Development Of qPCR And ddPCR Assays For Sensitive Detection Of Streptococcus iniae And Application In Studying Infection Dynamics In Tilapia (Oreochromis spp.)

Authors: Ma, Jie; Lafrentz, Benjamin

Submitted to: Annual Eastern Fish Health Workshop
Publication Type: Abstract Only
Publication Acceptance Date: 3/23/2026
Publication Date: 3/26/2026
Citation: Ma, J., Lafrentz, B.R. 2026. Development Of qPCR And ddPCR Assays For Sensitive Detection Of Streptococcus iniae And Application In Studying Infection Dynamics In Tilapia (Oreochromis spp.). Annual Eastern Fish Health Workshop [ABSRACT]. 49th Annual Eastern Fish Health Workshop

Interpretive Summary: Streptococcus iniae is a major pathogen in tilapia aquaculture, responsible for systemic infections and substantial economic losses. This study introduces highly sensitive molecular assays—real-time qPCR and droplet digital PCR (ddPCR)—targeting the conserved groEL gene of S. iniae. The ddPCR method demonstrated exceptional specificity, detecting as few as five genomic copies and effectively distinguishing S. iniae from other aquatic microbes and host tissues. Infection profiling revealed rapid colonization of the spleen, intense proliferation in the brain, and persistent presence in the kidney, suggesting organ-specific dynamics and potential reservoirs. Additionally, waterborne shedding peaked at 48 hours post-infection and persisted at low levels for seven days, indicating ongoing environmental contamination. These findings provide reliable tools for disease surveillance, outbreak tracking, and biosecurity enhancement in commercial fish farming.

Technical Abstract: Streptococcus iniae is a major bacterial pathogen in aquaculture, responsible for systemic infections and significant economic losses in different industries, including the global tilapia (Oreochromis spp.) industry. In this research, we developed and optimized real-time quantitative PCR (qPCR) and droplet digital PCR (ddPCR) assays, targeting the conserved groEL gene, for sensitive and specific quantification of S. iniae. Both qPCR and ddPCR reliably distinguished S. iniae isolates from other aquatic bacteria, with a detection limit of 100 fg/reaction (approximately 46 genomic copies) and 10 fg/reaction (approximately 5 genomic copies), respectively. Organ-level bacterial quantification revealed early and intense colonization of the spleen post-infection, followed by exponential proliferation in the brain. This suggests the spleen is a highly susceptible, infection prone organ during the initial systemic phase, and the brain is a major replication site during systemic infection. The liver exhibited minimal bacterial burden, while the kidney exhibited stable bacterial loads and retained detectable S. iniae DNA at 7 d post-challenge (dpc), suggesting its role as a potential long-term reservoir. Waterborne shedding of S. iniae was observed and peaked sharply at 48 h post-challenge, corresponding with elevated systemic bacterial loads. Low-level persistence of S. iniae DNA in tank water through 7 dpc suggests chronic environmental contamination from infected hosts. Together, the molecular assays and infection profiling established in this research provide effective tools for sensitive detection and surveillance of S. iniae in commercial tilapia production.