Journal of Microbiology Research 2013, 3(1): 11-18 DOI: 10.5923/j.microbiology.20130301.02 Characterization of Vibrio Isolates from Carpet Shell Clam (Ruditapes Decussatus ) Suffering from Brown Ring Disease (BRD) on Tunisian Coasts Monia El Bour 1,* , Fatma Lakhal 1 , Radhia Mraouna 1 , Annick Jacq 2 , Christine Paillard 3 , John Klena 4 1 Laboratoire de Pathologie, Institut National des Sciences et Technologies de La Mer (INSTM), 2025, Salammbô, Tunisie 2 Institut de Génétique et Microbiologie, Université Paris-Sud, CNRS, Orsay, France 3 LEMAR UMR, Institut Universitaire Européen de la Mer-CNRS, Plouzané, France 4 U.S. Centers for Disease, AU1 Control and Prevention, Unit 7300, Box 0066, DPO AP, US Abstract A total of 13 pathogenic Vibrio spp. bacteria were isolated from carpet shell clams (Ruditapes decussatus) with Brown Ring Disease (BRD) in Tunisia. Organisms were identified based on a combination of phenotypic and molecular methods (SSP-PCR and 16SrDNA sequencing). Virulence effects were determined by in vivo testing on R. decussatus and in vitro testing on the Manila clam (Ruditapes philippinarum). All isolates demonstrated biochemical profiles typical of Vibrio spp; nine different biotypes. Three isolates were identified as V.splendidus biovar II TAE2 but the remaining isolates fell into eight biotypes different from each other and from the V.tapetis biotype. Species delineation based on 16S rDNA sequencing indicated that the isolated Vibrio spp closely resembled V.chagasii (eight isolates), V.splendidus (two isolates), V.alginolyticus(one isolates), Psychrobacter spp. and Pseudoalteromonas mariniglutinosa (one isolates for each). In vitro cytotoxicity effects and mortalities could be induced by the isolates of V.chagasii, V.splendidus and V.alginolyticus at lower dosages than induced by V.tapetis (CECT4600). Keywords Vibrio, Brown Ring Disease, Biochemical Profiles, Sequencing, Toxicity, Ruditapes decussatus, Ruditapes Philippinarum 1. Introduction Many bacterial diseases in adult bivalves are caused by organisms in the genus Vibrio (Gram-negative, rod-shaped bacteria) that are widespread in coastal and estuarine environments ([6],[20],[23],[33]). Recent data have shown an expansion in the host range of aquatic organisms affected by pathogenic Vibrio species; these are primarily bivalve invertebrates ([7],[11],[27]). In addition to bivalve bacteriosis, Brown Ring Disease (BRD), a disruption of the periostracal lamina with abnormal conchiolin deposition, which affects main ly the Manila clam Ruditapes philippinarum and the carpet shell c la m Ruditapes decussatus. Infections of BRD have been documented in wild populations and cultured clams isolated from waters around France, England, Ireland, Spain, occasionally in Italy and even in Korea ([2],[33],[35],[37]). In most reports of BRD contaminations, Vibrio tapetis bacteria have been identified ([2],[10],[30]). V.tapetis – like * Corresponding author: monia.elbour@instm.rnrt.tn (Monia El Bour) Published online at http://journal.sapub.org/microbiology Copyright © 2013 Scientific & Academic Publishing. All Rights Reserved organisms, such as those identified in[35], have also been reported as causing BRD in Manila clams in Korea. BRD could be induced by exposing stocks of Manila clams to V. tapetis[13], resulting in high mortality. However, the number of Vibrio species negatively impacting R. decussatus and R. philippinarum has increased to more than 12 species, including Vibrio alginoyticus and the Vibrio splendidus clade ([7],[8],[9]). Vibrio splendidus and related species Vibrio celticus, Vibrio tasmaniensis, Vibrio neptunius and Vibrio furnissii have been characterized as pathogen inducing mortalities for a number of bivalve species including R. phillipinarum, Venerupis pullastra, Mya arenaria and Meretrix meretrix ([5],[23],[39],[40]). The carpet shell clam represents the most economically important bivalve in Tunisia and is widely distributed along the Tunisian shoreline where it is extensively harvested for local consumption and export to Europe. However, very few studies have been published documenting the health status of this species. In 2003 we reported the occurrence of BRD in several wild populations of carpet shell clam on the Tunisian coast (Southern Mediterranean Sea) with prevalence rates ranging between 1 and 40%, but with no mortalities[14]. More recently[17], we described the results of a 6-year surveillance of BRD in natural populations of carpet shell