Comparison of tracheal aspirates and bronchoalveolar lavage in racehorses. 2. Evaluation of the diagnostic significance of neutrophil percentage

OBJECTIVE: To determine whether diagnosis of airway inflammation, using cut-off percentages for neutrophils, differs when based on samples from tracheal aspirate (TA) and bronchoalveolar lavage (BAL) collected concomitantly from the same racehorse. DESIGN: Retrospective case series of 48 young Thoroughbred and Standardbred racehorses in race training, but showing poor performance. PROCEDURE TA and BAL samples were collected from all horses 1 to 2 h after high-speed treadmill exercise. Aliquots of the retrieved fluid were cytocentrifuged and smears stained with Diff-Quik. The mean percentage of neutrophils was calculated. Diagnostic cut-off points were set at 20% for TA samples and 5% for BAL samples. Agreement in the interpretations between the two techniques was analysed. RESULTS: In 19 of 51 paired samples (37%) there were differences in diagnostic interpretation between TA and BAL samples. Of these, airway inflammation was indicated only by the TA sample in 13 and only by the BAL in 6. CONCLUSION: TA and BAL samples give important information about different regions of the airway, but neither should be used alone for the diagnosis of inflammation of the entire lung. The limitations of these procedures mean that both samples should be collected when it is desired to cytologically evaluate the entire lower airway.     

Quantitative bacterial cultures and cytological examination of bronchoalveolar lavage specimens in dogs

Cytology and quantitative bacterial cultures of lower respiratory tract secretions are widely used in human medicine to differentiate airway infection from simple bacterial colonization. A retrospective study was conducted to determine the usefulness of quantitative aerobic cultures and Gram stain intracellular bacteria counts from bronchoalveolar lavage (BAL) specimens in dogs in diagnosing lower respiratory tract infection (LRTI) and to determine whether chronic bronchitis is associated with marked bacterial growth in dogs. The threshold determined to define clinically relevant bacterial growth was 1.7 x 10(3) colony-forming units per milliliter of BAL fluid. We used this threshold and found that diagnostic sensitivity and specificity were 86% and 100%, respectively. With a threshold for infection of >2 intracellular bacteria observed in any of 50 fields, microscopic examination of Gram stain BAL preparations had a sensitivity of 71% and a specificity of 97% in establishing LRTI. There was a high correlation between bacterial morphology on BAL Gram stain and bacterial cultures. Combining the results of intracellular bacteria counts from the BAL Gram stain with those from the quantitative cultures, the sensitivity in diagnosing LRTI was 87% and the specificity was 97%. BAL quantitative cultures as well as quantitating intracellular bacteria on Gram stain BAL cytology were revealed to be useful in identifying LRTI in dogs. Chronic bronchitis does not appear to be associated with marked bacterial growth in dogs.     

Comparison of bronchoalveolar lavage cytospins and smears in dogs and cats

Differences in the cytological interpretation of bronchoalveolar lavage fluid (BALF) after cytospin preparation (CP) or manual smearing of pelleted cells preparation (MSP) were investigated in client-owned dogs and cats with inflammatory or infectious lower respiratory disease. Bronchoalveolar lavage fluid from healthy cats was also examined. With MSP, cell lysis was more frequently observed, and cellular distribution was more heterogeneous throughout the slide. When samples from healthy and diseased animals were considered together, a significantly greater percentage of neutrophils was seen on CP than on MSP slides (P<0.002). Cytospin preparations were considered of better quality in all individual comparisons. Cytospin preparation is advised in the evaluation of BALF with low total cell count. When only MSPs are evaluated, clinicians should be aware that differential neutrophil counts may underestimate the counts found on CP slides.     

Effect of an external nasal dilator strip on cytologic characteristics of bronchoalveolar lavage fluid in Thoroughbred racehorses

OBJECTIVE: To determine the effects of an external nasal dilator strip on cytologic characteristics of bronchoalveolar lavage (BAL) fluid in racing Thoroughbreds. DESIGN: Clinical trial. ANIMALS: 23 Thoroughbred racehorses in active training. PROCEDURE: Each horse raced on 2 occasions: once while wearing an external nasal dilator strip and once while not. Bronchoalveolar lavage was performed 12 to 18 hours after each race, and BAL fluid was analyzed for RBC and leukocyte counts and hemosiderin content. RESULTS: Mean +/- SEM count of RBCs in BAL fluid when horses raced without the nasal dilator strip (84.6 +/- 275 cells/microL) was not significantly different from count when they raced with it (41.7 +/- 12.2 cells/microL). Horses were grouped as having mild or severe bleeding on the basis of RBC count in BAL fluid after horses raced without the nasal dilator strip. Mean count when horses with severe bleeding raced without the nasal dilator strip (271.0 +/- 63.7 cells/microL) was significantly higher than mean count when these horses raced with the strip (93.8 +/- 376 cells/microL). Mean count of lymphocytes in BAL fluid was significantly lower after horses raced with the external nasal dilator strip. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that use of an external nasal dilator strip in Thoroughbred racehorses may decrease pulmonary bleeding, particularly in horses with severe exercise-induced pulmonary hemorrhage.     

Cytologic analysis of tracheal wash specimens and bronchoalveolar lavage fluid in the diagnosis of mycotic infections in dogs

Tracheal wash and bronchoalveolar lavage fluid analyses were performed in 9 dogs that had mycotic infections with pulmonary involvement. Characteristic organisms were identified in tracheal wash fluid in 3 of 7 dogs with blastomycosis. Organisms were identified in bronchoalveolar lavage fluid in 5 of 7 dogs with blastomycosis and in one dog with histoplasmosis. Organisms were not found in either fluid in one dog with coccidioidomycosis. These procedures should be considered for dogs with suspected mycotic infections that involve the lungs and that cannot be diagnosed by less invasive means.     

Effect of sodium cromoglycate on light racehorses with elevated metachromatic cell numbers on bronchoalveolar lavage and reduced exercise tolerance

Some young horses with clinical signs of small airway disease demonstrate increased metachromatic cell numbers on bronchoalveolar lavage. The purpose of this study was to determine the effect of sodium cromoglycate treatment on clinical signs, bronchoalveolar lavage cytology and bronchoalveolar lavage histamine parameters in these horses. Twelve racehorses (age: 3.4 ± 1.6 years) with a history of respiratory embarrassment at exercise, clinical signs of obstructive airway disease and bronchoalveolar lavage metachromatic cell differential greater than 2% were selected. Horses were randomly assigned to receive either 200 mg sodium cromoglycate or saline placebo nebulized twice daily for 7 days. A clinical respiratory score was assigned and bronchoalveolar lavage was performed on each animal on days 0 and 7. Measurements were made of the following bronchoalveolar lavage fluid parameters: total nucleated cell concentration, differential cell percentage and concentration, supernatant and lysate histamine concentration, lysate: supernatant histamine ratio and metachromatic cell histamine content. Between the two evaluation periods, sodium cromoglycate treated horses demonstrated an improvement in respiratory score (P = 0.01) and a stabilizing of metachromatic cell histamine content (P = 0.04) when compared with placebo treated horses. We conclude that sodium cromoglycate is effective for the treatment of small airway disease in this population of young racehorses although the pharmacodynamics of this drug in the horse require further investigation.     

Ovine bronchoalveolar lavage cellularity: reproducibility and the effect of multiple repeated lavage.

We sought to determine the normal within-sheep variability of bronchoalveolar cellular parameters and to infer the numbers of sheep required to detect a defined significant change within the same. We further sought to examine the effect of repeated bronchoalveolar lavage (BAL) on those parameters. Neither the total cell number nor the absolute numbers of any given cell type changed significantly following repeated BAL. Additionally, there was no significant change in percentage differential cell populations over time in randomly sampled lobes. However for lobes sampled repeatedly, a significant change in the percentage of lymphocytes was detected. Although the percentages of neutrophils in repeatedly and randomly sampled lobes were significantly correlated, the percentage found in the former tended to be greater and more variable than in the latter. As a consequence, larger group sizes are required to detect given changes in neutrophil percentages in repeatedly sampled lobes over time when compared with detecting equivalent changes in randomly selected lobes.     

Effect of water vapor-saturated air therapy on bronchoalveolar lavage and tracheal mucus transport rate in clinically normal horses

Water vapor-saturated air was delivered to 12 healthy, housed horses for 2 hours daily for 5 days. Treatment had no effect on tracheal mucus transport rate, bronchoalveolar lavage total and differential cell counts, blood cell counts, or plasma fibrinogen concentration.     

Comparison of the antioxidant status in tracheal and bronchoalveolar epithelial lining fluids in recurrent airway obstruction

REASONS FOR PERFORMING STUDY: Following a period of airway inflammation the clearance of inflammatory cells along the mucociliary escalator may impose a considerable oxidant load on the trachea. OBJECTIVES: To determine the degree of oxidative stress in tracheal epithelial lining fluid (ELF) in comparison to that present in peripheral airways after an acute exposure to organic dust. METHODS: Tracheal wash fluid and bronchoalveolar lavage fluid (BALF) were collected for cytology and antioxidant analyses from 6 recurrent airway obstruction (RAO)-affected horses and 6 healthy control horses before and after stabling on straw bedding for 24 h. RESULTS: In RAO-affected horses, organic dust exposure resulted in a significant decrease in ascorbic acid concentration in tracheal ELF (P<0.0001), which was greater than the decrease in bronchoalveolar ELF (P = 0.0003). The percentage decrease in tracheal ELF ascorbic acid correlated with the percentage decrease in bronchoalveolar ELF ascorbic acid (r = 0.76; P = 0.004) following exposure. CONCLUSIONS: Acute organic dust exposure results in significant antioxidant depletion in the trachea, which may reflect inflammation and oxidative processes in peripheral airways. POTENTIAL RELEVANCE: Further work is required to evaluate the role of ascorbic acid depletion in the pathogenesis of RAO.     

Bronchoalveolar lavage for the diagnosis and treatment of pneumonia associated with transport in Thoroughbred racehorses.

To evaluate a hypothese that use of bronchoalveolar lavage (BAL) for early treatment of pneumonia would improve their prognosis by reducing bacterial numbers and excessive numbers of neutrophils in the lung, initial experiences with BAL in the diagnosis and treatment of pneumonia were performed in 36 racehorses that became ill within 24 hr of long distance travel (1,200-1,600 km, 26-32 hr) by road. Comparisons were made of the outcomes of the 36 horses and those of 42 horses (81.0% recovered, 50.0% returned to racing) treated for transport associated pneumonia without BAL. The total amount of BAL fluid injected during hospitalization varied from 700 to 3,700 ml and the duration of antibiotic treatments ranged from 5 to 40 days. Clinical symptoms after lavages showed good results with no side effects. None of the horses required thoracic drainage. Horses treated with BAL required shorter period of antibiotic therapy, a greater percentage recovered (100%, 36/36) and a greater percentage returned to racing (77.8%, 28/36). Eight (22.2%) never raced because of lameness or other considerations.     

Cytological analysis of equine bronchoalveolar lavage fluid. Part 2: Comparison of smear and cytocentrifuged preparations

The aim of this study was to develop a diagnostically useful smear method for preparation of equine bronchoalveolar lavage fluid (BALF) for use by practitioners. A smear method for equine BALF preparation which included the addition of serum was developed, and cell morphology, differential cell counts (DCC) and repeatability of counting DCC compared with those of cytocentrifuged BALF preparations. BALF samples (n = 21) were collected from 5 control horses and 5 heaves-susceptible horses. Smear preparations of BALF produced smaller, darker, staining cells, making cytological identification more difficult than on cytocentrifuged preparations. There was a significantly higher (P<0.01) macrophage DCC and lower lymphocyte DCC on cytocentrifuged compared to smear preparations. Mast cell and eosinophil DCC were significantly higher (P<0.05) on cytocentrifuged compared to smear preparations of BALF. Smear preparations were shown to be reliable for the cytological diagnosis of equine neutrophilic pulmonary disease and offer practitioners an alternative to sending equine BALF to a laboratory for processing and cytological analysis.     

Comparison of transtracheal aspirate and bronchoalveolar lavage cytology in 50 horses with chronic lung disease

Comparisons were made between transtracheal aspirate (TTA) and bronchoalveolar lavage (BAL) cytology obtained from 50 horses with chronic lung disease and from 10 control horses. There was no significant correlation between the TTA cytology and the BAL cytology, suggesting that the cell population in the trachea is not representative of the cell population in the lower airways. In control horses the range of differential cell counts obtained from TTA fluid was remarkably large, whereas the variability in cell populations observed in BAL fluid was smaller. In the principal horses the total and differential cell counts of the TTA and BAL fluids were within the 95 per cent confidence interval in 38 and 24 per cent of cases, respectively; and an increase in percentage neutrophils was most common. It was concluded that BAL may be a useful diagnostic aid when evaluating horses with chronic lung disease, but that the clinical usefulness of cytological evaluations of TTA fluid may be limited in these cases.     

Comparison of bronchoalveolar lavage and respiratory secretion cytology in horses with histologically diagnosed pulmonary disease

Equine bronchoalveolar lavage (BAL) fluid collected from 70 horses and respiratory secretions (RS) obtained from 61 of these horses were evaluated cytologically and grouped according to the histological diagnosis of the lungs from which they were obtained. The histological categories included: normal lung (8 horses); pulmonary eosinophilic infiltration (9 horses); interstitial pneumonia (5 horses); pulmonary hemorrhage (5 horses); and mild (12 horses), moderate (7 horses) and severe (24 horses) chronic small airway disease. In horses with pulmonary disease, all BAL samples and all but one RS sample differed cytologically to those obtained from normal horses; however, the type and severity of the pulmonary disease could not always be determined using either BAL or RS cytology. There was a positive association between the percentage of neutrophils in BAL and the neutrophil scores in RS specimens; there was no positive association between other cell types.     

Neutrophil migration induced by equine respiratory secretions, bronchoalveolar lavage fluids and culture supernatants of pulmonary lavage cells

Supernatants of equine respiratory secretions enhanced the migration of equine neutrophils into the lower compartments of Boyden chambers. Checkerboard analysis revealed that the neutrophil migration promoting activity (NMPA) of secretion specimens was in great part caused by chemokinesis, irrespective of the neutrophil score of the specimen. The NMPA of respiratory secretions was correlated neither with the neutrophil score of the secretion specimen nor with the severity of the chronic pulmonary disease. Respiratory secretions collected while horses were kept under low dust or under dusty housing conditions induced migration of neutrophils in the same order of magnitude. The number of migrated neutrophils and the procoagulant activity (PCA) within respiratory secretion specimens was positively correlated; however, the meaning of this finding is not yet clear. None of the nine cell-free supernatants of bronchoalveolar lavage fluid, which were assayed undiluted, induced significant neutrophil migration, although some samples contained up to 4.0 x 10(5) neutrophils/ml. In vitro culture of lung lavage cells, which mainly comprised macrophages and lymphocytes, without stimulation or with the addition of low doses of phytohemagglutinin (PHA) resulted in the secretion of NMPA which was in great part chemotactic. However, culture supernatants of lung cell preparations which were stimulated by lipopolysaccharide (LPS) or by PHA-prestimulated lymphocytes reduced the migration of neutrophils compared with the supernatants of control cells. NMPA within culture supernatants had a highly significant negative correlation with the PCA of macrophages within the lung cell preparations. Our results imply that a complicated and sophisticated regulation underlies neutrophil accumulation within the airways of horses affected with chronic pulmonary disease. Future experiments are required to assess the biological significance of the factors modulating neutrophil migration which are present in the respiratory secretions and in the culture supernatants of equine lung lavage cells.     

Comparison of bronchoalveolar lavage and respiratory secretion cytology in horses with clinically diagnosed chronic pulmonary disease

Thirty-nine horses and 3 ponies underwent a thorough respiratory examination and were grouped as follows: healthy (4 horses and 1 pony); mild chronic pulmonary disease (CPD 11 horses); moderate CPD (16 horses and 1 pony); and severe CPD (8 horses and 1 pony). Bronchoalveolar lavage (BAL) fluid collected from all animals and respiratory secretions (RS) obtained from 39 of these animals were evaluated cytologically and the results were compared. It was concluded that cytological examination of either BAL fluid or RS was useful in diagnosing various equine pulmonary diseases. The only advantage that BAL offered over RS sampling was in cases in which there was no RS available in the trachea. In addition, the severity of the CPD did not always correlate with either RS or BAL cytology.     

IgE-bearing cells in bronchoalveolar lavage fluid and allergen-specific IgE levels in sera from RAO-affected horses

Recurrent airway obstruction (RAO) is a common condition in stabled horses characterized by small airway inflammation, airway neutrophilia and obstruction following exposure of susceptible horses to mouldy hay and straw and is thus regarded as a hypersensitivity reaction to mould spores. However, the role of immunoglobulin E antibodies (IgE) in the pathogenesis of RAO is unclear. We hypothesized that the number of cells with receptor-bound IgE in bronchoalveolar lavage fluid (BALF) and IgE levels in serum would be higher in RAO-affected than in healthy horses living in the same environment. Therefore, IgE-positive (+) cells were identified by immunocytochemistry on cytospins from BALF and counted. IgE levels against the mould extracts Aspergillus fumigatus (Asp. f.) and Alternaria alternata (Alt. a.) and the recombinant mould allergen Aspergillus fumigatus 8 (rAsp f 8) were measured by enzyme-linked immunosorbent assay (ELISA) in the sera of seven RAO-affected and 22 clinically healthy mature horses housed in the same conventional stable environment. After correcting for the number of neutrophils, there were no significant differences in IgE+ cells on cytospins from BALF between both groups of horses (5% versus 7%, P > 0.1). Serum IgE levels against the mould extracts were significantly higher in RAO-affected than in clinically healthy horses [median = 119 versus 66 relative ELISA units (REU), P < 0.05]. Furthermore, significantly more RAO-affected than healthy horses had detectable serum IgE against the recombinant allergen rAsp f 8 (4/7 and 3/22, respectively, P < 0.05). Age had no significant effect on BALF cell ratios or on specific serum IgE levels. These results show that high IgE levels against mould antigens are associated with RAO under controlled environmental conditions but ranges of mould-specific serum IgE levels overlapped too much between diseased and clinically healthy animals to be of any diagnostic value. Further studies are needed to assess whether IgE-mediated reactions contribute to the pathogenesis of RAO.     

Arachidonate metabolites in bronchoalveolar lavage fluid from horses with and without COPD.

Arachidonate metabolites were measured in bronchoalveolar lavage fluid (BALF) from horses with (N = 4) and without (N = 7) chronic obstructive pulmonary disease (COPD). Prostaglandin (PG) D2, leukotriene (LT) B4 and LTC4 were present in highest concentrations in BALF from clinically normal horses. Concentrations of PGE2 and PGF were significantly higher in BALF from horses with COPD than in BALF from normal horses, but no differences were detected in thromboxane B2, 6-keto-PGF1 alpha, PGD2, LTB4 or LTC4.     

Comparison of bronchoalveolar lavage findings and measurements of gas exchange during exercise in horses with poor racing performance

Twenty-four Thoroughbred and twelve Standardbred racehorses aged between 2 and 6 years, presented for reported poor racing performance, underwent clinical exercise testing. During the last 10 s of exercise at each speed throughout an incremental speed exercise test on a treadmill inclined at a 10% slope, samples of arterial blood and expired gases were collected. Maximum oxygen uptake and the partial pressures of oxygen and carbon dioxide in arterial blood were determined. These values were compared between the two breeds of horses and also with reference to cytological findings of bronchoalveolar lavage samples, including neutrophil, erythrocyte and haemosiderophage percentage and the total nucleated cell concentration. The results revealed an inverse relationship (Spearman R = -0.45, p < 0.05) between the total nucleated cell count in bronchoalveolar lavage samples and arterial oxygen partial pressure during exercise at 11 m.s(-1). This result suggests that subclinical pulmonary disease may be a more important cause of poor racing performance than previously thought. Also of note was a positive correlation (Spearman R = 0.50, p < 0.05) between maximum oxygen uptake and the percentage of erythrocytes.     

Comparison of bronchoalveolar lavage findings and measurements of gas exchange during exercise in horses with poor racing performance

Twenty-four Thoroughbred and twelve Standardbred racehorses aged between 2 and 6 years, presented for reported poor racing performance, underwent clinical exercise testing. During the last 10 s of exercise at each speed throughout an incremental speed exercise test on a treadmill inclined at a 10% slope, samples of arterial blood and expired gases were collected. Maximum oxygen uptake and the partial pressures of oxygen and carbon dioxide in arterial blood were determined. These values were compared between the two breeds of horses and also with reference to cytological findings of bronchoalveolar lavage samples, including neutrophil, erythrocyte and haemosiderophage percentage and the total nucleated cell concentration. The results revealed an inverse relationship (Spearman R = -0.45, p<0.05) between the total nucleated cell count in bronchoalveolar lavage samples and arterial oxygen partial pressure during exercise at 11 m.s^-1. This result suggests that subclinical pulmonary disease may be a more important cause of poor racing performance than previously thought. Also of note was a positive correlation (Spearman R = 0.50, p<0.05) between maximum oxygen untake and the percentage of erythrocytes.