Isolation, identification and pathogenicity analysis of luminous Vibrio campbellii from Litopenaeus vannamei

A serious disease occurred in cultured white-leg shrimp, Litopenaeus vannamei, in greenhouse pond of mariculture farm in Taizhou, Zhejiang Province, in 2019. The diseased L. vannamei showed corkscrew swimming, anorexia, slow growth, hepatopancreas discoloured, with a mortality rate of more than 80% in 2-3 days. To study the pathogenic mechanism of PvL-1 in L. vannamei, the bacteria were isolated from morbid L. vannamei with typical symptoms by using thiosulfate citrate bile salts sucrose (TCBS) agar, with the light Green colony of 3-7 mm in diameter, luminous in dark and Gram negative rods. PvL-1 was identified as Vibrio campbellii with API20E biochemical bacteria identification, with similarity of 90% to reference strains CAIM249. PvL-1 belonged to the same cluster with V. campbellii CP020076 on phylogenetic analysis for fitZ, with 99.31% similarity to reference strains, and same cluster as V. campbellii EF596552, with 98.89% similarity to gapA. V. campbellii species-specific primers were used for PCR test, with 328 bp band for PvL-1 and negative for V. rotiferianus and V. harveyi. These results indicating that PvL-1 was a member of V. campbellii. PvL-1 can form typical β hemolyisis on sheep blood agar and transparent circle on the milk agar, indicating the existence of hemolytic and extracelllar protease toxin. Acute hepatopanergic necrosis, hemolysin gene, flagellate gene and other virulence genes of PvL-1 were analyzed, indicating that PvL-1 has virulence genes such as AP4, TLH, vcahHly, flaC, mukF, gloB, sodB and esrB. The healthy L. vannamei, infected with V. campbellii PvL-1, died with the similar symptoms to naturally morbid L. vannamei, with LD₅₀ of 2.94×10⁴ CFU/ind. Histopathologic examination showed that PvL-1 challenged L. vannamei hepatopancreas showed tubular epithelial cells massive shed and sloughing, hemocytic infiltration etc., which was similar to the naturally morbid L. vannamei. In summary, the luminescent V. campbellii PvL-1 was isolated, with high pathogenicity, which expanded our cognition of the luminescent V. campbellii of L. vannamei.