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\begin{document}

\title{Associations between clinical signs, endoscopic and cytological findings
in equine bronchoalveolar lavage samples}



\author[1]{Camilla Amstrup}%
\author[1]{Marian Larsen}%
\author[1]{Julie Fjeldborg}%
\author[1]{Nina Otten}%
\author[2]{Keith Baptiste}%
\author[3]{Sanni Hansen}%
\affil[1]{University of Copenhagen}%
\affil[2]{ Danish Medicines Agency }%
\affil[3]{Kobenhavns Universitet Institut for Produktionsdyr og Heste}%


\vspace{-1em}



  
  \date{June 14, 2024}


\begingroup
\let\center\flushleft
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\selectlanguage{english}
\begin{abstract}
Background: Endoscopy, including tracheal mucus score (TMS) and
bronchoalveolar lavage (BAL) fluid cytology, are commonly used to
diagnose equine asthma (EA). Large-scale studies comparing clinical
signs, TMS and BAL cytology are warranted. Objectives: Investigate
associations between clinical signs of EA, tracheal mucus score and BAL
cytology. Study design: cross-sectional study Methods: Data from 604
horses, each horse having one BAL cytology sample evaluated at the
laboratory service at the Large Animal Teaching Hospital in Denmark,
with further information provided by an owner questionnaire and
veterinarian performing the endoscopy and BAL. Results: Tracheal mucus
score increased significantly with EA severity, BAL neutrophilia and
owner reported coughing, but not with BAL mastocytosis. Owner complaint
of coughing was associated with an increasing amount of BAL neutrophils,
but not with an increase in BAL mast cells. Poor performance was not
correlated to BAL neutrophils, mast cells or TMS. Percentage of BAL
aspirated was found lower in the SEA group compared to the non-asthmatic
and MEA groups. Main limitations: Several different veterinarians
performed the BAL sampling, BAL samples were shipped and a 24 hour delay
in processing of the sample. Not able to adjust for season as these data
was not recorded. Conclusion: this large-scale study showed a
correlation between TMS and increasing severity of lower airway
inflammation, confirming TMS as a reliable diagnostic factor for EA.
Owner complaint of coughing, was found to be associated with TMS and BAL
neutrophilia, and poor performance without coughing is not a reliable
parameter for EA.%
\end{abstract}%



\sloppy


Original research paper

Title: Associations between clinical signs, endoscopic and cytological
findings in equine bronchoalveolar lavage samples

Running title: Endoscopic findings with equine asthma

Marian Larsen\textsuperscript{1} Camilla Amstrup\textsuperscript{1}Julie
Fjeldborg\textsuperscript{1}, Nina D. Otten\textsuperscript{2}Keith E
Baptiste\textsuperscript{3}, Sanni Hansen\textsuperscript{*1}

\textsuperscript{1}University of Copenhagen, Faculty of Health and
Medical Sciences, Department of Large Animal Sciences, Taastrup, Denmark

\textsuperscript{2}Department of Veterinary Clinical Sciences, Faculty
of Health and Medical Sciences, University of Copenhagen, DK-1870,
Frederiksberg C, Denmark

\textsuperscript{3}Department of Veterinary Medicine, Danish Medicines
Agency (L\selectlanguage{ngerman}ægemiddelstryelsen), Axel Heides gade 1, DK-2300 Copenhagen
South

*Corresponding author: Sanni Hansen, Department of Veterinary Clinical
Sciences, University of Copenhagen, Hoejbakkegaard Allé 5, DK-2630
Taastrup, Denmark.

E-mail address: sannih@sund.ku.dk (S. Hansen)

Summary

Background: Endoscopy, including tracheal mucus score (TMS) and
bronchoalveolar lavage (BAL) fluid cytology, are commonly used to
diagnose equine asthma (EA). Large-scale studies comparing clinical
signs, TMS and BAL cytology are warranted.

Objectives: Investigate associations between clinical signs of EA,
tracheal mucus score and BAL cytology.

Study design: cross-sectional study

Methods: Data from 604 horses, each horse having one BAL cytology sample
evaluated at the laboratory service at the Large Animal Teaching
Hospital in Denmark, with further information provided by an owner
questionnaire and veterinarian performing the endoscopy and BAL.

Results: Tracheal mucus score increased significantly with EA severity,
BAL neutrophilia and owner reported coughing, but not with BAL
mastocytosis. Owner complaint of coughing was associated with an
increasing amount of BAL neutrophils, but not with an increase in BAL
mast cells. Poor performance was not correlated to BAL neutrophils, mast
cells or TMS. Percentage of BAL aspirated was found lower in the SEA
group compared to the non-asthmatic and MEA groups.

Main limitations: Several different veterinarians performed the BAL
sampling, BAL samples were shipped and a 24 hour delay in processing of
the sample. Not able to adjust for season as these data was not
recorded.

Conclusion: this large-scale study showed a correlation between TMS and
increasing severity of lower airway inflammation, confirming TMS as a
reliable diagnostic factor for EA. Owner complaint of coughing, was
found to be associated with TMS and BAL neutrophilia, and poor
performance without coughing is not a reliable parameter for EA.

Key words: horse, severe equine asthma, mild-moderate equine asthma,
tracheal mucus score, poor performance, cough



\textbf{Introduction}

Equine Asthma (EA) is a highly prevalent disease worldwide (Allen et
al., 2006; Courouce-Malblanc et al., 2010; Robinson et al., 2006), and
can be further divided into two phenotypes; severe equine asthma (SEA),
affecting primarily older horses, and mild-moderate equine asthma (MEA)
affecting horses of all ages (Couetil et al., 2016). Horses diagnosed
with EA express different severities of coughing, poor performance and
increased respiratory effort, either at exercise and/or at rest (Couetil
et al., 2020; Koblinger et al., 2011; Robinson et al., 2003). A further
subdivision of the MEA phenotype has been proposed to include mild EA
for high performance horses with clinically unrecognized airway disease,
as well as moderate EA of horses with clinical signs such as coughing
and poor performance (Couetil et al., 2020).

A relationship between lower airway inflammation, poor performance,
Tracheal mucus score (TMS) and coughing has been noted in both small and
large-scale studies (Bedenice et al., 2008; Christley et al., 2001;
Fogarty and Buckley, 1991; Holcombe et al., 2006; Lavoie et al., 2011;
Widmer et al., 2009). Other, large-scale studies are warranted to
investigate associations between clinical signs and diagnostics.

Horse owner questionnaires have shown to be reliable for identifying
horses with SEA, whereby the horse owner assessed respiratory signs
index (HOARSI) has been validated (Hotchkiss et al., 2006) and used in
EA populations (Laumen et al., 2010). Besides owner reported clinical
signs, endoscopic examination including scoring of tracheal mucus
(Gerber et al., 2004b) and bronchoalveolar lavage (BAL) is considered
the gold standard in EA diagnostics (Bond et al., 2018; Couetil et al.,
2016). For example, tracheal mucus score is reported to increase with
both MEA and SEA cases, poor performance and coughing, and is thus a
reliable, important parameter of the endoscopic examination (Almeida et
al., 2015; Cardwell et al., 2011; Gerber et al., 2004a; Holcombe et al.,
2006; Koblinger et al., 2011; Pirie et al., 2016; Widmer et al., 2009).
Tracheal mucus score between MEA subtypes has been evaluated sparsely,
and an increased amount of mucus has been found associated only with the
mixed MEA subtype (Nolen-Walston et al., 2013).

The amount of BAL fluid obtained after instilling a volume of 250-500
ml, and manually aspiration, is expected to be between 50-70 percent of
the instilled fluid volume (Couetil et al., 2016). The volume aspirated
has been found to be lower in horses diagnosed with SEA compared to
healthy horses (Couëtil et al., 2005; Jean et al., 2011), but no
difference in volume aspirated has been found between MEA subtypes
(Richard et al., 2014). This present study investigated the associations
between stabling factors, clinical signs of EA, TMS and BAL cytology,
including a large group of horses. It was hypothesized that 1) Clinical
signs of coughing and poor performance would be associated with BAL
neutrophilia, BAL mastocytosis and an increased TMS. 2) The TMS score
will increase with an increase in BAL neutrophils and severity of EA
diagnosis, but not with an increase in BAL mast cells.3) Volume of BAL
aspirated in percentage of BAL instilled would decrease with EA disease
severity and lastly, 4) Stabling environment and roughage fed would be
associated with the EA diagnosis.

\textbf{Materials and methods}

\emph{Data}

A cross-sectional study was performed including 604 BAL samples, from
clinical cases, submitted by referring veterinarians all over Denmark
during the period between 2016-2019, to the diagnostic service at the
University of Copenhagen. A questionnaire and further information were
provided from both the horse owner and referring veterinarian about the
horse, clinical symptoms and BAL procedure. Duplicate samples were
excluded from the analysis. Furthermore, data were excluded if another
BAL volume than 200-250 ml was used, if shipping had been delayed or if
the sample was of a non-diagnostic quality.

\emph{Horse and housing variables}

Based on questionnaire data obtained from the horse owners, the
following variables were collected including age and breed of the horse,
along with the clinical history. For the analyses, horses were
categorized into three age-groups 0-6 years, 7-14 years and
\textgreater{}14 years.

The owners were also requested for information about the stabling
facilities/housing types (box, loose housing), bedding material (straw,
shavings) and roughage fed (dry hay, soaked hay, silage).

\emph{Clinical parameters}

Since the two main complaints reported by the owners were poor
performance and coughing, then the dataset further sub-divided horses
into four groups based on clinical symptoms:

Group 1: no clinical symptoms,

Group 2: poor performance,

Group 3: coughing,

Group 4: both poor performance and coughing (Table 1).

Referring veterinarians were requested for the clinical examination
(temperature, pulse, respiration and lung auscultation) and details of
the endoscopic examination, including: TMS, BAL volume as well as BAL
volume aspirated.

Referring veterinarians were given a protocol on how to perform the
endoscopic examination, including how to score mucus and the BAL volume
to use (Appendix 1).

Tracheal mucus score (TMS) for each horse was based on a qualitative
ordinal scoring system ranging 0-5 (Gerber et al., 2004b) (0 = no mucus,
1 = small blobs, 2 = multiple blobs, 3 = stream-forming, 4 =
pool-forming and 5 = profuse amount). A TMS \selectlanguage{english}[?]2 was considered acceptable
in healthy sports horses (Couetil et al., 2016). Initial BAL volume used
was standardized to 200-250 ml, and the amount aspirated as a percentage
of the volume instilled was calculated. Based on the results, horses
were sub-divided normal BAL cytology or diagnosed with either MEA or SEA
based on alternative BAL cytology reference values (Couetil et al.,
2016) and differences in the aspirated volume between EA diagnoses were
explored.

\emph{Laboratory procedure}

The laboratory analyses of the BAL samples shipped by veterinarians from
all over Denmark, including cytospin preparations and differential cell
counts, were performed by the diagnostic laboratory services at the
Large Animal Teaching Hospital in Denmark. The samples were shipped on
ice, stored in EDTA tubes, and received by the laboratory within 24
hours. A two-hundred \selectlanguage{greek}µ\selectlanguage{english}l aliquot of the BAL EDTA sample was instilled
into the funnel of a cytospin (StatSpin\selectlanguage{ngerman}® Cytofuge, USA) and centrifuged
at 93 g for seven minutes. The cytospin samples were fixed in methanol
(ChemSolute®, Th. Geyer, Denmark) and stained with May-Grünwald-Giemsa
(Merck, Germany). Slides were examined under light microscopy at 400 x
magnification using the five-field counting technique (Fernandez et al.,
2013) by experienced observers.

Four-hundred cells were counted and classified as percentages of
alveolar macrophages, lymphocytes, neutrophils, mast cells or
eosinophils.

\emph{Asthma types}

Based on the BAL differential cell counts, horses were diagnosed as
either with normal BAL cytology, MEA or SEA (Couetil et al., 2016).
Horses with normal BAL cytology were defined by a BAL neutrophil count
below 10\%, a mast cell or eosinophil cell count below 5\%; MEA horses
by a neutrophil cell count between 10-25\%, and/or a mast cell and/or
eosinophil cell count above 5\% and SEA horses by a neutrophil cell
count above 25\%. Mastocytic MEA was defined as horses with BAL mast
cells count above 5\% mast cells, and with BAL neutrophils below 10\%
and eosinophils below 5\%; neutrophilic MEA referred to horses with a
BAL neutrophil count between 10-25 \% with both BAL mast cells and
eosinophils being below 5\%.

\emph{Statistical analysis}

All data analyses were done using the program R version 4.0.2. All data
were examined and found to be non-parametric by Shapiro-Wilk's test,
visual evaluation of QQ plots and density plots. Descriptive analysis,
along with quantile sets, were calculated and box plots were created to
describe and visualize the data. The non-parametric Kruskal-Wallis test
and Games-Howell post hoc test were used to examine statistically
significant differences between the ordinal test groups for continuous
outcome variables (BAL cytology and BAL volume instilled and aspirated).
Associations between ordinal variables TMS, clinical signs, asthma
types, housing factors and age groups were assessed by Chi-square tests
of independence. All p values were assessed at a significance level of
5\%.

\textbf{Results}

Descriptive analyses

During the data collection period, a total of 737 samples were received
at the diagnostic laboratory. Data control was performed and duplicates
were removed, as only the first sample from a horse was included.
Finally, six hundred and four BAL samples were included, originating
from 70 different veterinary practices around Denmark. Forty-seven
different horse breeds were represented, with warmblood (n=108) and
Icelandic horse (n=89) being the two most common breeds. The mean age of
the total population was 10.5 (±4.5) years (Table 1). Bronchoalveolar
lavage differential cell counts were done for all horse samples included
(Table 2).

Clinical signs

An anamnesis of poor performance without concomitant coughing was not
associated with TMS (p=0.060). Horses without an anamnesis of coughing
and poor performance, and horses with only poor performance had
significantly lower levels of BAL neutrophils compared to the group of
horses with coughing (p=0.009 and p=0.001, respectively) and the group
of horses with both poor performance and coughing (p=0.005 and
p\textless{}0.001, respectively). No differences were found between
horses with clinical signs coughing or poor performance and the
percentage of BAL mast cells (p=0.129).

Tracheal mucus score

A TMS was available for 448 horses, and a significant association
between TMS and asthma types was found (p\textless{}0.001). The largest
contributions to the differences between the observed and expected
values in the Chi-square test were due to horses with normal BAL
cytology having a TMS of 1 (14.8\%), while horses with SEA had a higher
frequency of TMS score 4 (15.7\%) and score 5 (14\%). The TMS plotted
against the different BAL cytology diagnosis in shown in figure 1.

The further specification of horses diagnosed with MEA and
differentiated into MEA neutrophilic, MEA mastocytic, or MEA mixed
diagnoses revealed significant differences in TMS (p\textless{}0.001)
(Table 2). Horses with normal BAL cytology had a higher frequency of
lower TMS scores compared to horse with SEA, with a contribution of
horses with normal BAL cytology /TMS scores 0-1 of 13.8\% vs. SEA/TMS
score 4-5 of 26.7\%. Amongst the differentiated asthma types, the MEA
mastocytic accounted for the highest proportion of differences in TMS
scores with a total contribution of 9\%, followed by MEA neutrophilic
6\%, MEA eosinophilic 3\% and MEA mixed 1.6\%. No significant
association between TMS and BAL mast cell count was found.

BAL volume aspirated

The BAL volume aspirated was available for 540 horses. No significant
difference was found for the group of horses with normal BAL cytology
values compared with the group of horses with MEA (p=0.076), but
significantly higher compared to the group of horses with SEA (50.0\%
vs. 40.0\%, p=0.035) (table 2).

No significant difference between the BAL volume aspirated and the age
of the horse was found, neither when horses were sub-divided into age
groups (p=0.139).

Housing

Information on housing (single box or loose-housed in groups) was
collected for 523 horses. The Chi- square test revealed dependency
between housing and EA groups (p=0.011). Of the horses in loose-housed
groups 28\% had normal BAL cytology, 34\% had MEA and 38\% had SEA. Of
the horses housed in single boxes, 19\% had normal BAL cytology, 49\%
had MEA and 32\% had SEA diagnosis (Figure 2). Information on roughage
fed (divided into dry hay n=186, soaked hay n =56 or haylage n=254) was
available for 496 horses, the Chi-square test revealed dependency
between roughage fed and EA groups (p\textless{}0.001). Of the horses
with the SEA diagnosis, 56\% were fed hay, 14\% were fed soaked hay and
30\% were fed haylage at the point of examination. For the horses with
the MEA diagnosis, 27\% were fed hay, 10\% were fed soaked hay and 63\%
were fed haylage.

\textbf{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 T\textsubscript{2} 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 \selectlanguage{english}[?] 2/5
for sports horses and \selectlanguage{english}[?]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
and\emph{eosinophilic 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 Couetil 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.

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Figure legends

Figure 1: Distribution of tracheal mucus scores among horses with normal
bronchoalveolar lavage (BAL) cytology, with mild-moderate equine asthma
(MEA) diagnosis, based on reference values \textgreater{}10\%
neutrophils, \textgreater{}5\% mast cells or eosinophils, and severe
equine asthma (SEA) with reference values \textgreater{}25\%
neutrophils. Significant differences were found between all groups.

Figure 2: The distribution of horses with normal BAL cytology,
mild-moderate equine asthma (MEA) and severe equine asthma (SEA)
sub-divided into horses stabled in individual boxes and horses in
loose-housing systems.

Table 1: Horses included in the study, sub-divided into groups based on
owner complaints (coughing, poor performance). For each group, age and
bronchoalveolar lavage (BAL) cytology results are shown, cytological
diagnoses are based on reference values for mild-moderate equine asthma
\textgreater{}10\% neutrophils, \textgreater{}5\% mast cells or
eosinophils, and SEA \textgreater{}25\% neutrophils.\selectlanguage{english}
\begin{longtable}[]{@{}llllll@{}}
\toprule
& No coughing or poor performance & Poor performance & cough & Poor
performance and cough & Total\tabularnewline
\midrule
\endhead
Number of horses & 63 & 297 & 435 & 225 & 604\tabularnewline
Age & 9.4 (\selectlanguage{ngerman}±4.2) & 10.7 (\selectlanguage{ngerman}±4.4) & 10.8 (\selectlanguage{ngerman}±4.7) & 11.0 (\selectlanguage{ngerman}±4.5) & 10.5
(\selectlanguage{ngerman}±4.5)\tabularnewline
Normal BAL cytology & 24 & 60 & 83 & 43 & 122\tabularnewline
MEA & 46 & 141 & 186 & 96 & 283\tabularnewline
SEA & 24 & 93 & 165 & 83 & 199\tabularnewline
\bottomrule
\end{longtable}

BAL = bronchoalveolar lavage; MEA = mild-moderate equine asthma; SEA =
severe equine asthma. Age showed as mean and standard deviation.

Table 2: Bronchoalveolar lavage (BAL) cytology results for the included
horses, and divided into groups based on cytological diagnosis, based on
reference values for mild-moderate equine asthma \textgreater{}10\%
neutrophils, \textgreater{}5\% mast cells or eosinophils, and SEA
\textgreater{}25\% neutrophils.\selectlanguage{english}
\begin{longtable}[]{@{}lllllllll@{}}
\toprule
& Normal BAL cytology & MEA & Mastocytic MEA & Neutrophilic MEA & Mixed
MEA & Eosinophilic MEA & SEA & Total\tabularnewline
\midrule
\endhead
Number of horses & 122 & 283 & 79 & 136 & 64 & 4 & 199 &
604\tabularnewline
Age (mean std) & 10.1 (\selectlanguage{ngerman}±4.5) & 10.2 (\selectlanguage{ngerman}±4.8) & 9.4 (\selectlanguage{ngerman}±4.7) & 10.8 (\selectlanguage{ngerman}±5.0) &
9.9 (\selectlanguage{ngerman}±4.5) & 7.7 (\selectlanguage{ngerman}±2.7) & 11.9 (\selectlanguage{ngerman}±3.7) & 10.5 (\selectlanguage{ngerman}±4.5)\tabularnewline
\% BAL aspirated & 50.0 (32.7-60.0) & 44.0 (28.9-54.0) & 50.0
(30.0-55.6) & 41.7 (28.9-50.0) & 40.0-(25.0-50.0) & 30.0 (25.0-66.7) &
40.0 (30.0-50.0) & 43.3 (30.0-54.8)\tabularnewline
Mucus score (0-5) & 1 (1-2) & 2 (1-3) & 2 (1-3) & 2 (1-3) & 2 (1-3) & 2
(0-3) & 3 (2-4) & 2 (1-3)\tabularnewline
\% BAL macrophages & 68.8 (48.9-76.5) & 58.0 (52.0-65.0) & 66.0
(59.5-72.5) & 57.0 (52.5-62.0) & 52.5 (46.6-55.8) & 68.0 ( 59.8-72.9) &
35.5 (24.0-45.0) & 54.5 (43.0-64.5)\tabularnewline
\% BAL lymphocytes & 24.5 ( 16.9-32.5) & 22.5 (16.5-27.5) & 21.5
(15.0-27.5) & 23.0 (16.6-27.5) & 23.0 (19.0-29.4) & 10.7 (2.0-16.9) &
13.8 (7.5-20.0) & 20.0 (13.4-26.5)\tabularnewline
\% BAL neutrophils & 5.0 (2.0-7.0) & 14.0 (7.5-19.0) & 4.0 (2.0-7.0) &
17.0 (13.5-21.0) & 16.0 (13.0-19.0) & 3.3 ( 0.9-6.4) & 43.5 (33.0-62.5)
& 16.5 (7.0-33.0)\tabularnewline
\% BAL mast cells & 2.3 (1.0-3.5) & 5.0 (2.0-7.0) & 7.0 (6.0-9.0) & 2.0
(1.0-3.0) & 7.0 (5.5-8.8) & 3.5 ( 0.9-3.9) & 1.5 (0.0-2.5) & 2.5
(1.0-5.0)\tabularnewline
\% BAL eosinophils & 0.0 (0.0-0.5) & 0.5 (0.0-1.0) & 0.0 (0.0-0.5) & 0.5
(0.0-0.5) & 0.8 (0.0- & 12.5 ( 5.3-33.3) & 0.0 (0.0-0.6) & 0.0
(0.0-0.5)\tabularnewline
\bottomrule
\end{longtable}

BAL = bronchoalveolar lavage; MEA = mild-moderate equine asthma; SEA =
severe equine asthma. Age reported as mean and standard deviation, all
other parameters are reported by median and 25-75 interquartile range
due to data not being normally distributed.\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/Figure-mucus-clinical-signs/Figure-mucus-clinical-signs}

\end{center}
\end{figure}\selectlanguage{english}
\begin{figure}[H]
\begin{center}
\includegraphics[width=0.70\columnwidth]{figures/figure-2/figure-2}

\end{center}
\end{figure}

\selectlanguage{english}
\FloatBarrier
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