**Background:** Bacterial pathogens, particularly Aeromonas hydrophila, are major economic threats to aquaculture. The overuse of antibiotics has driven interest in alternative immunostimulatory feed additives. Chlorella vulgaris, a unicellular freshwater microalga, is rich in protein, polysaccharides, carotenoids, β-1,3 glucan, and phenolics with reported antibacterial, antiviral, antioxidant, and immunomodulatory activities. This study aimed to determine the optimum dietary level of Chlorella vulgaris as an immunostimulant and its protective role against A. hydrophila in Labeo rohita, a species contributing to two-thirds of carp polyculture production in India.
**Methods:** Crude ethanolic extract of Chlorella vulgaris was prepared and partially purified via silica gel column chromatography using ethyl acetate/hexane solvent systems, yielding nine fractions (CF1–CF9). Antibacterial activity of the crude extract and fractions was tested against four A. hydrophila strains (AH1–AH4), two Pseudomonas putida strains (PP1, PP2), and reference strains using the disc diffusion method (100 µg/disc) with streptomycin, tetracycline, and bacitracin as positive controls. For the feeding trial, 360 rohu fingerlings (25 ± 2 g) were acclimatized for 30 days and then allocated to four treatment groups in duplicate (45 fish per tank): C0 (control, 0 g/kg), C1 (0.1 g/kg), C2 (0.5 g/kg), and C3 (1.0 g/kg) Chlorella powder incorporated into basal feed for 90 days. Water quality was maintained at 28 ± 1 °C, pH 7.6, dissolved oxygen 6.0 ± 0.4 mg/L. Blood samples were collected at days 30, 60, 90, and 10 days post bacterial challenge. Non-specific immune parameters measured included serum bactericidal activity (viable colony count method), superoxide anion production (NBT assay), and lysozyme activity (turbidimetry method using M. luteus). Serum biochemical parameters (total protein, albumin, globulin) and haematological parameters (haemoglobin, total RBC and WBC counts) were assessed. Growth performance was evaluated using specific growth rate (SGR) and feed conversion ratio (FCR). At day 90, 20 fish per replicate were injected intraperitoneally with a lethal dose of A. hydrophila (1 × 10^5 CFU/fish) and mortality was recorded over 10 days. Statistical analysis used one-way ANOVA with Duncan's multiple range test (p < 0.05).
**Key Results:** Among the nine chromatographic fractions, CF2 showed maximum antibacterial activity against A. hydrophila AH4 (zone of inhibition: 15.67 ± 0.58 mm) and P. putida PP1 (13.0 ± 0 mm). CF5 showed the highest inhibition against P. putida PP2 (14.0 ± 1.73 mm). For immune parameters, superoxide anion production (NBT value) was significantly higher (p < 0.05) in the C3 group (0.045 ± 0.001) after bacterial challenge compared to control. Maximum lysozyme activity (750.00 ± 3.27 U/mL) was recorded in the C3 group on day 30, and all treated groups showed increased lysozyme activity after infection. Serum bactericidal activity was significantly enhanced (p < 0.05) in the C1 group (73.52%) on day 30 relative to control. Serum total protein was significantly elevated in C1 on day 30 and in all treated groups by day 90; post-infection, C2 showed significantly higher total protein. Globulin levels were significantly higher in all treatment groups on day 90, and C2 showed significantly increased immunoglobulin post-challenge. Haemoglobin was significantly increased in all treated groups on days 60 and 90 (except C1 on day 60). WBC count was significantly higher in treated groups on day 90 and post-challenge. RBC count was significantly higher in C2 and C3 on days 60 and 90. Growth performance improved with Chlorella supplementation: the highest SGR was in C3 (1.0 g/kg), and FCR values were 1.813 (C1), 1.692 (C2), and 1.609 (C3) versus control. Survival after A. hydrophila challenge was highest in C3 (86.5%), followed by C2 (81.65%), with zero cumulative mortality up to 24 h in all Chlorella-fed groups.
**Clinical Implications:** Dietary supplementation with Chlorella vulgaris at 0.5–1.0 g/kg of feed significantly enhances non-specific immune responses, growth performance, and survival against A. hydrophila infection in L. rohita. The improved bactericidal activity, lysozyme production, and respiratory burst activity indicate that Chlorella activates innate immune mechanisms, likely due to its bioactive compounds including β-1,3 glucan, carotenoids, and the chlorella growth factor (CGF). The 86.5% survival rate in the 1.0 g/kg group versus approximately 50% in controls demonstrates substantial protective efficacy. These findings support Chlorella vulgaris as a sustainable, antibiotic-free immunostimulant feed additive for aquaculture, particularly for carp polyculture systems. Further purification and characterization of the active antibacterial fractions (especially CF2 and CF5) could identify specific bioactive compounds for therapeutic development.