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Piscine lactococcosis is an emergent bacterial disease that is associated with high economic losses in many farmed and wild aquatic species worldwide. Early and accurate detection of the causative agent of piscine lactococcosis is essential for management of the disease in fish farms. In this study, a TaqMan quantitative polymerase chain reaction (qPCR) targeting the 16S-23S rRNA internal transcribed spacer region was developed and validated. Validation of the qPCR was performed with DNA of previously typed L. petauri and L. garvieae recovered from different aquatic hosts from distinct geographical locations, closely related bacterial species and common pathogens in trout aquaculture. Further diagnostic sensitivity and specificity was investigated by screening of fish, water and faecal samples. The developed qPCR assay showed high specificity, sensitivity and accuracy in detection of L. petauri and L. garvieae with lack of signals from non-target pathogens, and in screening of rainbow trout (Oncorhynchus mykiss) posterior kidney and environmental samples. The detection limit of the qPCR was four amplicon copies. Moreover, the sensitivity of the qPCR assay was not affected by presence of non-target DNA from either fish or environmental samples. The robustness, specificity and sensitivity of the developed qPCR will facilitate fast and accurate diagnosis of piscine lactococcosis to establish appropriate control measures in fish farms and aquaria.

The first isolation of a flavivirus from fish was made from moribund Chinook salmon (Oncorhynchus tshawytscha) from the Eel River, California, USA. Following the observation of cytopathic effect in a striped-snakehead fish cell line, 35-nm virions with flaviviral morphology were visualized using electron microcopy. Next-generation sequencing and rapid amplification of cDNA ends obtained the complete genome. Reverse transcriptase quantitative PCR (RT-qPCR) confirmed the presence of viral RNA in formalin-fixed tissues from the wild salmon. For the first time, in vivo replication of an aquatic flavivirus was demonstrated following intracoelomic injection in a Chinook salmon model of infection. RT-qPCR demonstrated viral replication in salmon brains up to 15 days postinjection. Infectious virus was then reisolated in culture, fulfilling Rivers’ postulates. Only limited replication occurred in the kidneys of Chinook salmon or in tissues of rainbow trout (Oncorhynchus mykiss). The proposed salmon flavivirus (SFV) has a 10.3-kb genome that encodes a rare dual open reading frame, a feature uncharacteristic of classical flaviviruses. Phylogenetic analysis places SFV in a basal position among a new subgroup of recently recognized aquatic and bat flaviviruses distinct from the established mosquito-borne, tick-borne, insect-only, and unknown-vector flavivirus groups. While the pathogenic potential of the virus remains to be fully elucidated, its basal phylogeny and the in vivo infection model will allow SFV to serve as a prototype for aquatic flaviviruses. Ongoing field and laboratory studies will facilitate better understanding of the potential impacts of SFV infection on ecologically and economically important salmonid species. IMPORTANCE Chinook salmon are a keystone fish species of great ecological and commercial significance in their native northern Pacific range and in regions to which they have been introduced. Threats to salmon populations include habitat degradation, climate change, and infectious agents, including viruses. While the first isolation of a flavivirus from wild migrating salmon may indicate an emerging disease threat, characterization of the genome provides insights into the ecology and long evolutionary history of this important group of viruses affecting humans and other animals and into an expanding group of recently discovered aquatic flaviviruses.