Characterization and pathogenicity of Vibrio splendidus strains associated with massive mortalities of commercial hatchery-reared larvae of scallop Argopecten purpuratus (Lamarck, 1819)

Abstract

Three strains (VPAP16, VPAP18 and VPAP23 strains) were isolated as the most predominant organisms from 3 different episodes of massive mortalities of larval cultures of the Chilean scallop Argopecten purpuratus occurred in different commercial hatcheries located in northern Chile. The main aims of this study were to identify the pathogenic strains and investigate their pathogenic activity. Based on selected phenotypic features and sequence identity of the 16S rRNA gene and the housekeeping gene, RNA polymerase α-chain rpoA, all pathogenic strains were identified as Vibrio splendidus. Healthy 10-day-old scallop larvae cultures exhibited mortality percentages of 69.61±3.35%, 79.78±6.11% and 61.73±3.71% after 48 h when were inoculated with 1×10(6) CFU (colony forming units)mL(-1) of VPAP16, VPAP18 and VPAP23 strains, respectively, and evidenced that concentrations ⩾10(4) CFU mL(-1) would probably be detrimental for the larval culture. The main clinical signs observed in challenged larvae for 24h were bacterial swarms on the margins of the larvae, extension and disruption of the velum, detachment of velum cilia cells and digestive tissue necrosis. Otherwise, challenge assays using pathogenic strains stained with 5-([4,6-dichlorotriazin-2-yl]amino)fluorescein hydrochloride (5-DTAF)evidenced that after 1h stained bacteria were detected in high density in the digestive gland and the margin of the shell. When larval cultures were inoculated with cell-free extracellular products (ECP) of V. splendidus strains, exhibited larval mortalities higher than 70% (VPAP16), 80% (VPAP18) and 50% (VPAP23) after 24 h, even when ECP were treated with proteinase K or heat, indicating that extracellular pathogenic activity is mainly mediated by non-proteic thermostable compounds. In this study all Koch's postulates were fulfilled and it was demonstrated for the first time the pathogenic activity of V. splendidus strains on reared-larvae of scallop A. purpuratus and prompt the necessity to maintain this species at concentrations lower than 10(4) CFU mL(-1) to avoid episodes of mass mortalities in scallop hatcheries.

Keywords: Argopecten purpuratus; Bacteria; Scallop larvae; Shellfish pathology; Vibrio splendidus; Vibriosis.