Isolation and Pathogenic Characterization of Vibrio bivalvicida Associated With a Massive Larval Mortality Event in a Commercial Hatchery of Scallop Argopecten purpuratus in Chile

Abstract

The VPAP30 strain was isolated as the highly predominant bacteria from an episode of massive larval mortality occurring in a commercial culture of the Chilean scallop Argopecten purpuratus. The main aims of this study were, to characterize and identify the pathogenic strain using biochemical and molecular methods, to demonstrate its pathogenic activity on scallop larvae, to characterize its pathogenic properties and to describe the chronology of the pathology. The pathogenic strain was identified as Vibrio bivalvicida based on its phenotypic properties, the multilocus sequence analysis (MLSA) of eight housekeeping genes (ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA) and different in silico genome-to-genome comparisons. When triplicate cultures of healthy 10 days old scallop larvae were challenged with 1 × 10⁵ colony forming units (CFU) mL^-1 of the VPAP30 strain, percentages of larval survival of 78.9 ± 3.3%, 34.3 ± 4.9%, and 0% were observed at 12, 2,4 and 36 h, respectively, whereas uninfected larval cultures showed survival rates of 97.4 ± 1.2% after of 48 h. Clinical symptoms exhibited by the scallop larvae infected with the VPAP30 strain include the accumulation of bacteria around the scallop larvae, velum disruption and necrosis of digestive gland. The 50% lethal dose (LD₅₀) of VPAP30 strain at 24 and 48 h was 1.3 × 10⁴ and 1.2 × 10³ CFU mL^-1, respectively. The invasive pathogenic activity of the VPAP30 strain was investigated with staining of the bacterial pathogen with 5-DTAF and analyzing bacterial invasion using epifluorescence, and a complete bacterial dissemination inside the larvae at 24 h post-infection was observed. When scallop larvae were inoculated with cell-free extracellular products (ECPs) of VPAP30, the larval survival rate was 59.5 ± 1.7%, significantly (P < 0.001) lower than the control group (97.4 ± 1.2%) whereas larvae treated with heat-treated ECPs exhibited a survival rate of 61.6 ± 1.8% after 48 h of exposure. V. bivalvicida VPAP30 exhibits high pathogenic activity on scallop larvae, mediated both by bacterial invasion and the production of toxigenic heat-stable compounds. This report constitutes the first isolation of V. bivalvicida out of Europe and extends the host range of this species, having demonstrated its pathogenic activity on the Chilean scallop larvae (A. purpuratus). These results supporting the pathogenic potential of V. bivalvicida to kill the larvae of a broad range of bivalve species reared in hatcheries located in the Atlantic and the Pacific coasts.

Keywords: Argopecten purpuratus; Chile; Vibrio bivalvicida; hatchery; scallop larvae; shellfish pathology; vibriosis.

FIGURE 1

Phylogenetic tree based on concatenated sequences of the housekeeping genes ftsZ, gapA, gyrB, mreB, pyrH, recA, rpoA, and topA obtained by the neighbor-joining method. Horizontal branch lengths are proportional to evolutionary divergences. Bootstrap from 1,000 replicates appears next to the corresponding branch. Photobacterium damselae was used as an outgroup.

FIGURE 2

Survival of 10 days old scallop larvae not challenged (control) and challenged with 1 × 10⁵ CFU mL^-1 of Vibrio bivalvicida VPAP30 and Vibrio pectenicida A365. Values are a mean (±SD) of three replicates. Asterisks indicate significant differences.

FIGURE 3

Main clinical signs exhibited by Argopecten purpuratus larvae infected with Vibrio bivalvicida VPAP30 after 24 h of exposure. (A) Bacterial swarms of bacteria on the margins of the larvae, (B) velum disruption, (C) detachment of ciliary cells of the velum (black arrows), and (D) necrosis of digestive tissue of scallop larvae stained with trypan blue. Scale bars: 30 μm.

FIGURE 4

Invasive activity of V. bivalvicida VPAP30 strain on larvae of scallop Argopecten purpuratus determined by epifluorescence microscopy. (A) Bacterial cells of V. bivalvicida VPAP30 stained with 5-DTAF (A); Stained V. bivalvicida in the digestive gland at 30 min post-infection (B); Bacterial cells of V. bivalvicida in the digestive gland at 1 h post-infection (C); Bacterial cells invading completely the larval body cavity and surrounding shell at 24 h post-infection. (Dg) Digestive gland; (Ve) Velum; (Mo) Mouth. Scale bars: 10 μm (A); 50 μm (B–D).

FIGURE 5

Survival of 10 days old scallop larvae not challenged (control) and challenged with extracellular products of V. bivalvicida VPAP30 and V. pectenicida A365. Values are a mean (±SD) of three replicates. Asterisks indicate significant differences.