Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Mon, 17 Dec 2018 21:12:58 INTERNATIONAL JOURNAL OF SYSTEMATIC BACTERIOLOGY, Apr. 1996, p. 480-484 Copyright zyxwvutsrq 0 1996, International Union of Microbiological Societies 0020-7713/96/$04.00+ 0 Vol. 46, No. 2 zy Vibrio tapetis zyxwv sp. nov., the Causative Agent of the Brown Ring Disease Affecting Cultured Clams JUAN J. BORREGO,'" DOLORES CASTRO,* ANTONIO LUQUE,' CHRISTINE PAILLARD,2 PHILIPPE MARIA T. GARCIA,3 AND ANTONIO VENTOSA3 Department of Microbiology, Faculty of Sciences, University of Malaga, 29071 Malaga, and Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, zyxwv 41 012 Seville, Spain, and zyxwvuts U. R.A. Centre National de la Recherche ScientiJique 0-1513, Institut d'Etudes Marines, Gorgeu, 29287 Brest Cedex, France2 A taxonomic characterization was carried out on strains of the bacteria that cause the brown ring disease of clams. On the basis of their phenotypic and genotypic characteristics, these strains can be considered to constitute a new taxonomic unit, distinct from other Ebrio species. The guanine-plus-cytosinecontent of the strains ranged between 42.9 and 45.5 mol% (43.2 mol%for the proposed type strain). DNA-DNA hybridization studies showed 100% intragroup relatedness, but levels of genetic relatedness to the reference strains of different Ebrio species tested ranged between 15 and 58%. The strains have all the properties characteristic of the genus vibrio and can be clearly differentiated from other species of this genus by their growth at 4°C and their negative responses for growth at 30°C and in 6% NaCl, arginine dehydrolase, lysine decarboxylase, ornithine decarboxylase, and Voges-Proskauer reaction. The name yibrio tapetis is proposed for the new species; strain B1090 (CECT 4600) is the type strain. Since 1980, the genus Vibrio has been subject to an extensive taxonomic revision, and 15 new species have been described (12, 22). At present, the genus Vibrio includes more than 35 species, most of which are of aquatic origin. Some of them have been demonstrated to be pathogenic for aquatic animals, including fish (Vibrio anguillarum, V. alginolyticus, K damsela, K Jischeri, K ordalii, K salmonicida, V. splendidus, and zyxwvu I/: vul- niJicus) and shellfish (V. haweyi, K pelagtus, K splendidus, and Il tubiashii) (1, 6, 9-11, 15, 18, 25, 30, 31). Brown ring disease is the first epizootic infection described in Europe which affects cultured and wild populations of adult clams, mainly manila clams (Tapes philippinarum) and fine clams (Tapes decussatus). This disease was firstly described in France by Paillard et al. (21), although further epidemiological studies have demonstrated its prevalence in other locations, mainly southwestern and northwestern Spain (2, 3, 23). The biochemical characterization of the causative agent of brown ring disease indicated that it belonged to the genus Kbrio, and it was tentatively named Vibrio group P1 (20). However, on the basis of the response to routine biochemical tests, Vibrio group P1 does not match any of the presently described species of the genus Vibrio (4). We carried out a phenotypic and genetic characterization of several strains belonging to Vibrio group P1 and compared them with other Vibrio strains frequently isolated from affected clams. The results obtained have alIowed us to propose a new species of the genus Vibrio, V. tapetis, on the basis of its phys- iological, biochemical, and genetic features. MATERIALS AND METHODS Bacterial strains. Twenty-four strains of Vibrio group P1 constituting the phenon 6 described by Castro (2), isolated from cultivated and natural popula- tions of diseased clams between 1988 and 1991, were studied (Table 1). They were maintained as working stocks at -20°C in basal medium (peptone, 0.4%; zyxwvutsr * Corresponding author. Mailing address: Departamento de Micro- biologia, Facultad de Ciencias, Universidad de Mhlaga, Campus Uni- versitario Teatinos, 29071 Mdaga, Spain. Phone: 34-5-2131893. Fax: 34- 5-2132000. Electronic mail address: JJBORREGO@CCUMA.UMA.ES. yeast extract, 0.1%; NaCI, 2%) supplemented with 20% glycerol (volivol). For comparative purposes, 15 culture collection strains of the genus Vibrio were used (Table 2). All of them are halophilic microorganisms frequently isolated from marine environment and/or from poikilotherms, including fish and shellfish (4), and they shared several biochemical and physiological characteristics with the strains of I/: tapetis. All strains were cultured on marine agar (Difco Laboratories, Detroit, Mich.) at 20 -C 2°C for 24 to 36 h. Phenotypic characterization. All biochemical tests were performed at 20 2 2°C using media supplemented with NaCl at a final concentration of 2%, except growth tests in NaCl. The isolates were identified by standard morphological, physiological, and biochemical tests (5, 14, 26, 33). Sensitivity to the vibriostatic agent 0/129 (2,4 diamino-6,7-diisopropylpteridine) was determined after 24 h on marine agar plates by using 0/129 discs (150 pg) (Oxoid Ltd., Basingstoke, Hampshire, England). The utilization of substrates as sole carbon and energy TABLE 1. Designation and geographical sources of K tapetis strains tested Origin Strain designation Source BlO9OT IS-1 IS-5 IS-7 IS-8 IS-9 2.3 8.1 8.2 8.3 8.4 8.5 8.6 8.7 8.17 8.19 9.3 9.4 9.5 9.7 11.1 11.2 11.3 11.4 Cultured Tapes philippinarum Cultured Tapes philippinarum Cultured Tapes philippinarum Cultured Tapes philippinarum Natural Venerupis aurea Natural Cerastodema edule Cultured Tapes philippinarum Cultured Tapes philippinarum Cultured Tapes philippinarum Cultured Tapes philippinarum Cultured Tapes philippinarum Cultured Tapes philippinarum Cultured Tapes philippinarum Cultured Tapes philippinarum Cultured Tapes philippinarum Cultured Tapes philippinarum Cultured Tapes philippinarum Cultured Tapes philippinarum Cultured Tapes philippinarum Cultured Tapes philippinarum Cultured Tapes philippinarum Cultured Tapes philippinarum Cultured Tapes philippinarum Cultured Tapes philippinarum Landeda, France Landeda, France Landeda, France Quiberon, France Quiberon, France Quiberon, France Landeda, France Landeda, France Landeda, France Landeda, France Landeda, France Landeda, France Landeda, France Landeda, France Landeda, France Landeda, France Landeda, France Landeda, France Landeda, France Landeda, France Landeda, France Landeda, France Landeda, France Landeda, France 480