Use of tuf gene sequencing for Identification of Lactococci isolated from diseased rainbow trout (Oncorhynchus mykiss)

Abstract

Lactococcosis is a bacterial infection that is characterized by the appearance of septicemia in fish. Traditionally, the identification of causes of disease outbreaks in aquaculture poses a major challenge due to their comparable characteristics. In this study, simple PCR and sequencing based on the tuf gene were developed to identify isolates belonging to the Streptococcaceae family. The outbreaks of lactococcosis in rainbow trout have been mainly associated with Lactococcus garvieae, but the emergence of closely related species, Lactococcus petauri and Lactococcus formosensis pose a challenge in accurate identification. The study aimed to identify Gram-positive catalase-negative cocci and differentiate between Lactococcosis-causing bacteria (LCB) in rainbow trout isolates associated with lactococcosis outbreaks in Iran. Seventy-five Gram-positive cocci strains from diseased rainbow trout were tested using a PCR and sequencing method targeting the tuf gene. Sequence and phylogenetic analyses were conducted to distinguish between closely related Lactococcus species. The PCR results revealed a successful amplification with tuf gene primers for all strains, allowing species identification. The sequence analysis showed that all 29 isolates initially identified as L.garvieae were 100% identical to L.petauri, indicating a previous misclassification. This work is the first report by L.petauri of Iran. Analysis of tuf gene sequences revealed different patterns of single nucleotide polymorphism (SNP) for accurate species differentiation and phylogenetic analysis confirmed different clusters for LCB. The study highlights the misidentification of L.petauri as L.garvieae in lactococcosis outbreaks and the importance of accurate species differentiation in disease management. The tuf gene sequencing method shows promise for practical, cost-effective, and accurate differentiation of closely related streptococcal species in aquaculture. These findings strongly indicate the need to re-evaluate the initial identification of isolates from fish with lactococcisis. The findings of this study also suggest that the tuf gene sequencing, established in this research for its practicality and cost-efficiency, could potentially be integrated into routine diagnostic procedures for Gram-positive, catalase-negative cocci associated with outbreaks of infections in aquatic animals; however, further experimental works are warranted.