First report on genetic characterization, cell-surface properties and pathogenicity of Lactococcus garvieae, emerging pathogen isolated from cage-cultured cobia (Rachycentron canadum).

Abstract

The diseased cage-cultured cobia (Rachycentron canadum) displayed clinical signs, haemorrhagic eyes, dorsal darkness and gross pathological lesions, enlargement of spleen and liver. Haemorrhages were found in brain, heart and liver with cumulative mortality rates ranging from 20% to 50%. Extensive congestion in the heart, liver, spleen, kidney, and brain were observed histopathologically. Epicarditis and meningitis were also revealed in diseased cobia. All isolates recovered from the organs (liver, spleen, head kidney, posterior kidney, brain, and muscle) of cobia were found to be gram-positive, non-motile, ovoid cocci, short-chain-forming (diplococci) and α-haemolytic. The API 32 strep system together with the polymerase chain reaction assay for species-specific primers (pLG1 & pLG2) and the internal transcribed spacer (ITS) region (G1 & L1 primers) confirmed all four selected isolates as L. garvieae. Partial 16S rDNA nucleotide sequence (~1,100 bp) of one representative L. garvieae isolate AOD109191 (GenBank accession number, MW328528.1) shared 99.9% identities with the 16S rDNA nucleotide sequence of L garvieae (GenBank accession numbers: MT604790.1). Transmission electron microscopy (TEM) evaluation of one representative L. garvieae isolate (AOD109191) and the results of multiplex PCR did not reveal the presence of the capsular gene cluster (CGC), thus categorizing the isolate as the KG+ phenotype. Capsule staining and TEM observations confirmed the presence of a hyaluronic acid-like capsule, a possible virulence factor in KG+ phenotype L. garvieae isolates. The pathogenic potential of the representative isolate (AOD109191) was assessed through intraperitoneal injection challenges in cobia. The gross lesions and histopathological changes found in experimentally infected cobia was similar to those seen in naturally infected fish. This is first report of confirms that L. garvieae-induced warm water lactococcsis can cause outbreaks of diseases in cage-cultured cobia.