Reduction of adhesion properties of Ruditapes philippinarum hemocytes exposed to Vibrio tapetis

Abstract

Vibrio tapetis is the causative agent of brown ring disease (BRD), which affects a species of clam, Ruditapes philippinarum. After incubation with V. tapetis, hemocytes lose filopods and become rounded, indicating cytotoxic activity of the bacterium. To rapidly quantify this cytotoxicity, a flow-cytometry test was developed based on the capacity of V. tapetis to inhibit adhesion of clam hemocytes to plastic. Several bacteria:hemocyte ratios, the cytotoxicity of other Vibrio spp. pathogenic to bivalves, and that of various V. tapetis isolates were tested. Inhibition of adherence is detectable with as few as 5 bacteria per hemocyte. The greater cytotoxic activity of V. tapetis compared to that of V. splendidus and V. pectenicida suggests a specific pathogenicity of V. tapetis to R. philippinarum hemocytes. Although all V. tapetis isolates inhibited adhesion, significant variations in cytotoxicity among isolates was demonstrated. KEY WORDS: Cytotoxicity · Flow cytometry · Vibrio tapetis · Ruditapes philippinarum · Bivalve hemocytes · Pathogenicity Resale or republication not permitted without written consent of the publisherDis Aquat Org 57: [109][110][111][112][113][114][115][116] 2003 external defense factors, such as phagocytosis by hemocytes in the pallial fluid as well as the epithelium.However, mechanisms of interactions between Vibrio tapetis and clam hemocytes remain poorly understood. Measurement of cytotoxicity of V. tapetis against hemocytes could be a good indicator of the virulence properties of this bacterium. In the literature, 2 assays using bacteria-hemocyte interaction have already been described: one was direct and employed microscopic observation, the other was indirect and used a chemiluminescence assay. The first used a time-lapse video recording to determine the toxic effect of V. anguillarum and V. alginolyticus on mussel Mytilus edulis hemocytes (Lane & Birkbeck 1999). These 2 Vibrio species cause cell-rounding of the hemocytes. The most toxic bacterium, V. anguillarum, was able to induce rounding in 50% of the hemocytes at a ratio of only 1 bacterium per hemocyte. Differences in cellrounding percentages permitted quantification of cytotoxicity. The second method was used to examine the effects of V. pectenicida and of its cytoplasmic extract on the respiratory burst of Pecten maximus hemocytes , Lambert et al. 2001). This indirect method used chemiluminescence to quantify production of reactive oxygen intermediates (ROIs). V. pectenicida, and in particular the cytoplasm extract, were responsible for the inhibition of chemiluminescence activity in oyster and scallop hemocytes.To better understand the cellular effects of Vibrio tapetis toward hemocytes, the present study was designed first to develop a cytotoxicity test using flow cytometry, and second to compare the pathogenicity of different V. tapetis strains on clam and oyster hemocytes. MATERIALS AND METHODSBacteria. The bacteria used in this study were generally isolated from BRD-diseased clams and asymptomatic cockles at ...

Metalloprotease Vsm Is the Major Determinant of Toxicity for Extracellular Products ofVibrio splendidus

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