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.     

Comparison of tracheal aspirates before and after high-speed treadmill exercise in racehorses

OBJECTIVE: To determine whether percentages of neutrophils in tracheal aspirate (TA) samples collected from racehorses are increased after exercise and whether interpretation of results from TA samples taken before and after exercise agree. DESIGN: Case series of 40 young Thoroughbred and Standardbred racehorses in race training presented for evaluation of poor performance. PROCEDURE: TA samples were collected endoscopically from racehorses presented for poor performance 24 h before and 1 to 2 h after high speed treadmill exercise testing. Aliquots of the retrieved fluid were cytocentrifuged and smears were stained with Diff-Quik. Mean neutrophil counts were expressed as percentages of the total number of inflammatory cells counted and subsequently were categorised as either above or below an accepted cut-off of 20%. Comparisons between percentages of neutrophils before and after exercise were made. RESULTS: Percentage of neutrophils from TA samples obtained from racehorses after exercise was significantly higher than neutrophil percentages from TA samples collected from the same horse before exercise. In horses with TA specimens that were categorised as having < or = 20% neutrophils before treadmill exercise, the percentage of neutrophils in their TA specimens after exercise was, on average, significantly higher and was greater than the cut-off value of 20%. CONCLUSION: Recent strenuous exercise may change the proportion of neutrophils in lower airways of racehorses and practitioners should be aware of this when collecting and interpreting the results from TA samples. The most practical time for collection of a TA sample to obtain the most diagnostically useful information might be after a suitable washout period of at least 1 to 2 h post-exercise.     

Comparison of four staining methods for detection of mast cells in equine bronchoalveolar lavage fluid

Mast cells normally are present in equine bronchoalveolar lavage fluid (BALF), but usually represent <2% of all cells in healthy horses. An increased percentage of mast cells has been associated with airway hyperactivity and inflammatory airway diseases, but marked differences are reported between studies in normal and diseased horses. Because an abnormal mast cell count may be of clinical relevance, we compared the ability of a fast Romanowsky method to stain mast cell granules with that of 3 metachromatic stains: automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains. The BALF cells from 24 horses were studied. A differential cell count was performed blindly on 400 cells. The percentages of mast cells obtained were analyzed by means of repeated-measures analysis of variance and Fischer's PLSD test. The Bland and Altman method was used to assess agreement among stains. The mean percentage of mast cells in BALF was significantly lower with the fast Romanowsky than with the automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains. With the fast Romanowsky stain, the metachromatic granules of mast cells were not stained, and their identification was based on morphologic criteria. Toluidine blue staining allowed detection of the highest mean percentage of mast cells, but was inadequate for performing a differential cell count on other cell types. In conclusion, fast Romanosky stain may be inadequate for detection of mast cells in equine BALF, whereas automated Romanowsky, May-Grünwald Giemsa, and toluidine blue stains provide metachromatic staining of mast cell granules.     

Equine bronchoalveolar lavage cytology: survey of Thoroughbred racehorses in training

SUMMARY Sixty-two Thoroughbred horses aged between 1 and 7 years in training in Sydney had bronchoalveolar lavage (BAL) samples collected for cytological examination. All horses, except the yearlings and those with a cough, had raced at the time of the examination and the trainers reported satisfactory performance. Free erythrocytes were found In 73% of samples and haemoslderophages In 90% of the samples, Indicating Immediate or past occurrences of exercise-Induced pulmonary haemorrhage (EIPH). Bronchoalveolar fluid from the yearlings contained significantly less (P / 0.05) erythrocytes and haemosiderophages than samples from horses In other age groups. In the older horses, there was also more haemosiderln within the macrophages. No differences In BAL cytology could be attributed to gender, and there was no relationship between BAL cytology and racing performance. The main cytological findings were (mean ± sd): total nucleated cells - 832 ± 578/μL with the main cell types being: macrophages - 59 ± 10% (haemosiderophages - 20 ± 24%); neutrophlls - 9 ± 6%; lymphocytes - 31 ± 9%. The erythrocyte count was 10.3 ± 17.7% of the total cell count. Horses with chronic coughing had a higher proportion of macrophages and a lower proportion of lymphocytes in the leucocytes obtained from BAL. There was a higher occurrence of EIPH detected In BAL findings than that previously reported when endoscopic examination has been used to diagnose EIPH. The occurrence and severity of EIPH as Indicated by the BAL findings was found to be related to exercise Intensity. The cytological findings were similar to those reported in horses in the northern hemisphere. We conclude that BAL cytology may be useful In the diagnosis of various lower respiratory tract disorders and that exercise-induced pulmonary haemorrhage occurs In virtually all horses In race training.     

Coughing in thoroughbred racehorses: risk factors and tracheal endoscopic and cytological findings

A matched case-control study was made of 100 thoroughbred horses which were coughing and 148 control horses which were free of clinical signs of respiratory tract disease. The variables identified by multivariable conditional logistic regression as being significantly associated with coughing included age (the risk decreased with age), the stage of training (horses in early training were at greatest risk), the time since the last race (horses that had never raced were at greatest risk) and the time since they were last transported (horses transported more than 14 days previously were more likely to cough than those transported within the last week). The coughing horses were significantly more likely to have high scores for upper and lower tracheal mucus and pharyngeal lymphoid hyperplasia. In addition, the tracheal aspirates of the coughing horses had increased odds of neutrophilia and were more likely to have intracellular bacteria than the control horses. However, a considerable proportion of the control horses had cytological and/or endoscopic evidence of airway inflammation.     

Effects of collecting serial tracheal aspirate and bronchoalveolar lavage samples on the cytological findings of subsequent fluid samples in healthy Standardbred horses

Objective To evaluate the effect of collecting serial tracheal aspirate (TA) and bronchoalveolar lavage (BAL) samples on the cytological findings of subsequent fluid samples obtained from horses without clinical signs of respiratory disease. Study design Experimental. Study population Six healthy Standardbred horses. Methods Endoscopically‐guided TA samples, and BAL samples collected using the blind field technique were obtained from the six horses on days 1, 2, 3, 4, 5, 12, and 17. On day 17, horses were sampled three times: at baseline and at 2.5 h and 4 h apart. The differential cytology of the fluid samples collected at each time point was expressed as percentages and compared statistically. Results There was a significant increase in neutrophil percentage in the TA samples taken at day 17 (at 2.5 h but not at 4 h apart). There was no significant change in the neutrophil percentages in the TA samples when repeated samples were taken ≥24 h apart. There was no significant change in the neutrophil percentages in the BAL fluid at any collection point. There were inconsistent changes in the percentages of lymphocytes and macrophages in the BAL fluid over time, but these remained within normal reference ranges and were considered clinically insignificant. Conclusions Serial TA and BAL samples can be taken at 24 h intervals without affecting the cytological findings of subsequent fluid samples collected using the techniques described.     

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.     

Cytological analysis of equine bronchoalveolar lavage fluid. Part 1: Comparison of sequential and pooled aliquots

The aim of this study was to investigate whether initial equine bronchoalveolar lavage fluid (BALF) aliquots were more representative of bronchial cytology that bronchiolar and alveolar cytology. Cell viability and total nucleated (TCC), differential (DCC) and absolute cell counts of cytocentrifuged preparations of 3 sequentially collected BALF aliquots (Aliquots 1-3) were compared with those of pooled BALF (Aliquot 4) to assess whether all aliquots were representative of the lavaged lung segment. BALF samples (n = 21) were collected from control horses (n = 5) or heaves-affected horses (n = 5). There were nonsignificant trends of increasing TCC and absolute macrophage count from Aliquot 1 to Aliquot 3 and significant differences in macrophage (P<0.05) and lymphocyte (P<0.01) DCC among aliquots of all horses; however, no linear trend in this DCC data was observed. There was a significant decrease in mast cell DCC (P<0.01) from Aliquot 1 to Aliquot 3 in control horses. Cell viability did not differ significantly among aliquots. There was no diagnostically significant difference in TCC, DCC, absolute cell counts or cell viability, among sequential and pooled BALF aliquots and, therefore, all aliquots can be considered to represent the cytology of the lavaged lung segment. This indicates that even if BALF recoveries are very low, cytological analysis of samples will be of diagnostic value.     

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.     

Evaluation of cytokine mRNA expression in bronchoalveolar lavage cells from horses with inflammatory airway disease

Inflammatory airway disease (IAD) is a common disorder of performance horses and is associated with poor performance and accumulation of mucus and inflammatory cells in lower airway secretions. Horses with IAD frequently have increased relative counts of neutrophils in bronchoalveolar lavage fluid (BALF); less commonly relative counts of eosinophils and/or mast cells may be increased. The aetiopathogenesis of IAD is unknown and may involve innate and/or acquired immune responses to various factors including respirable dust constituents, micro-organisms, noxious gases and unconditioned air. The molecular pathways and role of the immune system in the pathogenesis of IAD remain poorly defined and it is unknown whether polarised T cell responses occur in the disease, as have been reported to occur in equine recurrent airway obstruction and asthma in humans. Elucidating cytokine responses that develop in horses with IAD may allow a greater understanding of the possible aetiopathological pathway(s) involved and could contribute to development of novel treatments. We compared the mRNA expression of tumour necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukin (IL)-1β, IL-2, IL-4, IL-8, IL-13, IL-17 and IL-23 in cell pellets extracted from BALF of horses with IAD (n=21) and horses free of respiratory tract disease (n=17). Horses with IAD had significantly increased levels of TNF-α, IL-1β and IL-23 mRNA; no significant differences in the other cytokine mRNAs were detected. The results of this study indicate that IAD of horses is associated with increased mRNA expression of pro-inflammatory cytokines in BALF cells, which may reflect stimulation of the innate immune responses to inhaled antigens. There was no evidence of a polarised T-cell cytokine response suggesting hypersensitivity responses may not be involved in the aetiopathogenesis of IAD.     

Comparison of endoscopic tracheal washing, bronchoalveolar lavage and visual detection of blood following instillation of blood into the airways of horses

In an experimental study of endoscopic tracheal washings and bronchoalvveolar lavage specimens collected prior to and following instillation of 0,250, 500 or 1000 ml of autologous citrated blood into the airways of nonexercising, adult horses, there was no apparent correlation between the amount of blood or exudate seen endoscopically, the volume of washing fluid recovered, or the cytologic evaluation of numbers of erythrophages or siderophages. Tracheal washing and bronchoalveolar lavage specimens appear to be complementary for detection of erythrophages or siderophages in cytologic specimens since these cell types may be present in one, or the other, or both types of specimens. There may be an advantage to collecting two BAL specimens since more erythrophages or siderophages were seen in the second BAL in some horses. Further studies of experimentally-instilled blood and spontaneouslyoccurring exercise-induced pulmonary hemorrhage are needed to further characterize the mechanisms by which pulmonary hemorrhage is resolved and/or characterize the cytologic methods best suited for detection and/or quantitation of pulmonary hemorrhage.     

Bronchoalveolar lavage in the cat: cytological findings.

Cells recovered by bronchoalveolar lavage from the lungs of specific pathogen-free (SPF) cats from birth to maturity and from adult conventional cats were enumerated and identified. The predominant cell type recovered was the pulmonary alveolar macrophage from all ages of both SPF and conventional cats. Other cell types included eosinophils, neutrophils and lymphocytes. Lavage of conventional cats yielded significantly more eosinophils and neutrophils than were recovered from SPF cats.     

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.     

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 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.     

Influence of truck-transportation on the function of bronchoalveolar lavage fluid cells in cattle

The study sought to evaluate whether truck-transportation had an impact on the respiratory immune system of cattle. Six castrated 6-10-month-old Holstein calves were shipped approximately 100 km by road for 4 h. Plasma and bronchoalveolar lavage (BAL) fluid samples, collected immediately before transportation, at 4 h (soon after transportation), and on days 3 and 7 after transportation, were examined. A marked elevation of plasma cortisol concentration was observed at 4 h, but this level was unchanged in controls. The chemiluminescence (CL) response of phagocytes in BAL fluid cells, composed mainly of alveolar macrophage, decreased significantly after transportation (P<0.05). Transportation increased the CD3+ T cell population significantly (P<0.05), and a significant increase (P<0.05) in the ratio of CD4+ to CD8+ cells in BAL fluid was evident. We conclude that short-term road transportation alters pulmonary cells and their function, which may engender bovine respiratory disorders.     

Effect of tracheal mucus and tracheal cytology on racing performance in Thoroughbred racehorses

REASON FOR PERFORMING STUDY: Accumulations of mucus within the trachea are often found during endoscopic examinations of the airways of poorly performing racehorses, but the clinical importance of this finding is unknown. OBJECTIVES: To determine the effect of tracheal mucus, pharyngeal lymphoid hyperplasia (PLH) and cytological indices of tracheal aspirate on racing performance in Thoroughbred horses assessed by race place and whether the horse was raced. METHODS: Endoscopic examination of the nasopharynx, larynx and trachea was performed, and a tracheal aspirate obtained monthly at Thistledown racetrack from April to December, 2002 and 2003. Horses received a score of 0-4 for the degree of PLH and 0-4 for the amount of mucus visible in the trachea. The tracheal aspirate was assessed for turbidity, and total and differential cell counts. Generalised estimating equations models were used as repeated measures models for each risk factor and the level of association assessed through the risk factor's P value in the model. RESULTS: Moderate to severe tracheal mucus (2-4) was a risk factor for poor racing performance. There was no association between degree of PLH, cell counts or turbidity of tracheal wash fluid and racing performance. However, horses that raced had higher total neutrophil counts in tracheal wash aspirates than horses that did not race. CONCLUSIONS: Grades 2-4 tracheal mucus should be considered a potential cause of poor racing performance in Thoroughbred horses. CLINICAL RELEVANCE: Because moderate to severe tracheal mucus accumulation, and not increased tracheal neutrophils, was a risk factor for poor racing performance, functionally significant airway inflammation may best be confirmed by the presence of mucus rather than increased number of neutrophils in the trachea.