2.2. Examination of Affected Fish and Collection of Parasites

Necroscopy of the affected fish suggested a severe infestation of the trophonts stages of Piscinoodinium sp. in the gills, fins, and body surfaces. The gills along, with the arch, were carefully placed on a Petri plate and observed under a stereomicroscope (Olympus SZ61, Olympus, Tokyo, Japan) to examine the parasite burden. The sizes of the trophont stages of the parasites were measured using CellSens Imaging Software version 1.17 (Evident Corporation, Nagano, Japan). Subsequently, the gills were washed and aspirated with distilled water, and the parasites were carefully collected in a sterile Petri plate using a Pasteur pipette under the stereomicroscope. The trophonts were further washed in two changes of distilled water and collected in a 1.5-mL microcentrifuge tube (Eppendorf, Hamburg, Germany). The trophonts from all five fish were pooled together. The tube was centrifuged at 5000 rpm for 10 min and the supernatant water was discarded. The parasites were then preserved in 100% ethanol and stored at −20 °C for molecular analysis. Samples were also collected from the sampled fish for routine bacterial and viral diagnostics. Briefly, nucleic acid (DNA and RNA) was extracted from kidneys, gills, and brains of the infected animals. DNA was extracted using the salting-out method [14], and RNA was extracted using TRIzol reagent (Invitrogen Carlsbad, USA). The cDNA synthesis was performed from the extracted RNA using the Verso cDNA kit (Thermo Scientific, Vilnius, Lithuania). Subsequently, polymerase chain reaction (PCR) was conducted to check for the presence of viruses, such as cyprinid herpesvirus 2 (CyHV-2) [15], carp edema virus (CEV) [16], infectious-spleen-and-kidney-necrosis virus (ISKNV) [17], and viral nervous necrosis (VNN) [18]. The presence of bacterial infection was examined by aseptically collecting samples from the kidney and spleen of the animals and culturing them in the nutrient medium for bacterial growth.