Respiratory- asthma

Question 1

Severe asthma in horses is associated with increased airway innervation

Answer 1

In horses with severe asthma: Increased number/ area periobronchial nerves, number of smooth muscle associated nerves, airway and vascular smooth muscle area vs controls. No difference in pulmonary artery innervation. Unclear if this is casue/ sequelae of airway inflammation.

Question 2

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

Answer 2

• fever inconsistent but weight loss in 78%
• 24% 3 month survival, 59% to hospital discarge
• short term Px: lower max temp, lower band neuts, higher lymp:neut in BALF. Corticosteroid tx
• long term Px: institution, max rectal temp, blood lymphocyte count, BALF lymp % neut %

Question 3

Airway smooth muscle remodelling in mild and moderate equine asthma

Answer 3

• BALF inflammation in horses presenting MEA was mostly neutrophilic (n = 12), followed by an elevated percentage of mast cells (n = 9) and abnormal eosinophil count (n = 2)
• Expression of the (+)insert SMMHC isoform in airway smooth muscle was approximately 1.5 times greater in horses with MEA compared with controls.
• There were no differences between groups in the proliferation of airway smooth muscle cells (P = .4) or myocyte density (P = .3, mean difference −0.6),
• the percentage of proliferating myocytes (PCNA) was correlated to pulmonary neutrophilia in horses with neutrophilic inflammation (P = .01, r = .80) and to the expression of the (+)insert SMMHC isoform in asthmatic horses (P = .03, r = .66).

• Structural changes, such as hyperplasia or hypertrophy, were not prominent in MEA, differentiating it from severe equine asthma (SEA).

Role of the (+)Insert SMMHC Isoform:
• This isoform, associated with faster airway smooth muscle contraction velocity, may increase airway stiffness and contribute to AHR in MEA.
• Despite its overexpression in the asthma group, not all asthmatic horses displayed increased levels, suggesting variability in disease endotypes or stages.

Question 4

Pulmonary response of severely asthmatic horses after intra-articular administration of methylprednisolone

Answer 4

SEA horses
• IA MPA administration resulted in a significant but mild and transient improvement in pulmonary resistance (RL) in severe asthmatic horses, observable at Day 1 but not sustained.
• Serum cortisol levels decreased significantly within 6 hours of IA MPA administration and returned to baseline by Day 3, indicating a short systemic effect.
• IM administration of MPA showed no significant effects on pulmonary function or cortisol levels, suggesting lower systemic absorption and reduced efficacy compared to IA administration.

• Differences in pulmonary effects between TA and MPA likely stem from their pharmacokinetics. MPA has a shorter systemic duration (peak concentration at 6–7 hours post-injection, undetectable by Day 6).
• TA is known for a longer duration of systemic effects, maintaining significant cortisol suppression for up to 11 days post-administration.
• The short-term pulmonary benefits of IA MPA are unlikely to significantly impact equine performance beyond 48 hours post-administration.
• MPA’s limited efficacy in improving pulmonary function suggests it may not be a suitable alternative to TA for asthma management. Is less potent than TA for pulmonary dysfunction

Question 5

Clinical efficacy of bronchodilators in equine asthma: Looking for minimal important difference

Answer 5

Bronchodilator agents:
• reduced ΔPplmax and RL
• increased Cdyn.

• a change in RL of 0.63 cm H2O/L/s (95% CI 0.33–0.94) represents the MID leading to the MCDD in equine asthma clinical signs. Considering that from a statistical viewpoint these confidence intervals include the true population mean in 95% of cases, a change in ≥1 cm H2O/L/s (the higher 95% CI value) is the smallest change in RL that is clinically meaningful in equine asthma.
• The MID provides a practical threshold to assess the clinical efficacy of bronchodilators in equine asthma.
• Identifying MID allows clinicians to tailor treatments and predict outcomes for individual horses based on their lung function response.

• A significant (P<0.05) relationship was found between the changes in IDEASS score (Improved Detectable Equine Asthma Scoring System) and maximum change in transpulmonary pressure (ΔPplmax) or pulmonary resistance (RL). Cdyn was not correlated with the changes in IDEASS score.

Meta-Regression Insights:
• The meta-regression analysis highlighted the consistency of RL as a predictor of clinical improvement across studies.
• Sensitivity analysis confirmed that the heterogeneity in the included studies stemmed from variations in assessment times post-treatment.

Question 6

Evaluation of histamine-provoked changes in airflow using electrical impedance tomography in horses

Answer 6

• Electrical Impedance Tomography (EIT) was effective in detecting histamine-induced changes in airflow in horses.
• The study found that more than 80% of the variation in Δflow could be explained by changes in EIT-derived expiratory flow variables, emphasizing EIT’s diagnostic value.
• Significant increases in global inspiratory (InFglobal) and expiratory (ExFglobal) flow indices correlated with increasing histamine doses, confirming their sensitivity to airway changes.
• Multiple regression revealed that the variance in ExFglobal, and right and left ventral expiratory flow best explained the variance in Δflow (r 2 = .82).

The study revealed significant regional differences in airflow with histamine bronchoprovocation:
• Ventral regions of both lungs, particularly the right lung, showed more pronounced changes during inspiration.
• Dorsal regions were less affected, consistent with previous findings showing ventilation favoring the right lung in standing horses.
• The regional flow changed primarily in the ventral regions of the lungs and during inspiration. No changes were observed for any regional peak expiratory flow measurements after histamine provocation
• A significant increase in regional inspiratory flow was seen in the right and left ventral lung and dorsal right lung.
• This pattern aligns with the expected distribution of aerosolized particles in the lungs, suggesting that particle size and weight, along with ventilation, influence histamine deposition.

Question 7

Effects of a propriety oiled mixed hay feeding system on lung function, neutrophilic airway inflammation and oxidative stress in severe asthmatic horses

Answer 7

underpowered, unblinded…

• The Nutri-Foin Système (NFS) diet, involving soybean oil-treated hay, was as effective as pelleted hay in improving lung function and reducing airway inflammation in severe equine asthma (SEA).
• Both diets led to significant reductions in pulmonary resistance (RL), elastance (EL), airway neutrophilia, and mucus scores over a 3-month period.

Mechanism of Action:
• The NFS system is proposed to reduce inhaled antigens by amalgamating fungal spores, bacteria, and dust particles in hay with oil, thereby decreasing airway irritation and inflammation.
• Improvement in clinical signs is attributed to reduced antigenic and particulate exposure during feeding.

• Despite significant improvements, some horses in both groups showed residual airway obstruction, which may be due to:
-Persistent exposure to stable dust and irritants, even under controlled feeding conditions.
-Chronic airway remodeling associated with severe asthma.
• However, complete normalization of airway neutrophilia (defined as <5%) was not observed in most horses, consistent with previous findings.

Antioxidant Enzyme Activity:
• Serum antioxidant enzyme activity (SODCu-Zn and GPx) significantly decreased over time in both groups, suggesting reduced oxidative stress.
• The absence of group differences indicates that the decrease in oxidative stress was likely due to the remission of asthma rather than a specific effect of soybean oil.

Weight and Palatability:
• Both diets supported weight gain, correlating with improved lung function and reduced metabolic costs of breathing.
• The treated hay was palatable, making it a viable alternative to pelleted hay for horses with SEA.

Question 8

Efficacy of dexamethasone, salbutamol, and reduced respirable particulate concentration on aerobic capacity in horses with smoke-induced mild asthma

Answer 8

Message: drugs are of limites use in MEA, environment more important
• Improved ambient air quality (reduction in PM2.5) significantly enhanced aerobic capacity (VO2max) in horses with smoke-induced mild asthma, with an average increase of 13.2%.
• Dexamethasone administration provided no additional benefit beyond the effects of improved air quality.
• Salbutamol administration did not significantly improve compared to the effects of air quality improvement.

Role of Air Quality:
• Enhanced air quality was identified as the primary driver of reduced airway inflammation and improved respiratory performance.
• Reduction in PM2.5 was associated with a significant decrease in tracheal mucus scores and better airway function.
• The results emphasize the critical role of environmental management in treating and preventing mild asthma in horses.

Effects of Dexamethasone:
• Dexamethasone treatment resulted in no significant differences in BAL fluid cytology or mucus scores compared to the placebo (saline) group.
• These findings align with previous studies indicating that corticosteroids alone do not normalize airway neutrophilia without concurrent environmental modifications.
Effects of Salbutamol:
• While salbutamol improved mucociliary clearance and may have anti-inflammatory properties, its administration did not yield significant improvements in VO2max under the study conditions.
• Possible reasons for the lack of significant effects include:
-Maximal sympathetic drive during exercise may override the pharmacological benefits of bronchodilators.
-Limited study power to detect small improvements in aerobic capacity.
Tracheal Mucus and Airway Inflammation:
• Moderate-to-severe tracheal mucus accumulation is associated with poor performance in Thoroughbred racehorses.
• Although horses in the study had lower mucus scores, they exhibited significant performance and clinical improvements following the reduction in PM2.5 and airway inflammation.

Study Strengths:
• Field-based evaluation of aerobic capacity (VO2max) provided practical insights into real-world conditions.
• Controlled, randomized, and double-blinded study design ensured robust data collection and analysis.

Question 9

Tracheal microbial populations in horses with moderate asthma

Answer 9

• Horses with moderate asthma exhibited a significantly lower bacterial load in tracheal aspirates compared to healthy controls, as measured by 16S rRNA gene quantification.
• Corynebacterium spp., considered commensal bacteria, were more frequently found in control horses, suggesting their role in maintaining a healthy airway microbiome.
• No significant differences in the presence or abundance of pathogenic bacteria, such as Streptococcus spp. or Pasteurellaceae, were found between asthmatic and control horses.

Airway Dysbiosis in Equine Asthma:
• The findings suggest that bacterial dysbiosis, rather than overgrowth, is characteristic of moderate equine asthma.
• Dysbiosis could be either a consequence of chronic inflammation or a perpetuating factor, but further studies are needed to clarify this relationship.
• In humans, airway dysbiosis has been associated with asthma exacerbations, severity, and corticosteroid resistance, emphasizing parallels between species.

Role of Commensal Bacteria:
• Corynebacterium spp., identified more frequently in control horses, may play a protective role by competing with potential pathogens and maintaining microbial balance.
• The findings align with human studies where commensal bacteria like Corynebacterium accolens show antagonistic interactions with respiratory pathogens.

• The presence of Streptococcus spp., the most common bacteria detected, did not correlate with clinical signs, tracheal mucus scores, or inflammation in bronchoalveolar lavage fluid (BALF). This lack of association suggests that these bacteria may not play a central role in the pathogenesis of moderate equine asthma.
• There was no association between microbial populations and clinical signs, tracheal mucus or BALF inflammation.
• There was an overall poor concordance between positive Streptococcus spp. qPCR results and growth
• The presence or absence of Streptococcus spp. or Chlamydophila spp. was not associated with asthma, or specific clinical signs, high mucus scores and BALF inflammation

Question 10

Dust exposure and pulmonary inflammation in Standardbred racehorses fed dry hay or haylage: A pilot study

Answer 10

Healthy horses

• Feeding haylage instead of hay significantly reduced exposure to respirable dust and β-glucan concentrations.
• Horses fed haylage showed reduced bronchoalveolar lavage fluid (BALF) neutrophilia within two weeks, with effects persisting through six weeks.
• BALF interleukin-4 (IL-4) levels increased significantly in horses fed hay but remained stable in those fed haylage, indicating reduced airway inflammation with haylage.

Question 11

Inhaled ciclesonide is efficacious and well tolerated in the treatment of severe equine asthma

Answer 11

• The treatment success rate (30% decrease in WCS) in ciclesonide-treated horses was 73.4% after 10 days of treatment- significantly higher than in the placebo group with 43.2%
• The mean WCS reduction after 10 days of treatment was 7.2 ± 4.8 in the ciclesonide-treated group, compared to 3.8 ± 4.5 in the placebo group (P < 0.0001)
• the reduction in WCS after ciclesonide administration was greater in horses with severe clinical signs at the time of enrolment compared with horses with moderate clinical signs
• owners perceived an improved Quality of Life after 5 and 10 days of treatment in 60.2% and 69.3% of ciclesonide-treated horses, compared to 32.7% and 43.4% of placebo-treated horses, This equated to a positive and significant (P ≤ .0001) risk difference
of 27.5% and 26.1% after 5 and 10 days of treatment respectively.

Mechanism of Action:
• Ciclesonide is a glucocorticoid prodrug converted in the lungs to its active form, desisobutyryl-ciclesonide (des-CIC), which has high glucocorticoid receptor affinity.
• The localized activation and deposition in the lungs minimize systemic absorption and reduce potential adverse effects, such as hypothalamic-pituitary-adrenal (HPA) axis suppression.

Ciclesonide showed a favorable safety profile:
• Low incidence of adverse events, similar to placebo.
• No significant deviations in hematological or biochemical parameters, confirming systemic safety.
• Absence of serum cortisol suppression reported in other studies.

Study Design and Validity:
• Large sample size (224 horses) and rigorous randomized, double-blinded methodology ensure high-quality evidence.
• Weighted Clinical Score (WCS) used as a primary endpoint reflects clinical relevance and was validated as a proxy for pulmonary function in previous studies.
• Owners’ perceptions of improved QoL aligned with clinical outcomes, further substantiating results.
• but selected for horses with treatable asthma, only 10 days and selected for those that would tolerate the equihaler

Question 12

Airway remodeling in horses with mild and moderate asthma

Answer 12

Horses with mild and moderate asthma (MMA) exhibit significant airway remodeling in the central airways compared to control horses.
• Increased lamina propria thickness, positively correlated with extracellular matrix (ECM) area.
• Epithelial hyperplasia, measured through histomorphometry and histologic scoring.
• Increased smooth muscle fibrosis, although the total smooth muscle area did not differ between groups.

Comparison with Severe Asthma (SA):
• Similar remodeling features (e.g., epithelial hyperplasia and smooth muscle fibrosis) were observed in both MMA and SA, indicating shared pathophysiological mechanisms.
• Unlike SA, smooth muscle hypertrophy was not prominent in MMA, possibly reflecting disease severity or differences in biopsy sampling.

Question 13

Nebulized dexamethasone sodium phosphate in the treatment of horses with severe asthma

Answer 13

• Nebulized dexamethasone did not significantly improve lung function in horses with severe asthma, aligning with previous findings using different nebulizers.
• Both nebulized (NE) and oral (OR) dexamethasone significantly suppressed serum cortisol concentrations, indicating systemic absorption despite the lack of clinical efficacy in the nebulized group.

Altered drug deposition and absorption in diseased airways may explain the ineffectiveness of nebulized dexamethasone in asthmatic horses:
• Diseased horses exhibit altered breathing patterns, cough, bronchoconstriction, and mucus accumulation, impeding drug delivery to distal airways.
• Inflammation and airway obstruction shift deposition to central airways, reducing local therapeutic effects.
• The low dose and injectable formulation may contribute to the observed inefficacy, as higher doses or tailored formulations might achieve better clinical outcomes.

• Cortisol suppression occurred in both treatment groups but was more pronounced in the oral group, where several horses had cortisol levels below the detection threshold.
• Ciclesonide remains the only inhaled corticosteroid studied in horses that effectively improves lung function without systemic cortisol suppression, underscoring its unique pharmacokinetics.

Question 14

Effects of low-dust forages on dust exposure, airway cytology, and plasma omega-3 concentrations in Thoroughbred racehorses

Answer 14

• Horses fed haylage showed lower neutrophil proportions in bronchoalveolar lavage fluid (BALF) within three weeks, with further reductions after six weeks.
• Lower BALF neutrophil proportions were strongly associated with reduced dust exposure in horses fed haylage.
• Haylage-fed horses demonstrated a rapid reduction in neutrophil proportions, reaching clinically relevant levels for improved respiratory performance.
• No significant changes in mast cell or eosinophil proportions were observed, except for a transient reduction in mast cells at week 3.

Dust and Particulate Matter (PM) Exposure:
• Haylage feeding resulted in a 66% reduction in PM10 exposure compared to dry hay and a 44% reduction compared to steamed hay.
• Respirable dust exposure in haylage-fed horses was comparable to levels observed in horses on pasture
• Reduced dust exposure directly correlated with lower tracheal mucus accumulation and BALF neutrophil proportions

Role of Omega-3 Fatty Acids:
• Plasma EPA to arachidonic acid ratios increased significantly in haylage-fed horses
• No significant changes were observed in other omega-3 or omega-6 polyunsaturated fatty acids (PUFAs) or specialized pro-resolving mediators (SPMs), suggesting that additional factors may influence inflammation resolution.

Comparison of Forage Types:
• Steamed hay reduced dust exposure and mucus accumulation compared to dry hay but did not significantly improve BALF cytology or plasma omega-3 profiles.
• Dry hay resulted in the highest dust and particulate exposure, contributing to elevated neutrophil proportions and poorer airway health.

Question 15

Protein microarray allergen profiling in bronchoalveolar lavage fluid and serum of horses with asthma

Answer 15

• BALF allergen-specific IgE profiles were better at distinguishing asthmatic horses from controls than serum profiles, with higher predictive accuracy.
• Serum IgE concentrations reflected environmental exposure rather than true sensitization, limiting their diagnostic utility for asthma.
• Weak correlations between BALF and serum allergen-specific IgE concentrations highlight the need for lung-localized testing.
• Only 9 allergens were significant in both BALF and serum models, with varying levels of correlation (e.g., strong correlation for Blattella germanica Bla g 1, weak for Hevea brasiliensis allergens).

Sensitization Profiles (BALF IgE):
• Aspergillus fumigatus
• Latex (Hevea brasiliensis) allergens- mild to moderate cases
• Insect allergens (Culicoides spp.)

Cross-Reactivity Concerns:
• Possible cross-reactivity between latex and grass pollen allergens (e.g., Phleum pratense) requires further study to confirm clinical relevance.
• BALF filtering may result in the loss of IgE bound to mucus, potentially underestimating allergen-specific concentrations.

Question 16

Effects of soaked hay on lung function and airway inflammation in horses with severe asthma

Answer 16

Soaked Hay Benefits:
• Improved lung function and clinical signs in horses with severe asthma, comparable to feeding alfalfa pellets.
• Reduced tracheal mucus scores over six weeks.
• Lung resistance (RL) and lung elastance (EL) improved significantly in the soaked hay group compared to baseline. Were normal by week 6.
Pellets:
• Reduced pulmonary neutrophilia more effectively than soaked hay.
• showed improvements as well but had residual bronchoconstriction at study end, suggesting less comprehensive control.
• no significant change in tracheal mucus

• Pulmonary neutrophilia improved in both groups, although more pronounced in the pellet group, potentially due to higher baseline neutrophil counts.

Weight Loss in Soaked Hay Group:
• Soaked hay reduced caloric intake due to loss of water-soluble carbohydrates, proteins, and minerals during soaking.
• Weight loss (averaging 20.5 kg) was not clinically significant but highlights the need for monitoring and dietary adjustments.

Question 17

Microarray molecular mapping of horses with severe asthma

Answer 17

Regional Variability:
• Horses from Canada and France demonstrated strong discrimination between severe equine asthma (SEA) and control groups using partial least squares discriminant analysis (PLS-DA) (AUC: 0.995 and 0.867, respectively).
• Horses from the USA exhibited poor discrimination (AUC: 0.38), likely due to regional differences in sensitization profiles and environmental factors.

Four allergens were consistently associated with SEA classification across all regions:
• Hev b 5.0101 (latex allergen).
• Cyn D (Bermuda grass pollen).
• Der p 2 (house dust mite allergen).
• Rum cr (common curled dock pollen).
• These allergens are implicated in respiratory allergies, emphasizing their global significance in SEA pathogenesis.

• Sensitization profiles varied significantly between regions, influenced by local climates, vegetation, and stable conditions.
For example:
• Pollen sensitization was higher in the USA due to elevated temperatures and ubiquitous grass allergens.
• Dust mite allergens (Der p, Der f) were prominent in all groups, reflecting environmental humidity and forage storage practices.

Question 18

A CONSORT-guided randomized controlled clinical trial of nebulized dexamethasone and saline

Answer 18

• Nebulized dexamethasone (15 mg) was associated with upregulation of several inflammatory cytokines, including IL-1β, IL-6, IL-8, IL-17, and TNF-α.
-Downregulation of IL-10, an anti-inflammatory cytokine, was noted, suggesting a paradoxical pro-inflammatory effect.
-Cytological airway inflammation did not improve, highlighting limited therapeutic benefit for this route and dosage.
- serum cortisol decreased at all time points.

• Nebulized saline resulted in downregulation of multiple inflammatory cytokines compared to both the dexamethasone-treated and untreated groups.
-The absence of significant changes in inflammatory cytology suggests that saline alone is insufficient for treating airway inflammation.

• All horses were stabled in a high-dust environment, contributing to the development of airway inflammation in control horses.
• Control horses developed airway inflammation after 14 days of dust exposure- Shift from T2 to T1 cytokine response in controls reflects a normal adaptive response to antigenic exposure.

Question 19

Alveolar macrophage phenotypes in severe equine asthma

Answer 19

Protocol 1 demonstrated that polarization in control horses generated distinct transcriptional signatures.
• M1 polarization (via IFNg + LPS) induced pro-inflammatory markers (e.g., TNFα, IL-8, IL-12p40, CD80).
• M2 polarization (via IL-4) showed a more anti-inflammatory profile, marked by elevated CD206 and suppressed IL-10.
• Equine AMs differ from other species in their IL-10 response; IL-4 suppressed IL-10 expression instead of enhancing it.

Non-Canonical AM Phenotypes:
• Horses with SEA at pasture exhibited increased IL-10 expression, indicating a unique phenotype that persists despite controlled inflammation.
• Both SEA and control horses displayed a mixed phenotype with features of M1 and M2 during NC (e.g., co-expression of CD206 and IL-10).

• M1 polarization enhanced pro-inflammatory responses (e.g., IL-1β, IL-8, IL-6), while M2 polarization suppressed these responses.
• SEA AMs maintained an altered phenotype with exaggerated inflammatory responses to HD, characterized by elevated IL-10 and IL-8.
• Increased IL-10 in SEA may have dual roles: suppressing inflammation and contributing to airway remodeling and mucus production.
• Elevated CD206 during NC, independent of other M2-associated changes, suggests a context-specific function not driven solely by IL-4.

Question 20

Epidemiology of Chlamydia psittaci infections in pregnant Thoroughbred mares and foals

Answer 20

• No foal loss or maternal cases of Chlamydia psittaci (C. psittaci) or equine herpesvirus-1 (EHV-1) were recorded among study cohort mares, despite some foals testing positive for both pathogens.
• Foals born with C. psittaci or EHV-1 were primarily asymptomatic, indicating low shedding levels and a limited clinical impact under study conditions.

Risk Factor Analysis:
• Foals born during the winter months had a significantly higher risk of C. psittaci infection (adjusted odds ratio = 15.83; p < 0.001).
• Co-infection with EHV-1 was not associated with a higher risk of C. psittaci infection, suggesting independent transmission pathways.
• Avian fecal samples from study farms revealed a C. psittaci prevalence of 5.3%, consistent with prior Australian studies.
• Observations linked infection patterns in foals and birds to winter months, but not to avian breeding seasons, suggesting other environmental factors may influence shedding.

Pathogen Transmission and In Utero Dynamics:
• The absence of C. psittaci in pregnant mares indicates that maternal infection may not directly predict foal infection. Environmental sources or asymptomatic carriers are likely contributors.
• In utero transmission remains a plausible route, supported by clinical cases of neonatal pneumonia and late-term abortion associated with C. psittaci.

Question 21

Neutrophil gelatinase-associated lipocalin as a potential biomarker for equine asthma

Answer 21

• NGAL concentrations in bronchoalveolar lavage (BAL) fluid increased with disease severity, distinguishing between control horses, mild-moderate equine asthma (MEA), and severe equine asthma (SEA).
• Serum NGAL concentrations did not show significant differences between groups, suggesting BAL NGAL as a more specific biomarker for equine asthma (EA).

BAL NGAL as a Biomarker:
• BAL NGAL was significantly higher in horses with EA compared to controls and correlated strongly with BAL neutrophil counts (rho = 0.6; p < 0.001).
• NGAL concentrations in BAL fluid differentiated SEA from MEA, providing insights into disease severity.
• Three ROC curves and concomitant AUC was calculated for BAL NGAL concentrations in control horses versus EA horses (AUC = 0.65, less accuracy), for control versus MEA horses; AUC = 0.72, moderate accuracy, and for control versus SEA horses; AUC = 0.88, moderate accuracy
• Serum NGAL concentrations did not correlate with BAL NGAL or BAL neutrophil counts, nor did they significantly differ between EA groups and controls.

Comparative Insights with Human Asthma:
• Increased NGAL concentrations in BAL fluid are consistent with findings in human asthma, where NGAL is associated with airway inflammation and remodeling.
• The correlation between BAL neutrophil counts and NGAL aligns with human studies linking NGAL to neutrophil activity and inflammation.

Question 22

Intra-articular triamcinolone acetonide improves lung function in horses with severe asthma

Answer 22

• Intra-articular (IA) and intramuscular (IM) triamcinolone acetonide (TA) administrations were equally effective in improving lung function (similar AUC) in severely asthmatic horses over a 4-week period.
• The effects on pulmonary resistance (RL) and elastance (EL) were initially more pronounced following IA administration but were of shorter duration compared to IM administration.

Mechanisms and Pharmacokinetics:
• IA administration resulted in higher serum TA concentrations at earlier time points compared to IM administration. This difference likely arises from TA’s lipophilic properties and its interaction with the synovial membrane.
• Serum TA concentrations dropped below the threshold set by the International Association of Racing Commissioners (ARCI) by day 7 in most IA-treated horses, but lung function improvements persisted beyond this point, indicating sustained systemic anti-inflammatory effects.
• The kinetic of the changes after IM and IA administrations differed, however.
• IM:
-RL and EL values were significantly decreased from day 14 until day 28
- Pulmonary resistance and elastance values had normalised (RL<1 cm H2O/L/s and EL<1 cm H2O/L) in 2 horses on day 21 and EL values had normalised in a third horse from the same group on day 28.
• IA:
- RL values were significantly decreased from baseline starting on day 7 and until day 21.
- EL values were also decreased from day 7 to day 28.
-Pulmonary resistance and elastance had normalised in 4 and 3 horses, respectively, on day 7 in this group

Question 23

Effects of nebulized dexamethasone on the respiratory microbiota and mycobiota and relative equine herpesvirus-1, 2, 4, 5 in an equine model of asthma

Answer 23

• Nebulized dexamethasone reduced microbial diversity in the upper respiratory tract but had no significant impact on the mycobiota, which was predominantly influenced by the environment.
• Notable increases in certain fungal genera, including Alternaria (an allergen linked to asthma exacerbation in humans), were observed post-treatment.
• Relative expression of equine herpesviruses (EHV-2 and EHV-5) was altered, with EHV-2 levels increasing and EHV-5 decreasing following dexamethasone treatment.

•Four phyla represented 99.25% of the total abundance in nasal swab samples and 98.29% in tracheal wash samples: Proteobacteria, Bacteroidetes, Firmicutes, and Actinobacteria
• 2 phyla representing 99.78% of the total abundance in nasal swab samples and 99.42% in tracheal wash samples: Ascomycota and Basidiomycota
• In the upper respiratory tract, dexamethasone treatment resulted in a significant decrease in microbiota diversity based on Chao1 (P = .004) and Shannon (P = .004) indices
• No significant differences were found in either beta or alpha diversity at the lower respiratory tract level with treatment
• Alysiella, Bordetella̧ Acinetobacter, Staphylococcus, and Pedobacter being increased with treatment, whereas Brevundimonas, Pigmentiphaga, and an OTU assigned to the genus Corynebacterium_1 decreased with treatment
• The mycobiota was dominated by time point effects; when the interaction between treatment group and time point was tested, no significant genera were detected
• Alternaria, an opportunistic pathogen and allergen in humans recognized as a risk factor for asthma, asthma severity, and exacerbations, was increased with treatment.

• Treatment affected relative quantification of the equine gamma herpesviruses (EHV2 and -5); EHV-2 DNA levels increased and those of EHV-5 decreased. In the lower respiratory tract, relative EHV-4 DNA levels decreased 3.25-fold with treatment

Environmental Effects:
• Prolonged exposure to a dusty stable environment had a dominant influence on both the microbiota and mycobiota, overriding treatment effects in some cases.
• Even healthy control horses developed airway inflammation under these environmental conditions, emphasizing the critical role of environmental management in respiratory health.

Mechanisms and Clinical Implications:
• Dexamethasone’s effects on the microbiota may reflect its immunosuppressive properties, potentially facilitating shifts in microbial populations.
• The increase in Alternaria, a major aeroallergen, raises concerns about its potential role in exacerbating clinical signs or contributing to treatment resistance in equine asthma.
• Alterations in herpesvirus expression may indicate a complex interaction between corticosteroid use and latent viral activity, with potential implications for the recurrence of clinical signs post-treatment.

Question 24

Fecal microbiota in horses with asthma

Answer 24

Probably SEA- poor case definition

• Healthy horses displayed predictable increases in cellulose-degrading bacteria (Fibrobacter) and decreases in Prevotella and unclassified Clostridiales upon hay feeding.
• Asthmatic horses lacked these adaptations, suggesting impaired microbiota plasticity during environmental and dietary changes.

Asthmatic horses
• overrepresentation of Firmicutes during exacerbation.
• Low-abundance taxa, including those with potential immune-modulating effects, were more impacted-> higher alpha diversity at family level
• Lack expected increases in Fibrobacter (impaired adaptation of cellulolytic bacteria to dietary changes)
• dysbiosis exacerbated by dusty hay and poor-quality environments
• more pronounced differences in bacterial community membership and structure during environment change

• Healthy horses exhibited more stable alpha diversity (richness and evenness) across environments, indicating resilient microbiota.
• Beta diversity clustering was more influenced by environment
• Hay feeding consistently increased Fibrobacter in healthy horses, associated with cellulolytic activity and diet adaptation.

• Dysbiosis may exacerbate airway inflammation by:
-Reduced production of beneficial metabolites like short-chain fatty acids (SCFAs).
-Increased absorption of pro-inflammatory bacterial components (e.g., endotoxins).
-Altered immune responses due to shifts in microbial populations.

Question 25

Exercise-induced airflow changes in horses with asthma measured by electrical impedance tomography

Answer 25

small sample size and SEA not in acute exacerbation.

• EIT successfully measured airflow changes in nonsedated horses both at rest and after exercise, offering a noninvasive and field-applicable method for evaluating lung function in equine asthma (EA).
• The method detected significant differences in airflow indices between healthy horses and those with mild-to-moderate (MEA) or severe equine asthma (SEA) after exercise, but not at rest.
• EIT’s ability to provide real-time, dynamic lung function data positions it as a valuable alternative to traditional pulmonary function tests that are impractical in field settings.

Bronchoconstriction and Asthma Severity:
• Exercise significantly increased both peak expiratory flow (PEF) and peak inspiratory flow (PIF) in horses with EA compared to healthy controls, reflecting exercise-induced airway narrowing.
• The increase in PEF was more pronounced in expiration than inspiration.
• Differences between MEA and SEA were not observed, potentially due to the small sample size or the chronic state of SEA in study subjects.

Regional Ventilation Insights:
• EIT revealed more pronounced airflow changes in ventral lung regions compared to dorsal regions after exercise
• Left and right lung flows increased equally in asthmatic horses post-exercise, reflecting the systemic nature of equine asthma affecting both lungs.

Tidal Volume and Ventilation Distribution:
• No significant changes in tidal volume or center of ventilation (CoV) were observed across groups, suggesting that exercise-induced airflow changes were driven by airway narrowing rather than shifts in lung volume or ventilation patterns.
• Ventilation distribution remained symmetric, indicating no preferential regional airflow obstruction in asthmatic horses.

Question 26

Bronchial angiogenesis in horses with severe asthma and its response to corticosteroids

Answer 26

• Bronchial angiogenesis is a significant pathological feature in horses with severe asthma, contributing to airway wall thickening.
• Increased numbers of blood vessels and vascular area were observed in the bronchial walls of asthmatic horses during both exacerbation and remission phases compared to healthy controls.

Response to 2 weeks Corticosteroid Treatment:
• Contrary to expectations, corticosteroids did not reduce the number of vessels in asthmatic horses.

Study 1
• The number of vessels and vascular area were increased in the airway walls of asthmatic horses in exacerbation (P = .01 and P = .02, respectively) and in remission (P = .02 and P = .04, respectively) when compared to controls.
• vascular area was significantly increased in asthmatic horses in exacerbation when compared to controls or to horses in remission.
Study 2
• The number/ size of vessels in the lamina propria in asthmatic horses was unaffected by weeks 0.06mg/kg dex PO

Question 27

Nicoletella semolina in the airways of healthy horses and horses with severe asthma

Answer 27

• Oral and nasal loads were not significantly different between control and SEA horses in either environment
• there was significantly more N. semolina in the nose of horses kept indoors on hay rather than on pasture (P = .008), but not in their mouth
• There was no significant correlation between BALF neutrophils percentage or lung function and load
• no significant effect of asthma status
• Pulmonary loads were not different between antigen exposures
• % N. semolina-positive horses in the lungs was not significantly different between remission (5/10, 50%) and exacerbation (1/10, 10%; P = .13).
• There was no significant correlation between nasal and pulmonary loads
• A significant negative correlation was observed between nasal loads and age in healthy horses
• High rate of ID

Summary
• Nicoletella semolina (N. semolina) was found in a high proportion of nasal, oral, and bronchoalveolar lavage fluid (BALF) samples in both healthy and asthmatic horses.
• The bacterium was more prevalent in horses housed indoors and fed hay, suggesting environmental and management factors influence its presence.
• No significant differences in bacterial loads were found between asthmatic and healthy horses, or between exacerbation and remission states in asthmatic horses.

Prevalence and Distribution:
• Molecular detection methods (qPCR) showed higher detection rates of N. semolina compared to previous culture-based studies.
• High nasal loads were detected in horses living in close proximity
• Negative correlation between nasal loads and age in healthy horses
• Indoor housing and hay feeding were associated with increased nasal loads of N. semolina.
• No significant effect of diet (dry hay vs. soaked hay vs. alfalfa pellets) on nasal or pulmonary loads was observed
• The lack of correlation between nasal and BALF loads suggests the bacterium may colonize the lungs independently or through mechanisms beyond simple contamination during sampling.
• Higher pulmonary loads in remission compared to exacerbation may result from improved fluid recovery rather than true increases in bacterial presence.

Question 28

Breath characteristics and adventitious lung sounds in healthy and asthmatic horses

Answer 28

• Adventitious sounds were consistently more frequent in horses with severe equine asthma during exacerbation (sEA+).

Adventitious Sounds in Asthma:
• Wheezes:
-Strongly associated with severe asthma exacerbations (median 68.6% of breaths in sEA+).
-Almost absent in healthy horses (ctl), mild-to-moderate asthma (mEA), and horses in remission (sEA−).
-Positively correlated with tracheal mucus scores and the 23-point Weighted Clinical Score (WCS23).
• Crackles and Rattles:
-Present in lower proportions (7%-17%) in sEA+ but less discriminatory for asthma severity.
-Not significantly associated with mucus scores or airway cytology findings.
• Breath Intensity:
- Increased progressively from healthy controls to severe asthma exacerbations.
- Associated with WCS23, highlighting its potential as a diagnostic marker.

Comparisons Between Groups
• Healthy Controls (ctl): Minimal adventitious sounds and normal breath intensity.
• Mild-to-Moderate Asthma (mEA): Slight increase in adventitious sounds, though not statistically significant. Limited ability to ID
• Severe Asthma in Remission (sEA−): Low levels of adventitious sounds, similar to mEA. Limited ability to ID
• Severe Asthma in Exacerbation (sEA+): Markedly higher rates of adventitious sounds and breath intensity, reflecting severe airway obstruction and inflammation.

Question 29

Comparative study of the bronchodilator efficacy and adverse effects of salbutamol and hyoscine butylbromide in horses with severe asthma

Answer 29

• Both salbutamol and hyoscine butylbromide (HBB) demonstrated significant bronchodilator effects in horses with severe asthma (SA).
• Salbutamol provided longer-lasting bronchodilation (up to 180 minutes) compared to HBB (approximately 60 minutes).
• HBB’s shorter duration likely results from its rapid systemic metabolism due to intravenous administration.

Adverse Effects:
• Salbutamol showed no significant adverse effects.
• HBB caused:
-Tachycardia, lasting 30 minutes post-administration, which could exacerbate cardiovascular complications in asthmatic horses.
-Reduced gastrointestinal motility, with a significant decrease in borborygmi lasting 30 minutes.

Pulmonary resistance
• at 3 Hz (small airways): improved from 5 to 180 minutes (not significant at 120 minutes) after salbutamol inhalation and at 10 and 15 minutes after HBB administration
• At 7 Hz (large airways): With salbutamol, the pulmonary resistance at 7 Hz significantly increased at 5 and 10 minutes compared to baseline whereas it remained unchanged with HBB
• The R3/R7 ratio, a variable that deteriorates with small airway diseases such as asthma, significantly improved over time in both groups (time effect, P < .0001)

Pulmonary reactance:
• 3Hz: It increased from 5 to 30 minutes with salbutamol and from 5 to 15 minutes with HBB
• 7Hz: It increased starting 5 minutes after the administration of both bronchodilators and lasted 180 minutes with salbutamol and 15 minutes with HBB

Question 30

Bilateral bronchoalveolar lavage cytology profiles in a warmblood horse population during a 1‐year period

Answer 30

• Higher mast cell counts were observed in the left lung compared to the right lung and pooled samples.
• No significant differences in neutrophil or eosinophil counts between the left and right lungs were detected, though there was strong correlation for neutrophils and weaker correlation for eosinophils.
• Pooled BAL samples showed the lowest accuracy for diagnosing airway inflammation (AI), frequently underestimating mast cell counts.

Unilateral vs. Bilateral Inflammation:
• 14%-16% of horses exhibited unilateral AI, highlighting the need for bilateral sampling to avoid underdiagnosis.
• Some horses transitioned between unilateral and bilateral AI over the 1-year study period, underscoring the dynamic nature of inflammation.

Longitudinal Findings:
• Over one year, mast cell counts generally increased, while eosinophil counts decreased.
• Seasonal or environmental factors, such as dust exposure or weather, may explain these variations despite stable management practices.

Diagnostic Considerations:
• Single-sided BAL sampling carries a risk of false negatives, especially in mild-moderate asthma cases.
• BAL from the left lung had the highest sensitivity of 84%, a specificity of 100%, an AUC of 0.92 and was not significant different. BAL from right lung and the pooled BAL sample both had a lower sensitivity and a significant difference was found when compared to final AI diagnosis
• Pooled samples underestimated inflammation in cases of unilateral AI and showed reduced sensitivity compared to individual lung samples. Their use in clinical settings should be reconsidered, especially when diagnosing mild or localized asthma.

Question 31

Post-exercise endoscopic and cytologic diagnosis of equine asthma syndrome in asymptomatic Brazilian pacers

Answer 31

This study evaluates the occurrence of Equine Asthma Syndrome (EAS) in asymptomatic Campolina and Mangalarga Marchador horses using post-exercise endoscopy and bronchoalveolar lavage fluid (BALF) cytology.
Focuses on the effectiveness of diagnostic criteria, including cell counts, mucus scoring, and breed-specific differences.

• 56.7% of the sampled horses were diagnosed with EAS based on BALF cytology, despite the absence of clinical signs.
• Most cases were classified as mild EAS, with only one horse (3%) meeting the criteria for moderate EAS.

Key Diagnostic Criteria:
• Total Cell Count (TCC): The most consistent marker for EAS diagnosis, present in all positive cases.
• Mucus Score: Elevated in 52.9% of EAS-positive horses, reflecting airway inflammation.
• Eosinophilia and Neutrophilia: Observed in 47% and 35.3% of cases, respectively, though eosinophilia was more common than expected for subclinical EAS.

Breed and Age Comparisons:
• No significant differences in EAS occurrence were found between Campolina and Mangalarga Marchador breeds, or between younger and older horses.
• However, differences in macrophage (higher in Mangalarga) and lymphocyte (higher in Campolina) percentages were noted, suggesting potential environmental or management influences.

Cytological Insights:
• Post-exercise BALF analysis showed that TCC above 530 cells/µL was a reliable diagnostic criterion.
• Cytological profiles demonstrated increased recruitment of inflammatory cells, consistent with airway inflammation.