exacerbation was evaluated in relation to aeroallergen counts (20 types of fungal spores and 28 types of pollen). Results—Seasonal pattern of clinical exacerbation of SPA-RAO was associated with increases in temperature (heat) and dew-point temperature (humidity), counts of fungal spores, and counts of grass pollen grains. Seasonal pattern of clinical exacerbation paralleled and was associated with increases in counts of specific types of fungal spores, particularly Basidiospore, Nigrospora, and Curvularia spp. Conclusions and Clinical Relevance—Although a causal relationship could not be determined, the seasonal pattern of clinical exacerbation of SPA-RAO was associated with hot and humid conditions and high environmental counts for fungal spores and grass pollen grains. It is not known yet whether these are aeroallergens that cause clinical exacerbation of the disease.

An 11-month-old, beefmaster bull presented with anorexia and signs of respiratory disease. Physical examination, thoracic ultrasonography and radiography, and pleural fluid analysis indicated pericarditis and septic neutrophilic pleuropneumonia. Postmortem findings were abomasal adherence to the diaphragm, a fibrotic fistulous tract connecting the abomasum and pleural cavity, granulomatous abomasitis, granulomatous pericarditis, and fibrinonecrotic pleuritis.