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.     

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.     

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.     

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     

The effects of sodium cromoglycate on antigen inhalation challenge in two horses affected with chronic obstructive pulmonary disease (COPD)

80 mg sodium cromoglycate (SCG) was administered by inhalation to two COPD-affected animals known to have respiratory hypersensitivity to Micropolyspora faeni. SCG treatment 20-30 minutes prior to inhalation challenge with M. faeni prevented exacerbation of respiratory disease, usually seen 4-8 hours after challenge. duration of protection against antigen challenge after a single SCG treatment was 4-5 days. The duration of protection was not prolonged by reducing the frequency of antigen challenge. Multiple antigen challenge, using M. faeni and Aspergillus fumigatus, shortened the protective period of SCG to 3 days.     

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.     

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.     

Detection of adenovirus precipitating antibodies in the sera of Polo horses in Nigeria.

Serum samples obtained from 107 Polo horses showing clinical signs of viral respiratory disease were tested for precipitating antibodies to adenovirus by agar gel precipitation test and counter-immunoelectrophoresis method. The results obtained demonstrate serological evidence of adenovirus infection in Polo horses in Nigeria. The counter-immunoelectrophoresis method was observed to be about 3 times more sensitive than the agar gel precipitation test with 19.3 vs 64.5%. It could thus be used to screen a large number of serum samples within a short period.     

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.     

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.     

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.     

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.     

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.     

Influenza hemagglutination inhibiting activity in respiratory mucus from horses with chronic obstructive pulmonary disorders (heaves syndrome).

Samples of mucus from the lower trachea were collected from 53 horses with chronic obstructive pulmonary disease and from 24 clinically normal horses. Serum samples were collected from 35 of the horses with chronic obstructive pulmonary disease and from the 24 normal horses. Samples were tested for inhibition of hemagglutination by influenza A equine 1 and 2 viruses. There were high levels of hemagglutination inhibiting activity against influenza A equine 1 in mucus samples from horses with chronic obstructive pulmonary disease.     

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.     

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.     

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.     

Efficacy of montelukast in the treatment of chronic obstructive pulmonary disease in five horses

Five horses with moderate to severe chronic obstructive pulmonary disease (COPD) were treated with 0.11 (0.01) mg/kg bodyweight of montelukast, a cysteinyl leukotriene receptor antagonist, once a day for 26 days. The horses were evaluated clinically and endoscopically and subjected to arterial blood gas analysis and lung function tests before and after the period of treatment, and the plasma concentrations of montelukast were determined by high-performance liquid chromatography with fluorescence detection. The treatment did not result in statistically significant differences in the total scores of clinical and endoscopical signs, or in the difference in the arterioalveolar partial pressure of oxygen, maximal changes in pleural pressure, pulmonary resistance or dynamic compliance. The mean (sd) peak plasma concentration (C(max0) of montelukast was 12 (4) ng/ml and was reached 66 (13) minutes (t(max)) after its oral administration. The dose of montelukast per kg bodyweight was approximately the same as that for human beings, but the C(max) in the horses was 28 times lower and the t(max) was reached in one-fifth of the time, suggesting that its oral bioavailability may be lower.     

Climatic and aeroallergen risk factors for chronic obstructive pulmonary disease in horses

OBJECTIVE: To estimate the association between climate and airborne pollen and fungal factors and chronic obstructive pulmonary disease (COPD) in horses. SAMPLE POPULATION: Data from 1,444 horses with a diagnosis of COPD. PROCEDURE: The Veterinary Medical Database was used to identify records of horses admitted to veterinary teaching hospitals in the United States and Canada between 1990 and 1999. Rainfall, mean minimum and maximum temperature, and maximum monthly pollen and fungal spore (mold) counts recorded at the city closest to where the hospital is located were identified for each month data were reported to the Veterinary Medical Database. Associations between climatic and aeroallergen data and monthly prevalence of COPD were estimated by use of cross-correlation and logistic regression models. RESULTS: Significant positive correlations were found between prevalence of COPD and rainfall 3 months previously, minimum temperature 1 and 2 months previously, total pollen counts measured 3 months previously, and total mold counts measured during the same month and 1 month previously. CONCLUSIONS AND CLINICAL RELEVANCE: Outdoor aeroallergens and climatic factors may contribute to the occurrence of COPD in horses.     

Monthly, daily, and circadian variations of measurements of pulmonary mechanics in horses with chronic obstructive pulmonary disease

OBJECTIVES: To determine temporal variations of pulmonary function in horses without respiratory tract disease (controls) and horses with chronic obstructive pulmonary disease (COPD) and determine whether reversibility of airway obstruction after environmental control can be predicted by response to atropine administration. ANIMALS: 7 COPD-affected and 5 control horses. PROCEDURES: Pulmonary function testing was performed monthly during 3 consecutive months, daily for 5 consecutive days, and at 6-hour intervals for 24 hours before and after administration of atropine (0.02 mg/kg of body weight, i.v.) and after 5 consecutive months at pasture. Respiratory rate, tidal volume (VT), minute ventilation (VE), maximal change in transpulmonary pressure (deltaP(L)), pulmonary resistance (R(L)), and pulmonary elastance (E(L)) were calculated. RESULTS: COPD-affected horses had a significantly higher expiratory to inspiratory time ratio (T(E)/T(I)) and deltaP(L), E(L), and R(L) than horses without respiratory tract diseases during all periods and higher V(E) during monthly and daily evaluations. Daily variation in VT and monthly and circadian variation in E(L) were significant in COPD-affected horses. In control horses, significant changes were apparent only in T(E)/T(I) during daily recordings. In COPD-affected horses, reduction in deltaP(L), R(L), and E(L) was significant after atropine administration and after maintenance on pasture. CONCLUSIONS AND CLINICAL RELEVANCE: Despite variations in measurements of respiratory mechanics in both groups of horses, values remained significantly different between groups over time. Despite individual variation, measurements were repeatable during short and long periods. Response to administration of atropine to COPD-affected horses underestimated improvement in respiratory tract function that resulted from maintenance on pasture.