Profiling of Differentially Expressed Genes Using Suppression Subtractive Hybridization in an Equine Model of Chronic Asthma

Background :\ud Gene expression analyses are used to investigate signaling pathways involved in diseases. In asthma, they have been primarily derived from the analysis of bronchial biopsies harvested from mild to moderate asthmatic subjects and controls. Due to ethical considerations, there is currently limited information on the transcriptome profile of the peripheral lung tissues in asthma.\ud \ud Objective :\ud To identify genes contributing to chronic inflammation and remodeling in the peripheral lung tissue of horses with heaves, a naturally occurring asthma-like condition.\ud \ud Methods :\ud Eleven adult horses (6 heaves-affected and 5 controls) were studied while horses with heaves were in clinical remission (Pasture), and during disease exacerbation induced by a 30-day natural antigen challenge during stabling (Challenge). Large peripheral lung biopsies were obtained by thoracoscopy at both time points. Using suppression subtractive hybridization (SSH), lung cDNAs of controls (Pasture and Challenge) and asymptomatic heaves-affected horses (Pasture) were subtracted from cDNAs of horses with heaves in clinical exacerbation (Challenge). The differential expression of selected genes of interest was confirmed using quantitative PCR assay.\ud \ud Results :\ud Horses with heaves, but not controls, developed airway obstruction when challenged. Nine hundred and fifty cDNA clones isolated from the subtracted library were screened by dot blot array and 224 of those showing the most marked expression differences were sequenced. The gene expression pattern was confirmed by quantitative PCR in 15 of 22 selected genes. Novel genes and genes with an already defined function in asthma were identified in the subtracted cDNA library. Genes of particular interest associated with asthmatic airway inflammation and remodeling included those related to PPP3CB/NFAT, RhoA, and LTB4/GPR44 signaling pathways.\ud \ud Conclusions :\ud Pathways representing new possible targets for anti-inflammatory and anti-remodeling therapies for asthma were identified. The findings of genes previously associated with asthma validate this equine model for gene expression studies

PREVALENCE DE LA COQUELUCHE CHEZ L'ADULTE PRESENTANT UNE TOUX PERSISTANTE ET CONSULTANT EN MEDECINE GENERALE

PARIS-BIUM (751062103) / SudocCentre Technique Livre Ens. Sup. (774682301) / SudocSudocFranceF

Species-Level Gut Microbiota Analysis after Antibiotic-Induced Dysbiosis in Horses

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

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

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

Severe asthma is associated with a remodeling of the pulmonary arteries in horses.

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 α-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

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

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

Differential hybridization screening.

<p>Representative differential screening results of macroarrays of the SH-Ctls library. Four identical membranes were dot-blotted with PCR products obtained by SSH. The membranes were then hybridized with four different probes: SH-Ctls subtracted cDNAs (A), SH unsubtracted cDNAs (B), Ctls-SH subtracted cDNAs (C) and Ctls unsubtracted cDNAs. The arrow in the top left corner indicates the positive control (<i>LCN2</i>). The arrow head indicates an example of differentially expressed genes in SH compare with Ctls.</p

Evaluation of subtraction efficiency.

<p>A: Reduction of <i>GAPDH</i> cDNA following subtraction in the SH-Ctls sample. PCR was performed on SH-Ctls subtracted and SH unsubtracted samples. <i>GAPDH</i> PCR products (760 pb) were detectable 10 cycles earlier in the unsubtracted sample (18 cycles) than in the subtracted sample (28 cycles). B: Enrichment of <i>LCN2</i> cDNA following subtraction in the SH-Ctls sample. PCR was performed on SH-Ctls and Ctls-SH subtracted samples as well as SH and Ctls unsubtracted samples. <i>LCN2</i> PCR products (210 pb) were detected after 20 cycles for both SH unsubtracted and SH-Ctls subtracted samples, the difference in the intensity of the 2 bands indicate the enrichment compare to Ctls unsubtracted and Ctls-SH subtracted samples.</p