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et al. 2010; Mazan 2018), but it has not yet been described in Brazilian pacer (‘Marchadores’) breeds such as Campolina and Mangalarga Marchador. These horses participate in a specific format of competition designed to test their resistance, riding comfort, manoeuvrability and ability to execute specific farm-related tasks. The competitions typically last between 2 and 4 h of continuous pacing with judging based on criteria elaborated by the Breeders Associations (ABCCMM and ABCCC). This study investigated the occurrence of subclinical EAS
in Campolina and Mangalarga Marchador horses through endoscopy and BALF collection for cytology conducted after a standardised exercise test in the field. Statistical queries were made to determine effects related to age group, sex and breed. Efficiency of different diagnostic criteria was also demonstrated.
Materials and methods
This study was approved by the Universidade Federal Fluminense Ethics Committee (CEUA-UFF) under the number 964/18. All privately owned horses had informed written consent provided by the responsible person before the start of the experiment.
Horses Thirty Brazilian pacers (‘Marchadores’) from two Campolina (Campo) and four Mangalarga Marchador (MM) stud farms were included in this study. Fifteen Campo and 15 MM, all in training and considered fit to participate in competitions, aged 3–18 years, with no recent history (6 months) of respiratory illness and no clinical signs of active respiratory disease, were chosen by their owners to participate in this research. Horses were picked due to their clinical and athletic status, based on owner convenience. Apart from the inclusion diagnostic criteria described above, horses had to be sound (no lameness), free of systemic diseases, with a normal haematology examination and not pregnant if female.
Physical examination, endoscopic mucus scoring and bronchoalveolar lavage fluid collection Each horse underwent a complete clinical examination by an experienced equine veterinarian before being authorised to participate in the study. Venous blood for a complete blood count and differential leucocyte count was collected prior to the study in EDTA vacuum tubes (BD Vacutainer)1. Horses allowed to participate in the study were subjected
to a standardised ridden exercise programme consisting of a brief warm-up at the walk and 30 min of fast pacing (between 12 and 14 km/h), alternating left and right reins. All exercise tests were conducted on sand at the properties where the animals were housed. Immediately before, immediately after and 30 min after the exercise, heart rate (HR), respiratory rate (RR) and rectal temperature (T) were measured and recorded. Thirty minutes after the end of the standardised exercise
programme, horses were sedated with 10 µg/kg of intravenous detomidine and respiratory endoscopy and BALF collection were conducted. Endoscopy was done according to prescribed techniques (Gerber et al. 2004b) using an Olympus Fibrescope model CF-10L2. Visible mucus in the trachea was classified by the same observer according to
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Gerber (Gerber et al. 2004a) as follows: 0 (no visible mucus), 1 (single blob or multiple small nonconfluent blobs), 3 (confluent or stream forming mucus), 4 (pool-forming mucus) and 5 (profuse amounts of mucus). Immediately after the endoscopy, BALF was collected
according to prescribed techniques (Hoffman 2008) using 250 mL of warmed saline for infusion through a silicone BAL tube (MILA, 300 cm, 2.5 mm)3. BALF was processed and analysed according to the Consensus guidelines (Cou€
etil
et al. 2016). In brief, samples were collected into sterile laboratory grade glass vials, placed on ice and immediately an aliquot of 250 µL was placed on a cytocentrifuge (Serocito, Fanem)4 and spun at 110 g for 5 min to produce cytology slides. Slides were stained with Romanowsky and Giemsa stains for differential cell counts (300 cells counted at 10009 magnification in a Bioval L2000 optic microscope)5. For the investigation of the effect of age on the diagnosis
of EAS, the horses were subdivided into two groups: those up to and including 7 years of age and those older than 7 years of age. This division was based on criteria published in the Consensus for EAS (Cou€
etil et al. 2016). Occurrence of EAS in males in comparison with females
and in Campolina horses in comparison with Mangalarga horses was also investigated.
EAS diagnostic criteria All horses with one of more of the following findings were considered EAS-positive: BALF neutrophils above 5%, eosinophils above 1%, mast cells above 2%, total nucleated cell count (TCC) above 530 cells/µL and tracheal mucus above grade 2. Only horses with all values below these cut- off points were considered healthy. The authors chose neutrophil percentages in the BALF as the criteria for classification into mild (5–10%), moderate (11–25%) and severe (above 25%) EAS, given the Consensus does not provide a guideline for such classification.
Statistical analyses Statistical analyses were conducted using Minitab 1.8 and GraphPad 8 software6. Individual results as well as averages and standard deviations for BALF cytology, HR and RR were analysed, and descriptive statistics are presented. For comparison of cell percentages in the BALF in each of
the breeds, a Student t test was used. For comparison of number of EAS-positive horses by age group, Fisher’s exact test was used. For comparison of mucus score between breeds, age groups, sex and state (healthy vs. EAS-positive), Mann–Whitney test was chosen. ANOVA was used to compare HR, RR and T between groups and state, followed by Tukey’s test for multiple comparisons. For comparison between diagnostic criteria, a chi-square test was used. For all statistical tests, a significance level of 95% (P<0.05) was used.
Results
Mean values and standard deviation (s.d.) for age; volume of fluid recovered during BAL procedure (mL); TCC in BALF (cells/µL); and percentages of lymphocytes, macrophages, neutrophils, mast cells and epithelial cells in BALF are shown in Table 2. There was no significant difference between the ages of the Mangalarga and Campolina horses when grouped (P = 0.12) or separated by sex (P = 0.27). A Student t
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