Special examination

Endoscopic examination of the respiratory tract is a simple, well tolerated by most horses and a valuable diagnostic tool for IAD. Horses with normal respiratory tract have at most a few flecks of mucus visible in the trachea by endoscopy.29,33 Increased mucopurulent exudate in the tracheobronchial tree is detected in the majority of horses with IAD but tracheal exudate is non-specific and is also present in the majority of pulmonary diseases other than IAD.³³ A semi-quantitative mucus scoring system using a scale between 0 and 5 has been developed and validated for use in the horse (Fig. 28.1).^19,34 This scale is useful to grade severity of IAD since a positive association has been found between tracheal mucus score, BAL neutrophilia and poor performance.^8,19,35 Tracheal septum thickness increases with age but is not greater in IAD as compared to healthy horses.^19,36

Cytologic examination of respiratory secretions collected from the tracheobronchial tree is important when trying to confirm a diagnosis of pulmonary disease. Tracheal secretions can be collected by direct aspiration or wash of tracheal lumen via transcutaneous catheterization or through an endoscope.37–39 Normal tracheal fluid cytology usually contains neutrophils representing less than 20% of total nucleated cells.^40,41 Several studies have shown an association between tracheal neutrophilia (>20%), coughing and IAD.^21,30,42,43 A relationship between tracheal mucus accumulation and neutrophilia was found by some investigators but not by others.^43,44 However, cytological examination of tracheal mucus in horses free of respiratory disease is highly variable, with neutrophil percentages ranging between 3 and 83% of total nucleated cells^45,46 and it correlates poorly with pulmonary histopathology.^45,47 Furthermore, mucus score but, not the proportion of neutrophils in tracheal wash, correlates with performance in racehorses as well as in sports horses.^8,35 Therefore, the preponderance of evidence suggests that neutrophil accumulation in tracheal lumen represents a physiological response to contamination of the respiratory tract by inhaled particulates or bacteria in otherwise healthy horses. In some cases, contamination can overwhelm local defense mechanisms (mucociliary clearance, immune response) and result in clinical signs such as cough and excess mucus accumulation.

In contrast, neutrophils represent a small proportion of total nucleated cells present in BAL fluid with most studies reporting neutrophils as <5% of cells in healthy horses (Table 28.2). Furthermore, BAL cytology reportedly correlates well with histopathology of the lungs⁴⁸ and does not correlate with tracheal wash cytology.^46,49 A BAL can be easily performed in field conditions using either a flexible endoscope (≥2 meters long) or an equine BAL catheter at least 2.5 meters long and 10 mm diameter with inflatable cuff at the end (Bivona, Gary, IN; Cook, Bloomington; IN, Jorgensen Laboratories, Loveland, CO). The technique has been described in detail previously.^50,51 It is important to always use the same technique during a BAL because the volume of fluid used as well as the number of boluses administered have a significant effect on cell count and differential (see Table 28.2). Fluid samples should be processed within 1–2 hours or stored at 4°C if sample shipping to the laboratory is to be delayed. Normal BAL fluid should appear slightly turbid with a layer of white foam on the surface (surfactant). Between 50 and 90% of the volume infused is expected to be retrieved.

Measurement of lung function can be done in a variety of ways including assessment of gas exchanges, determination of lung volumes (spirometry), and evaluation of the movement of air in and out the respiratory tract (lung mechanics). Racehorses with IAD exhibit more pronounced exercise-induced hypoxemia than healthy controls during standardized run to fatigue on a high-speed treadmill (Fig. 28.2).^52–54 Both groups of horses had comparable PaCO₂, suggesting that impaired gas exchanges were likely a result of ventilation–perfusion mismatching and not hypoventilation. Spirometry is useful in severe obstructive diseases such as RAO but not in milder diseases like IAD. On the contrary, lung mechanics allows quantification of the degree of airflow obstruction present in horses’ airways. Airflow obstruction is the basic mechanism responsible, at least in part, for some of the manifestation of IAD such as exercise intolerance and cough. The advantage of lung mechanics is quantification of airflow obstruction, which is often subclinical in horses with IAD. Measurement of the degree of airway obstruction allows the clinician to determine the severity of the disease process and provides objective means of assessing response to therapy. The disadvantage is that sophisticated equipment and a sound understanding of respiratory physiology are required. Therefore, tests of lung mechanics are currently used mainly in the research arena and in some referral clinics. However, one commercially available test (Open Pleth™, Ambulatory Monitoring, Inc., Ardsley, NY) allows lung function testing in the field, in particular measurement of airway responsiveness.^55,56

There are four main methods used in the horse to evaluate lung mechanics:

Conventional lung mechanics has been the first test of lung mechanics adapted to the horse ⁵⁷ and is still commonly used in research.^58–60 However, this test has not been used extensively in clinical settings because it does not permit detection of airway obstruction until it is severe and clinical signs are then evident.^18,61 Recently, some researchers showed that the addition of dead space to the respiratory system increases CO₂ rebreathing and ventilation which is accompanied by a significant rise in lung resistance in horses with IAD as compared to healthy controls.⁶² Forced expiration (FE) can be performed in standing, sedated horses.⁶³ As in people, FE is a sensitive test of airflow obstruction where a reduction in forced expiratory flow during late FE (i.e. FEF_95%) is an early indicator of mild airway obstruction such as in IAD (Fig. 28.3).¹⁸ Forced or impulse oscillometry techniques have the advantage of being non-invasive and have been used both in research and clinical cases. Horses with IAD often show frequency dependence of resistance with higher values obtained at the lower frequencies (<10 Hz) suggestive of heterogeneous airway obstruction.^64,65 Finally, flowmetrics or inductance plethysmography (Open Pleth™, Ambulatory Monitoring, Inc., Ardsley, NY, Fig. 28.4) is non-invasive, portable in the field and shows satisfactory correlation with standard lung mechanics.^56,66

Another means of detecting airway obstruction is by testing airway reactivity in response to inhaled irritant such as histamine. Exaggerated airway narrowing in response to an irritant is called airway hyperresponsiveness. Airway reactivity can be quantified using the four tests of lung mechanics described above.63,66–68 Airway hyperresponsiveness is a prominent feature of IAD in horses with increased BALF eosinophil and mast cell counts.^31,65,68,69 The sensitivity of forced oscillometry and flowmetrics is enhanced when used to detect airway hyperresponsiveness. This increased bronchoconstriction in response to inhaled irritants plays an important role in the pathogenesis of the cough and presumably exercise intolerance.

Laboratory examination

Hematology and serum biochemistry of horses with IAD are usually within normal limits.

Cytologic specimens of BALF are prepared by centrifugation and processed with Wright’s stain. Total cell count in healthy horses is usually less than 400 cells/µl (Table 28.2). Differential cell counts should be determined by examination of at least 200 cells per slide and preferably 500 cells. Cytological analysis of BAL fluid allows recognition of 3 main types of inflammatory profiles in IAD (Table 28.2).⁵⁰ The most commonly encountered profile is characterized by an increased total nucleated cell count with mild neutrophilia (5–20 % cells), lymphocytosis, and monocytosis (Fig. 28.5A).^14,16,18,31 Two other cytological profiles characterized by increased percentages of mast cells (>2%, Fig. 28.5B) and eosinophils (>1%, Fig. 28.5C) are also observed in some horses with IAD.^31,69,70 Some horses can also present with a combination of BALF abnormalities such as elevated neutrophils and mast cells, mast cells and eosinophils or all three cell types.^71,72 In contrast, BAL of horses with RAO shows moderate-to-severe neutrophilia (>20% cells), lymphopenia, and decreased alveolar macrophages.^18,73,74 Cytology of BALF collected from horses with RAO in clinical remission can be normal if there has been sufficient time free of exposure to inciting allergens.⁷⁵ Some RAO cases can be in clinical remission but still exhibit some degree of pulmonary neutrophilia and, therefore, can be difficult to differentiate from IAD. One way to discriminate RAO from IAD is by performing a hay challenge and monitoring clinical signs of respiratory disease, which should develop within a few hours to a few days in RAO-affected horses.^59,76 Horses with IAD exposed to moldy hay exhibit a worsening of coughing and pulmonary neutrophilia; however, they do not develop increased respiratory effort or nostrils flaring like RAO-affected horses do. Although practical, this challenge might not be feasible in client-owned horses and in such situations, pulmonary function testing is indicated.

Tracheal secretions can be collected by direct wash of tracheal lumen with sterile saline via a catheter introduced through the endoscope’s working channel or by percutaneous transtracheal aspiration. If microbiologic culture of the fluid is being pursued, tracheal wash (TW) should be performed using a sterile, multiple lumen guarded catheter (double or triple lumen) introduced through the endoscope working channel or via percutaneous catheterization of the trachea in mid-cervical region using a sterile technique. The latter technique requires sedation, whereas the former one can be conducted using only simple restraint such as application of a nose twitch. An abundant commensal flora inhabits nasal passages and nasopharynx (e.g. Streptococcus zooepidemicus). Therefore, TW via the instrument channel of the endoscope without a multiple lumen guarded catheter will result in contamination of the sample and will be inappropriate for microbiologic culture and render any such results uninterpretable.⁷⁷ Furthermore, coughing during the procedure increases the chances of oropharyngeal contamination of the sample.

A smear of the TW sample should be made as soon as possible after collection. Total nucleated cell count in healthy horses is usually less than 500 cells/µL with few to no red blood cells.11,78 Differential count should be determined based on 200–300 consecutive cells. Cells in TW are more difficult to identify and count than in BAL because they often are more degenerate and are trapped in abundant mucus strands. Normal TW fluid should contain a majority of macrophages (30–70%), low numbers of epithelial cells (higher if collected with catheter through the endoscope), <20% neutrophils, <10% lymphocytes, <1% eosinophils and very few mast cells (<0.2%).^11,49,77,78 Importantly, several studies have documented the fact that there is no correlation between TW and BAL cytology, indicating that one is not a substitute for the other.^49,79 Tracheal wash is the sample of choice when suspecting an infectious etiology and BAL is preferred otherwise because of the high correlation with lung lesions (as determined by histopathology).

Horses that cough during training more than 4 times in a 10-min period are more likely to have >20% neutrophils in TW, large numbers of bacteria (>10³ cfu/mL) and intracellular bacteria.⁴⁰ The likelihood of isolating bacteria from tracheal wash samples collected from racehorses in training is strongly associated with the TW inflammation and mucus accumulation, in particular in young horses.^15,23,30 But there is no apparent association between TW neutrophilia and mucus in older racehorses.⁴⁴ Isolation of more than 10³ colony-forming units of pathogenic Streptococcus spp. is also strongly associated with coughing.³⁰ Therefore, tracheal inflammation and high bacterial counts are common in racehorses but only excess tracheal mucus is associated with decreased performance, not TW neutrophilia.⁷

Necropsy examination

Histopathological examination of the lungs can be conducted ante-mortem by collecting tissue samples using transcutaneous lung biopsy needles and endobronchial biopsies forceps via the endoscope, or at post-mortem examination. Horses with IAD present similar histopathology findings as horses with RAO but with less severe and chronic changes.⁴⁸ Typical morphological findings include peribronchiolitis, bronchiolitis, bronchiolar epithelial hyperplasia, goblet cell metaplasia, luminal mucus, and in some cases alveolitis.^80,81 Severity of histopathologic changes assessed by a scoring system appears to be negatively correlated to various indices of respiratory and cardiovascular function in horses with IAD.⁸⁰ Consequently, as pulmonary lesions become more severe, horses become more exercise intolerant. However, histopathology findings obtained from transcutaneous lung biopsies or endobronchial biopsies are highly variable and the potential diagnostic benefit should be weighed against the potential risks associated with transthoracic biopsy such as hemorrhage and pneumothorax.

Diagnostic confirmation

In horses presenting with clinical signs including cough, increased respiratory secretions, and poor performance, detailed history, physical examination, and diagnostic tests should help eliminate differential diagnoses (Fig. 28.5B). Using a validated questionnaire administered to clients is useful to identify horses with heaves;⁸² however, the same questionnaire has been unable to differentiate IAD from healthy horses.¹⁶ Horses with IAD are not febrile and presence of a fever suggests infectious respiratory diseases such as viral diseases, bronchopneumonia, pleuropneumonia, or pulmonary abscess. Hematology findings can be helpful to confirm a suspicion of respiratory infection; however, they are often non-specific. Leukocytosis with neutrophilia is commonly found with bacterial respiratory infections and during the acute phase of a bacterial infection, increased numbers of immature neutrophils (‘left shift’) may be observed. Neutrophilia can also accompany non-infectious inflammatory diseases (such as caused by toxins), neoplasia, and mycotic and parasitic infections. Hematologic changes during the early phase of a viral respiratory infection, such as influenza, are often characterized by normocytic, normochromic anemia, lymphopenia or lymphocytosis, and sometimes neutropenia.^83,84 Neutrophilia can follow within a week of initial clinical signs, particularly in cases of secondary bacterial infection. Monocytosis can develop during the recovery phase of a viral infection.

Coughing is often chronic, intermittent (>3 weeks) in horses with IAD. Main differential diagnoses are mild cases of heaves (RAO), and parasitic pneumonitis (Fig. 28.6). Horses with RAO typically display exercise intolerance and increased respiratory efforts during periods of disease exacerbation; however, these signs can be subtle during periods of disease remission. In those cases, pulmonary function testing or more simply, moldy hay challenge, will help reaching a definitive diagnosis. Neutrophilia is commonly observed in BAL fluid from horses with RAO and IAD.^14,73,85 The neutrophilia is usually more pronounced (>20%) with RAO than IAD; however, there is significant overlap between diseases.^16,18,86 Eosinophilic inflammation can be associated with IAD, parasitic pneumonitis (Parascaris equorum, Dictyocaulus arnfieldi),⁸⁷ hypersensitivity pneumonitis, fungal pneumonia, and cutaneous habronemiasis.^70,88,89 Clinical signs of parasitic pneumonitis are non-specific. Fecal flotation (Baermann technique) is often not diagnostic of P. equorum infection because migration through the lungs occurs during the pre-patent period.⁹⁰D. arnfieldi follows a complete cycle in donkeys, mules, and asses; however, the infection is usually not patent in horses. Therefore, the Baermann fecal flotation is not useful either. P. equorum pneumonitis is more commonly detected in foals less than 6 month of age. D. arnfieldi usually occurs in horses in contact with infected donkeys and rarely by ingesting larvae excreted by infected horses.⁹¹ A presumptive diagnosis can be reached when respiratory secretions reveal eosinophilic inflammation with sometimes evidence of parasite eggs or larvae, exposure to donkeys exists, and anthelmintic therapy results in clinical improvement.⁹⁰ Increased numbers of metachromatic cells (mast cells, basophils) are common in horses with IAD but have not been associated with other types of respiratory disease.^70,92