**Background:** Saprolegniosis, caused primarily by Saprolegnia parasitica, is a fungal disease causing significant economic losses in freshwater aquaculture. Current control relies on chemical treatments with environmental and human health concerns. Probiotics have emerged as a biological alternative, but their efficacy must be evaluated for specific host-pathogen combinations. Three bacterial strains—Enterococcus gallinarum L1 (isolated from sea bass gut), Vagococcus fluvialis L21 (isolated from sole gut), and Lactobacillus plantarum CLFP3 (isolated from rainbow trout cutaneous mucus)—had previously demonstrated protective effects against Vibrio anguillarum in sea bass or Lactococcus garvieae in rainbow trout. This study investigated their potential for biocontrol of saprolegniosis in rainbow trout.
**Methods:** In vitro assays assessed inhibition of S. parasitica hyphal growth on solid BHI agar (plate assay) and in liquid broth (hemp seed test), inhibition of cyst germination, fungicidal effects, and activity of bacterial culture supernatants. Adhesion of bacteria to brown trout cutaneous mucus and their ability to reduce S. parasitica cyst adhesion under exclusion, competition, and displacement conditions were quantified using fluorescent staining. Pathogenicity of L1 and L21 for rainbow trout was tested via intraperitoneal and intramuscular injection (10^7 cells ml^-1). For in vivo biocontrol, rainbow trout (mean weight 28.79–29.29 g) were infected with S. parasitica zoospores (3 × 10^2 spores ml^-1) after skin scarification (ami momi treatment). Bacteria were administered either in tank water (10^6 CFU ml^-1, repeated every 24 h for 14 days) or in feed (10^8 CFU g^-1, fed at 2% biomass daily for 14 days pre-infection and 10 days post-infection). Survival was analyzed by Kaplan-Meier and log-rank tests (p ≤ 0.05).
**Key Results:** In solid medium, all three bacteria inhibited mycelial growth, with L. plantarum CLFP3 showing the strongest effect (zero colony diameter from 3-day bacterial culture onward; control colonies reached 5.17–5.70 cm). In liquid broth, E. gallinarum L1 and V. fluvialis L21 partially inhibited mycelial growth at ≥2 × 10^4 bacteria ml^-1, while L. plantarum CLFP3 did not inhibit mycelial growth at any concentration. All three inhibited cyst germination: L1 and L21 at >4 × 10^3 bacteria ml^-1, CLFP3 only at 4 × 10^5 ml^-1. Fungicidal effects were concentration- and time-dependent; with 3 days incubation, lethality was observed up to 2 × 10^3 bacteria ml^-1. Culture supernatants only inhibited mycelial growth when undiluted (L1 also at 1:2 dilution) and did not inhibit cyst germination. Adhesion to cutaneous mucus was low (L1: 9.92%, L21: 14.60%, CLFP3: 7.54%) with no significant differences versus control substrates. In competition assays, all three bacteria reduced cyst adhesion at lower concentrations than in exclusion or displacement tests; V. fluvialis L21 at 2.5 × 10^7 cells ml^-1 reduced adhesion by 73.86% (competition) and 93.24% (displacement). L. plantarum CLFP3 at 2.5 × 10^7 cells ml^-1 reduced adhesion by 87.50% in exclusion. Both L1 and L21 were non-pathogenic for rainbow trout. In vivo, none of the three probiotics provided protection. First macroscopic lesions appeared 24–48 h post-infection, first deaths on day 3, and cumulative mortality reached 100% between days 6 and 9 in both treated and untreated groups, with no statistically significant differences (log-rank test, p > 0.05). Control groups (uninfected) had 100% survival except for one trout in the L1 water experiment.
**Clinical Implications:** This study demonstrates that probiotics effective against one disease (vibriosis, lactococcosis) may be ineffective against another (saprolegniosis) even in the same or related host species. Despite promising in vitro inhibition of pathogen growth and adhesion, none of the three strains conferred in vivo protection. The results highlight a critical limitation: in vitro assays, while useful for screening, cannot reliably predict in vivo efficacy for saprolegniosis biocontrol. The findings suggest that successful probiotic selection must be empirically validated for each specific host-pathogen system, and that mechanisms of action (e.g., competitive exclusion, siderophore production, immune stimulation) may differ between target diseases. The study reinforces the need for disease-specific probiotic screening and cautions against extrapolating efficacy across different pathogens based on in vitro data alone.