Immunoproteomics enable broad identification of new Aspergillus fumigatus antigens in severe equine asthma.

Introduction: Severe equine asthma (SEA) is a common chronic disease of adult horses with characteristic recurrent airway obstruction and similarities to neutrophilic asthma in humans. As an extrinsic stimulus, hay dust exposure is a major risk factor and induces acute exacerbation in susceptible horses. However, single inducing agents of SEA have hardly been identified on a molecular basis. Aspergillus fumigatus (A. fumigatus) is a common mold species in hay and has been described as a major provoking agent of SEA. Methods: Aiming to identify disease-relevant antigens, we analyzed A. fumigatus using an immunoproteomics approach on two-dimensional immunoblots of A. fumigatus protein probed with serum from environmentally matched asthmatic and healthy horses (n=5 pairs). A. fumigatus binding serum immunoglobulins (Pan-Ig), and the isotypes IgG4/7 and IgG3/5 were quantified for each protein spot and then compared between asthmatic and healthy horses. Results and discussion: For 21 out of 289 spots serum immunoglobulin (Ig) binding was different between the two groups for Pan-Ig or the isotypes. If differences were detected, Pan-Ig and IgG4/7 binding to the proteins were lower, while IgG3/5 binding was higher in asthmatic than healthy horse sera. Proteins were extracted from the 21 spots of interest and analyzed by liquid chromatography mass spectrometry. Eight prioritized proteins (candidate antigens) were expressed as recombinant proteins. Some of these have been previously described as major or minor A. fumigatus allergens, alongside other proteins, most with hydrolase activity. Recombinant candidate antigens were tested on 1D immunoblots to confirm their relevance as antigens by serum antibody binding. Four proteins (beta-hexosaminidase, class II aldolase/adducin domain protein, glucoamylase, peptide hydrolase B0XX53) showed different antibody binding characteristics between asthmatic and healthy horses and are likely relevant antigens in SEA. Their identification can provide the basis for innovative diagnostics, prevention, or therapeutic approaches. Additionally, a more profound understanding of SEA and its potential underlying mechanisms can be established. Elevated serum IgG3/5 antibodies correlate with T helper cell 2 responses in other equine pathologies, and the recombinant SEA antigens developed here can become instrumental in analyzing the involvement of SEA-specific T cell responses and Ig responses in future studies.

Equine pastern dermatitis: a narrative review on clinical presentation, diagnosis, risk factors, prevention, and therapeutic approaches.

Equine pastern dermatitis (EPD) is a nonspecific cutaneous reaction pattern on the distal extremities, typically in the palmar/plantar area of the pastern. Although EPD is commonly seen in equine practice and can be a debilitating condition, peer-reviewed original studies on many aspects of this multifactorial syndrome are still scarce. This narrative review aims to give an overview of the clinical presentation (forms of EPD and clinical scores and differential diagnoses), risk factors, and therapeutic approaches. The emphasis is on intrinsic and extrinsic risk factors as most of the original work has been published on these aspects. The available data supports the effects of age, breed, and breed-related phenotypical traits (draft breeds with feathers and large cannon circumference) on the frequency and severity of EPD manifestations. Hind legs and unpigmented limbs are also more frequently affected. Genetic effects in draft breeds appear to be complex, and no commercial genetic tests currently exist. Evidence for meteorological effects like rainfall and humidity is inconclusive. Associations with Chorioptes infestation and bacterial microbiota imbalances but not with fungal infections have been consistently shown. Causality has not been demonstrated for specific infectious agents. Original studies have investigated the effects of antibacterial agents (Kunzea oil, phytosphingosines, and triclosan), fatty acids, aromatic oils, and humectants as well as therapeutic approaches to Chorioptes infestation in EPD-affected equids. While therapy remains largely empirical, knowledge of investigated risk factors for this multifactorial syndrome can inform diagnostic and therapeutic approaches. Raising owner awareness of EPD could be key to improving the welfare of affected horses.

Immunoproteomics reveal increased serum IgG3/5 binding to Dermatophagoides and yeast protein antigens in severe equine asthma in a preliminary study.

Introduction: Severe equine asthma (SEA) is a common, chronic respiratory disease of horses characterized by hyperreactivity to hay dust which has many similarities to severe neutrophilic asthma in humans. SEA-provoking antigens have not been comprehensively characterized, but molds and mites have been suggested as relevant sources. Here, we identified relevant antigen candidates using immunoproteomics with IgG isotype-binding analyses. Methods: Proteins from Dermatophagoides pteronyssinus (Der p) were separated by two-dimensional gel electrophoresis followed by immunoblotting (2D immunoblots) resulting in a characteristic pattern of 440 spots. After serum incubation, antibody (Ig)-binding of all Ig (Pan-Ig) and IgG isotypes (type-2-associated IgG3/5, type-1-associated IgG4/7) was quantified per each spot and compared between asthmatic and healthy horses' sera (n=5 per group). Results: Ig binding differences were detected in 30 spots. Pan-Ig binding was higher with asthmatics compared to healthy horses' sera on four spots, and IgG3/5 binding was higher on 18 spots. Small IgG4/7 binding differences were detected on 10 spots with higher binding with asthmatics' sera on four but higher binding with healthy horses' sera on six spots. Proteins from the spots with group differences including mite and yeast proteins were identified by liquid chromatography mass spectrometry. The latter likely originated from the feeding substrate of the Der p culture. Prioritized antigen candidates amongst the proteins identified were Der p 1, Der p 11, group 15 allergens, myosin heavy chain, and uncharacterized Der p proteins. Additionally, yeast enolases, alcohol dehydrogenase (ADH), phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase, and heat shock proteins were prioritized. Eleven antigen candidates were tested for confirmation by ELISAs using the respective proteins separately. Differences in asthmatics vs. healthy horses' serum Ig binding to Der p 1, Der p 18, and three yeast enzymes (enolase, ADH, and PGK) confirmed these as promising antigens of immune responses in SEA. Discussion: Antigens with relevance in SEA were newly identified by immunoproteomics, and yeast antigens were considered for SEA for the first time. Serum IgG3/5 binding to relevant antigens was increased in SEA and is a novel feature that points to increased type-2 responses in SEA but requires confirmation of the corresponding cellular responses.

Prevalence and WGS-based characteristics of Staphylococcus aureus in the nasal mucosa and pastern of horses with equine pastern dermatitis.

BACKGROUND: Many contributing factors are involved in the development of equine pastern dermatitis (EPD). Among the most frequently suspected is Staphylococcus aureus, known for its pathogenic potential in skin and soft tissue infections. We therefore investigated the association between S. aureus carriage and EPD. RESULTS: One hundred five EPD-affected horses and 95 unaffected controls were examined for the presence of methicillin-resistant and -susceptible Staphylococcus aureus (MRSA and MSSA) on the pastern skin and in the nostrils. S. aureus isolates were cultivated from swab samples on selective MSSA and MRSA chromogenic agar and identified using MALDI-TOF MS. Isolates were analysed by Illumina whole genome sequencing for genetic relatedness (cgMLST, spa typing), and for the presence of antimicrobial resistance and virulence determinants. A markedly higher proportion of samples from EPD-affected horses proved positive for S. aureus, both from the pastern (59.0 % vs. 6.3 % in unaffected horses; P<0.001), and from the nose (59.0 % vs. 8.4 %; P<0.001). Isolates belonged to 20 sequence types (ST) with lineages ST15-t084 (spa) (18 %), ST1-t127 (13 %), and ST1-t1508 (12 %) being predominant. Eight S. aureus were MRSA ST398-t011 and ST6239-t1456, and contained the staphylococcal cassette chromosome SCCmecIVa. Antimicrobial resistance genes were almost equally frequent in pastern and in nasal samples, whereas some virulence factors such as the beta-hemolysin, ESAT-6 secretion system, and some enterotoxins were more abundant in isolates from pastern samples, possibly enhancing their pathogenic potential. CONCLUSIONS: The markedly higher prevalence of S. aureus containing specific virulence factors in affected skin suggests their contribution in the development and course of EPD.

The relationship between equine pastern dermatitis, meteorological factors, and the skin microbiota.

BACKGROUND: Equine pastern dermatitis (EPD) is a multifactorial syndrome, with prolonged exposure to moisture assumed to be a predisposing or primary factor. HYPOTHESIS/OBJECTIVES: To examine the course of EPD lesion severity, changes in bacterial skin microbiota, and the influence of meteorological factors. ANIMALS: Prospective, longitudinal cohort study over a one-year period, with six Franches-Montagnes stallions, four affected by EPD and two unaffected, that were kept under the same conditions. METHODS AND MATERIALS: Pasterns were scored for lesion severity and sampled once a month for 12 consecutive months. Lesion severity, the skin microbiota and meteorological factors were examined for associations. RESULTS: EPD lesions tended to worsen in autumn and at the beginning of spring. The relationship between lesion severity and the meteorological factor precipitation was not clearly evident; high scores were preceded by both low or high rates of precipitation. Microbiota in affected pasterns appeared to have experienced a reduction in alpha diversity. Beta diversity analyses demonstrated that bacterial community structures were altered in affected versus unaffected pasterns, and that alterations were more pronounced with higher EPD scores (P = 0.005). Meteorological factors also had considerable influences on the bacterial composition, whereby these influences appeared to be more marked in the affected pasterns (P = 0.001, F = 3.19) than in unaffected ones (P = 0.005, F = 1.83). CONCLUSIONS AND CLINICAL RELEVANCE: Our study provides preliminary observations of the relationships between lesion severity, meteorological factors and cutaneous bacteria. The population was too small to draw firm conclusions, and further studies on environmental factors and the involvement of bacteria in this multifactorial disease are needed.

The skin microbiota in equine pastern dermatitis: a case-control study of horses in Switzerland.

BACKGROUND: Equine pastern dermatitis (EPD), a multifactorial syndrome, manifests as skin lesions of variable severity in the pastern area. Despite the widespread use of antibacterial therapy for treating this condition, little is known about the contributing bacteria. HYPOTHESIS/OBJECTIVES: To investigate the bacterial skin microbiota in EPD-affected and unaffected (control) pasterns. ANIMALS: Case-control study with 80 client-owned horses; each with at least one EPD-affected and one control pastern. METHODS AND MATERIALS: Horses were grouped by the form of EPD (mild, exudative or proliferative), the assigned severity grade and type of pretreatment (disinfectant, topical antibacterial or no antibacterial pretreatment). Skin swabs were obtained, and the microbiota composition was compared between the groups. RESULTS: Bacterial alpha diversity was reduced in affected pasterns (P < 0.001) and this reduction was significantly associated with the EPD forms (P < 0.001), and not with the type of pretreatment (P > 0.14). Analyses of beta-diversity confirmed a disordering of the skin microbiota (P = 0.004) in affected versus control pasterns, that was particularly profound in more severe lesions. The type of pretreatment was not significantly associated with this disordering. Four differentially abundant families were detected, of which Staphylococcaceae was the most distinct. The relative abundance of staphylococci was significantly increased in affected pasterns (P = 0.011), particularly in those that had received antibacterial treatment previously. CONCLUSIONS AND CLINICAL RELEVANCE: Changes in the microbiota are associated with the EPD form or severity of lesions. The role of bacteria in the pathogenesis of EPD as well as the propriety and consequences of antibacterial treatment should therefore be further investigated.

The influence of clinical severity and topical antimicrobial treatment on bacteriological culture and the microbiota of equine pastern dermatitis.

BACKGROUND: Equine pastern dermatitis (EPD) is a common dermatological problem in horses, yet its aetiology and pathogenesis are poorly understood. OBJECTIVES: This study aimed to investigate the effects of lesion severity and topical antimicrobial treatment on bacterial flora of EPD-affected skin. ANIMALS: Sixteen horses with EPD were investigated. METHODS AND MATERIALS: An observational study was conducted by assigning a clinical severity score ranging from 0 (macroscopically nonlesional) to 21 (severe), and sampling the most and least severely affected limbs of 16 horses (32 limbs) for bacteriological culture and 16S rRNA sequencing. Topical antimicrobial treatment in the month before sampling was recorded. The limbs were allocated to a nonlesional or mildly affected group (Group A, score 0-3) and a moderate to severely affected group (Group B, score 4-21). RESULTS: The most commonly cultured bacterial species was Staphylococcus aureus (one of 15 Group A versus nine of 17 Group B). Within Group B, S. aureus was found in three of six limbs treated with topical antimicrobials and in six of 11 untreated limbs. ß-haemolytic streptococci (three of 32) and Trueperella pyogenes (two of 32) also were cultured exclusively in the untreated limbs of Group B. Staphylococci and streptococci were found more often by 16S rRNA sequencing than in culture. Limbs with higher lesion severity and topical antimicrobial treatment appeared to have a lower alpha diversity and different beta diversity compared to milder and untreated lesions. CONCLUSIONS AND CLINICAL IMPORTANCE: Observed differences in microbiota of equine skin are likely to be linked to the presence and severity of EPD and topical antimicrobial treatment. Further research is needed to establish causal bacteria.