Isolation and characterization of a toxic intracellular protein from Vibrio parahaemolyticus

A toxic factor released from disrupted cells of Vibrio parahaemolyticus was partially purified by gel filtration after precipitation with (NH₄)₂SO₄ at 40% saturation. The factor, which was a thermostable protein of 63 kDa, lysed human erythrocytes at a concentration of 0.15 g ml^-1. Its LD₅₀ by intravenous injection into mice was 6.4 g. Fluid accumulated in suckling mice force-fed with the toxic material (1 to 25 g). Haemolytic activity, which occurred maximall at 37°C and pH 7.0 was enhanced by Ca²⁺, Cu²⁺ and Zn²⁺, each at 1 mm. Anti-toxic-factor serum agglutinated V. parahaemolyticus cells. The factor may play a role in the pathogenesis of V. parahaemolyticus infections and in the host's defence mechanisms against infection by the microorganism.     

Isolation and partial characterization of a compound with siderophore activity from Vibrio parahaemolyticus

A compound with siderophore activity was purified by successive column and thin layer chromatographic procedures from Dowex 1 x 8 extracts of culture supernatants of Vibrio parahaemolyticus AQ 3354. The strain synthesized the compound in culture media containing less than 2 microM added FeCl3. Hydrolysis of the compound yielded alanine, ethanolamine, citric acid and 2-ketoglutaric acid. The 1H-NMR spectrum exhibited the presence of a residue from each of these components in the intact molecule. The fast-atom bombardment mass spectrum of the methyl ester derivative indicated a prominent ion at m/z 477, probably corresponding to [M + 1] ion. Other strains of V. parahaemolyticus were also found to produce this compound when grown in an iron-limited medium.     

Age and growth of an Iberian cyprinodont, Aphanius iberus (Cuv. & Val.), in its most northerly population

Age and growth of an Iberian cyprinodont, Aphanius iberus (Cuv. & Val.), in its most northerly population, La Rubina lagoon (Alt Empordà wetlands, NE Spain), was studied. The age groups for this Mediterranean population (O +, 1 + and 2 +) agree with those reported for the Atlantic population (R. Guadalquivir marshes), in the southern limit of the species distribution in the Iberian peninsula. Differences in age structure between these populations and the one previously studied in La Tancada lagoon (R. Ebro delta marshes) can be attributed to age estimation bias. Male A. iberus data appear to fit similarly to a Gompertz growth function and to a von Bertalanffy growth function, but L _x, was clearly underestimated. A Gompertz growth function was not a good fit for females, perhaps because female growth is clearly faster and less attenuated than male growth. The life history and growth pattern ofthis species is similar to that in other cyprinodontiform fishes, typified by a short longevity. The growth model of this species does not seem to fit types I and II of Sebens's classification, which are characteristic of fish.     

Isolation and molecular characterization of toxigenic Vibrio parahaemolyticus from the Kii Channel, Japan

Studies were conducted on the ecology of potentially pathogenic Vibrio parahaemolyticus in three coastal areas of Kii Channel, Tokushima, Japan. Seawater and seaweed samples were collected seasonally between June 2003 and May 2004. Total and toxigenic strains of V. parahaemolyticus were isolated using most probable number culture and colony blot hybridization. Toxigenic strains were serotyped and further characterized by random amplified polymorphic DNA (RAPD) and ribotyping. Six thousand strains of V. parahaemolyticus were isolated and 18 were found positive for tdh. V. parahaemolyticus were detected in all samples during summer and autumn, and from some samples during winter and spring. Among the toxigenic strains seven serotypes, five ribotypes and RAPD patterns were observed. Seven strains belonged to O3:K6 clone with identical ribotypes and RAPD patterns to that of a pandemic reference strain. The presence of toxigenic V. parahaemolyticus with pandemic potential might indicate a human health risk due to consumption of marine food sources.     

Reproduction of the Endangered Killifish Aphanius iberus at Different Salinities

Adult fish of a freshwater population of the Iberian endangered cyprinodontid Aphanius iberus, were induced to reproduce at salinities of 5, 15, 30, 45 and 60ppt. For each salinity five 30l aquaria were used, each one including a male and two females. Maturity and spawning outside the natural season, were obtained at conditions of 22 to 28°C and a photoperiod of 14L:10D. The larvae were fed with rotifers Brachionus plicatilis and Synchaeta cecilia valentina. Experiment lasted 40 days. The first spawning occurred on the 17th day at 45ppt of salinity and the first embryos hatched on the 34th day at 5 and 15ppt salinity. The final average number of larvae per aquarium ranged from 5.2 (45ppt salinity) to 10.8 (15ppt salinity). No significant differences were found between the average values at different salinities (p<0.01).     

Isolation and characterization of Vibrio parahaemolyticus from seafoods along the southwest coast of India

The work was aimed to study the microbial quality of the seafood sold in the domestic markets and incidence of Vibrio parahaemolyticus. Samples comprising of shellfish, finfish, and cephalopods were collected from various fish markets in and around Cochin. Presumed V. parahaemolyticus were identified by standard biochemical tests, and further confirmed by polymerase chain reaction targeting species-specific tl gene (450 bp). About 81% of the samples were found to exceed the limits specified for total plate count while total presumptive V. parahaemolyticus count was above the limit in 71% of the samples ranging from 5.5 × 10⁵ to 9.7 × 10⁷ and 0.31 × 10² to 7.8 × 10⁶ cfu/g, respectively. Pathogenicity of the identified isolates was confirmed by Kanagawa phenomenon and urease activity. A total of 10% of the isolates exhibited weak haemolysis on Wagatsuma agar, and 1% of the isolates showed urease activity using Christensen’s urea agar. Random amplified polymorphic DNA analysis revealed two major clusters based on the species rather than seasonality. The gel pattern revealed 8–10 bands ranging from 0.45 to 3.0 kb. Antibiogram results revealed 85% of the strains sensitive to chloramphenicol and nitrofurantoin. Multiple antibiotic resistance index was found to be 0.4 thus suggesting the risk potential involved in consuming seafoods. The present study has clearly demonstrated the need to adopt seafood safety measures for the products meant for human consumption.     

Isolation of Vibrio parahaemolyticus from Salt Springs in Florida

Vibrio parahaemolyticus was isolated from various locations in two salt springs of Florida and appears to be a normal inhabitant of these artesian waters.     

Modified Selective and Differential Isolation Medium for Vibrio parahaemolyticus

The semiselective salt-starch-agar formulation of Baross and Liston was modified as the result of a systematic study of the effect of each constituent on the growth of Vibrio parahaemolyticus and competitive species characteristic of the marine environment. The selection of nutrient constituents depended on an analysis of their effect on generation time. The addition of inhibitors depended on an analysis of minimal inhibitory concentrations. The modified formulation included: peptone, 2.0%; yeast extract, 0.2%; corn starch, 0.5%; NaCl, 3.0%; agar, 1.5% (pH 8.0). Penicillin at 2 to 5 units/ml increased selectivity without significantly inhibiting Vibrio in pure suspensions. Over 62% of the most sensitive strain (YM-K33) was recovered at a concentration of 5 units of penicillin per ml. The per cent recovery of V. parahaemolyticus from fish homogenate compared favorably with other selective formulations. At an initial concentration of 10(5) cells/ml, recovery varied with the strain used from 60 to 119%, whereas at 10(2) cells/ml the range was 36 to 94%. Applications of the medium for Vibrio quantification are discussed.     

Isolation of Vibrio parahaemolyticus and Vibrio alginolyticus from Estuarine Areas of Southeastern Alaska

The first reported isolations of halophilic vibrios, including Vibrio parahaemolyticus, from three seafood processing areas in Southeastern Alaska are described.     

Isolation of Pandemic Vibrio parahaemolyticus from UK Water and Shellfish Produce

Vibrio parahaemolyticus is a Gram-negative, halophilic bacterium found commonly in temperate and warm estuarine waters worldwide. V. parahaemolyticus is considered an emerging bacterial pathogen in Europe and has been responsible for several recent seafood-associated outbreaks. During ad hoc testing of raw shellfish produce in May 2012, pandemic group (O3:K6) V. parahaemolyticus was isolated from Pacific oysters (Crassostrea gigas), harvested in Southern England. Follow-on testing of water and shellfish, encompassing a small number geographically diverse sites, also retrieved pandemic group isolates. These strains are amongst the most northerly pandemic strains described to date and represent the first instance of pandemic V. parahaemolyticus isolated in the UK, highlighting the expanding geographical distribution of these foodborne pathogens in the environment.     

Purification and characterization of membrane-bound 5'-nucleotidase of Vibrio parahaemolyticus

Membrane-bound 5'-nucleotidase from Vibrio parahaemolyticus was solubilized and purified using a nonionic detergent, heptyl-beta-D-thioglucoside, and was characterized. This enzyme required Mg2+ for activity, maximum activity being observed at 5 and 20 mM Mg2+ with AMP and ATP, respectively, as substrates. Of the divalent cations tested, Mn2+ and Co2+ were able to replace Mg2+ partially, whereas Ca2+ was ineffective. Zinc strongly inhibited the enzyme activity and Ni2+ caused partial inhibition. This enzyme required Cl- for activity, the optimal concentration being 20 mM or more. The order of effectiveness of anions was Cl- greater than Br- greater than I- approximately NO3-. Sulfate and acetate were ineffective. The optimal pH was 8.0. The activity of the purified enzyme was stimulated by the addition of lipid to the assay mixture. This enzyme hydrolyzed all 5'-nucleotides tested, but did not hydrolyze 3'-nucleotides or ribose 5-phosphate. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme appeared to be a single polypeptide, with a molecular weight of 72 kDa.     

Electrophoretic and chemical characterization of lipopolysaccharides of Vibrio parahaemolyticus.

Lipopolysaccharides (LPSs) isolated from three Kanagawa-positive and three negative strains of Vibrio parahaemolyticus were characterized by using electrophoretic, immunochemical, and chemical methods. The results of this study indicated that the LPSs of all six strains of V. parahaemolyticus examined did not have an O-specific side chain. These V. parahaemolyticus LPSs appeared to have molecular weights similar to that of the rough-type (Ra) LPS of Salmonella typhimurium TV-119 and might just contain lipid A and a core region. However, the microheterogeneity of V. parahaemolyticus LPS observed was greater than that of S. typhimurium LPS. The profile of V. parahaemolyticus LPS consisted of closely spaced triplet or quadruplet bands, but that of S. typhimurium consisted of doublet bands. Slower-moving bands appeared on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels only when large amounts of V. parahaemolyticus LPS were loaded. These bands were proven to be the aggregates of the fastest-moving low-molecular-weight bands by re-electrophoresis. The banding pattern of V. parahaemolyticus LPSs produced on nitrocellulose membranes by immunoblotting indicated that the V. parahaemolyticus LPSs did not have an O-specific side chain. The low ratio of total carbohydrate to lipid A of V. parahaemolyticus LPSs also suggested that they were like rough-type LPS. The mobility and profile of V. parahaemolyticus LPS on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel and its chemical composition were closely related to the serotype of a specific strain but not with the Kanagawa phenomenon.     

Toxin Isolation from a Kanagawa-Phenomenon Negative Strain of Vibrio parahaemolyticus

Production of a toxin by Vibrio parahaemolyticus Kanagawa-phenomenon negative strains was examined. Ammonium sulfate fractions of broth culture filtrates were dialyzed, concentrated by lyophilization, and tested for toxic effects by mouse intraperitoneal injection. One fraction, which we think is a toxin, was isolated from a broth culture filtrate of V. parahaemolyticus FC 1011 (a Kanagawa-phenomenon negative strain) and consistently produced lethal effects in mice at high concentrations and diarrhea in lower concentrations. The toxin was assayed for mouse LD50 and ability to produce diarrhea via forced feeding in mice. V. parahaemolyticus FC 1011 toxin was found to be protein, to be inactivated by heat or trypsin hydrolysis, and to produce positive skin permeability reactions in rabbits. However, it failed to induce fluid accumulation in ligated ileal loops in rabbits.     

Expression and characterization of a metalloprotease from a Vibrio parahaemolyticus isolate

The extracellular zinc metalloprotease from Vibrio parahaemolyticus (VPM) is a putative virulence factor for host infection. It is synthesized from the vpm gene of V. parahaemolyticus as a polypeptide of 814 amino acids with an estimated molecular mass of 89,833 Da, containing a zinc metalloprotease HEXXH consensus motif. To investigate the enzymatic properties of V. parahaemolyticus metalloprotease, the mature vpm gene was overexpressed in Escherichia coli, and the recombinant protein (rVPM) was purified by a His-binding metal affinity column (>95% purity). The activity of the recombinant protease produced in E. coli was examined by gelatin activity staining and proteolytic activity assays using gelatin and azocasein as substrates. rVPM showed maximum activity at about 37 degrees C and pH 8. The cytotoxicity against flounder gill cells and fish pathogenicity indicated a potential role in pathogenesis.     

Food of an endangered cyprinodont (Aphanius iberus): ontogenetic diet shift and prey electivity

Synopsis We studied the ontogenetic diet shift and prey electivity of an endangered cyprinodontid fish endemic to the Iberian Peninsula, the Spanish toothcarp (Aphanius iberus). The toothcarp’s diet was omnivorous, dominated by harpacticoid copepods (Mesochra lilljeborgi and Tisbe longicornis), copepod nauplii and detritus. Diet composition varied greatly among habitats, depending on prey availability. In a rarely inundated habitat (glasswort), there was more consumption of the isopod Protracheoniscus occidentalis and the harpacticoid copepod Mesochra lilljeborgi, while in algal mats another harpacticoid (Tisbe longicornis), chironomid dipterans and invertebrate eggs were more important in diet. Although a benthic feeding habitat has previously been suggested, in our study the diet was based rather on water column organisms for both glasswort and algal mat habitats. There was also an ontogenetic diet shift, with an increase of mean prey length with fish length, clearly linked to a microhabitat change. Smaller fish showed positive electivity and greater reliance on planktonic prey (e.g. copepod nauplii, the harpacticoid copepods Mesochra lilljeborgi and Tisbe longicornis, the rotifer Brachionus plicatilis, and ostracods), while larger fish elected and preyed on more benthic organisms (e.g. Canuella perplexa, Mesochra rapiens, and ephydrid dipterans).