**Background:** Free-living amoebae (FLA) are ubiquitous protozoa that can cause disease in fish, particularly nodular gill disease (NGD) in freshwater species. Acanthamoeba is a known pathogen in humans, causing granulomatous amoebic encephalitis and keratitis, but its role in ornamental fish diseases is poorly understood. Discus fish (Symphysodon aequifasciatus) are popular ornamental fish, and outbreaks of NGD have been reported globally. This study aimed to identify and characterize FLA from diseased discus fish in Iran using clinical, histopathological, and molecular methods.
**Methods:** From February 2020 to February 2022, 40 discus fish (8 per farm, including 2 apparently normal and 6 with clinical signs) were collected from five freshwater ornamental fish farms in different regions of Iran. Fish exhibited lethargy, anorexia, increased opercular ventilation, mucus hypersecretion on gills and skin, and up to 70% monthly mortality. Wet smears of skin, fins, and gills were examined microscopically. After euthanasia, necropsy was performed, and bacterial and fungal cultures from kidney, spleen, and liver were negative. Amoeba culture was performed on 1% non-nutrient agar from skin, gills, and intestinal mucosa, incubated at 25–30°C for up to 60 days. Histopathology of skin, gills, and internal organs was conducted using H&E, PAS, and Masson's trichrome stains. DNA was extracted from skin and gill tissue homogenates, and PCR was performed using specific primers for Acanthamoeba (JDP1 & 2 targeting SSU rRNA, ~500 bp), Vahlkampfiids (ITS1 & 2), and Vermamoeba (Hv1227F & Hv1728R). Positive amplicons were sequenced, and phylogenetic analysis was performed using MEGA 7.0 with 1000 bootstrap replications.
**Key Results:** Wet smears revealed numerous amoebic-like organisms on skin and gills. No bacterial or fungal growth was observed. Histopathology of gills showed lamellar epithelial hyperplasia, extensive fusion of secondary lamellae, inflammatory cell infiltration (mainly lymphocytes), and round to oval interlamellar vesicles. Amoebic trophozoites (20–25 μm) and cysts (12–16 μm, thin to thick-walled with round nucleus) were embedded in hyperplastic epithelium. Cysts stained positively with Masson's trichrome. Skin showed hyperplasia of goblet cells, PAS-positive. Intestinal tissue revealed trophozoites with foamy cytoplasm and eosinophilic granular cells, indicating amoebic enteritis. Other internal organs were normal. PCR using JDP1 & 2 primers yielded ~500 bp amplicons from skin and gill homogenates, confirming Acanthamoeba. Sequencing of five isolates (GenBank accession numbers OP910247, OP910249, OP910251, OP910248, OP910250) showed 99.5–100% identity with Acanthamoeba genotype T4 sequences. Phylogenetic analysis clustered all isolates with genotype T4. Treatment with chloramine-T (0.01 g/L for 8 h every other day) reduced mortality and infection intensity but did not eliminate infestation; other treatments (salt bath, formalin, metronidazole, antibiotics) were ineffective.
**Clinical Implications:** This is the first molecular identification of Acanthamoeba genotype T4 in discus fish in Iran, confirming its role in NGD. The presence of cysts in gills, not previously reported in fish NGD, resembles human Acanthamoeba infections. The zoonotic potential is significant, as Acanthamoeba T4 is a common human pathogen. The failure of standard treatments highlights the need for effective control measures, such as chloramine-T. The study underscores the importance of monitoring FLA in ornamental fish farms and implementing biosecurity protocols to prevent transmission to humans.