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.