Infectious gastroenteritis caused by Vibrio harveyi (V. carchariae) in cultured red drum, Sciaenops ocellatus

Summary An outbreak of serious mortality among the cultured red drum Sciaenops ocellatus (L.) characterized by a swollen intestine containing transparent yellow fluid (ascites and gastroenteritis) occurred in July 2000 in Taiwan. A motile strain Rd 0700 was isolated from head kidney and/or the intestinal yellow fluid on tryptone soya agar (TSA) supplemented with 2% (w/v) NaCl and/or thiosulfate citrate bile salt (TCBS) sucrose agar plates. Applying biochemical characteristics, this strain was characterized and identified as Vibrio harveyi (V. carchariae). The bacteria could be re‐isolated from kidney, liver, and the transparent yellow fluid of swollen intestine of fish after bacterial challenge. The LD₅₀ values of the organism and its extracellular products (ECP) were 2.9×10⁷ colony forming units (CFU) and 3.85 μg protein g^?1 fish body weight, respectively. All moribund/dead fish exhibited gastroenteritis except those killed within 12 h. This is a first report showing that intraperitoneal (i.p.) injection of the ECP from V. carchariae is lethal to red drum and can reproduce gastroenteritis in the fish.     

Isolation and Characterization of Pathogenic Vibrio harveyi(V. carchariae) From the Farmed Marine Cobia Fish Rachycentron canadum L. with Gastroenteritis Syndrome

An outbreak of serious mortality among the cultivated juvenile cobia Rachycentron canadum L. (weighing 8–10 g) characterized by a swollen intestine containing transparent yellow fluid (ascites and gastroenteritis) occurred in August 2001 in Taiwan. Ten motile bacterial strains, C3d1–C3d10, were isolated from head kidney (an organ located near the head of the fish) and/or the intestinal yellow fluid on tryptic soy agar supplemented with 1% NaCl (TSA1) and/or thiosulphate citrate bile salt sucrose (TCBS) agar plates. These strains were characterized and identified as Vibrio harveyi(V. carchariae) on the basis of biochemical characteristics, and comparisons with those of three reference strains, originally identified as V. harveyior V. carchariae. The strain C3d1 was selected as a representative strain for virulence tests and was found lethal to the cobia with an LD₅₀ value of 7.48 × 10⁴ colony forming units g^–1 fish body weight. All the moribund/dead fish exhibited gastroenteritis as that observed in natural outbreak. The same bacteria could be reisolated from kidney and the transparent yellow fluid of swollen intestine of fish after bacterial challenge using TSA1 and TCBS plates. This is a first report showing that V. harveyi(V. carchariae) is the causative agent of gastroenteritis in the cobia.     

Pathogenesis of Gastroenteritis Caused by Vibrio carchariae in Cultured Marine Fish

Serious mortality among the cultured grouper Epinephelus coioides, characterized by a swollen intestine containing yellow fluid (gastroenteritis), occurred in 1993 in Taiwan. A bacterium isolated from the intestinal fluid and head kidney of moribund groupers was identified as Vibrio carchariae. Since then, the same Vibrio species has also been isolated from moribund black sea bream Acanthopagrus schlegeli, yellowfin sea bream A. latus, Japanese sea bass Lateolabrax japonicus, and red drum Sciaenops ocellatus suffering from the same syndrome. Each isolate was virulent to the respective fish. Recently, a similar syndrome, flounder infectious necrotizing enteritis, also caused by V. carchariae in summer flounder Paralichthys dentatus, was reported in Rhode Island. The extracellular products (ECPs) of V. carchariae strains EmI82KL (from grouper), Rd (from red drum), and SfUSA (from summer flounder, U.S.A.) were virulent to the grouper or red drum. A 33-kDa serine protease partially purified from the ECP of strain EmI82KL was lethal to the fish. All the moribund or killed fish exhibited gastroenteritis except those killed within 12 hours. This report is the first to show that intraperitoneal injection of the ECP or protease in the fish is virulent and can reproduce gastroenteritis. The serine protease was suggested as a major toxin in the grouper or red drum secreted by V. carchariae.     

News & Notes: Virulence of Vibrio alginolyticus Isolated from Diseased Tiger Prawn,Penaeus monodon

Outbreaks of mass mortality among cultured tiger prawns (Penaeus monodon) with white spotted syndrome (WSS) in the carapace occurred in the summer of 1994 in I-Lan, Taiwan. A swarming strain Val was isolated from hemolymph of the moribund prawns with tryptic soy agar (TSA, supplemented with 1% NaCl, Oxoid) and/or thiosulfate citrate bile salt sucrose (TCBS, Difco) agar. This strain was characterized and identified to be Vibrio alginolyticus. The strain was susceptible to antibiotics such as chloramphenicol, ciprofloxacin, doxycycline hydrochloride, nalidixic acid, oxolinic acid, and oxytetracycline while resistant to ampicillin, novobiocin, penicillin G, sulfisoxazole, and sulfonamide. The bacteria and their extracellular products (ECP) were lethal to both tiger prawns (P. monodon) and kuruma prawns (P. japonicus) with LD₅₀ values of 1.13 × 10⁵, 2.46 × 10⁵ CFU/g, and 0.23, 0.63 μg protein/g prawn body weight, respectively.     

New microsatellite resources for groupers (Serranidae)

We characterized nine microsatellite loci and identified an additional 60 genomic regions containing microsatellites in the red hind grouper, Epinephelus guttatus. The nine loci were highly polymorphic, and primers designed from red hind genomic DNA produced a strong amplification product in a test panel of 16 other groupers in the genera Epinephelus and Mycteroperca collected from across the world. Most of the amplified regions were homologous to the red hind locus and a well‐defined microsatellite repeat was generally evident. The nine loci, together with the 60 uncharacterized microsatellite‐containing regions, provide a powerful tool for ecological and evolutionary studies in groupers.     

Characterization of polymorphic microsatellite loci from black grouper,Mycteroperca bonaci (Teleostei: Serranidae)

Many groupers in the western Atlantic are vulnerable to overfishing, and appropriate management requires knowledge of the population structure of these fish. To this end microsatellite markers were developed from black grouper, Mycteroperca bonaci, using an enrichment procedure. Five loci with levels of polymorphism ranging from 0.5 to 0.9 were selected. These markers also proved to be useful in scamp, M. phenax; red grouper, Epinephelus morio; and goliath grouper, E. itajara. Conformation to Hardy–Weinberg equilibrium was observed except for heterozygote deficiencies noted for E. morio at two of the loci used for this species.     

Isolation of mutants with altered pigments after irradiating haploid protoplasts from Datura innoxia Mill. with X-rays

Summary Ten different mutants with altered pigment patterns were isolated following X-irradiation of approximately 10⁵ haploid protoplasts of Datura innoxia Mill. Seven of the selected strains gave rise to shoots and 3 to leaves only. The mutants were selected from light green or white calli, which had developed 4 weeks after transfer of developing cell clusters onto B₅ agar medium containing 0.5 mg/l BAP (Gamborg et al., 1968). Of the 10 mutant strains 5 were light green, two were yellow, one was pale yellow and one was white. One additional strain does not possess anthocyanin in its stems; a feature chracteristic of the wildtype is the possession of anthocyanin. This strain is able to grow in soil and has now flowered. None of the mutants obtained is haploid. Nine are diploid and the other is tetraploid. The chlorophyll deficient strains can be propagated on B₅ agar medium supplemented with higher concentration of sucrose than normally required for the growth of the wild-type.     

The Association of Physical and Environmental Factors with Abundance and Distribution Patterns of Groupers around Kolombangara Island, Solomon Islands

I collected baseline abundance data on groupers from the reefs of Vavanga village on Kolombangara Island, Solomon Islands. Physical and environmental factors significantly affected the abundance and distribution patterns of groupers. Size and age distribution of groupers varied with depth. Power analysis on the abundance data showed that although power could be calculated to within an acceptable level from a management perspective for abundant species such as the coral hind, Cephalopholis miniata, the same could not be done for larger and naturally scarce species, such as the humpback grouper, Cromileptes altivelis.     

Respiratory responses and tolerance to hypoxia in two marine teleosts,Epinephelus akaara (Temminck & Schlegel) and Mylio macrocephalus (Basilewsky)

The respiratory responses and tolerance of hypoxia were studied in two marine teleosts, the red grouper (Epinephelus akaara, a sluggish species) and the black sea bream (Mylio macrocephalus, an active species). Neither species showed abnormal behaviour or mortality when exposed to 2 mg O₂ l^–1 for 7 h. The black sea bream was, however, comparatively more tolerant when exposed to 1 mg O₂ l^–1, but tolerance of both species became similar under extremely hypoxic conditions (i.e. 0.5 mg O₂ l^–1). In contrast to most other teleosts, both species showed a reduction in opercular beating rate during hypoxia, and oxygen conformity was found in the range of 0.5 to 7.0 mg O₂l ^–1. O₂ dissociation curves were constructed, and the P₅₀ value of the black sea breams (27 ± 5.6 mm Hg) was found to be much lower than that of the red groupers (50 ± 2.5 mm Hg). For both species, the general levels of venous PO₂ showed a direct relationship to ambient PO₂, and were markedly reduced after 1 h exposure to various levels of hypoxia. Compared with the red groupers, the black sea breams appeared to be more able to maintain its venous PO₂ levels during prolonged hypoxic exposure.     

Characterization of <Emphasis Type="Italic">Microbulbifer</Emphasis> Strain CMC-5, a New Biochemical Variant of <Emphasis Type="Italic">Microbulbifer elongatus</Emphasis> Type Strain DSM6810<Superscript>T</Superscript> Isolated from Decomposing Seaweeds

A Gram-negative, rod-shaped, non-spore forming, non-motile and moderate halophilic bacteria designated as strain CMC-5 was isolated from decomposing seaweeds by enrichment culture. The growth of strain CMC-5 was assessed in synthetic seawater-based medium containing polysaccharide. The bacterium degraded and utilized agar, alginate, carrageenan, xylan, carboxymethyl cellulose and chitin. The strain was characterized using a polyphasic approach for taxonomic identification. Cellular fatty acid analysis showed the presence of iso-C_15:0 as major fatty acid and significant amounts of iso-C_17:1ω9c and C_18:1ω7c . Phylogenetic analysis based on 16S rDNA sequence indicated that strain CMC-5 is phylogenetically related to Microbulbifer genus and 99% similar to type strain Microbulbifer elongatus DSM6810^T. However in contrast to Microbulbifer elongatus DSM6810^T, strain CMC-5 is non-motile, utilizes glucose, galactose, inositol and xylan, does not utilize fructose and succinate nor does it produce H₂S. Further growth of bacterial strain CMC-5 was observed when inoculated in seawater-based medium containing sterile pieces of Gracilaria corticata thalli. The bacterial growth was associated with release of reducing sugar in the broth suggesting its role in carbon recycling of polysaccharides from seaweeds in marine ecosystem.     

News & Notes: Isolation of Vibrio harveyi from Diseased Kuruma Prawns Penaeus japonicus

Outbreaks of high mortality among the cultured kuruma prawn Penaeus japonicus without overt gross signs occurred during August and December of 1994 in I-Lan, Taiwan. Eleven luminous bacterial strains were isolated from the hepatopancreas of moribund prawns from five different farms by use of tryptic soy agar (TSA, supplemented with 2% NaCl) and/or thiosulfate citrate bile salt sucrose (TCBS) agar. These strains, together with our two previously unpublished isolates, were characterized and identified to be Vibrio harveyi in comparison with two ATCC Type strains and one strain previously isolated from the tiger prawn, P. monodon. Received: 22 November 1995 / Accepted: 24 February 1996     

Treponema bryantii sp. nov., a rumen spirochete that interacts with cellulolytic bacteria

A saccharolytic spirochete that associated and interacted with cellulolytic bacteria was isolated from bovine rumen fluid. Isolation was accomplished by means of a procedure involving serial dilution of a sample of rumen fluid into a cellulose-containing agar medium. Clear zones appeared within the medium as a result of cellulose hydrolysis by rumen bacteria. The saccharolytic spirochete and a cellulolytic bacterium later identified as a strain of Bacteroides succinogenes were isolated from the clear zones. The spirochete did not utilize cellulose, but grew in coculture with the cellulolytic bacterium in cellulose-containing media. When cocultured in these media the spirochete used, as fermentable substrates, soluble sugars released from cellulose by the cellulolytic bacterium. In cellulosecontaining agar medium the spirochete enhanced cellulose breakdown by the B. succinogenes strain. Electron microscopy showed that the helical spirochete cells possessed an outer sheath, a protoplasmic cylinder, and two periplasmic fibrils. Under a CO₂ atmosphere, in a reduced medium containing inorganic salts, rumen fluid, glucose, and NaHCO₃, the spirochete grew to a final density of 1.9×10⁹ cells/ml. Succinate, acetate, and formate were products of the fermentation of glucose by growing cells. CO₂ (HCO₃ ^-), branched short-chain fatty acids, folic acid, biotin, niacinamide, thiamine, pyridoxal, and a carbohydrate were required for growth of the spirochete. The results of this study indicated that the rumen spirochete represents a new species of Treponema. It is proposed that the new species be named Treponema bryantii.Abbreviations cpm counts per minute - GC guanine plus cytosine - T_m melting temperature - PC protoplasmic cylinder - PF pertplasmic fibrils (axial fibrils) - OS outer sheath - ID insertion disk     

Geodermatophilus nigrescens sp. nov., isolated from a dry-hot valley

A novel actinomycete, designated as strain YIM 75980^T, was isolated from a soil sample collected from a dry-hot river valley in Dongchuan county, Yunnan province, south-west China and was subjected to polyphasic taxonomic characterization. The organism produced circular, smooth, red to black coloured colonies comprising coccoid-shaped cells. Colonies on agar medium lacked mycelia and cells adhered to the agar. Strain YIM 75980^T contained meso-diaminopimelic acid as the diagnostic diamino acid in the cell-wall peptidoglycan and contained galactose, arabinose and glucosamine as the main sugars in the whole-cell hydrolysates. The predominant menaquinone was MK-9 (H₄) and the major fatty acids were iso-C_15:0, iso-C_16:0 and C_16:0. The DNA G + C content of strain YIM 75980^T was 73.1 mol%. Phylogenetic analyses based on 16S rRNA gene sequences clearly showed that strain YIM 75980^T formed a distinct clade within the genus Geodermatophilus and was closely related to Geodermatophilus obscurus DSM 43160^T (level of similarity, 97.9%). Furthermore, the result of DNA–DNA hybridization between strain YIM 75980^T and G. obscurus 43160^T demonstrated that this isolate represented a different genomic species in the genus Geodermatophilus. Moreover, the physiological and biochemical data showed the differentiation of strain YIM 75980^T from its closest phylogenetic neighbour. Therefore, it is proposed that strain YIM 75980^T represents a novel species of the genus Geodermatophilus, for which the name Geodermatophilus nigrescens sp. nov. is proposed. The type strain is YIM 75980^T (=CCTCC AA 2011015^T =JCM 18056^T).     

<Emphasis Type="Italic">Gaetbulibacter jejuensis</Emphasis> sp. nov., isolated from seawater

A novel marine bacterium, designated strain CNURIC014^T was isolated from coastal seawater of Jeju Island in Korea. Strain CNURIC014^T formed yellow colonies on marine agar 2216 and the cells were Gram-negative, non-motile, strictly aerobic, rod-shaped. The temperature, pH and NaCl ranges for growth were 15–37°C, pH 6.0–9.0 and 1.0–7.0% NaCl. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain CNURIC014^T was most closely related to Gaetbulibacter marinus and Gaetbulibacter saemankumensis, with a sequence similarity of 95.1% and 94.6%, respectively. The DNA G+C content of the strain was 33.1 mol% and the major respiratory quinone was menaquinone-6. The major cellular fatty acids were iso-C_15:1 (22.8%), iso-C_15:0 (18.8%), summed feature 3 (iso-C_15:0 2-OH/C_16:1 ω7c, 12.9%) and iso-C_17:0 3-OH (11.5%). On the basis of phenotypic, phylogenetic, and genotypic data, strain CNURIC014^T represents a novel species within the genus Geatbulibacter, for which the name Gaetbulibacter jejuensis sp. nov. is proposed. The type strain is CNURIC014^T(=KCTC 22615^T =JCM 15976^T).     

Streptomyces daliensis sp. nov. from soil

A novel actinomycete strain YIM 31724^T was isolated from a soil sample collected from Dali, Yunnan Province, People’s Republic of China. The strain is characterized by white to yellow white aerial mycelia, spiral spore chains and smooth spore surface. The cell wall of strain YIM 31724^T contained LL-diaminopimelic acid (A₂pm) and traces of meso-A₂pm. Whole-cell hydrolysates contained mainly glucose and small amounts of galactose and xylose. The menaquinones were MK-9(H₆) (31%) and MK-9(H₈) (69%). Phosphatidylethanolamine was the diagnostic phospholipid. The DNA G+C content of strain YIM 31724^T was 67.2 mol%. Phylogenetic analysis indicated that the strain belongs to the genus Streptomyces, with highest similarity to Streptomyces rimosus subsp. rimosus JCM 4667^T (rRNA gene sequence similarity value of 98.9%) and Streptomyces erumpens DSM 40941^T (rRNA gene sequence similarity value of 98.7%). Based on its phenotypic and genotypic characteristics, including low DNA–DNA hybridization results, strain is proposed as the type strain of a novel species, Streptomyces daliensis sp. nov.     

Establishment,Characterization, and Virus Susceptibility of a New Marine Cell Line from Red Spotted Grouper (<Emphasis Type="Italic">Epinephelus akaara</Emphasis>)

A marine fish cell line from the snout of red spotted grouper Epinephelus akaara, a protogynous hermaphrodite, was established, characterized, and subcultured with more than 60 passages. The grouper snout cell line (GSC) cells multiplied well in Dulbecco’s modified Eagle’s medium (DMEM) medium supplemented with 10% fetal bovine serum. The optimal growth temperature was 25°C, and morphologically the cells were fibroblastic. Chromosome analysis revealed that the GSC cell line has a normal diploid karyotype with . A virus titration study indicated that the cells were susceptible to turbot Scophthalmus Maximus rhabdovirus (SMRV) (10^8.5 TCID₅₀ ml⁻¹), while the viral titer of frog Rana grylio virus 9807 (RGV₉₈₀₇) reached 10^3.5 TCID₅₀ ml⁻¹. The infection was confirmed by cytopathic effect (CPE), immunofluorescence, and electron microscopy experiments, which detected the viral particles in the cytoplasm of virus-infected cells, respectively. Further, significant fluorescent signals were observed when the GSC cells were transfected with pEGFP vector DNA, indicating their potential utility for transgenic and genetic manipulation studies.     

Laboratory video recordings and underwater visual observations combined to reveal activity rhythm of red-spotted grouper and banded wrasse, and their natural assemblages

The diel activity rhythm of red-spotted grouper Epinephelus akaara was studied both in captivity and in the wild. Behavior of solitary grouper (58 to 397?mm in total length) in a tank was video recorded using infrared illuminators under 11L/10D and two 1.5-h twilight transition periods, and was compared to that of banded wrasse Halichoeres poecilopterus, a typical diurnal fish. Underwater observations using SCUBA were also conducted in their natural habitat to reveal the behavioral activity together with a visual census of adjacent fish and crustacean assemblages. Red-spotted grouper showed a strong nocturnal activity in a tank regardless of body size as opposed to the strongly diurnal banded wrasse. Activity of groupers in natural waters was high at dawn and dusk, low at noon, and only a few individuals were observed at night. Visual census in the habitat revealed that fish abundance and species richness was highest at noon, lowest at night, and intermediate at dawn and dusk. The opposite trend was found in crustacean assemblages. Absence of groupers at night may reflect their nocturnal feeding migration away from the study area. Alternatively, the crepuscular activity of groupers in the wild is suggested to be an adaptation to feed on small fishes that shift between daytime activity and nighttime rest and/or on nocturnal crustaceans that show the opposite activity pattern.     

Kineococcus glutineturens sp. nov., isolated from soil in Yunnan, south-west China

An orange-coloured, non-spore-forming, motile and coccus-shaped actinobacterium, designated YIM 75677^T, was isolated from a soil sample collected from a dry-hot river valley in Dongchuan county, Yunnan Province, south-west China and its taxonomic position was investigated. Growth of strain YIM 75677^T occurred at 12–55 °C, pH 6.0–9.0 and NaCl tolerance up to 2 % (w/v). Cells adhered to agar media and were agglutinated tightly together. The peptidoglycan contained meso-diaminopimelic acid, alanine and glutamic acid. The whole-cell hydrolysates mainly contained glucose, galactose, mannose and ribose. The predominant menaquinone was MK-9 (H₂) and the major fatty acids were anteiso-C_15:0 and iso-C_15:0. Mycolic acids were not present. The DNA G+C content of strain YIM 75677^T was 74.8 mol%. Phylogenetic analyses based on 16S rRNA gene sequence comparisons clearly revealed that strain YIM 75677^T represents a novel member of the genus Kineococcus and is closely related to Kineococcus xinjiangensis S2-20^T (level of similarity, 98.6 %). Meanwhile, the result of DNA–DNA hybridization between strain YIM 75677^T and K. xinjiangensis S2-20^T demonstrated that this isolate represented a different genomic species in the genus Kineococcus. On the basis of phylogenetic, phenotypic and chemotaxonomic characteristics, strain YIM 75677^T represents a novel species of the genus Kineococcus, for which the name Kineococcus glutineturens sp. nov. is proposed. The type strain is YIM 75677^T (=CCTCC AA 209075^T = JCM 18126^T).     

Molecular phylogenetic relationships of China Seas groupers based on cytochrome <Emphasis Type="Italic">b</Emphasis> gene fragment sequences

The classification and evolutionary relationships are important issues in the study of the groupers. Cytochrome b gene fragment of twenty-eight grouper species within six genera of subfamily Epinephelinae was amplified using PCR techniques and the sequences were analyzed to derive the phylogenetic relationships of the groupers from the China Seas. Genetic information indexes, including Kimura-2 parameter genetic distance and T _S/T _V ratios, were generated by using a variety of biology softwares. With Niphon spinosus, Pagrus major and Pagrus auriga as the designated outgroups, phylogenetic trees, which invoke additional homologous sequences of other Epinephelus fishes from GenBank, were constructed based on the neighbor-joining (NJ), maximum-parsimony (MP), maximum-likelihood (ML) and minimum-evolution (ME) methods. Several conclusions were drawn from the DNA sequences analysis: (1) genus Plectropomus, which was early diverged, is the most primitive group in the subfamily Epinephelinae; (2) genus Variola is more closely related to genus Cephalopolis than the other four genera; (3) genus Cephalopolis is a monophyletic group and more primitive than genus Epinephelus; (4) Promicrops lanceolatus and Cromileptes altivelis should be included in genus Epinephelus; (5) there exist two sister groups in genus Epinephelus.     

Utilization of seagrass habitats by juvenile groupers and snappers in Banten Bay,Banten Province,Indonesia

Coastal development in Banten Bay, Indonesia, decreased seagrass coverage to only 1.5% of its surface area. We investigated the importance of seagrass as habitat for juvenile groupers (Serranidae) and snappers (Lutjanidae), by performing beam trawl hauls on a weekly basis in two seagrass locations and one mudflat area, and monthly trawl hauls in three different microhabitats (dense, mixed and patchy seagrass) in one of the seagrass locations. We studied the effects of location and microhabitat, as well as temporal patterns (diel, weekly and monthly) on the probability of occurrence and abundance of the most abundant grouper (Orange-spotted grouper, Epinephelus coioides) and snapper (Russell’s snapper, Lutjanus russellii). We found that both species were almost exclusively found in seagrass locations, with a preference for microhabitats of high complexity (dense and mixed microhabitats). L. russellii had a higher probability of catch and abundance during the night, most probably because of its ability to avoid the beam trawl during daytime sampling. In addition there was an effect of week and month on the presence and abundance of both species, but patterns were unclear, probably because of high fishing pressure on juvenile groupers and snappers by push net fishermen. Groupers and snappers mainly fed on abundant shrimps, and to a lesser extent on fish. Moreover, juveniles find protection against predators in seagrass, which confirmed the critical role of quantity and quality of seagrass areas for juvenile groupers and snappers in Banten Bay.