Earle s salts

The neutralising antibody assay was performed using the sera of all 8 ponies collected at D − 5 and D + 1 to D + 20 according to the OIE method [58 ]. Briefly, seven twofold serial dilutions of each serum from 1:4 to 1:256 were performed in duplicate on 96-well plates using complemented MEM with Earle’s salts (Eurobio). The EHV-1 strain KyD (ATCC^® VR-700^TM) was added to each dilution at a concentration of 100 TCID₅₀/well, and plates were incubated at 37 °C for one hour. Virus and sera mixtures were then transferred on RK13 monolayer cells seeded in 96-well plates and incubated at 37 °C and 5% CO₂. After 3 days of culture, cells monolayers were examined using an inverted microscope. The SN titre was estimated as the last dilution of serum neutralising the virus (no cytopathic effect formation).

Nasopharyngeal samples were collected at D − 5, each day from D + 1 to D + 10 and at D + 12, D + 14, D + 16, D + 18 and D + 20 using 745 mm sterile swabs for mares (IMV Technologies, L’Aigle, France). Swabs were inserted through the nostrils up to the nasopharynx, alternating the left and right nostril each day. Swabs were then placed directly into polypropylene tubes containing 3 mL of minimum essential medium (MEM) with Earle’s salts (Eurobio) complemented with 1% L-glutamine (Eurobio), 100 IU/mL penicillin, 0.1 mg/mL streptomycin and 0.25 μg/mL amphotericin B (Eurobio). Nasopharyngeal swabs were then vortexed and centrifuged for clarification, and the supernatant was frozen at −20 °C for viral titre determination and viral quantitation.

RK13 cells were cultivated in MEM with Earle’s salts (Eurobio) complemented with 10% foetal bovine serum (Eurobio), 1% L-glutamine (Eurobio), 100 IU/mL penicillin, 0.1 mg/mL streptomycin and 0.25 μg/mL amphotericin B (Eurobio) at 37 °C and 5% CO₂. Subsequently, 1 × 10⁶ PBMCs were seeded on RK13 monolayer cells on 6-well plates and incubated at 37 °C for 72 h. The presence of viral cytopathic effects was observed using an inverted microscope, and plates were frozen at −80 °C for viral quantitation. Eight tenfold serial dilutions of swab samples were inoculated in triplicate on RK13 monolayer cells on 96-well plates and incubated at 37 °C for 7 days. Wells with cytopathic effects were recorded, and the TCID₅₀ of each sample was determined using the Spearman–Kärber method [54 (link)].