Retrospective diagnosis of archived marine fish experienced unexplained mortality reveals dual infections of Nocardia seriolae and Streptococcus iniae

Abstract

Due to the limitation of accessible laboratory facilities for disease diagnosis at the time of the events, clinical samples of snubnose pompano (Trachinotus blochii) and orange-spotted grouper (Epinephelus coioides) were frozen preserved for retrospective investigation. This study employed a simple challenged test using crude homogenate prepared from the long-term frozen preserved tissues (~ 16 months) to infect Asian sea bass (Lates calcarifer) model. The experimental fish became sick and exhibited visceral white spots similar clinical signs previously observed in natural cases. Two morphologically different Gram-positive bacteria were concurrently recovered from the experimentally sick fish but not directly from the archived frozen tissue. Based on sequence homology of 16S rRNA, the bacterial strains were identified as Nocardia seriolae and Streptococcus iniae, respectively. Subsequent specific PCR assays confirmed co-infections of N. seriolae and S. iniae in the majority of naturally diseased fish preserved. Concurrent infection of N. seriolae and S. iniae is new to both pompano and grouper. This study also suggests that frozen preservation combined with challenged assay might be useful for retrospective diagnosis and recovery of infectious agents in under-equipped laboratories in Southeast Asia or elsewhere.