Identification of new Vibrio campbellii strains harboring the pVA1 plasmid isolated from Penaeus vannamei postlarvae affected by outbreaks of acute hepatopancreatic necrosis disease (AHPND) in Mexico

Abstract

At present, AHPND continues to produce large losses in Mexican shrimp farms, but no scientific report on mortality in commercial hatcheries has been published. Here, we present the clinical signs, histopathological lesions, bacteriological analyses and identification through whole-genome sequencing of the isolates obtained in three natural outbreaks of AHPND in Penaeus vannamei postlarvae obtained from three local hatcheries. After three to four days of acclimation, mortality was recorded and clinical signs such as an empty digestive tract, whitish hepatopancreas, anorexia and lethargy were observed in shrimp postlarvae. Histopathological analysis and detection of pirAB genes using the PCR AP4 method after sample enrichment were used as presumptive diagnoses of AHPND. Significantly more Vibrio-like density (p = 0.045) and green colonies (p = 0.021) were found in the hepatopancreas of postlarvae affected by AHPND compared to apparently healthy organisms in outbreak 1 and 2, respectively. Finally, 29 isolates from the hepatopancreas and stomachs of shrimps with or without AHPND clinical signs were sequenced, and two new Vibrio species were obtained, one closely related to the Harveyi clade and one to the Orientalis clade. Vibrio campbellii were the most abundant species followed by V. parahaemolyticus. The six genomes identified as V. campbellii harbour the pVA1-like plasmid carrying the pirAB genes, responsible for producing the delta-endotoxin that causes AHPND. The pVA1-like plasmids were similar to V. parahaemolyticus strains from the Americas. pVA1 plasmid hosts the Tn3 transposon like the V. campbellii strain from Ecuador, but absent in the China strain, suggesting a horizontal plasmid transfer between coexisting V. parahaemolyticus and V. campbellii within a geographic region. These findings provided the first report of V. campbellii strains carrying a pVA1-like plasmid that causes AHPND in postlarvae from commercial hatcheries in Mexico and may help in the adoption of active genomic surveillance of this disease.