Characterization of strains related to brown ring disease outbreaks in southwestern Spain

Several characteristics, including biochemical, serological, drug resistance and plasmid profiles, of strains isolated from clams in southwestern Spain affected with brown ring disease have been comparatively studied. On the basis of 36 standard physiological and biochemical tests, all strains isolated were included in the genus Vibno and further divided into 6 groups. The groups were differentiated on the basis of only 8 phenotypic traits: growth at 35 "C, arginine dihydrolase, gelatinase production, Voges-Proskauer and ONPG tests, and acid production from sucrose, amygdalin and mannitol. Applying these characteristics, the strains resembled Vibno pelagius and V. splendidus species. However, the isolates of each group showed no cross-reactions with the antisera raised against several reference strains of different species of Vibrio, including V. angu~llarum, V. tubiashii, V. damsela, V. pelagius, V. splendidus and the unclassified Vibno PI. Although only 57.7 % of the strains tested harbored one or more plasmids, the majority of the plasmid-containing strains (93.3 %) carried a large plasmid band of 34.4 MDa. A high number of isolates, regardless of their taxonomic group, were resistant to ampicillin and erythromycin. However, all the Vibrio strains were sensitive to chloramphenicol, tetracycline, gentamicin, nitrofurantoin, nalidixic acid and trimethoprim sulphamethoxazole. No correlation between plasmid content and drug resistance was observed. INTRODUCTION flora associated with this disease composed mainly of Vibrio species. Since summer 1988 several epizootic outbreaks causIn this study we have compared the biochemical and ing high mortalities of manila clams Tapes philipserological characteristics of the different strains isopinarum have appeared in the clam beds of Cadiz lated from affected clams in southwestern Spain. In (southwestern Spain). The symptoms associated with addition, drug resistance and plasmid profiles of these these outbreaks are characterized by the presence of a strains were also analyzed. brown deposit of organic matter on the inner surface of the shell, between the pallial line and the shell margins. The populations affected with brown ring disease MATERIALS AND METHODS (BRD) generally showed low development, shell deformities and mortality rates over 60 %. This disease was Sample processing. Samples of manila clams Tapes first reported in clam beds of Landeda (Finisterre, philippinarum affected with BRD symptoms were colFrance) by Paillard et al. (1989), and has been extenlected in different zones of the Cadiz Bay (Spain). In all sively studied in France (Flassch 1989, Paillard & Maes the samplings, 100 specimens or more were collected 1990, Maes et al. 1992) and in Spain (Castro et al. and the incidence of the condition was recorded. Later, 1990). the clams with symptoms of BDR were microbiologiPaillard & Maes (1990) proposed an infectious origin cally analyzed following the scheme described in of the disease, the causative agent being identified as a Fig. 1. All the general and selective media employed Vibrio strain called PI. However, studies carried out in for bacterial isolation were supplied by Difco. For longour laboratory indicate the presence of a varied microterm preservation, cultures were frozen at -80 "C in a O Inter-Research 1992 230 Dis. aquat. Org. 14: 229-236, 1992