Czapek yeast agar

Acetonitrile, ethyl acetate, formic acid and methanol were purchased from Kefo (Sisak, Croatia); Czapek Yeast Agar (CYA) was purchased from Oxoid (Hampshire, UK). Medium RPMI 1640, fetal bovine serum (FBS), phosphate buffered saline (PBS; without Ca²⁺ and Mg²⁺), trypsin-EDTA, antibiotics penicillin and streptomycin were from Lonza (Basel, Switzerland). The MTT reagent [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], acridine orange, ethidium bromide, normal melting point agarose (NMP), low melting point agarose (LMP), phorbol 12-myristate 13-acetate (PMA), Triton X-100 and Tris buffer were purchased from Sigma-Aldrich (Deisenhofen, Germany) and molecular biology grade dimethyl sulfoxide (DMSO), used for dissolving the extracts and for the control treatment, was purchased from Sigma-Aldrich (Deisenhofen, Germany); technical grade DMSO, used for dissolving the formazane, ethanol, sodium chloride, disodium-EDTA and sodium hydroxide were from Kemika (Zagreb, Croatia).

Tracheal wash, BALF specimens, serum, and material collected from guttural pouches and necropsy were examined at the Public Health Institute in Ostrava (Czechia) and the Czech Academy of Sciences. Aspergillus spp. isolates were grown on Sabouraud dextrose agar (Merck, Darmstadt, Germany), malt extract agar (Oxoid, Basingstoke, UK), or Czapek yeast agar (Oxoid, Basingstoke, UK) at 26 and 37 °C. BDG (Fungitell^®, Associates of Cape Cod, Falmouth, MA, USA) and GM (Aspergillus EIA, Bio-Rad Platelia™, Marnes-la-Coquette, France) were quantified following the manufacturer’s instructions for use with human samples. BDG concentration was determined in pg/mL, GM was set as a routinely widely used positivity index (PI). Cut-off values for horses have not been previously described.