Improving the diagnosis of Streptococcus iniae using a novel probe-based qPCR assay combined with an enrichment step.

Streptococcus iniae is a bacterial pathogen that causes streptococcosis, leading to significant losses in fish aquaculture globally. This study reported a newly developed probe-based quantitative polymerase chain reaction (qPCR) method for the detection of S. iniae. The primers and probes were designed to target the lactate oxidase gene. The optimized method demonstrated a detection limit of 20 copies per reaction and was specific to S. iniae, as evidenced by no cross-reactivity when assayed against genetic materials extracted from 23 known aquatic animal pathogens, and fish samples infected with Streptococcus agalactiae or Streptococcus dysgalactiae. To validate the newly developed qPCR protocol with field samples, fish specimens were systematically investigated following the Food and Agriculture Organization of the United Nations & Network of Aquaculture Centres in Asia-Pacific three diagnostic levels approach, which integrated basic and advanced techniques for disease diagnosis, including observation of gross signs (level I), bacterial isolation (level II), qPCR and 16S rDNA sequencing (level III). The result showed that 7/7 affected farms (three Asian seabass farms and four tilapia farms) experiencing clinical signs of streptococcosis were diagnosed positive for S. iniae. qPCR assays using DNA extracted directly from fish tissue detected S. iniae in 11 out of 36 fish samples (30.6%), while 24 out of 36 samples (66.7%) tested positive after an enrichment step, including apparently healthy fish from affected farms. Bacterial isolation of S. iniae was only successful in a proportion of clinically diseased fish but not in healthy-looking fish from the same farm. Overall, the newly developed qPCR protocol combined with enrichment would be a useful tool for the diagnosis and surveillance of S. iniae infections in fish populations, thereby aiding in the disease control and prevention.