Airway Diagnostics: Bronchoalveolar Lavage, Tracheal Wash, and Pleural Fluid.

Indications for bronchoalveolar lavage, tracheal wash, and thoracocentesis for the diagnosis of respiratory diseases are discussed. Each technique is described in detail and illustrated by videos. Sample handling, preparation and evaluation are reviewed. The advantages and limitations of bronchoalveolar lavage and tracheal wash procedures as well as a critical comparison between the 2 techniques for equine asthma diagnosis are presented. Finally, validated cut-off values for equine asthma diagnosis are reviewed.

Association between exercise-induced pulmonary hemorrhage and inflammatory airway disease in polo ponies.

The respiratory system is essential for health and high athletic performance in horses. Respiratory diseases have been recognized as having a major impact on training equine animals and are commonly cited as the second most common cause of wasted training time. Inflammatory airway disease (IAD) is an important cause of poor performance in young racehorses. Exercise-induced pulmonary hemorrhage (EIPH) is considered a major issue for the equine industry because of its high prevalence and association with reduced athletic performance. In Brazil, polo is a growing equestrian sport, but studies on it are still scarce. The aim of this study was to evaluate the occurrence of EIPH, the association between EIPH and IAD, and EIPH influence on the tracheal cytological profile of polo ponies. Thirty-seven horses regularly used for polo were included in this study. Endoscopic examination was performed every 30 to 90 min after practice, and tracheal lavage was performed after 18 to 24 hr. Sixteen animals (43.2%) presented a score of 0 for mucus in endoscopy; twelve animals (32.4%) presented a score for 1 and nine animals (24.3%) presented score 2 of mucus. IAD was characterized by tracheal cytology in 12 animals (32.4%). The occurrence of EIPH in this study was 29.7% (11/37). No significant difference was found in the cell types in tracheal cytology when EIPH-positive and EIPH-negative horses were compared. Polo ponies are affected by IAD and EIPH in relevant proportions, but there was no association between EIPH and tracheal cytological profile.

Multiple molecular detection of respiratory viruses and associated signs of airway inflammation in racehorses.

BACKGROUND: The potential involvement of viruses in inflammatory airway disease (IAD) was previously investigated through either serology or PCR from nasopharyngeal swabs (NS). The aims of this study were to determine the prevalence and incidence of viral genome detection by qPCR in the equine airways, and their association with respiratory clinical signs. METHODS: Both NS and tracheal washes (TW) were collected monthly on 52 Standardbred racehorses at training, over 27 consecutive months (581 samples). Equid herpesviruses (EHV)-1, -4, -2 and -5, equine rhinitis virus-A and -B (ERBV), equine adenovirus-1 and -2, equine coronavirus and equine influenza virus were systematically investigated in both NS and TW. Nasal discharge, coughing, tracheal mucus score and TW neutrophil proportions were simultaneously recorded. RESULTS: Genome for 7/10 viruses were detected at least once throughout the study; up to 4 different viruses being also concomitantly detected. Monthly incidence in TW was respectively 27.9% (EHV-5), 24.8% (EHV-2), 7.1% (ERBV), 3.8% (EHV-4), 1.9% (EAdV1) and 0.2% (EHV-1; ERAV). Neither agreement nor correlation between NS and TW was found for respectively genome detection and viral loads. Detection of viral genome in NS was not associated with any clinical sign. Coughing was significantly associated with TW detection of EHV-2 DNA (OR 3.1; P = 0.01) and ERBV RNA (OR 5.3; P < 0.001). Detection of EHV-2 DNA in TW was also significantly associated with excess tracheal mucus (OR 2.1; P = 0.02). CONCLUSIONS: Detection and quantification of EHV-2 and ERBV by qPCR in TW, but not in NS, should be considered when investigating horses with IAD.

Lower airway ultrasonographic, endoscopic, and cytological changes in horses undergoing inhalation anesthesia with controlled ventilation for minor elective surgeries.

Effects of general anesthesia with controlled ventilation on the respiratory system have had limited evaluation in horses. A prospective observational study was performed with eleven client-owned horses undergoing elective surgery. Physical examination, auscultation with a rebreathing bag, complete blood cell count, lung ultrasound imaging, tracheal endoscopy imaging and transendoscopic tracheal wash were conducted before and 24 hours after anesthesia. Lung ultrasound imaging was also repeated just after recovery. A significant increase in blood neutrophil count between pre- and post-anesthesia (P=0.004) was observed. There was an increase in ultrasonographic score of the lungs at recovery (left P=0.007, right P=0.017). The score of the dependent lung was higher than the independent lung at recovery time (P=0.026) but no difference was observed 24 hours after anesthesia. The tracheal mucus score was higher after anesthesia (P=0.001); severe local inflammation was present in several horses at the site of endotracheal tube cuff. Neutrophil count was significantly higher on tracheal wash fluid cytology after anesthesia (P=0.016), without any significant changes on bacterial load. Increased tracheal mucus score and neutrophil count in tracheal wash samples were observed after general anesthesia in healthy horses without clinical evidence of pneumonia (fever, cough). Tracheal inflammation secondary to endotracheal intubation and cuff inflation was, therefore, suspected. Elective surgery without complications can induce inflammation of the trachea and changes in ultrasound images of the lungs in healthy horses and should be considered when evaluating respiratory system after a general anesthesia.

Increased freedom of head movement mitigates stress and bacterial load in the airways of horses during transport.

Introduction: Protection of horse welfare during transport is crucial. The aim of this study was to determine the effect of head and neck restraint on behavior and airway bacteria. Methods: In a randomized crossover study, six healthy Thoroughbreds were transported by road for 22 h in an individual bay with tight head restraint (50 cm short-rope) or loose head restraint (95 cm long-rope). Behavioral parameters relating to head position, eating, and stress were monitored during transportation. Tracheal wash samples were obtained 6 days before and immediately after transport for bacterial culture and metagenomic analysis. Results and discussion: Compared to before transport, bacterial load (CFU/mL) after transport was significantly increased in the short-rope group (p = 0.04), whereas no changes were observed in the long-rope group. Transport significantly reduced Simpson index at phylum, class, order, and family levels in both groups (p < 0.001) of tracheal microbiota. In both groups, this reduction was associated with increases in the dominant members of relative abundance at phylum (Firmicutes: +24% in long-rope and +14% in short-rope), class (Bacilli: +20% in long-rope and +22% in short-rope) and family (Streptococcaceae: +22% in long-rope and +23% in short-rope) levels. Licking behavior during transportation with short-rope restraint was more frequent than in horses with long-rope restraint. These results suggest loose head restraint during transportation is likely to ameliorate stress and mitigate the associated increased bacterial load in the lower airways associated with transport. Further, head position during transportation is likely a more important determinant of airway hygiene and distress than duration of travel.

Clinical findings and outcome predictors for multinodular pulmonary fibrosis in horses: 46 cases (2009-2019).

BACKGROUND: Prognostic indicators for equine multinodular pulmonary fibrosis (EMPF), an interstitial fibrosing lung disease, are poorly described. HYPOTHESIS/OBJECTIVES: Describe diagnostic findings and outcome predictors for EMPF. ANIMALS: Forty-six adult horses with EMPF. METHODS: Retrospective multicenter case series from 2009 to 2019. Radiographic (n = 27) and ultrasonographic studies (n = 19) from EMPF horses and bronchoalveolar lavage fluid (BALF) cytology from 6 EMPF and 13 asthma cases were independently reviewed and blinded to diagnosis and outcome. Associations between predictor variables and survival were assessed by predictor screening followed by Fisher's exact and Wilcoxon rank sum tests. RESULTS: Primary clinical findings were weight loss (36/46, 78%), increased respiratory effort (33/46, 72%), tachypnea (32/46, 70%), and fever (18/46, 39%). Macrophage atypia was seen in more EMPF than asthmatic horse BALF (67% vs. 8%; P = .02). Equine herpesvirus 5 (EHV-5) was detected in 24 of 30 (80%) and hyperfibrinogenemia in 25 of 28 (89%) cases. Twenty-seven of 46 horses (59%) and 11 of 45 (24%) survived to discharge and to 3 months, respectively. Three-month survival was associated with lower median (range) respiratory rates (30 [24-36] vs. 41 [30-60] breaths per minute; P = .04), and higher BALF lymphocyte:neutrophil ratios (4.7 [1.4-22] vs. 0.47 [0.11-1.9]; P = .01) and blood lymphocyte counts (1.25 [0.93-2.55] vs. 0.90 [0.70-1.24] × 109/L; P = .03). Imaging findings, EHV-5 detection, and corticosteroid treatment were not associated with survival. CONCLUSIONS AND CLINICAL IMPORTANCE: Fever is not a sensitive clinical sign of EMPF. Diagnostic testing should be pursued for horses with increased respiratory rate and effort and weight loss. The prognosis for EMPF horses is poor. Corticosteroid treatment does not improve 3-month survival.

Diagnosis of Aspergillosis in Horses.

Invasive pulmonary aspergillosis (IPA) may be a rare cause of granulomatous pneumonia in horses. The mortality of IPA is almost 100%; direct diagnostic tools in horses are needed. Bronchoalveolar lavage fluid (BALF) and serum samples were collected from 18 horses, including individuals suffering from IPA (n = 1), equine asthma (EA, n = 12), and 5 healthy controls. Serum samples were collected from another 6 healthy controls. Samples of BALF (n = 18) were analyzed for Aspergillus spp. DNA, fungal galactomannan (GM), ferricrocin (Fc), triacetylfusarinin C (TafC), and gliotoxin (Gtx). Analysis of 24 serum samples for (1,3)-ß-D-glucan (BDG) and GM was performed. Median serum BDG levels were 131 pg/mL in controls and 1142 pg/mL in IPA. Similar trends were observed in BALF samples for GM (Area under the Curve (AUC) = 0.941) and DNA (AUC = 0.941). The fungal secondary metabolite Gtx was detected in IPA BALF and lung tissue samples (86 ng/mL and 2.17 ng/mg, AUC = 1).

Molecular detection of equid herpesvirus in bronchoalveolar lavage fluid from asymptomatic horses in Southern Brazil.

Background and Aim: Molecular approaches to diagnose respiratory viruses have provided an opportunity for early and subclinical pathogen detection, particularly in samples from the upper respiratory tract. This study aimed to investigate the presence of herpesviruses, particularly equid herpesvirus (EHV)-2 and EHV-5, in samples from the lower respiratory tract of healthy racehorses from Southern Brazil. Materials and Methods: Samples from the lower respiratory tract (i.e., bronchoalveolar lavage fluid [BALF]) were assessed by video endoscopy, cytological evaluation of BALF, and tracheal aspirates (TA), along with quantitative polymerase chain reaction (qPCR), to detect equine herpesvirus infection in the lower respiratory tract samples and compare corresponding cytological and endoscopic findings. Results: At least one abnormality per horse during endoscopy examination was observed, including, but not limited to, mucous secretion in the airways and pharyngeal lymphoid hyperplasia. The presence of EHV-2 and/or EHV-5 was detected by qPCR in 3/10 animals. One horse was positive for EHV-2 alone, one for EHV-5 alone, and one for both. Conclusion: To the authors' knowledge, this is the first molecular detection of EHV-2 and EHV-5 in Brazilian thoroughbred horses. These findings may provide new insights into the epidemiology of EHV-2 and EHV-5 in Brazilian horses, evidencing the importance of the molecular investigation, early detection, and prevention of respiratory diseases.

Diagnostics of Infectious Respiratory Pathogens in Reptiles.

Methods for the detection of pathogens associated with respiratory disease in reptiles, including viruses, bacteria, fungi, and parasites, are constantly evolving as is the understanding of the specific roles played by various pathogens in disease processes. Some are known to be primary pathogens with high prevalence in captive reptiles, for example, serpentoviruses in pythons or mycoplasma in tortoises. Others are very commonly found in reptiles with respiratory disease but are most often considered secondary, for example, gram-negative bacteria. Detection methods as well as specific pathogens associated with upper- and lower-respiratory disease are discussed.

Cytological analysis of tracheal wash and bronchoalveolar lavage fluid in health and respiratory disease in dromedary camels.

BACKGROUND: Tracheal wash (TW) and bronchoalveolar lavage (BAL) have proven to be useful tools for the identification of disease-associated changes in the respiratory tract in human and different animal species. In the dromedary camel, little is known about cytological analysis of TW and BAL in health and disease. The aim of the present study was to evaluate the cytological composition of TW and BAL in health and respiratory disease in dromedary camels. METHODS: TW and BAL samples were collected from dromedary camels and cytological analysis was performed by microscopic examination of prepared smears. Camels with clinical respiratory disease (n = 18) were compared with apparently healthy (control) camels (n = 9). RESULTS: In the apparently healthy camels, differential cytological analysis of TW samples identified macrophages and neutrophils as the main cell populations with lesser proportions of lymphocytes and epithelial cells and very rare abundance of eosinophils and mast cells. In the TW of camels with respiratory disease, neutrophils were the most abundant cells followed by macrophages and lymphocytes. In the BAL of healthy camels, macrophages represented the main cell type followed by lymphocytes and neutrophils. In respiratory-diseased camels, BAL samples contained higher percentages of neutrophils with reduced percentages of macrophages and lymphocytes in comparison to camels from the control group. Collectively, the results of the current study revealed higher abundance of neutrophils in the TW and BAL from dromedary camels than many other veterinary species. The cytological patterns of TW and BAL from camels with respiratory diseases were characterized by increased proportion of neutrophils and decreased proportion of macrophages in comparison to healthy camels. The proportion of lymphocytes was also decreased in TW samples from diseased camels.

Bacterial infection before and after stent placement in dogs with tracheal collapse syndrome.

BACKGROUND: Dogs with tracheal stents often have positive airway bacterial cultures. The pathogenicity of these organisms and risk factors for infection have not been investigated. OBJECTIVE: Describe bacterial infection in dogs with tracheal collapse before and after tracheal stent placement. ANIMALS: Fifty-three client-owned dogs. METHODS: Retrospective review of medical records of dogs receiving tracheal stents with thoracic radiographs, tracheoscopy, and endotracheal lavage. RESULTS: There was no difference between the overall prevalence of dogs with positive bacterial cultures before (31/38; 82%) or after stent placement (24/31; 77%) (P = .67). An increased number of geriatric (17/28; 61%) and traditional-type collapse (TTC) (16/26; 62%) dogs had positive pathogenic airway infections before stent placement, compared to young (8/25; 32%; P = .04) and malformation-type collapse (MTC) dogs (9/27; 33%; P = .04). After tracheal stent placement, geriatric dogs had a 52% reduction in pathogenic bacteria infection frequency (P = .02) and dogs with TTC had a 56% reduction in pathogenic bacteria infection frequency (P = .01). Significant risk factors for pathogenic infection included a history of pneumonia (OR = 3.6; 95% CI, 0.28-43.36) and cardiac disease (OR = 1.25; 95% CI, 0.16-9.92) in geriatric dogs, and hepatomegaly in young dogs (OR = 1.5; 95% CI, 0.12-19.44). CONCLUSIONS AND CLINICAL IMPORTANCE: Tracheal stent placement does not increase the overall rate of pathogenic bacterial infection in dogs with tracheal collapse and can decrease the rate of subsequent pathogenic infections in geriatric dogs and dogs with TTC that require tracheal stenting. Airway culture and cytology should be performed in all dogs undergoing tracheal stent placement.

Effect of inclusion or exclusion of epithelial cells in equine respiratory cytology analysis.

Published studies vary as to whether epithelial cells are included in differential counts for tracheal wash (TW) and bronchoalveolar lavage (BAL) cytology in horses. The aim of this study was to determine whether inclusion or exclusion of epithelial cells affects interpretation of airway cytology. Using criteria of >20% TW neutrophils, >10% BAL neutrophils and/or >5% BAL mast cells to indicate airway inflammation, there was a change in categorisation from 'normal' to 'abnormal' in 21%, 4% and 8% horses, respectively, when epithelial cells were excluded from differential counts. It is recommended that future equine respiratory research studies explicitly state whether epithelial cells are included or excluded in differential counts. A consensus on epithelial cell inclusion during cytology reporting is required.

Fungi in respiratory samples of horses with inflammatory airway disease.

BACKGROUND: Fungi contribute to the inflammatory response of lungs in horses with recurrent airway obstruction and in some forms of asthma in humans. The role of fungi in inflammatory airway disease (IAD) has not been assessed. OBJECTIVES: Evaluate the prevalence of fungi in the respiratory samples of horses diagnosed with IAD, describe clinical signs associated with the presence of fungi in respiratory samples, and assess the risk factors associated with IAD and with the presence of fungi in the airways. ANIMALS: Seven-hundred thirty-one active horses referred to a specialized ambulatory practice for signs of respiratory disease or poor performance. METHODS: A prospective observational study was performed, collecting clinical data, environmental conditions, and results of a tracheal wash (TW; cytology, fungal culture, and bacterial culture), and bronchoalveolar lavage (cytology). RESULTS: A positive fungal culture was obtained in 55% (402/731) of horses. Horses with fungal elements observed on the TW cytology had 2 times greater chance of having IAD than horses without fungi (odds ratio [OR] = 2.1; 95% CI 1.08-3.33; P = .0003). Risks of being diagnosed with IAD and likelihood of fungi in TW were higher when horses were bedded on straw (OR = 2.0; 95% CI 1.2-3.2 and OR = 1.9; 95% CI 1.3-2.6, respectively) or fed dry hay (OR = 2.7; 95% CI 1.7-4.4 and OR = 2.6; 95% CI 1.6-3.4, respectively). CONCLUSIONS AND CLINICAL IMPORTANCE: Horses inhaling aerosolized fungal particles are at a significantly higher risk of developing IAD. The type of bedding and forage represent significant risk factors for IAD and fungal contamination of equine airways.

Comparison of Tracheal Wash and Bronchoalveolar Lavage Cytology in 154 Horses With and Without Respiratory Signs in a Referral Hospital Over 2009-2015.

Most equine lower respiratory diseases present as increased airway neutrophilia, which can be detected in tracheal wash (TW) or bronchoalveolar lavage fluid (BALF) cytology samples. The aim was to compare the TW and BALF results in a population of client-owned horses with and without clinical respiratory disease signs. A secondary aim was to determine the sensitivity (Se) and specificity (Sp) of TW and BALF neutrophilia in detecting respiratory disease. The cutoff values for neutrophils were also evaluated. Retrospective data from 154 horses of various breeds that had been subject to both TW and bronchoalveolar lavage (BAL) sampling at rest during 2009-2015 were used. The horses were divided into three groups based on the presenting signs, physical examination, and endoscopy mucus score. Neutrophil counts of >20% in TW and >5% in BAL were considered abnormal. Cytology results between groups, correlations between TW and BALF cell types, and tracheal mucus score were analyzed. Two graph receiving operating characteristic (ROC) curves of the neutrophil percentage values of TW and BALF were created to determine the optimal cutoff values and to calculate the diagnostic Se and Sp for diagnosing airway inflammation in horses with and without clinical respiratory signs. The Se and Sp of TW and BALF neutrophil percentages were further estimated using a two-test one-population Bayesian latent class model. The two tests showed substantial agreement, and only 17.5% of the horses were classified differently (healthy vs. diseased). The neutrophil percentage was found to correlate between TW and BALF. The Se and Sp of TW were generally higher than for BAL when estimated with area under the curve or Bayesian model. Cutoff values of 17.7% for TW and 7% for BALF were indicated by the ROCs. We conclude that TW is a more sensitive and specific method in our patient population. We suggest that the current neutrophil cutoff values of 20% for TW and 5% for BALF would still be appropriate to use in clinical diagnosis of airway inflammation. However, further studies with other cell types and in other populations are warranted to determine the best sampling method for individual horses.

Association of neutrophil morphology with bacterial isolates in equine tracheal wash samples.

BACKGROUND: Tracheal wash sample neutrophilia is common in lower airway inflammation of various causes; however, relevance of cytomorphological features to culture of bacterial pathogens has not been established. OBJECTIVES: To investigate whether the presence of nondegenerate or degenerate neutrophils in tracheal washes is associated with culture of bacteria and, if so, whether this is influenced by age or temporal factors. STUDY DESIGN: Cross-sectional study. METHODS: Tracheal wash samples submitted to Rossdales LLP from 1/1/2013 to 31/7/2015 were evaluated using set criteria. Neutrophilia and degenerate neutrophilia (graded ≥2/4 on Rossdales cytological scale [0-4]) were analysed in relation to bacterial isolates considered potentially pathogenic in respiratory disease. Statistical analyses included multivariable logistic regression to identify associations between two separate outcomes: 1) the presence of neutrophilia compared with no neutrophilia and 2) the presence of degenerate neutrophilia compared with nondegenerate neutrophilia and four independent variables. RESULTS: Sufficient data for inclusion in the multivariable model for nondegenerate neutrophilia were available from 1100 horses. Culture of potentially pathogenic bacteria was associated with increased odds of degenerate neutrophilia compared with samples with negative culture (OR 4.5, 95% CI 3.1, 6.4, P-value<0.001). Horses over 9 years old had lower odds of having degenerate neutrophilia than those aged 1-3 years (OR 0.6, 95% CI 0.4, 0.9, P-value<0.02). In the spring/summer, horses had reduced odds of a degenerate neutrophilia compared with winter (OR 0.4, 95% CI 0.3, 0.7, P-value <0.001). MAIN LIMITATIONS: The study relied on routine laboratory submissions, with no control over sample collection or submitted clinical history. CONCLUSIONS: Cytological evaluation of tracheal washes should include cytomorphological features of the neutrophil response. The presence of degenerate neutrophils, especially in young horses, indicates added value of culture and sensitivity for antimicrobial therapeutics. The absence of degenerate changes, in combination with clinical factors, can help support diagnosis of a nonseptic cause of airway neutrophilia.

Association of tracheal mucus or blood and airway neutrophilia with racing performance in Thoroughbred horses in an Australian racing yard.

OBJECTIVES: To determine the variation of tracheal mucus scores, tracheal blood scores and transendoscopic tracheal wash (TW) cytology in a population of Thoroughbred (TB) racehorses and assess their association with racing performance. METHODS: A total of 220 endoscopic examinations were performed and TWs obtained from 155 TB racehorses. Samples were collected 60-120 min following gallop work. Tracheal mucus score, tracheal blood score and TW cytology were analysed and their association with racing performance assessed. RESULTS: Of the total examinations and samples, 194 from 135 horses fitted the criteria for inclusion. The overall prevalence of visible tracheal mucus was 2.5% (5/194) and of increased tracheal mucus was 0%. The prevalence of visible tracheal blood was 8.8% (17/194) and of increased tracheal blood was 4.6% (9/194). A total of 36% (70/194) of TWs contained elevated percentages of neutrophils and of these, 96% (67/70) occurred in the absence of any visible tracheal mucus. There was no significant association between tracheal mucus score or TW cytology and subsequent racing performance. There was a statistically significant association (P = 0.004) between increased tracheal blood scores and poor racing performance. CONCLUSIONS: Visible tracheal blood seen after strenuous exercise in clinically normal TB racehorses was a risk factor for poor racing performance, but the presence of airway neutrophilia was not. No horses in this study were found to have increased tracheal mucus, so the association of increased tracheal mucus with racing performance could not be assessed.