Mixed infection of Pseudomonas aeruginosa and Non-O1/O139 Vibrio cholerae in tilapia eggs: Pathogen confirmation, synergistic virulence, and effective control measures

Nile tilapia ( Oreochromis niloticus ) egg survival is critical for hatchery productivity. In March 2023, a commercial hatchery in Hainan, China, experienced severe egg mortality, with hatching rates below 10%. This study identified the causative pathogens, evaluated pathogenicity, and developed control strategies. Eight bacterial isolates from two species were recovered from diseased eggs; the most virulent isolate of each species was selected via challenge testing (three replicates, 50 eggs each), yielding two dominant strains identified as Pseudomonas aeruginosa TLV-01 and non-O1/O139 Vibrio cholerae LHV-07 based on physiological, biochemical, and 16S rRNA analyses. Both isolates showed β-haemolytic activity, and LHV-07 tested negative for major cholera toxin genes (ctxA, toxR). Artificial infection assays showed both strains were pathogenic, with 7-day LD₅₀ values of 2.0 × 10⁸ CFU/mL (TLV-01) and 2.0 × 10⁶ CFU/mL (LHV-07). Co-infection markedly enhanced lethality, lowering the LD₅₀ to 2.0 × 10⁵ CFU/mL—1,000-fold lower than TLV-01 alone and 10-fold lower than LHV-07 alone. To assess deviation from expected independent effects, the Bliss independence model was applied as the primary analytical tool, with dose-response curves fitted using a four-parameter log-logistic model and forward interpolation used to estimate expected combined mortality. Results indicated weak synergism at low concentrations (1.0×10⁴–1.0×10⁵ CFU/mL), additive effects at 1.0×10⁶ CFU/mL, and a lethality saturation effect at high concentrations (1.0×10⁷–1.0×10⁸ CFU/mL), likely due to microenvironmental resource limitation. The Loewe additive model, used for cross-validation, showed combination index (CI) values below 1 (0.021–0.662) across 1.0×10⁴–1.0×10⁷ CFU/mL, exceeding 1 at 1.0×10⁸ CFU/mL—consistent with the Bliss model trend. These findings confirm a potentiating effect of co-infection on lethality, providing a quantitative basis for hatchery risk assessment. Antibiotic susceptibility testing revealed multidrug resistance in TLV-01, while LHV-07 remained broadly susceptible. Disinfection trials identified chloramine-T (4 mg/L) as most effective, raising hatchability to 65.2 ± 2.5% without larval deformities. This study provides the first evidence of concurrent co-infection with P. aeruginosa and non-O1/O139 V. cholerae in tilapia eggs, linked to increased mortality, and offers practical antimicrobial and disinfection strategies to improve hatchery biosecurity and the sustainability of seed production.