Chronic obstructive pulmonary disease (COPD) in horses: aetiological studies: responses to intradermal and inhalation antigenic challenge.

Micropolyspora faeni and Aspergillus fumigatus were identified as common causes of respiratory hypersensitivity in horses affected with chronic obstructive pulmonary disease (COPD). Rye grass pollen and an Actinomycete evoked respiratory allergy in a few horses. Not infrequently, individual horses were found to have respiratory hypersensitivity to two or more antigens. The methods used to examine for allergy were intradermal testing and inhalation challenge with environmental antigens. An intradermal test using an M faeni extract was demonstrated to be suitable for diagnostic use in horses previously accurately diagnosed as suffering from COPD. In contrast, the A fumigatus antigen used proved unsatisfactory for such a purpose. Skin reaction to M faeni and A fumigatus extracts by horses affected with COPD indicated that the hypersensitivity was a dual one--a weak response shortly after injection followed by an Arthus-like response 4 to 8 hours later. As a parameter for monitoring responses to inhalation challenge, maximum intrathoracic pressure change (max delta Ppl) proved satisfactory, whereas changes in partial pressure of arterial oxygen (PaO2) did not.     

Pulmonary artery pressures in normal horses and in horses affected with chronic obstructive pulmonary disease

Horses clinically affected with chronic obstructive pulmonary disease (COPD) were found to have pulmonary artery hypertension which was associated with systemic arterial hypoxia. The pulmonary hypertension in symptomatic COPD-affected horses was partially reversible upon remission of clinical signs or by oxygen administration. The induction of acute hypoxaemia caused an increase in pulmonary artery pressure in both normal and COPD-affected horses.     

Effects of environmental control on pulmonary function of horses affected with chronic obstructive pulmonary disease

The effects of environmental control on horses affected with chronic obstructive pulmonary disease was assessed by clinical examination and pulmonary function tests, ie, maximum change in intrathoracic pressure, tidal volume, minute volume, non-elastic work of breathing, dynamic compliance, inspiratory and expiratory flow rates and arterial blood gas analysis. A controlled environment (ie, bedding horses on shredded paper and feeding a complete cubed diet) caused symptomatic COPD affected horses to become asymptomatic within four to 24 days (mean ± sd 8.4 ± 4.8 days). When asymptomatic, their pulmonary function values did not differ significantly from those of normal horses, which indicates that the pathophysiological changes occurring in equine COPD are reversible. The time taken for horses to become asymptomatic correlated significantly with age, duration of illness and severity of disease as adjudged by the non-elastic work of breathing.     

Effect of antigen challenge on the activation of peripheral blood neutrophils from horses with chronic obstructive pulmonary disease

The effect of antigen challenge on the state of activation of peripheral blood neutrophils from horses with chronic obstructive pulmonary disease ( ) has been determined by measuring neutrophil superoxide anion formation. Prior to a seven-hour antigen challenge superoxide anion production by neutrophils from asymptomatic horses with and normal horses in response to platelet activating factor ( ) (with and without cytochalasin B), serum treated zymosan ( ) and phorbol myristate acetate ( ) was similar. Agonist-induced superoxide production by neutrophils from symptomatic and normal horses remained unchanged five and 24 hours after antigen challenge. Interestingly, however, superoxide production by neutrophils from symptomatic horses was significantly increased 24 hours after antigen challenge in the control samples for each agonist (basal superoxide production), a five-fold increase being measured in the presence of cytochalasin B. There was a small increase in superoxide production by neutrophils from normal horses but this only reached significance in one set of control samples. The change in activation state of circulating neutrophils during antigen challenge may facilitate the lung neutrophilia and subsequent tissue damage which occur in .     

The presence of precipitating antibodies in the sera of horses with chronic obstructive pulmonary disease (COPD).

The sera of horses affected and not affected with chronic obstructive pulmonary disease (COPD) were examined for precipitins to Micropolyspora faeni and Aspergillus fumigatus. Precipitins to both antigens were not restricted to COPD cases but occurred more frequently in animals affected with COPD. Many animals without detectable precipitins responded clinically to inhalation challenge with these antigens.     

Antigen challenge increases adherence of circulating neutrophils in horses with chronic obstructive pulmonary disease

Activation of circulating neutrophils has been observed following challenge of horses with chronic obstructive pulmonary disease (COPD) and may facilitate the accumulation of these cells in the airways. In this study, no significant difference was observed between adherence to protein coated plastic of blood neutrophils from asymptomatic COPD-susceptible and normal horses stimulated by the mediators PAF, human recombinant (hr)IL-8 and hrC5a. Twenty-four hours after the start of a 7 h antigen challenge, adherence of unstimulated neutrophils from COPD-susceptible horses increased from 2.5 (0.5-4.1)% and 3.4 (0.6-6.6)% to 19.6 (16.9-20.3)% and 21.8 (10.6-23.1)% adherence for cells in medium containing 0.1% or 0.2% BSA, respectively; (median [range]; n = 4). Adherence of cells from normal horses remained unchanged. Addition of an anti-CD18 monoclonal antibody, H20A, inhibited the increase in adherence at 24 h by 96 (45-100)%, n = 3. The percentage of neutrophils in bronchoalveolar lavage fluid at 24 h increased from 1 (0-2) to 80 (65-94), (median (range), n = 4). These results suggest that antigen challenge results in exposure of circulating equine neutrophils to one or more factors that prime, or activate, these cells, which may enhance their recruitment to the lungs. Inhibition of circulating neutrophil activation may therefore represent a therapeutic target.     

A study of the effect of a platelet activating factor (PAF) receptor antagonist on antigen challenge of horses with chronic obstructive pulmonary disease

Antigen challenge involving exposure to straw and mouldy hay for 7 h produced lung function changes and neutrophil recruitment to the lungs in horses with chronic obstructive pulmonary disease (COPD). During the challenge, an increase in radiolabelled neutrophils in the lungs occurred, together with increased respiratory rate and pleural pressure. The role of platelet activating factor (PAF) in antigen-induced neutrophil accumulation, and increased pleural pressure and respiratory rate was investigated by administering the PAF receptor antagonist WEB 2086 to asymptomatic COPD horses prior to antigen challenge. WEB 2086 (3 mg/kg i.v.) did not affect antigen-induced changes in either neutrophil accumulation or respiratory function. These results suggest that PAF may not be an important mediator of the response to antigen in equine COPD.     

Alveolar clearance in horses with chronic obstructive pulmonary disease

OBJECTIVE: To assess sensitivity of scintigraphic alveolar clearance rate as an indicator of alveolar epithelium damage in horses. ANIMALS: 5 healthy horses (group A) and 5 with chronic obstructive pulmonary disease (COPD; group B). PROCEDURE: Horses underwent clearance rate (k [%/min]) determination. Clearance rate of group-B horses was determined after remission of the disease following 2 months at pasture (remission 1), stabling in a controlled environment (remission 2), and during crisis induced by exposure to moldy hay and straw. Methacholine challenge test was performed at each investigation period to determine nonspecific pulmonary airway hyperresponsiveness. Pulmonary function tests (PFT) also were performed, and cell populations in bronchoalveolar lavage (BAL) fluid were determined on another occasion. RESULTS: Group-B horses had significantly faster mean clearance rate during crisis (k = 4.30+/-0.95%/min), compared with that for remission 1(k = 1.98+/-0.55%/min), which did not differ from the rate in group-A horses (k = 1.95+/-0.33%/min). Despite lack of clinical signs of COPD during remission when stabled in a controlled environment, an intermediate value was found (k = 3.20+/-0.72%/min). CONCLUSIONS: This technique allowed grading of lung damage induced by COPD, whereas use of PFT and determination of BAL fluid cell populations failed to differentiate between remission 1 and remission 2. CLINICAL RELEVANCE: Determination of alveolar clearance rate by use of scintigraphy is a sensitive indicator of lung damage. A modified clearance rate was found despite the lack of clinical and functional changes.     

An investigation of the incidence of chronic obstructive pulmonary disease (COPD) in random populations of Swiss horses

The incidence of Chronic Obstructive Pulmonary Disease (COPD) in Switzerland was determined in three groups of horses selected at random. Group A (97 horses) and Group B (93 horses) had no history of respiratory disease, but Group C (113 horses) had a history of lower respiratory tract disease. All horses were examined by auscultation of the respiratory tract under forced breathing, endoscopic examination of the upper and lower respiratory tract, arterial blood gas analysis at rest and cytological examination of respiratory secretions (RS). Fifty-four per cent of the horses in Group A and 54.8 per cent of Group B were diagnosed as suffering clinical COPD and only 12.4 per cent of horses in Group A and 8.6 per cent in Group B had no detectable abnormalities in their lower respiratory tracts. In Group B, 19.4 per cent were considered to be suffering from parasitic pulmonary disease indicated by elevated numbers of eosinophils in their RS. Pharyngeal lymphoid hyperplasia was exhibited in 70.1 per cent of Group A and 74.2 per cent of Group B. Cytological examination of RS was useful to determine if the secretions found in the trachea originated from the upper or lower respiratory tract. Arterial blood gas analysis revealed no significant differences between healthy horses and those with subclinical or mild COPD.     

Effects of cromolyn in horses with chronic obstructive pulmonary disease

The effect of disodium cromoglycate (cromolyn) in preventing the pulmonary dysfunction caused by the inhalation of barn and hay dust was studied in 5 horses with confirmed chronic obstructive pulmonary disease (COPD). The horses were studied before (Con) and after exposure to hay and dust allergens (Expos) and after pretreatment with cromolyn followed by exposure (Cr-Expos).There was a significant reduction in PaO₂ from 86.8±8.3 to 73.1±8.8 when the horses were exposed to hay and dust allergens. The PaO₂ after pretreatment with cromolyn and exposures was 78.1±5.5. There were no significant changes in PaCO₂, FRC, pH and A-aDO₂ when the Con, Expos, and Cr-Expos periods were compared.There were significant increases in V_E from a control value of 77.9±18.2 to 128.7±55.1 and 133.7±17.1 L/min during the exposures, which was due primarily to increases in respiratory frequency. Respiratory dead space (V_D/V_T) increased from 0.55±0.10 to 0.71±0.07 and 0.65±0.05, and alveolar ventilation (V_E) remained constant. Pulmonary resistance (R_L) and transpulmonary pressure (P_tr) increased from a control of 0.77±0.28 cm H₂O/L/sec and 7.73±3.38 cm H₂O to 2.93±1.01 and 20.17±4.81 during the Expos period and tidal volume (V_T) fell from 7.5±1.0 to 5.7±1.3 L. The pre-treatment with cromolyn before exposures significantly reduced the increase in R_L and P_tr and returned V_T to Con levels.Supported by Fisons, Ltd, Pharmaceutical Division and the Pennsylvania State Horse Racing Commission     

Ultrastructural findings in horses with chronic obstructive pulmonary disease (COPD). I: Alterations of the larger conducting airways

Extensive light and electron microscope studies of the conducting airways were carried out in 28 horses with varying degrees of clinically manifested chronic obstructive pulmonary disease (COPD) and in 8 horses with normal lungs. The principal ultrastructural changes were found in the ciliated cells. There was focal loss of ciliated cells, which were replaced by undifferentiated cells in a largely hyperplastic epithelium, and some horses, independent of the degree of severity of the disease, showed various types of ciliary malformation. The finding of dilated intercellular clefts and accumulations of mast cells was interpreted as morphological evidence of non-specific mucosal hyperreactivity. Interstitial cells with intracytoplasmal crystal inclusions, the cause of which is not clear, were seen in many horses. Comparison between the clinical diagnosis and the morphological findings showed partial correlation. The ciliary loss, the appearance of peribronchial inflammatory processes and the occurrence of intracytoplasmic crystalline inclusions showed a positive correlation. In 12 of 28 cases, the severity of clinical diagnosis agreed with the degree of the morphological findings. The changes in the conducting airways were interpreted pathogenetically as reactive processes to changes in the small airways in the course of equine COPD.     

Phosphodiesterase activity in neutrophils from horses with chronic obstructive pulmonary disease

Neutrophils are recruited to the lungs of horses with chronic obstructive pulmonary disease (COPD) and exhibit increased activity after antigen challenge. Phosphodiesterase type4 (PDE4) inhibitors have been shown to attenuate human neutrophil activation. The aim of this study was to establish the PDE isoenzyme profile of equine neutrophils using isoenzyme selective inhibitors to determine if these compounds should be evaluated in horses with COPD. Total cAMP and cGMP dependent PDE activity was no different in neutrophils from normal (156.2+/-7.1 and 6.8+/-0.6 pmol/min/mg for cAMP and cGMP, respectively) and COPD susceptible horses (146.0+/-10.2 and 5.5+/-0.6 pmol/min/mg for cAMP and cGMP, respectively). The PDE4 inhibitors, CDP840 and rolipram, caused significant, concentration related and almost complete inhibition of PDE activity (IC(50) values=8.8+/-0.1 x 10(-9) and 7.3+/-0.2 x 10(-9)M for CDP840; 1.2+/-0.1 x 10(-6) and 1.1+/-0.1 x 10(-6)M for rolipram in normal and COPD susceptible horses, respectively). The inhibitory effects of the mixed PDE3/ PDE4 inhibitor, zardaverine were of similar magnitude and potency to rolipram. However, the limited inhibitory effects of the PDE3 inhibitor, siguazodan, suggest that zardaverine is acting primarily via PDE4 inhibition. These results indicate that PDE4 is the predominant isoenzyme present in the equine neutrophil and inhibition of PDE activity using selective PDE4 inhibitors may, therefore, modulate equine neutrophil activation in horses with COPD.     

Influence of clenbuterol on mucociliary transport in healthy horses and horses with chronic obstructive pulmonary disease

The mucociliary tracheal transport rate and clearance index were measured in 16 healthy horses and 16 horses suffering from chronic obstructive pulmonary disease. A marker, Indian ink in syrup, was placed in the mucus layer of the interior wall of the trachea, just in front of the carina, and its displacement was measured after 30 and 60 minutes. The same procedure was repeated 30 minutes after the intravenous administration of 0.8 microgram clenbuterol/kg bodyweight. In the diseased horses the mucociliary transport rate was significantly lower than in the healthy horses. After clenbuterol administration there was a significant increase in the transport rate and a similar increase in the clearance index. Sixty and 90 minutes after clenbuterol there was a significant increase of mucus secretion in both the healthy and diseased horses. Measurements in five healthy horses and five diseased horses on two different days gave similar results, showing that the technique provided reproducible data.     

Results of intradermal tests in horses without atopy and horses with chronic obstructive pulmonary disease

OBJECTIVE: To compare results of intradermal tests (IDT), conducted using environmental allergens, in horses without atopy and horses with chronic obstructive pulmonary disease (COPD). ANIMALS: 38 horses (22 horses without atopy and 16 horses with COPD). PROCEDURE: All horses were examined (physical examination, hematologic examination, serum biochemical analyses, examination of bronchoalveolar lavage fluid). An IDT was conducted, using a full panel of 73 allergens consisting of grasses, weeds, trees, molds, and insects. Results of the IDT were evaluated 30 minutes and 4, 6, and 24 hours after injection of allergens. Horses without atopy were euthanatized, and gross and histologic changes of lung parenchyma were assessed. RESULTS: Horses without atopy had a greater number of positive immediate and late-phase reactions than did horses with COPD. Horses with COPD did not have a significantly greater number of positive reactions than horses without atopy at any time period for any allergen group (grasses, weeds, trees, molds, and insects). CONCLUSIONS AND CLINICAL RELEVANCE: Positive results of IDT document allergen-specific hypersensitivity but do not necessarily distinguish clinically relevant reactions from subclinical reactivity in horses with COPD. Interpreting the clinical relevance of results of IDT requires a thorough knowledge of the medical history, physical examination findings, and environment of each animal.     

Histamine inhalation challenge in normal horses and in horses with small airway disease

A histamine inhalation challenge (HIC) procedure was developed to assess hyperreactive states in horses. Following clinical evaluation, percutaneous lung biopsies were performed on nine light breed mares aged 6 to 15 years. Five horses, with normal small airways, were classified as group A and four subjects with small airway disease (SAD) lesions formed group B. Pulmonary mechanics parameters were monitored following an aerosol of 0.9% saline and every 5 min for up to 30 min after HIC with 0.5% w/v of histamine diphosphate, administered through a face mask for 2.5 min. Tidal volume (VT) and airflow (V) values were obtained with a pneumotachograph. Transpulmonary pressure (delta Ppl) was measured by the esophageal balloon catheter method. Dynamic compliance (Cdyn), total pulmonary resistance (RL), end expiratory work of breathing (EEW) and respiratory rate (f) were calculated by a pulmonary mechanics computer. Group A horses had increases in RL, and decreases in Cdyn whereas horses in group B were hyperreactive and showed greater changes in EEW, Cdyn, and delta Ppl but with a relatively lower variation of RL. One horse in clinical remission from SAD, but with a high biopsy score (group B), and one clinically normal horse belonging to group A showed marked hyperreactivity as shown by increases in EEW, maximum change in delta Ppl and RL and decreases in Cdyn. These results suggest that the HIC described can be used as a method to investigate airway hyperreactivity and SAD in horses.     

Comparison of bronchodilators in alleviating clinical signs in horses with chronic obstructive pulmonary disease

A representative of each of 3 types of bronchodilators was given iv to horses with clinical signs of chronic obstructive pulmonary disease. We used atropine (0.01 mg/kg of body weight) as an anticholinergic, isoproterenol HCl as a beta-adrenergic, and aminophylline, the soluble salt of theophylline, as a methylxanthine. Response to these drugs was evaluated by measuring maximal change in intrathoracic pressures (delta Ppl) and observing clinical signs. Atropine caused a significant (P less than 0.01) reduction in delta Ppl, compared with that caused by aminophylline. Atropine caused the delta Ppl to decrease an average of 83 +/- 15% toward normal. Clinical improvement was noticed in 87% of the cases. Maximal changes in pleural pressures were reduced by an average of 21 +/- 14 mm of Hg, with 27% returning completely to normal. Isoproterenol helped 77% of horses clinically, and caused delta Ppl to decrease an average of 68 +/- 31% toward normal. The average reduction in delta Ppl was 16 +/- 10 mm of Hg. Aminophylline helped alleviate clinical signs of 50% of the horses and caused delta Ppl to decrease an average of 41 +/- 36% toward normal. The average reduction in delta Ppl was 10 +/- 11 mm of Hg.     

Ultrastructural findings in horses with chronic obstructive pulmonary disease (COPD). II: Pathomorphological changes of the terminal airways and the alveolar region

Extensive light and electron microscope studies (transmission and scanning electron microscopy) of the bronchioles and alveolar region, in 28 horses suffering chronic obstructive pulmonary disease (COPD) and eight control horses, revealed good correlation between clinical severity and morphological changes. In the bronchiolar epithelium the non-ciliated bronchiolar epithelial (Clara) cells, in particular, showed ultrastructural alterations and, even in the mild stages of disease, these presented degenerative changes and lack of differentiation. Together with loss of granulation in the Clara cells and metaplasia of the goblet cells, cells were seen with unusual intracytoplasmic lamellar inclusion, the number of which increased sharply with clinical severity. The focal changes in the alveolar region were necrosis of type I epithelial cells, alveolar fibrosis of varying degrees with type II epithelial transformation and emphysema or hyperinflation, with an increase in Kohn's pores. Some horse also showed morphological signs of interference with the surfactant system, in the form of marked cysts with lamellar structure. The alveolar changes were mostly in the peribronchiolar region and were, therefore, interpreted as reactive processes. No conclusions as to the aetiology of equine COPD can be derived from these morphological investigations.     

Intradermal testing of horses with chronic obstructive pulmonary disease and recurrent urticaria.

Six horses with chronic obstructive pulmonary disease (COPD) and 8 horses with recurrent urticaria were skin tested with 67 extracts from 58 allergens, including pollens, epidermals, cultivated farm plants, dusts, molds, and insects. Reactions were evaluated 3 times over a 24-hour period immediately after the injections. Results were compared with those obtained from 11 clinically normal horses. All horses had positive skin test reactions. Significant differences was evident between horses with COPD and clinically normal horses for only 3.0% of the possible extract reactions, and between horses with urticaria and clinically normal horses for only 4.5% of the possible extract reactions. Horses with COPD or urticaria had greater total percentage of allergen extract reactions than did clinically normal horses. Positive reactions were observed at all 3 evaluation periods, and late-onset reactions were not always preceded by positive reaction at earlier periods. All horses with COPD or urticaria had at least 1 skin test reaction that exceeded the mean +/- 2 SD, as calculated for each of the 67 extracts for the group of clinically normal horses.     

Interleukin-4 and interferon-gamma gene expression in summer pasture-associated obstructive pulmonary disease affected horses

We hypothesised that horses affected with summer pasture-associated obstructive pulmonary disease (SPAOPD) react to an allergen or allergens in their summer environment that is either absent or present at lower levels in their winter environment; and that such allergens stimulate SPAOPD-affected horses to produce a different T helper lymphocyte cytokine profile from that of control horses. The primary objective of this study was to determine the cytokine mRNA profile of T helper lymphocytes obtained from summer pasture-associated obstructive pulmonary disease (SPAOPD) affected horses when 1) the horses were showing signs of disease (summer) and 2) they were in clinical remission (winter). A further objective was to determine the differences between cytokine mRNA T helper lymphocyte profiles of control and affected horses in the summer and winter seasons. Interleukin 4 (IL-4) and interferon-gamma (IFN-gamma) mRNA expression levels were increased in bronchoalveolar lavage fluid (BALF) and peripheral blood mononuclear cell (PBMC) samples of affected horses during disease expression. No significant amounts of IL-5 mRNA were detected in any of the samples. These results suggest that there is an allergic component to SPAOPD of horses and that appropriate manipulation of the immune system could offer hope for treatment and prevention of the disease in the future. Further research studies will be needed to determine the most appropriate treatments to use to alter the antigen-stimulated cytokine profile being expressed by SPAOPD-affected horses or to alter the effects that these cytokines produce.     

Concentrations of elastinolytic metalloproteinases in respiratory tract secretions of healthy horses and horses with chronic obstructive pulmonary disease

OBJECTIVES: To determine whether samples of tracheal epithelial lining fluid (TELF) obtained from horses have elastinolytic activity characteristic of metalloproteinases, to compare elastinolytic activity in TELF obtained from healthy horses and horses with chronic obstructive pulmonary disease (COPD), and to determine whether chemically modified tetracycline-3 (CMT-3) inhibits elastinolytic activity in TELF ANIMALS: 10 horses with COPD and 10 healthy control horses. PROCEDURE: Zymography and fluorometry were used to measure elastinolytic activity, and EDTA was used to inhibit elastinolytic activity and verify that the activity was attributable to metalloproteinases. Possible inhibition of elastinolytic activity with CMT-3 was studied in vitro. RESULTS: Elastinolytic activity was found in TELF obtained from all horses, and this activity was significantly higher in TELF obtained from horses with COPD than in TELF obtained from healthy horses. For all samples, EDTA and CMT-3 inhibited elastinolytic activity. CONCLUSIONS AND CLINICAL RELEVANCE: Elastinolytic activity is detectable in TELF obtained from horses and seems to be attributable to metalloproteinases. Elastinolytic activity in TELF is significantly inhibited by CMT-3. Elastinolytic activity in TELF can be detected by means of zymography or fluorometry. Increased elastinolytic activity may reflect destruction of pulmonary tissue in horses with COPD. Chemically modified tetracyclines such as CMT-3 may provide an additional treatment possibility for horses with COPD.