Isolation, identification and pathogenicity studies on visceral granulomatous disease in Acanthopagrus schlegelii

A serious disease occurred in black seabream, Acanthopagrus schlegelii, cultivated in runway fish culture pond of mariculture farm in Taizhou, Zhejiang province, in April, 2017. The diseased fish showed sluggish swimming, anorexia and ulceration on surface. Moribund fish were dissected with symptoms of massive ascites and swelling of liver, spleen and kidney, with white granulomatous nodules, especially in spleen. The bacteria were isolated with tryptone soybean agar, and all the fish with typical symptoms showed the pure culture of grayish white colony with Gram negative rods. The bacterial strain AS15, from the spleen of fish with most serious granulomatous, was chosen for challenging test to healthy black seabream by injection intraperitoneally. Mortality was recorded with similar symptoms to native infected seabream, with the median death dose of 6.5×10⁴ CFU/ind. AS15 was identified as Edwardsiella tarda with API 20E biochemical bacteria identification system as well as 16S rDNA sequences blasted on NCBI, with 99% similarity to reference strains. With phylogenetic analysis for 16S rDNA, AS15 belonged to the same cluster with ET^T883, LADL05-105, E. piscicida according to new classification for genus E.dsiella, and same cluster to LADL05-105 and NCIM2056, E. piscicida-like, the new recommended species of the genus, with 100% similarity on gyrB phylogenetic tree. The species-specific primers for genus E. dsiella were used for PCR test, and AS15 showed positive to EPL, the species primer for E. piscicida-like, and PCR negative to all other three primers of E. tarda, E. piscicida and E. ictalur. Ten pathogenic genes, including four fimbrial genes, and sodB, citC, esrB, mukF, katB, the related virulence genes were analyzed by PCR. The strain AS15 showed positive to all four fimbrial genes and other virulence genes except gene gadB. It could be considered that AS15, E. piscicida-like bacteria, the first pathogenic strain isolated and reported from marine cultivated seabream, was the pathogen of serious outbreak case of visceral granulomatous disease in black seabream in runway pond culture system.