Translucent post-larvae disease (TPD) has emerged as a significant new disease in shrimp since it was first identified in Pacific white shrimp (Penaeus vannamei) hatcheries in China in 2020. Two Vibrio spp. with distinct phenotypes, AG1 (green colonies in TCBS agar) and AY7 (yellow colonies in TCBS agar), were isolated from shrimp experiencing sudden death in an anonymous hatchery suspected to be caused by TPD. Both isolates tested PCR-negative for Vibrio parahaemolyticus causing acute hepatopancreatic necrosis disease based on the absence of the PirA and PirB toxin genes. Moreover, initial PCR screening detected three candidate virulence genes, Vibrio high virulent protein (vhvp)-1, vhvp-2, and vhvp-3 in AG1 but not in AY7 isolate, which are believed to be key virulence factors contributing to TPD.
The characterization of two bacterial isolates, AG1 and AY7, were performed using a combination of the Analytical Profile Index (API) 20E and 20NE systems and whole genome sequencing, which identified AG1 as V. parahaemolyticus and AY7 as V. harveyi. Immersion challenge using P. vannamei post-larvae (~0.2g ± 0.05) with doses rangeing from 10² to 10⁶ CFU/mL showed AG1 isolate is highly virulent with an LD50 of 8.51 × 10² CFU/mL compared to AY7 isolate which which had a predicted LD50 of 1.95 × 10⁸ CFU/mL at 96 hr post-challenge. Histological examination of the hepatopancreas from shrimp infected with AG1 isolate revealed the loss of hepatopancreatic tubule structure, with epithelial cells detaching and sloughing off. Additionally, these shrimp exhibited reduced intestinal contents, damaged intestinal walls, and some individuals showed signs of hemocytic enteritis. In contrast, shrimp infected with the AY7 isolate typically displayed abnormal cytoplasmic vacuolization in E-type cells within the hepatopancreatic tubules.
An additional bioassay using larger P. vannamei juveniles (~0.5g ± 0.1) revealed the virulence of these bacterial isolates is dose-dependent and significantly influenced by the size of the shrimp. Based on the LD50 from the first experiment, bioassay via immersion was conducted at doses of 2.42 × 10², 10³, and 10⁴ CFU/mL for AG1 isolate, and 1.89 × 10⁶, 10⁷, and 10⁸ CFU/mL for AY7 isolate over a 7-day period. No LD50 was determined for the AG1 strain since even the highest dose resulted in only 40% mortality. For the AY7 strain, there was no mortality, even at the highest dose of 10⁸ CFU/mL. These results confirm that V. parahaemolyticus causing TPD is highly lethal to post-larval shrimp, particularly when harboring the vhvp-1, vhvp-2, and vhvp-3 virulence genes. This study provides valuable insights into the etiologic agent of TPD and open avenues for exploring the pathogenic mechanisms of Vibrio strains associated with TPD in P. vannamei.