The genotypic and phenotypic properties of six strains that were isolated during two unrelated incidents of a bacterial disease of bivalve mollusk larvae were compared with phenotypically similar Vibrio species. The strains of this bivalve mollusk larval pathogen are distinct from other Vibrio spp. phenotypically and as determined by deoxyribonucleic acid-deoxyribonucleic acid hybridization and are described here as Vibrio tubiashii sp. nov. The base composition of the overall deoxyribonucleic acid is 43 to 45 mol% guanine plus cytosine. All strains of V . tubiashii degrade xanthine and tyrosine extracellularly. Strain ATCC 19109 is designated the type strain of V . tubiashii.Tubiash et al. (7) described strains of Vibrio spp. that were pathogenic for the larvae of bivalve mollusks. These organisms were tentatively identified as Vibrio anguillarum and were deposited in the American Type Culture Collection as strains ATCC 19105, ATCC 19106, and ATCC 19109T (T = type strain). In the last decade, knowledge of the taxonomy of the genus Vibrio has advanced rapidly, and many bacteria that would have been identified as V . anguillarum previously can now be allocated to genotypically and phenotypically distinct species, including V . anguillarum sensu stricto, Vibrio ordalii, Vibrio nereis, Vibrio fluvialis, Vibrio diazotrophicus, and Vibrio splendidus (1,9).In a numerical taxonomic study of 237 strains of the Vibrionaceae, including type and reference strains of most of the species of Vibrio and over 50 wild isolates of V . anguillarum, strains ATCC 19105, ATCC 19106, and ATCC 19109T were shown to be closely related to one another but phenotypically distinct from V . anguillarum and all other Vibrio spp. (10). Recently, Jeffries (3) isolated strains of Vibrio spp. in England which were pathogenic for oyster larvae and were phenotypically similar to the strains which Tubiash et al. isolated from diseased hardshell clams on the east coast of the United States (7).In view of the potential economic importance of these vibrios in the cultivation of bivalve mollusk larvae, the work described here was undertaken to establish the genetic relationship between the larval bivalve pathogens and phenotypically similar Vibrio spp. MATERIALS AND METHODSBacterial strains and phenotypic characterization. The strains which we examined and their sources are listed in Table 1. The methods used for phenotypic characterization of each strain have been described previously (9, 10). All preparations were incubated at 25°C.DNA extraction. Deoxyribonucleic acid (DNA) was isolated essentially by the methods of Marmur and Doty (5). per ml and 50 pg of pronase (Calbiochem-Behring Corp., La Jolla, Calif.) per ml for 30 min at room temperature, and lysed by adding 6 mg of sodium dodecyl sulfate per ml. A 0.2 volume of TES-saturated, distilled phenol was added, and the mixture was shaken for 30 min; this was followed by centrifugation at 6,000 x g for 15 min. The upper aqueous layer was collected, mixed with 30 ml of a chloroformisoamyl alcohol mixtu...
Two urease-positiveVibrio spp. were isolated from a brown shark (Carcharhinus plumbeus) that died in captivity at a national aquarium. Morphological, biochemical, and molecular genetic studies revealed one of the isolates to beV. damsela; the other isolate was unique and has been classified asV. carchariae sp. nov. BothV. damsela andV. carchariae were found to be virulent for spiny dogfish (Squalus acanthias), causing death in less than 18 hours after intraperitoneal injection of ca. 4×10(6) cells.V. damsela was strongly cytotoxic for Y1 adrenal cell monolayers;V. carchariae exhibited weak cytotoxicity for Y1 cells.V. damsela contained cryptic plasmids and both isolates were urease positive.V. carchariae was able to utilize urea as sole source of carbon and nitrogen.
Two Vibrio species identified as V. damsela and a new sucrose-positive Vibrio sp., V. carchariae sp. nov., were simultaneously isolated from a brown shark which died while being held in captivity at a large aquarium. Pathogenicity studies were subsequently conducted using a variety of elasmobranchs, including smooth dogfish and lemon sharks. Both bacterial strains proved pathogenic, causing death in nearly all of the elasmobranch hosts challenged. Virulence studies revealed that both bacterial strains were cytotoxic for Y-I mouse adrenal cells. The V. damsela strain was highly cytotoxic, causing Y-1 cellular damage at culture supernatant dilutions up to 1 : 128. Both strains were hemolytic, but neither exhibited the Kanagawa phenomenon. They were both capable of urea hydrolysis, an interesting trait, considering that elasmobranchs retain large (ca 300 milliosmolal) urea concentration in their tissue.
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