Bronchoalveolar lavage
The BAL was performed using a flexible 320 cm long videoendoscope (AOHUA, Medimage off-site; OLYMPUS in-house). After visualization of the carina, the endoscope was inserted into the right main bronchus until wedged. A total of 500 mL (two boluses of 250 mL each) of warmed (37°C) sterile isotonic saline solution was instilled through the endoscope biopsy channel, using 60 mL pre-filled syringes. After each bolus, the liquid was aspirated with the same syringes, and the first 20 mL corresponding to the endoscope channel volume that did not reach the lung was systematically discarded. At the end of lavage of the first lung side, the endoscope instrument channel was cleaned with a 60 mL bolus of sterile isotonic saline; then the endoscope was moved back to the carina and introduced in the contralateral lung and lavage procedure was repeated.
The volume of liquid collected and macroscopic assessment were recorded. A proportional volume of BALF from each lung was pooled in a jar to obtain a pooled BALF.
Endoscopes were sterilized between each horse by flushing the channels and soaking the endoscopes in disinfectant (Hexanios G+R, Anios Laboratories) as per manufacturer’s recommendations. Left, right and pooled samples were then placed in a tube containing EDTA for cytology, and another aliquot of pooled samples was placed in a sterile tube for mycology. All samples were kept chilled until handled by the laboratory, and all analyses were performed within 24 hours of sampling.