Isolation, Identification, Virulence and Pathogenic Features of Lactococcus garvieae from Cage-Cultured Tilapia...

Lactococcosis caused by Lactococcus garvieae is an emerging threat to warmwater aquaculture, yet evidence integrating field outbreaks with robust molecular confirmation and controlled virulence testing remains limited for Thailand's cage-cultured tilapia. From May to October 2025, acute mortality events were investigated in cage-cultured Nile tilapia (Oreochromis niloticus) in a reservoir in Ubon Ratchathani Province, Thailand. Suspected outbreaks were defined by abrupt daily mortality exceeding 5% accompanied by septicemia-like clinical signs. Water quality during sampling covered the following ranges: temperature 28.6-31.9 °C, pH 6.5-7.0, salinity 0.02-0.03 ppt, electrical conductivity 0.036-0.046 mS/cm, TDS 22.20-26.50 mg/L, total alkalinity 17.0-34.0 mg/L as CaCO₃, total hardness 12.0-60.0 mg/L as CaCO₃, dissolved oxygen 6.5-7.0 mg/L, and NH₃ were below the limit of detection. Full-length 16S rRNA tissue profiling revealed strong tissue partitioning: blood microbiomes were consistently dominated by Lactococcus and L. garvieae at the species level, whereas gills showed higher richness and mixed communities with multiple opportunistic taxa. Culture isolation was more reliable from blood than gills, yielding 16 Gram-positive, catalase-negative isolates (AAHM-LG2501-AAHM-LG2516) that clustered within the L. garvieae clade in near full-length 16S rRNA phylogenetic analysis and were separated from closely related Lactococcus lineages. A representative blood isolate (AAHM-LG2501) showed dose-dependent virulence in controlled challenges, with an LD₅₀ of ~1.05 × 10⁵ CFU/fish by intraperitoneal injection and an LC₅₀ of ~1.20 × 10⁶ CFU/mL by immersion. Histopathology supported systemic dissemination, with injection producing more consistent multi-organ lesions than immersion, particularly in head kidney, liver, and spleen, while gills exhibited route-associated epithelial and vascular alterations. Together, these findings confirm L. garvieae as a major etiological agent of septicemic outbreaks in cage-cultured tilapia in Thailand and support a practical surveillance framework prioritizing blood sampling, molecular confirmation, and risk-based monitoring to guide biosecurity and vaccine-oriented prevention.