Discussion
The results from this large-scale study including BAL samples from 604 horses adds to existing knowledge that coughing is the most important owner reported clinical sign with regard to EA, as coughing was found to be associated both with an increase in both TMS and BAL neutrophils. Furthermore, TMS showed, alongside with coughing, significant associations with both EA diagnosis, and BAL neutrophils.
Horses with an anamnesis of coughing, or a combination of coughing and poor performance, had a significantly higher TMS and a higher percentage of BAL neutrophils than horses reported solely with poor performance. Several studies have found associations between coughing, lower airway neutrophilia and TMS in both sport horses and racehorses (Almeida et al., 2015; Bedenice et al., 2008; Bosshard and Gerber, 2014; Leguillette et al., 2016; Wasko et al., 2011). In the present study, poor performance was found to have significantly lower TMS than the groups with complaints of coughing; this can be explained by the multifactorial nature of poor performance (Ellis et al., 2022), as well as uncertainties regarding both the poor performance definition and owner reliability (Richard et al., 2010; Salz et al., 2016). BAL mast cells were not associated with neither coughing nor poor performance in this study. Mastocytic MEA has been described as an allergic asthma type, with primarily a T2 helper cell response (Beekman et al., 2012; Hansen et al., 2020), and with clinical signs primarily associated with airway hyper-responsiveness (Secombe et al., 2019). Mastocytic MEA was previously found not to be associated with coughing (Bedenice et al., 2008), in line with the results from this study/our present findings.
Owner questionnaires have been widely included in studies of lower airway inflammation, and the owner reliability to detect SEA was found to be excellent, whereas owners’ ability to detect MEA was limited to a degree that they could not differentiate between MEA and healthy horses (Laumen et al., 2010; Wasko et al., 2011). In the present study, 24 horses diagnosed with SEA were not reported to cough; this can reflect a bias, as some of the horses were only used for pasture pets with limited owner recognition of coughing.
The TMS used in this study is based on a published study (Gerber et al., 2004b) and validated (Leguillette et al., 2016), as well as widely used in the EA literature (Holcombe et al., 2006; Robinson et al., 2006). Furthermore, the latest consensus statement on MEA includes a TMS ≥ 2/5 for sports horses and ≥1/5 for racehorses as one of the diagnostic criteria for MEA (Couetil et al., 2016).
In this study, TMS increased from the group of horses with normal BAL cytology, to horses with the MEA and SEA diagnoses. Furthermore, TMS increased with elevated BAL neutrophil cell percentages, in agreement with previous studies (Koblinger et al., 2011; Wysocka and Klucinski, 2014, 2015). A recent study reported poor sensitivity of the TMS as diagnostic criteria for MEA (Dauvillier et al., 2019).This in line with our findings, as no correlation was identified between TMS and the amount of BAL mast cells. In this study, differences were found between the MEA subtypes, in line with a study by Nolen-Walston et al. (2013) whom found that horses with the mixed MEA diagnosis were more likely to have an increased TMS than control horses; likewise, a study by Wysocka and Klucinski (2015) reported a negative correlation between TMS andeosinophilic MEA .
This study found a significantly lower volume aspirated in SEA horses than horses with normal BAL cytology and the MEA diagnosis, this is in line with a study by Couëtil et al. (2005) that found significant less volume aspirated before treatment than after treatment for a group of EA horses. In the present study, no age-related effect was found with the BAL volume aspirated. Using a larger instilled volume with subsequent manual aspiration of BAL fluid compared to mechanical suction would both significantly increase aspirated BAL volume (Orard et al., 2016), whereas the use of N-butylscopolammonium bromide did not have any effect on BAL volume aspirated (Bowser et al., 2018).
In this study, a positive correlation was found between higher BAL neutrophil cell percentages and increasing age. This highly likely reflect that SEA is predominantly a disease of older horses as this is in agreement with the literature reporting SEA predominantly as a disease of older horses (Couetil et al., 2016; Jocelyn et al., 2018; Robinson, 2001). The population of horses with BAL cytology within normal limits, no age-related changes in BAL differential cell count has been identified (Christmann et al., 2009; Hansen et al., 2013), except one single study showing a decrease in BAL lymphocyte count with increasing age (Gerber et al., 2003). Mild-moderate equine asthma is a more prevalent disease in high performance horses and pleasure horses of all ages (Couetil et al., 2016); in line with this, the results from our study found no age-related changes with regard to BAL mast cells.
Results from this study demonstrated that housing type and roughage fed were dependent on the stabling environment. Of hoses with the SEA diagnosis, 32% were stabled in a single box and 56% were fed hay. Feeding hay has been associated with airway inflammation (Olave et al., 2021), whereby soaking or steaming of the hay, or changing the roughage to haylage has been recommended (Clements and Pirie, 2007a, b; Dauvillier et al., 2019). Loose-housing is often recommended for horses with airway inflammation, but the specific design of the actual loose housing system needs to be considered in order to determine if such conditions are beneficial (Hansen et al., 2019).
Limitations of the study included the heterogeneity of the sampling, several different veterinarians included, shipping of the samples as well as the inability to adjust for year and month of sampling (data not available) add to limitations. With regard to year and month of sampling, a previous study found no effect of season on TMS (Nolen-Walston et al., 2013) and with equally high MEA diagnosis year round (Davis and Sheats, 2019).
In conclusion, this large-scale study found that owner reported coughing was significantly associated with TMS and BAL neutrophilia. Furthermore, TMS increased with severity of BAL neutrophils and EA diagnosis. BAL mast cells and mastocytic EA was not associated with either owner complaints, TMS, or ageing. Although, several of the study results have been reported previously, none of these studies included a sample size and diversity of horse population matching the present study.
Author contributions: M. Larsen, C. Amstrup, J. Fjeldborg, N.D. Otten, K.E. Baptiste and S. Hansen all contributed to the study design, data analysis and interpretation, preparation and final approval of the manuscript. M. Larsen and C. Amstrup collected the data. All authors gave their final approval of the manuscript.
Acknowledgements: All the practitioners contributing to this study by sending bronchoalveolar lavage sample for cytology to the laboratory at the Large Animal Teaching Hospital, University of Copenhagen.
Conflict of interest: no conflict of interest to declare.
Ethical approval: Research ethics committee approval was granted from the University of Copenhagen
Informed consent: Explicit owner informed consent for inclusion of samples from animals in this study was not sought but owners were aware that excess material from clinical samples would be retained for research; in general, all owners have the option to opt out of research.