25 cm2 cell culture flasks

Rainbow trout RBCs were obtained and purified as previously described [6 (link)]. Briefly, RBCs obtained from the caudal vein were purified by Ficoll density gradient centrifugation (Ficoll 1.007; Sigma-Aldrich, Madrid, Spain). Purified RBCs were cultured in 25 cm² cell culture flasks (Nunc, Roskilde, Denmark) with RPMI-1640 medium (Dutch modification) (Gibco, Thermo Fischer Scientific, Waltham, MA, USA) supplemented with gamma- irradiated 10% FBS (fetal bovine serum) (Cultek, Madrid, Spain), 1 mM pyruvate (Gibco), 2 mM l-glutamine (Gibco), 50 μg/mL gentamicin (Gibco), 2 μg/mL fungizone (Gibco), 100 U/mL penicillin, and 100 μg/mL streptomycin (Sigma-Aldrich) at 14 °C for 24 h before experimentation.
The fish cell line TPS-2 (rainbow trout stromal pronephros cell line) [17 ], donated by Dr. AJ Villena, was also used in this work. TPS-2 cells were maintained in RPMI medium containing 20% FBS, 1 mM pyruvate, 2 mM l-glutamine, 50 µg/mL gentamicin and 2 µg/mL fungizone at 21 °C.
The EPC (Epithelioma Papulosum Cyprini) cell line [18 (link)] was purchased from the Americal Type Culture Collection (ATCC, CRL-2872). EPC cells were maintained in RPMI medium containing 10% FBS, 1 mM pyruvate, 2 mM l-glutamine, 50 μg/mL gentamicin, and 2 μg/mL fungizone at 28 °C.

Axenic culture was established in 6 Acanthamoeba spp. isolates among 10 culture positive isolates following the procedure as described earlier [21 ]. We were unable to establish axenic culture in other four positive isolates due to fungal contamination. Briefly, Petri plates containing 2 % non-nutrient agar medium overlaid with live E. coli were treated under UV light in a laminar hood for 30 mins to kill the live bacteria. A small piece of culture of Acanthamoeba spp. from the previously cultured plate was placed face down on the surface of the UV treated plate and incubated at 30 °C for 7 days. Subsequently a small piece of agar containing amoebas from the UV-treated plate was transferred to 25 cm² cell culture flasks (Nunc) containing 10 ml of PYG growth medium [proteosep peptone (0.75 %), yeast extract (0.75 %) and glucose (1.5 %)] (pH = 7.4) with antibiotics (penicillin and streptomycin) in bactericidal concentrations and incubated at 30 °C for 5 days [21 ]. Flasks were examined daily for 5–10 days under an inverted microscope (Nikon) until full growth of amoebas was seen in the medium. Initial culture flasks were subsequently sub cultured in 25 cm² cell culture flasks containing PYG medium with antibiotics for at least 3 consecutive passages to achieve complete axenization.

Vero CCL-81 (ATCC CCL-81) cells were seeded in 24-well tissue culture plates (Nunc, Roskilde, Denmark) to 80% confluence in Gibco Dulbecco’s modified Eagle’s medium (DMEM) containing 10% Gibco fetal bovine serum (FBS), 2% Gibco l-glutamine (200 mM), 2% Gibco antibiotic-antimycotic (100×). The cells were rinsed with Gibco PBS, pH 7.4, and 50 μL serum was added followed by incubation at 37°C, 5% CO₂ (New Brunswick Galaxy 170 R CO₂ Incubator Series, Eppendorf, USA) for 1 h, rocking after every 15 min to allow virus adsorption. After incubation, Gibco DMEM maintenance medium (MM) with 5% Gibco FBS, 2% Gibco l-glutamine (200 mM), and 2% Gibco Antibiotic-Antimycotic (100×) was added. Cells were incubated at 37°C, 5% CO₂ (New Brunswick Galaxy 170 R CO₂ Incubator Series, Eppendorf, USA) and observed daily for signs of cytopathic effects (CPE). The CPE positive sample was passaged in 25-cm² cell culture flasks (Nunc, Roskilde, Denmark) and frozen at –80°C before harvesting by thawing and centrifuging at 3,000 rpm for 10 min. The infectious supernatant was stored at –80°C until further use.