Piscine lactococcosis poses a significant threat to a wide range of cultured and wild fish populations on a global scale . The disease routinely manifests as per-acute to acute hemorrhagic septicemia and is often associated with high levels of morbidity and mortality. Until recently, Lactococcus garvieae was thought to be the only causative agent of the piscine lactococcosis; however, further research has proven that the disease episodes are attributed not only to L. garvieae but also to the other two highly pathogenic Lactococcus sp. known as L. petauri and L. formosensis. These three bacterial species share phenotypic and genetic similarities that often lead to diagnostic challenges due to substantial misidentification using traditional microbiological and molecular methods . Thus, rapid, sensitive, and specific molecular diagnostic tools for correct differential diagnosis of the a etiologies of lactococcosis i n fish are warranted.
In the current study, we successfully developed a novel TaqMan species-specific multiplex qPCR for simultaneous identification and quantification of L. garvieae , L. petauri and L. formosensis following pangenome analysis of publicly available genomes of these bacterial species. The newly developed assay showed high sensitivity and specificity when tested with 139 bacterial isolates, representing the three bacterial species, recovered from multiple cultured fish species, originated from various geographical locations , including Mediterranean area (Turkey, Italy, Spain, Greece), Northern Europe (Norway), North America (USA) and South America (Brazil), along with a representative panel of closely related and non-related bacterial species. In addition, a successful detection and identification of the bacterial species was achieved using the novel multiplex assay on 126 field tissue samples. from clinical lactococcosis outbreaks, including tissues preserved in RNAlater, archived formalin fixed paraffin embedded (FFPE) tissues and tissues fixed on FTA cards . T he new molecular assay performed better at diagnosing piscine lactococcosis than the gold standard protocols currently in use in laboratories .
Overall, the multiplex qPCR assay developed in the current study provides a reliable, rapid, sensitive, and species- specific molecular method for diagnosis, genomic typing, and differentiation of the closely related etiological agents of piscine lactococcosis. Using this robust technique will contribute to the accurate diagnosis of the infection and will assist in the establishment of effective management strategies against lactococcosis in aquaculture.