Abnormalities in lung surfactant in horses clinically affected with recurrent airway obstruction (RAO)

Background: Abnormalities in lung surfactant are well described in human respiratory diseases including asthma, but are poorly described in horses. Hypothesis: Lung surfactant is abnormal in horses with clinical signs of recurrent airway obstruction (RAO). Animals: Six healthy horses and 5 horses with RAO. Methods: Bronchoalveolar lavage fluid (BALF) was obtained from all horses by standard procedures. Cell‐free BALF was separated into crude surfactant pellets (CSP) and supernatant via ultracentrifugation. Phospholipid and protein content was analyzed from both of these fractions. Phospholipid composition of CSP was determined using high‐performance liquid chromatography with an evaporative light scatter detector. Surface tension of CSP was measured with a pulsating bubble surfactometer. Results: Compared with healthy horses, surfactant from RAO‐affected horses was characterized by significantly decreased phospholipid content in total surfactant (median; range: 23.2; 14.7–62.2 μg/mL BALF versus 172; 111–267 μg/mL BALF, P= .0062) and CSP (20.2; 6.4–48.9 μL/mL BALF versus 155; 94.4–248 μg/mL BALF, P= .0062), and a significantly lower percentage of phosphatidylglycerol (PG) (4.5; 3.6–5.6% versus 6.6; 4.1–7.6%, P= .028). Furthermore, the ratio between the percentages of phosphatidylcholine and PG was significantly higher in RAO‐affected horses than in healthy horses (20.9; 16.6: 25.9 versus 13.9; 11.8–22.8, P= .045). Conclusions and Clinical Importance: This study demonstrates that surfactant from RAO‐affected horses is abnormal. Further studies are needed to determine if these abnormalities are related to an increased tendency for bronchoconstriction and to a decreased ability to clear airway mucus in RAO‐affected horses.     

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

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

Stimulation of airway neutrophils following dexamethasone administration and equid herpesvirus-2 challenge in horses

The aim of this study was to investigate neutrophil stimulation following experimentally-induced airway inflammation in healthy horses. Six horses received dexamethasone and four were then inoculated with equid herpesvirus-2 (EHV-2). Significant neutrophilia was detected in tracheal wash and bronchoalveolar lavage fluid for up to 6 days. Concentrations of neutrophil elastase (NE) and myeloperoxidase (MPO) were significantly increased compared to baseline for up to 14 days in tracheal washes and both markers were significantly correlated with neutrophil counts. Serum levels of surfactant protein D were not significantly modified throughout the study. These results suggest that dexamethasone administration with or without EHV-2 inoculation is associated with a sustainable activation and degranulation of neutrophils in the trachea along with moderate modifications detectable in the lower airways.     

Cardiovascular effects of acute pulmonary obstruction in horses with recurrent airway obstruction

BACKGROUND: Recurrent airway obstruction (RAO) is common in horses. Although pulmonary artery (PA) pressure increases during RAO, cardiac function in horses with RAO has received limited attention. HYPOTHESIS: The purpose of this study was to noninvasively determine the cardiovascular effects of acute pulmonary obstruction (APO) in horses with RAO and their reversibility. ANIMALS: Five geldings with RAO, inducible by exposure to moldy hay, were studied. METHODS: Pulmonary mechanics, echocardiography, serum troponin I concentrations, arterial blood gases, and hematocrit were obtained before and after 7 days of APO. Heart rate, PA diameter and flow characteristics, right and left ventricular luminal dimensions and wall thicknesses, global cardiac performance, and evidence of myocardial damage were evaluated. Pulmonary mechanics and echocardiography were reevaluated during remission. RRESULTS: Severe, transient APO did not induce chronic cor pulmonale in horses, because cardiac anatomy and function were normal between episodes. An acute episode of APO produced anatomical and functional cardiac changes in both the right and left heart (including increased PA diameter, abnormal septal motion, and decreased left ventricular diameter and estimated stroke volume), possibly because of the development of pulmonary hypertension, without apparent myocardial damage. The decrease in stroke volume was offset by the increase in heart rate. CONCLUSIONS AND CLINICAL IMPORTANCE: With APO of 7 days' duration, cardiovascular abnormalities and the functional airway changes that produce them are reversible when the offending allergens are removed.     

Equine recurrent airway obstruction and insect bite hypersensitivity: understanding the diseases and uncovering possible new therapeutic approaches

Recurrent airway obstruction (RAO) and insect bite hypersensitivity (IBH) are allergic conditions that are commonly encountered in the horse. Whilst complete allergen avoidance is an effective management strategy for both diseases, this may not be achievable in all cases and treatment options are therefore required. The inflammatory response is the main therapeutic target for glucocorticoids given to horses with RAO and severe cases of IBH, whilst the bronchodilators used in RAO primarily target airway smooth muscle. Such drugs are effective in most but not all individuals and there may be unwanted adverse effects. This article will review how knowledge of drug action and the pathogenesis of RAO and IBH can be utilised to identify potential targets for novel therapeutic agents that, in the longer term, may be safer and/or more effective in managing the allergic horse.     

Relationship of equine housing to large airway inflammation

The pasture, as compared to stall confinement, has been considered a more desirable environment for maintaining the health of the respiratory system in the horse. This conclusion is based on reports which showed that ventilation in most barns was generally poor, and that horses bedded on straw and fed hay were exposed to many forms of respirable debris. In this study, six normal horses were evaluated for evidence of airway mucosal inflammation after being housed on pasture or stabled in a barn for one month. The response of the horses' airways was measured by assigning scores for the degree of tracheal mucosal secretions that were observed by endoscopic visualization. Cytological examination of transtracheal wash secretions was also performed, as well as histologic evaluation of tracheobronchial tissues obtained by a transendoscopic epithelial biopsy technique. Samples were collected at three time points; the initial collection occurred after the horses were housed on pasture for one month. The horses were subsequently moved to a barn for an equal length of time and samples were obtained at the end of this period. The horses were then returned to their original pasture and final sampling was performed after they were housed in this environment for two months. There were no significant changes in any of the parameters evaluated, regardless of the environment in which the horses were maintained. These findings indicate that housing horses in a barn for four weeks does not cause tracheobronchial mucosal inflammation in a manner that could be detected using the methods employed in this study.     

The role of alveolar macrophages in the pathogenesis of recurrent airway obstruction in horses

When challenged with allergens and pro-inflammatory agents, such as Aspergillus fumigatus (AF), hay dust solution (HDS) and lipopolysaccharide (LPS), the innate immune response will not only activate the immune system but also increase the amount of pro-inflammatory cytokines in the bronchoalveolar space. The aim of this study was to assess the response of equine alveolar macrophages to different aerosolized challenges and to investigate the differences in this response between horses susceptible or nonsusceptible to recurrent airway obstruction (RAO). Seven susceptible and 5 nonsusceptible horses were challenged with saline, LPS, HDS, or AF, and bronchoalveolar lavage (BAL) cytology, total cell counts, and lung function were assessed. In addition, alveolar macrophages were isolated 6 and 24 hours after challenge, and macrophage mRNA expression of tumor necrosis factor (TNF)-alpha and interleukins (IL) IL-1beta, IL-6, IL-8, and IL-10 were measured by means of real-time (RT) polymerase chain reaction (PCR). There was a significant difference in lung function, neutrophil ratios, and total cell counts in the bronchoalveolar lavage fluid between RAO-susceptible and nonsusceptible horses. In addition, the expression of TNF-alpha, IL-1beta, and IL-8 by alveolar macrophages after challenges were higher in susceptible horses, than in nonsusceptible horses. In contrast, I1-6, considered an anti-inflammatory cytokine, showed a higher expression in nonsusceptible horses 6 hours after inhalation challenge with allergens and pro-inflammatory antigens. These data suggest that the differences between susceptible and nonsusceptible horses to RAO are not only dependent on adaptive immunity but also start with an innate immune response.     

Multiple Hypersensitivities Including Recurrent Airway Obstruction,Insect Bite Hypersensitivity,and Urticaria in 2 Warmblood Horse Populations

Background

Multiple hypersensitivities (MHS) have been described in humans, cats, and dogs, but not horses.

Hypotheses

Horses suffering from recurrent airway obstruction (RAO), insect bite hypersensitivity (IBH), or urticaria (URT) will have an increased risk of also being affected by another one of these hypersensitivities. This predisposition for MHS also will be associated with decreased shedding of strongylid eggs in feces and with a single nucleotide polymorphism (SNP BIEC2‐224511), previously shown to be associated with RAO.

Animals

The first population (P1) included 119 randomly sampled horses representative of the Swiss sporthorse population; the replication population (P2) included 210 RAO‐affected Warmblood horses and 264 RAO‐unaffected controls. All horses were Warmbloods, 14 years or older.

Methods

Associations between disease phenotypes (RAO, IBH, URT, MHS) fecal egg counts, the SNP BIEC2‐224511 as well as management and environmental factors were investigated.

Results

In P1, RAO‐affected horses had a 13.1 times higher odds ratio (OR) of also suffering from IBH (P = .004). In P2, the respective OR was 7.4 (P = .002) and IBH‐affected horses also showed a 7.1 times increased OR of concomitantly suffering from URT (P < .001). IBH, URT, and MHS phenotypes were significantly associated with the absence of nematode eggs in the feces.

Conclusions and Clinical Importance

This is the first report of MHS in horses. Specifically, an increased risk for IBH should be expected in RAO‐affected horses.     

The anti-inflammatory effects of IV administered clenbuterol in horses with recurrent airway obstruction

Cyclic AMP elevating agents have been shown to exhibit anti-inflammatory properties in addition to functions such as bronchodilation. The aim of this study was to investigate this dual action of clenbuterol (CB; Ventipulmin) on horses affected with recurrent airway obstruction (RAO). Seven RAO susceptible horses received inhalation challenges with aerosolised lipopolysaccharide (LPS), hay dust suspension (HDS) and Aspergillus fumigatus antigen (AF) with and without prior treatment with intravenous CB. Data showed that CB exerted significant beneficial effects on lung function, total cell count (TCC) and bronchoalveolar lavage neutrophil influx. In addition, CB significantly decreased the expression of several pro-inflammatory cytokines and chemokines in the alveolar macrophages of RAO-susceptible horses after challenge with LPS and HDS, and increased the expression of interleukin-6, known to act as a pro-and anti-inflammatory cytokine, following different challenges. This anti-inflammatory activity of CB is of additive value to its currently recognised use in equine RAO.     

The activity and expression of chitinase in the equine lung and its activity in normal horses and animals with recurrent airway obstruction

Recurrent airway obstruction (RAO) is a chronic inflammatory condition in equine lung, which may share a common immunological basis with human asthma, in which dysregulated Th2 responses occur. Mammals express chitinases and chitinase-like proteins, two of which are active enzymes, chitotriosidase and acidic mammalian chitinase (AMCase). Both enzymes are upregulated in a range of inflammatory conditions, including asthma. We investigated the activity of chitinase in bronchoalveolar lavage fluid from horses with and without RAO in response to organic dust challenges. No significant differences were found in activity, although in one study RAO animals had elevated chitinase activity that fell short of statistical significance. The pH optimum and pH lability of the activity was consistent with the presence of chitotriosidase. RT-PCR amplification of the mRNA encoding chitotriosidase and AMCase in normal equine lung showed that chitotriosidase, but not AMCase, is expressed in trachea, bronchi, and peripheral lung tissue. The gene for chitotriosidase was identified from the Equus caballus (horse) genome 1.1 database and its similarity to the same genes from other species was determined. The results of this study indicate that the involvement of chitotriosidase in RAO is uncertain.     

Impact of environmental temperature on response of neonatal pigs to an endotoxin challenge

OBJECTIVE: To evaluate the effect of various environmental temperatures (ET) on the ability of neonatal pigs to cope with an endotoxin challenge. ANIMALS: 28 crossbred male pigs that were 24 hours old. PROCEDURE: At 24 hours of age, pigs were placed in environmentally controlled chambers maintained at 18 or 34 C (14 pigs/ET). Rectal temperatures (RT) were recorded at 15-minute intervals for 3 hours following an IP injection of 0.9% NaCl (7 control pigs/ET) or lipopolysaccharide (LPS; 150 microg/kg of body weight; 7 LPS-treated pigs/ET). Tissue specimens and blood samples were collected following the 3-hour challenge period. RESULTS: LPS-treated pigs exposed to 18 C had a period of hypothermia whereas RT for LPS-treated pigs at 34 C did not differ from control pigs. The LPS-treated pigs maintained at 18 C lost the most body weight during the 3-hour period and also had the greatest increase in serum cortisol concentration. Serum prolactin (PRL) concentration was decreased in pigs at 18 C, compared with pigs at 34 C. Challenge with LPS resulted in an increase in serum PRL concentration at 18 C but had no effect on serum PRL at 34 C. Challenge with LPS resulted in an increase in expression of tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-6 receptor mRNA in the hypothalamus. CONCLUSIONS AND CLINICAL RELEVANCE: Exposure to a cold ET can inhibit the ability of neonatal pigs to cope with an exogenous endotoxin challenge. When combined, cold stress and exposure to exogenous endotoxin induces a rapid and potentially dangerous loss of body temperature in neonatal pigs.     

Rapid calcitonin response to experimental hypercalcemia in healthy horses

Calcium has important physiological functions, and disorders of calcium homeostasis are frequent in horses. We have made important progress understanding equine calcium homeostasis; however, limited information on equine calcitonin (CT) is available, in part because of the lack of validated CT assays. To determine the CT response to high ionized calcium (Ca²⁺) concentrations in healthy horses, we induced hypercalcemia in 10 healthy horses using a calcium gluconate 23% solution (5 mg/kg; 120 mL/500 kg horse) infused over 4 min. Four horses were infused with 120 mL of 0.9% NaCl and used as controls. We validated a human-specific CT radioimmunoassay for use in horses. Serum Ca²⁺ concentrations increased from 6.2 ± 0.3 mg/dL to 9.9 ± 0.5 mg/dL (4 min; P < 0.01). Serum CT increased from 16.7 ± 8.0 pg/mL to 87.1 ± 55.8 pg/mL at 2 min, and 102.5 ± 51.1 pg/mL at 4 min (P < 0.01). Serum CT returned to baseline by 20 min, whereas serum Ca²⁺ returned to baseline by 40 min. Of interest, CT concentrations returned to baseline despite hypercalcemia, suggesting thyroid gland C-cell CT depletion. Resting CT values higher than 40 pg/mL were considered abnormally elevated. No significant changes in serum Ca²⁺ or CT concentrations were found in control horses. The coefficients of variation for the CT radioimmunoassay were lower than 11.9%. We conclude that the equine thyroid gland C-cell responds quickly to changes in extracellular Ca²⁺ concentrations by secreting large quantities of CT into the systemic circulation, indicating that CT is important in equine calcium homeostasis. The human CT radioimmunoassay can be used to measure changes in equine CT.     

Serum iron and plasma fibrinogen concentrations as indicators of systemic inflammatory diseases in horses

BACKGROUND: Detection of systemic inflammation, which is important for proper diagnosis and prompt treatment, can be challenging. HYPOTHESIS: Measurement of plasma iron concentration is a sensitive method for detecting systemic inflammation in horses compared with measurements of plasma fibrinogen concentration, a traditional marker for inflammation in the horse. ANIMALS: Ninety-seven horses hospitalized with diseases causing systemic inflammation, 22 horses with localized inflammation, and 12 clinically normal horses were included in this study. METHODS: A retrospective study was made on hospitalized horses that had both plasma iron and fibrinogen concentrations measured on hospital admission. RESULTS: Plasma iron concentration was lower in horses with systemic inflammation (64 +/- 45 microg/dL) than the reference interval minimum (105 microg/dL) and were significantly lower (P = .001) than the value in a group of horses with local inflammation (123 +/- 45 microg/dL) and in healthy transported horses (143 +/- 29 microg/dL). Low plasma iron and high fibrinogen concentrations were both sensitive indicators of systemic inflammation in horses with sensitivity of 90 and 82%, respectively. There was a similar correlation between either continued decreases in iron concentration (Rsp of 0.239) or increases in fibrinogen concentration (Rsp of 0.280) during hospitalization and a worse prognosis. CONCLUSIONS AND CLINICAL IMPORTANCE: Measurement of plasma iron concentration better reflected acute inflammation than did fibrinogen concentration.