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Abstract Background Gastrointestinal microbiota can be influenced by several factors, including diet and systemic inflammation, and in turn could act as a modulator of the allergic response. Fecal microbiota of horses with asthma has not been described. Hypothesis/Objectives Analyze the bacterial fecal microbiota of horses with and without asthma under different environment and diet conditions, during both remission and exacerbation. Methods Prospective observational study. Feces from 6 asthmatic and 6 healthy horses were collected under 3 different conditions: on pasture, housed indoors receiving good quality hay (“good hay”), and housed indoors receiving poor quality hay (“dusty hay”). Sequencing was performed using an Illumina MiSeq platform and data were processed using the software mothur v.1.41.3 and LEfSe. Results In horses with asthma, low-abundance bacteria were more affected by changes in environment and diet (ie, when horses were experiencing an exacerbation), as shown by changes in membership and results from the LEfSe analysis. There was a significant increase in the relative abundance of Fibrobacter in healthy horses eating hay, a change that was not observed in horses with asthma. Conclusions and Clinical Importance The intestinal microbiota of horses with asthma does not adapt in the same way to changes in diet and environment compared to the microbiota of healthy horses. Mechanisms explaining how airway obstruction and inflammation could influence the intestinal microbiota and how in turn this microbiota could modulate systemic inflammation in asthmatic horses deserves further investigation.

Pulmonary hypertension and cor pulmonale are complications of severe equine asthma, as a consequence of pulmonary hypoxic vasoconstriction. However, as pulmonary hypertension is only partially reversible by oxygen administration, other etiological factors are likely involved. In human chronic obstructive pulmonary disease, pulmonary artery remodeling contributes to the development of pulmonary hypertension. In rodent models, pulmonary vascular remodeling is present as a consequence of allergic airway inflammation. The present study investigated the presence of remodeling of the pulmonary arteries in severe equine asthma, its distribution throughout the lungs, and its reversibility following long-term antigen avoidance strategies and inhaled corticosteroid administration. Using histomorphometry, the total wall area of pulmonary arteries from different regions of the lungs of asthmatic horses and controls was measured. The smooth muscle mass of pulmonary arteries was also estimated on lung sections stained for [alpha]-smooth muscle actin. Reversibility of vascular changes in asthmatic horses was assessed after 1 year of antigen avoidance alone or treatment with inhaled fluticasone. Pulmonary arteries showed increased wall area in apical and caudodorsal lung regions of asthmatic horses in both exacerbation and remission. The pulmonary arteries smooth muscle mass was similarly increased. Both treatments reversed the increase in wall area. However, a trend for normalization of the vascular smooth muscle mass was observed only after treatment with antigen avoidance, but not with fluticasone. In conclusion, severe equine asthma is associated with remodeling of the pulmonary arteries consisting in an increased smooth muscle mass. The resulting narrowing of the artery lumen could enhance hypoxic vasoconstriction, contributing to pulmonary hypertension. In our study population, the antigen avoidance strategy appeared more promising than inhaled corticosteroids in controlling vascular remodeling. However, further studies are needed to support the reversibility of vascular smooth muscle mass remodeling after asthma treatment.

Abstract Background There are limited data on potential dysbiosis of the airway microbiota in horses with asthma. Hypothesis/Objectives We hypothesized that the respiratory microbiota of horses with moderate asthma is altered. Our objectives were (a) to quantify tracheal bacterial populations using culture and qPCR, (2) to compare aerobic culture and qPCR, and (c) to correlate bacterial populations with bronchoalveolar lavage fluid (BALF) cytology. Animals Eighteen horses with moderate asthma from a hospital population and 10 controls. Methods Prospective case-control study. Aerobic culture was performed on tracheal aspirates, and streptococci, Pasteurella multocida, Chlamydophila spp., Mycoplasma spp., as well as 16S (bacterial) and 18S (fungal) rRNA subunits were quantified by qPCR. Results Potential pathogens such as Streptococcus spp., Actinobacillus spp., and Pasteurellaceae were isolated from 8, 5, and 6 horses with asthma and 3, 0, and 2 controls, respectively. There was a positive correlation between Streptococcus spp. DNA and 16S rRNA gene (r ≥ 0.7, P ≤ 0.02 in both groups), but the overall bacterial load (16S) was lower in asthma (1.5 ± 1.3 versus 2.5 ± 0.8 × 104 copy/μL, P < 0.05). There was no association between microbial populations and clinical signs, tracheal mucus or BALF inflammation. Conclusions and Clinical Importance This study does not support that bacterial overgrowth is a common feature of chronic moderate asthma in horses. Lower bacterial load could suggest dysbiosis of the lower airways, either as a consequence of chronic inflammation or previous treatments, or as a perpetuating factor of inflammation.

Abstract Background Asthma in horses is associated with nonspecific respiratory clinical signs and may be manifested only as exercise intolerance. Its diagnosis relies on bronchoalveolar lavage fluid (BALF) cytology in the presence of compatible clinical signs. The identification of blood biomarkers for this condition would facilitate diagnosis in the field, because there are regional areas where BAL is not routinely performed in clinical practice. Objective Identification of blood biomarkers for the diagnosis of asthma in horses. Animals Fourteen horses with asthma with increased neutrophil numbers in BALF (neutrophilic asthma), 9 healthy control horses, and 10 horses with other pathologic conditions (pathologic controls). Methods Physical examination, clinical score, hematology, and BALF cytology (in a subset of horses) were performed. Serum concentrations of surfactant protein D (SP-D), haptoglobin, and secretoglobin (SCGB) were measured using commercial ELISA assays. Results Serum concentration of SP-D > 43 ng/mL, serum concentration of haptoglobin >5730 ng/mL, and serum concentration of SCGB <19 ng/mL allowed differentiation of horses with neutrophilic asthma from horses of the control groups (healthy and pathologic) with sensitivity of 55, 95, and 75%, and specificity of 67, 28, and 60%, respectively. Specificity of 100% and sensitivity of 45% were obtained with the combination of SP-D, haptoglobin, and SCGB at the serum concentrations indicated above. Specificity of 95% and sensitivity of 45% were obtained with the combination of SP-D and SCGB serum concentrations. Conclusions and Clinical Importance Haptoglobin, SCGB, and SP-D may be diagnostic aids in horses with clinical signs of lower airway disease and neutrophilic pulmonary inflammation.

Abstract Background Reducing inhaled dust particles improves lung function in horses with severe asthma. Soaked hay is commonly used by owners, but its efficacy in improving lung function and inflammation has not been documented. Objectives To measure the effects of soaked hay and alfalfa pellets in horses with severe asthma. Animals Ten adult horses with severe asthma from a research colony. Methods Prospective controlled trial. Horses in clinical exacerbation were housed indoors and allocated to be fed either soaked hay (n = 5) or alfalfa pellets (n = 5) for 6 weeks. Soaked hay was immersed for 45 minutes and dried out hay was discarded between meals. Pulmonary function and clinical scores were measured before and after 2, 4, and 6 weeks. Tracheal mucus scores and bronchoalveolar lavages were performed before and after 6 weeks. Lung function was analyzed with a linear mixed model using log-transformed data. Results Lung resistance decreased from (median (range)) 2.47 (1.54-3.95) to 1.59 (0.52-2.10) cmH2O/L/s in the pellets group and from 1.89 (1.2-3.54) to 0.61 (0.42-2.08) cmH2O/L/s in the soaked hay group over the 6-week period for an average difference of 1.06 cmH2O/L/s for pellets (95% confidence interval [95% CI]: 0.09-2.04, P = .03, not significant after correction) and 1.31 cmH2O/L/s for soaked hay (95% CI: −0.23 to 2.85, P < .001, significant). Conclusion and Clinical Importance Soaked hay can control airway obstruction in horses with severe asthma. The strict protocol for soaking and discarding dried-out hay in this study could however be considered too great of an inconvenience by owners.

Abstract Background After-hours or out-of-clinic crossmatches are often limited by the lack of access to specialized material and technical expertise. Hypothesis/Objectives The goal was to adapt a stall-side crossmatch test for pretransfusion evaluation in horses. Animals Twelve healthy mares (plasma and blood donors, teaching mares). Methods In a prospective study, blood from 12 mares was used to compare the results of 132 crossmatches performed with a rapid gel assay to crossmatches performed with a microgel column assay, and with predicted compatibilities based on blood types and detection of antibodies at a reference laboratory (microplate assay). The rapid gel assay protocol for dogs was adapted to decrease the formation of rouleaux that initially precluded equine erythrocytes migration through the gel. Results There was a good agreement between the rapid gel assay and the microgel assay as well as with the predicted compatibilities (κ > .6 for both). Agreement was higher between the microgel assay and the predicted compatibilities (κ = .8). The rapid gel assay failed to detect 6 predicted Aa incompatibilities (agglutinins-related), 3 of which were also not detected with the microgel assay. Conclusions and Clinical Importance Based on these results, the modified rapid gel assay could be useful in settings when access to the microgel assay is not available. Discrepancies between both gel techniques and predicted compatibilities were most often low-grade agglutination, which warrants further investigation to assess their clinical importance.

Abstract Background Nicoletella semolina was identified in the airways of horses and its low prevalence could be because of its difficult differentiation from other Pasteurellaceae. Objectives To develop a molecular method for the identification of N. semolina and to evaluate its prevalence in the mouth and the airways of healthy and severe asthmatic horses. Animals Six healthy and 6 severely asthmatic horses in phase I, 10 severely asthmatic horses in phase II, and 10 healthy horses in phase III. Methods Cohort (phases I and II) and cross-sectional (phase III) studies. Quantitative polymerase chain reaction primers targeting the sodA gene were optimized. N. semolina was quantified in oral and nasal washes and in bronchoalveolar lavage fluid (BALF; phase I, sampled twice), in nasal washes and BALF (phase II, sampled twice), and in nasal washes (phase III). Results N. semolina was found in the nose of 5, 10, and 9 horses in phases I, II, and III, respectively (first sampling for phases I and II). Six BALF from 5 different horses were positive for N. semolina in phase II. In phase I, there was no significant difference in the nasal loads of healthy horses (median (range): 2.04 × 104 copies/mL (0-2.44 × 105)) and asthmatic horses in exacerbation (3.75 × 102 (0-4.84 × 106); Wilcoxon's rank sum test, P = .57). Conclusions and Clinical Importance N. semolina is commonly found in the airways of horses. The potential pathogenicity of N. semolina remains to be elucidated, but the molecular technique we developed will facilitate future studies.

All current studies have used Illumina short-read sequencing to characterize the equine intestinal microbiota. Long-read sequencing can classify bacteria at the species level. The objectives of this study were to characterize the gut microbiota of horses at the species level before and after trimethoprim sulfadiazine (TMS) administration and to compare results with Illumina sequencing. Nine horses received TMS (30 mg/kg) orally for 5 days twice a day to induce dysbiosis. Illumina sequencing of the V4 region or full-length PacBio sequencing of the 16S rRNA gene was performed in fecal samples collected before and after antibiotic administration. The relative abundance and alpha diversity were compared between the two technologies. PacBio failed to classify the equine intestinal microbiota at the species level but confirmed Bacteroidetes as the most abundant bacteria in the feces of the studied horses, followed by Firmicutes and Fibrobacteres. An unknown species of the Bacteroidales order was highly abundant (13%) and deserves further investigation. In conclusion, PacBio was not suitable to classify the equine microbiota species but detected greater richness and less unclassified bacteria. Further efforts in improving current databanks to be used in equine studies are necessary.

Understanding the importance of intestinal microbiota in horses and the factors influencing its composition have been the focus of many studies over the past few years. Factors such as age, diet, antibiotic administration, and geographic location can affect the gut microbiota. The intra- and inter-individual variability of fecal microbiota in horses complicates its interpretation and has hindered the establishment of a clear definition for dysbiosis. Although a definitive causal relationship between gut dysbiosis in horses and diseases has not been clearly identified, recent research suggests that dysbiosis may play a role in the pathogenesis of various conditions, such as colitis and asthma. Prebiotics, probiotics, and fecal microbiota transplantation to modulate the horse’s gastrointestinal tract may eventually be considered a valuable tool for preventing or treating diseases, such as antibiotic-induced colitis. This article aims to summarize the current knowledge on the importance of intestinal microbiota in horses and factors influencing its composition, and also to review the published literature on methods for detecting dysbiosis while discussing the efficacy of gut microbiota manipulation in horses.

Both microbe–microbe and host–microbe interactions can have effects beyond the local environment and influence immunological responses in remote organs such as the lungs. The crosstalk between the gut and the lungs, which is supported by complex connections and intricate pathways, is defined as the gut–lung axis. This review aimed to report on the potential role of the gut–lung gut–lung axis in the development and persistence of equine asthma. We summarized significant determinants in the development of asthma in horses and humans. The article discusses the gut–lung axis and proposes an integrative view of the relationship between gut microbiota and asthma. It also explores therapies for modulating the gut microbiota in horses with asthma. Improving our understanding of the horse gut–lung axis could lead to the development of techniques such as fecal microbiota transplants, probiotics, or prebiotics to manipulate the gut microbiota specifically for improving the management of asthma in horses.