Heterodisaccharide 4-O-(N-acetyl-β-D-glucosaminyl)-D-glucosamine is an effective chemotactic attractant for Vibrio bacteria that produce chitin oligosaccharide deacetylase

Aims: To investigate the attractant effect of 4‐O‐(N‐acetyl‐β‐d ‐glucosaminyl)‐d ‐glucosamine (GlcNAc‐GlcN) in the chemotaxis of Vibrio bacteria that produce carbohydrate esterase (CE) family 4 chitin oligosaccharide deacetylase (COD), an enzyme that catalyzes the production of GlcNAc‐GlcN from N,N′‐diacetylchitobiose (GlcNAc)₂. Methods and Results: The chemotactic effect of disaccharides from chitin on several strains of Vibrio bacteria was investigated using an agar gel lane‐migration method. The results demonstrated that GlcNAc‐GlcN functions as an effective chemoattractant in the CE family 4 COD‐producing vibrios, Vibrio parahaemolyticus and Vibrio alginolyticus. In contrast, this phenomenon was not observed in Vibrio nereis or Vibrio furnissii, which lack genes encoding this enzyme. From transmission electron microscope observation of V. parahaemolyticus cells following the chemotaxis assay, GlcNAc‐GlcN appears to stimulate polar flagellum rotation. Conclusions: GlcNAc‐GlcN is a specific chemoattractant for the CE family 4 COD‐producing vibrios, V. parahaemolyticus and V. alginolyticus. Significance and Impact of the Study: It was clarified for the first time that GlcNAc‐GlcN functions as a signalling molecule in the chemotaxis of Vibrio bacteria that have an ability to produce CE family 4 COD, which generate GlcNAc‐GlcN from (GlcNAc)₂.     

Occurrence of <Emphasis Type="Italic">Vibrio parahaemolyticus</Emphasis> and <Emphasis Type="Italic">Vibrio alginolyticus</Emphasis> in the German Bight over a seasonal cycle

Bacteria of the genus Vibrio are an important component of marine ecosystems worldwide. The genus harbors several human pathogens, for instance the species Vibrio parahaemolyticus, a main cause for foodborne gastroenteritis in Asia and the USA. Pathogenic V. parahaemolyticus strains emerged also in Europe, but little is known about the abundance, pathogenicity and ecology of V. parahaemolyticus especially in Northern European waters. This study focuses on V. parahaemolyticus and its close relative Vibrio alginolyticus in the North Sea (Helgoland Roads, Germany). Free-living, plankton-attached and shellfish-associated Vibrio spp. were quantified between May 2008 and January 2010. CFUs up to 4.3 × 10³ N l⁻¹ and MPNs up to 240 N g⁻¹ were determined. Phylogenetic classification based on rpoB gene sequencing revealed V. alginolyticus as the dominant Vibrio species at Helgoland Roads, followed by V. parahaemolyticus. We investigated the intraspecific diversity of V. parahaemolyticus and V. alginolyticus using ERIC-PCR. The fingerprinting disclosed three distinct groups at Helgoland Roads, representing V. parahaemolyticus, V. alginolyticus and one group in between. The species V. parahaemolyticus occurred mainly in summer months. None of the strains carried the virulence-associated genes tdh or trh. We further analyzed the influence of nutrients, secchi depth, temperature, salinity, chlorophyll a and phytoplankton on the abundance of Vibrio spp. and the population structure of V. parahaemolyticus. Spearman Rank analysis revealed that particularly temperature correlated significantly with Vibrio spp. numbers. Based on multivariate statistical analyses we report that the V. parahaemolyticus population was structured by a complex combination of environmental parameters. To further investigate these influences is the key to understanding the dynamics of Vibrio spp. in temperate European waters, where this microbial group and especially the pathogenic species, are likely to gain in importance.     

Physiological and Biochemical Characteristics of the Halophilic Bacteria Vibrio parahaemolyticus and V. alginolyticus Isolated from Marine Invertebrates of Peter the Great Bay, Sea of Japan

One hundred strains of halophilic vibrios were isolated from 16 species of marine invertebrates of Peter the Great Bay. Based on their morphological and biochemical characteristics, the bacteria were identified as Vibrio parahaemolyticus and Vibrio alginolyticus. Bacterial isolates possessed virulence enzymes (DNAase, lecithinase, catalase) and were characterized by a high enterotoxigenicity. It was determined that 76% of the V. parahaemolyticus strains and 43% of the V. alginolyticus strains were Kanagawa-positive. The isolates showed a high adhesive capability, the average adhesion index was 18.06 cells per erythrocyte for V. parahaemolyticus and 12.55 for V. alginolyticus. The results of this study suggest a high pathogenic potential of the isolated halophilic vibrios, which are an epidemic hazard to marine invertebrates and to humans.     

Simultaneous differential detection of human pathogenic and nonpathogenic Vibrio species using a multiplex PCR based on gyrB and pntA genes

Aims: To develop a multiplex PCR targeting the gyrB and pntA genes for Vibrio species differentiation. Methods and Results: Four pairs of primers targeting gyrB gene of Vibrios at genus level and pntA gene of Vibrio cholerae, Vibrio parahaemolyticus, Vibrio vulnificus were designed. This PCR method precisely identified 250 Vibrio species and demonstrated sensitivity in the range of 4 × 10⁴ CFU ml^?1 (c. 200 CFU per PCR) to 2 × 10³ CFU ml^?1 (c. 10 CFU per PCR). Overall, the gyrB gene marker showed a higher specificity than the dnaJ gene marker for Vibrio detection and was able to distinguish Aeromonas from Vibrio species. Conclusions: The multiplex PCR based on combined gyrB and pntA provides a high discriminatory power in the differentiation between Vibrio alginolyticus and V. parahaemolyticus, and between V. cholerae and Vibrio mimicus. Significance and Impact of the Study: This assay will be useful for rapid differentiation of various Vibrio species from clinical and environmental sources and significantly overcomes the limitations of the conventional methods.     

Spatiotemporal dynamics of the total and active Vibrio spp. populations throughout the Changjiang estuary in China

Vibrio is ubiquitously distributed in marine environments and is the most extensively characterized group within Gammaproteobacteria. Studies have investigated Vibrio spp. worldwide, but mostly focused on pathogenic vibrios and based on cultivation methods. Here, using a combination of molecular and culturing methods, we investigated the dynamics of the total and active Vibrio spp. throughout the Changjiang estuary in China. The total Vibrio abundance was higher in summer (~6.59 × 10³ copies ml⁻¹) than in winter (~1.85 × 10³ copies ml⁻¹) and increased from freshwater to saltwater (e.g. 8.04 × 10¹ to 9.39 × 10³ copies ml⁻¹ in summer). The ratio of active to total Vibrio (Va/Vt) revealed a high activity of vibrios, with remarkable differences between freshwater and saltwater (p < 0.05). Based on the community compositions of the culturable, total and active Vibrio, Vibrio atlanticus and Vibrio owensii were the dominant and active species in winter and summer, respectively. The distribution of Vibrio was governed by the effects of diverse environmental factors, such as temperature, salinity, pH, dissolved oxygen and SiO₃²⁻. Our study clearly demonstrates the spatiotemporal dynamics of total and active Vibrio spp. and lays a foundation for fully understanding the ecological roles of marine Vibrio.     

Enhanced immune defences in Pacific white shrimp (Litopenaeus vannamei) post-exposure to a vibrio vaccine

This study was conducted to determine if exposure of shrimp, Litopenaeus vannamei, to a commercial anti-vibrio vaccine caused changes in antibacterial and cellular (phagocytosis) defences. Shrimp post-larvae were administered either Vibromax™ vaccine or a blank preparation. Whole body homogenates were prepared before (day 0), during (day 10) and after (day 20) vaccination and incubated with a selection of pathogenic vibrios. Homogenate from day 0 animals showed natural antibacterial activity towards Vibrioanguillarum which was significantly enhanced for bacteria-exposed shrimp at 10 days post-challenge. This effect of the vaccine was short-term in its duration. No antibacterial activity was observed in day 0 shrimp homogenate against Vibrio alginolyticus but it was significantly enhanced for both vaccinated and blank-vaccinated shrimp by day 10. No natural or inducible antibacterial activity was observed against Vibrio harveyi at 0, 10 or 20 days post-challenge. To determine if prior exposure of shrimp to inactivated vibrios results in elevated hemocyte phagocytic activity, juveniles were injected with either a mixture of formalin-inactivated vibrios or saline. Hemocyte monolayers made from these shrimp were overlaid with a 1:1 mix of Bacillus subtilis and these vibrios. Hemocytes from vibrio-exposed animals showed elevated levels of internalised vibrios compared with those from the saline injected group. These studies show selectively enhanced cellular defences of shrimp following ‘vaccination’.     

Inhibition of pathogenic Vibrio by the microalgae Isochrysis galbana

Vibrio alginolyticus, Vibrio campbellii, and Vibrio harveyi were inhibited by Isochrysis galbana in batch cultures. I. galbana reduced the V. alginolyticus, V. campbellii, and V. harveyi counts to undetectable levels in 2, 4, and 7 days (<0.01 Vibrio spp. mL^?1), respectively, remaining so until the end of the experiment on day 15. Other heterotrophic bacteria reached counts of 10⁶ CFU mL^?1 on ZoBell medium at the end of the experiment. Vibrio parahaemolyticus was not inhibited by I. galbana. In all mixed I. galbana and Vibrio spp. cultures, the algal density increased from 3.5 to 4.0?×?10⁷ cells mL^?1, higher than that in I. galbana cultures alone, indicating a lack of an inhibitory effect on microalgae in the mixed cultures. The predominant fatty acids (>82 %) of I. galbana during the stationary growth phase were estearidonic (24.3 %), oleic (15.7 %), myristic (13.8 %), docosahexaenoic (11.0 %), palmitic (10.3 %), and α-linolenic (7.2 %) acids. These results demonstrate that I. galbana synthesizes antibacterial fatty acids that inhibit the growth of pathogenic bacteria such as V. alginolyticus, V. campbellii, and V. harveyi.     

Loop‐mediated isothermal amplification method for rapid detection of Vibrio alginolyticus,the causative agent of vibriosis in mariculture fish

Aims: The purpose of this study was to develop a loop‐mediated isothermal amplification (LAMP) method for the rapid, sensitive and simple detection of Vibrio alginolyticus in mariculture fish. Methods and Results: LAMP primers were designed by targeting the gyrB gene. With Bst DNA polymerase, the target DNA can be clearly amplified for 60 min at 64°C in a simple water bath. The detection sensitivity of the LAMP assay for the detection of V. alginolyticus is about 3·7 × 10² CFU ml^?1 (3·7 CFU per reaction). LAMP products could be judged with agar gel or naked eye after the addition of SYBR Green I. There were no cross‐reactions with other bacterial strains indicating a high specificity of the LAMP. The LAMP method was applied to detect V. alginolyticus‐infected fish tissues effectively. Conclusions: The LAMP established in this study is a simple, sensitive, specific, inexpensive and rapid protocol for the detection of V. alginolyticus. Significance and Impact of the Study: This LAMP method provides an important diagnostic tool for the detection of V. alginolyticus infection both in the laboratory and field.     

Characterization of Vibrios Diversity in the Mucus of the Polychaete Myxicola infundibulum (Annellida,Polichaeta)

Vibrios are among the most abundant culturable microbes in aquatic environments. They can be either free-living in the water column or associated with several marine organisms as mutualists, saprophytes, or parasites. In the present study we analysed vibrios abundance and diversity in the mucus of the polychaete Myxicola infundibulum, complementing culture-based with molecular methods. Vibrios reached 4.6?×?10³ CFU mL^?1 thus representing a conspicuous component of the heterotrophic culturable bacteria. In addition, luminous vibrios accounted for about 60 % of the total culturable vibrios in the mucus. The isolates were assigned to: Vibrio gigantis, Vibrio fischeri, Vibrio jasicida, Vibrio crassostreae, Vibrio kanaloae, and Vibrio xuii. Two Vibrio isolates (MI-13 and MI-15) may belong to a new species. We also tested the ability of the Vibrio isolates to grow on M. infundibulum mucus as the sole carbon source. All strains showed appreciable growth in the presence of mucus, leading us to conclude that this matrix, which is abundant and covers the animal entirely, may represent a microcosm and a food source for some bacteria, playing a crucial role in the structuring of a mucus-associated beneficial microbial community. Moreover, the trophic relationship between vibrios and M. infundibulum mucus could be enhanced by the protection that mucus offers to vibrios. The results of this study represent a contribution to the growing evidence for complex and dynamic invertebrate-microbe associations present in nature and highlight the importance of exploring relationships that Vibrio species establish with marine invertebrates.     

Administration of the hot-water extract of Spirulina platensis enhanced the immune response of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus

White shrimp Litopenaeus vannamei which had been injected with the hot-water extract of Spirulina platensis at 6, 10, and 20 μg g^?1, or immersed in aerated seawater containing extract at 200, 400, and 600 mg L^?1 were challenged with Vibrio alginolyticus at 1.5 × 10⁶ or 1.4 × 10⁶ colony-forming units (cfu) shrimp^?1, and then placed in seawater. Survival rates of shrimp that received the extract of S. platensis at 6–20 μg g^?1, and those of shrimp immersed in seawater containing the extract at 400 and 600 mg L^?1 were significantly higher than those of control shrimp after 24–96 and 48–96 h, respectively. In a separate experiment, the hyaline cell (HC) count, granular cell (GC, including semi-granular cell) count, total haemocyte count (THC), phenoloxidase (PO) activity, respiratory burst (RB), superoxide dismutase (SOD) activity, glutathione peroxidase (GPx) activity, and lysozyme activity were measured when shrimp were injected with the extract at 6, 10, and 20 μg g^?1, and immersed in seawater containing the extract at 200, 400, and 600 mg L^?1. These parameters directly increased with the concentration, and significantly increased when shrimp were immersed in the seawater containing the extract at 0.5–4 h. L. vannamei that received all doses of the extract via injection or via immersion all had increased phagocytic activity and clearance efficiency to V. alginolyticus at 12–72 h and 3–4 h, respectively. It was concluded that L. vannamei that received the hot-water extract of S. platensis had enhanced innate immunity and increased resistance against V. alginolyticus infection.     

White shrimp Litopenaeus vannamei that received the hot-water extract of Gracilaria tenuistipitata showed earlier recovery in immunity after a Vibrio alginolyticus injection

White shrimp Litopenaeus vannamei which had been immersed in seawater containing the hot-water extract of Gracilaria tenuistipitata at 0 (control), 200, 400, and 600 mg L^?1 for 3 h, were challenged with Vibrio alginolyticus at 4.6 × 10⁶ colony-forming units (CFU) shrimp^?1 and then placed in normal seawater (34‰). The survival rates of shrimp immersed in 200, 400, and 600 mg L^?1 of the hot-water extract were significantly higher than those of control shrimp over 48–120 h. In another experiment, L. vannamei which had been immersed in the hot-water extract at 0, 200, 400, and 600 mg L^?1 for 3 h, were challenged with V. alginolyticus at 4.0 × 10⁶ CFU shrimp^?1, and the immune parameters examined included the haemocyte count, phenoloxidase (PO) activity, respiratory burst (RB), and superoxide dismutase (SOD) activity at 12–120 h post-challenge after shrimp had been released into normal seawater. Shrimp not exposed to the hot-water extract or V. alginolyticus served as the background control. Results indicated that the haemocyte count, PO activity, RB, and SOD activity of shrimp immersed in 600 mg L^?1 were significantly higher than those of control shrimp at 12–72 h post-challenge. Results also indicated that total haemocyte count (THC), PO activity, RB and SOD activity of shrimp immersed in 400 and 600 mg L^?1 of the hot-water extract returned to the background values at 96, 48, 48, and 72 h, whereas these parameters of control shrimp returned to the original values at >120, >120, 96, and 96 h post-challenge, respectively. It was therefore concluded that L. vannamei that had been immersed in seawater containing the hot-water extract of G. tenuistipitata exhibited protection against V. alginolyticus as evidenced by the earlier recovery of immune parameters.     

Predicting the Distribution of Vibrio spp. in the Chesapeake Bay: A Vibrio cholerae Case Study

Vibrio cholerae, the causative agent of cholera, is a naturally occurring inhabitant of the Chesapeake Bay and serves as a predictor for other clinically important vibrios, including Vibrio parahaemolyticus and Vibrio vulnificus. A system was constructed to predict the likelihood of the presence of V. cholerae in surface waters of the Chesapeake Bay, with the goal to provide forecasts of the occurrence of this and related pathogenic Vibrio spp. Prediction was achieved by driving an available multivariate empirical habitat model estimating the probability of V. cholerae within a range of temperatures and salinities in the Bay, with hydrodynamically generated predictions of ambient temperature and salinity. The experimental predictions provided both an improved understanding of the in situ variability of V. cholerae, including identification of potential hotspots of occurrence, and usefulness as an early warning system. With further development of the system, prediction of the probability of the occurrence of related pathogenic vibrios in the Chesapeake Bay, notably V. parahaemolyticus and V. vulnificus, will be possible, as well as its transport to any geographical location where sufficient relevant data are available.     

The application of bioflocs technology to protect brine shrimp (Artemia franciscana) from pathogenic Vibrio harveyi

Aims: To study the potential biocontrol activity of bioflocs technology. Methods and Results: Glycerol‐grown bioflocs were investigated for their antimicrobial and antipathogenic properties against the opportunistic pathogen Vibrio harveyi. The bioflocs did not produce growth‐inhibitory substances. However, bioflocs and biofloc supernatants decreased quorum sensing‐regulated bioluminescence of V. harveyi. This suggested that the bioflocs had biocontrol activity against this pathogen because quorum sensing regulates virulence of vibrios towards different hosts. Interestingly, the addition of live bioflocs significantly increased the survival of gnotobiotic brine shrimp (Artemia franciscana) larvae challenged to V. harveyi. Conclusions: Bioflocs grown on glycerol as carbon source inhibit quorum sensing‐regulated bioluminescence in V. harveyi and protect brine shrimp larvae from vibriosis. Significance and Impact of the Study: The results presented in this study indicate that in addition to water quality control and in situ feed production, bioflocs technology could help in controlling bacterial infections within the aquaculture pond.     

<Emphasis Type="Italic">Vibrio cholerae</Emphasis> in waters of the Sunderban mangrove: relationship with biogeochemical parameters and chitin in seston size fractions

Wetland dynamics are probably linked to cholera endemicity in South Asia. We focus on links between Vibrio cholerae abundance, chitin content and suspended particle load in size fractions of suspended particulate matter (SPM) along the salinity gradient of Sunderban mangrove waters. SPM decreased downstream, while salinity increased from 0.2 to 4. Particulate organic carbon (90 ± 25 μM) and nitrogen (9.1 ± 3.3 μM) highly correlated with SPM and turbidity, suggesting a significant contribution of fine particles to organic matter. Total chitin ranged 1–2 mg/l and decreased downstream. The distribution among size fractions of SPM, chitin and V. cholerae O1 (the bacterial serogroup mainly associated with cholera epidemics) was similar, with ~98% of the total in the fraction <20 μm. In comparison, the number of V. cholerae O1 attached to zooplankton and microplankton size classes >20 μm was almost negligible, in contrast to usual assumptions. Thus, microdetritus, nanoplankton and fungal cells in size classes <20 μm represent a chitinaceous substrate on which V. cholerae can grow and survive. Total bacteria, cultivable vibrios and V. cholera O1 increased 5–10 times downstream, together with salinity and nitrite concentration. Overall, nitrate and silicate concentrations were relatively constant (>22 μM N and 100 μM Si). However, nitrite increased ~9 times in the outer sector, reaching ~1.2 μM N, probably as a result of increased abundance of nitrate-reducing vibrios. A characterization of Vibrio habitats that takes account of the presence of nitrate-reducing bacteria could improve the understanding of both mangrove nitrogen cycling and cholera seasonality.     

A shared antigen among Vibrio species: Outer membrane protein-OmpK as a versatile Vibriosis vaccine candidate in Orange-spotted grouper (Epinephelus coioides)

The outer membrane protein-OmpK has been considered as a vaccine candidate for the prevention of infections due to Vibrio harveyi, Vibrio alginolyticus and Vibrio parahaemolyticus in fish. Interestingly, the polyclonal antibody raised against the recombinant OmpK from V. harveyi strain EcGs020802 recognized the OmpK homologues from other strains of Vibrio species by immunoblotting. The ompK genes from 19 Vibrio strains including V. harveyi (11), V. alginolyticus (6) and V. parahaemolyticus (2) were then cloned and sequenced. Alignment analysis based on the amino acid sequences indicated that the OmpK from V. harveyi strain EcGs020802 had 71.7–99.2% of identities with those from V. harveyi, V. alginolyticus and V. parahaemolyticus. Western blot analysis revealed that the corresponding native proteins ranged between 28 and 31 kDa, consistent with predicated molecular weight of OmpK in Vibrio strains. Furthermore, the cross-protective property of recombinant OmpK was evaluated through challenge with heterogeneous virulent Vibrio strains in Orange-spotted groupers (Epinephelus coioides). Orange-spotted groupers vaccinated with recombinant OmpK were more tolerant of the infection by virulent Vibrio strains and their relative percentage survival (RPS) was correlative with the degree of the identity of deduced amino acid sequences of their OmpK. Taken together, the OmpK is a conserved protective antigen among tested Vibrio species and might be a potentially versatile vaccine candidate for the prevention of infections due to V. harveyi, V. alginolyticus and V. parahaemolyticus.     

Development of a single base extension-tag microarray for the detection of pathogenic Vibrio species in seafood

In this study, a single base extension-tag array on glass slides (SBE-TAGS) microarray was established to detect the seven leading seafood-borne pathogens, including Vibrio parahaemolyticus, Vibrio cholerae, Vibrio vulnificus, Vibrio mimicus, Vibrio alginolyticus, Vibrio anguillarum, and Vibrio harveyi. Three multiplex PCR assays were developed to specifically target the following species with individual gene markers, which are aadS, tdh, and trh for V. parahaemolyticus; col, toxR, and vvh for V. alginolyticus, V. mimicus, and V. vulnificus; and empA, vhh1, and tcpA for V. anguillarum, V. harveyi, and V. cholerae, respectively. The purified PCR products were used as template DNA for single base extension-tag reactions, labeled with Cy3 fluorescent dye and hybridized to DNA microarrays. The detection specificity of this microarray method was 100%, with the sensitivity for pure genomic DNA at 200 fg to 2 pg per reaction. Application of the DNA microarray methodology to 55 naturally contaminated seafood samples (shrimp, fish, and oysters) revealed the presence of V. parahaemolyticus at 50.9% and V. alginolyticus at 32.7%. This corresponds with traditional assays (microbiological and biochemical tests) except one sample which was identified as negative in V. parahaemolyticus by the microarray assay but as positive by the conventional method. Therefore, a combination of multiplex PCR with DNA microarray hybridization based on SBE-TAGS ensures rapid and accurate detection of pathogenic Vibrio species in seafood, thereby providing safer seafood products for consumers at a low financial burden to the aquaculture industry.     

Fucoidan effectively provokes the innate immunity of white shrimp Litopenaeus vannamei and its resistance against experimental Vibrio alginolyticus infection

In this study, we examined the effect of fucoidan on the immune response of white shrimp Litopenaeus vannamei and its resistance against Vibrio alginolyticus infection. Fucoidan induced degranulation, caused changes in the cell morphology, and increased activation of prophenoloxidase (proPO) and the production of superoxide anions in vitro. Shrimp that received fucoidan via immersion at 100, 200, and 400 mg l^?1 after 3 h showed haemocyte proliferation and a higher mitotic index of haematopoietic tissue. In another experiment, the haemocyte count, phenoloxidase (PO) activity, and respiratory bursts (RBs) were examined after the shrimp had been fed diets containing fucoidan at 0 (control), 0.5, 1.0, and 2.0 g kg^?1 for 7–21 days. Results indicated that these parameters directly increased with time. The immune parameters of shrimp fed the 1.0 g kg^?1 diet were significantly higher than those of shrimp fed the 2.0 g kg^?1 diet after 14 and 21 days. Phagocytic activity and the clearance efficiency against V. alginolyticus were significantly higher in shrimp fed the 1.0 g kg^?1 diet compared to those of shrimp fed the 0, 0.5 and 2.0 g kg^?1 diets. In a separate experiment, shrimp that had been fed diets containing fucoidan for 21 days were challenged with V. alginolyticus at 10⁶ colony-forming units shrimp^?1. Survival rates of shrimp fed the 1.0 and 2.0 g kg^?1 diets were significantly higher than those of shrimp fed the 0 and 0.5 g kg^?1 diets for 96–120 h. We concluded that fucoidan provokes innate immunity of shrimp as evidenced by haemocyte degranulation, proPO activation, and the mitotic index of haematopoietic tissue, and that dietary administration of fucoidan at 1.0 g kg^?1 enhanced the immune response of shrimp and their resistance against V. alginolyticus infection.     

Experimental Vibriosis Induction with Vibrio alginolyticus of Larvae of the Catarina Scallop (Argopecten ventricosus =circularis) (Sowerby II, 1842)

Abstract Six-day-old larvae of the catarina scallop, Argopecten ventricosus (=circularis), were infected with different concentrations of Vibrio alginolyticus to determine virulence and to describe vibriosis in this species. The development of vibriosis was compared to the effect of the supernatant of a 24-h V. alginolyticus culture. An experimental larvae culture system (ELCS) yielded a maximum survival of 80% from the 6th to the 19th day (control and low concentrations of V. alginolyticus). No effect was shown with concentrations of V. alginolyticus below 0.5 × 10⁵ CFU ml⁻¹. At concentrations higher than 5.0 × 10⁵ CFU ml⁻¹, swimming depletion, empty stomachs, lipidic granules in the digestive system, velum degradation, and massive mortality were observed. The supernatant of V. alginolyticus culture showed similar effects to the highest concentrations of V. alginolyticus cells. Received: 13 November 1996; Accepted 28 March 1997     

The survival of different vibrios in association with a laboratory culture of the red-tide-causing organism Amphidinium carterae

The survival of different vibrios in association with a red-tide-causing organism Amphidinium carterae was studied in the laboratory. Vibrio alginolyticus and V. harveyi could not survive beyond 14 days in an actively growing culture of A. carterae. On the other hand, V. parahaemolyticus could be detected up to 40 days.     

Evolutionary relationships in Vibrio and Photobacterium as determined by immunological studies of superoxide dismutase

The amino acid sequence divergence of superoxide dismutases (SODs) from 22 species and five groups of Vibrio, Photobacterium, and a number of related organisms was determined by means of the microcomplement fixation technique and the Ouchterlony double diffusion procedure. Five reference antisera were used which had been prepared against the purified SODs from V. alginolyticus, V. splendidus II, V. fischeri, V. cholerae, and P. leiognathi. With a few exceptions the results were in agreement with past studies of other informational molecules and provided a comprehensive overview of evolutionary relationships in Vibrio and Photobacterium. The genus Vibrio was found to consist of a major group of primarily marine species which included V. fischeri, V. logei, V. splendidus, V. pelagius, V. nereis, V. campbellii, V. harveyi, V. natriegens, V. alginolyticus, V. parahaemolyticus, V. proteolyticus, V. fluvialis, V. vulnificus, V. nigripulchritudo, and V. anguillarum. On the outskirts of this large and relatively heterogeneous group were the fresh water and estuarine species V. cholerae and V. metschnikovii as well as the marine species V. gazogenes. A considerable distance from Vibrio were the related species of Photobacterium: P. phosphoreum, P. leiognathi, and P. angustum. Both genera were distant from species of Aeromonas as well as from Plesiomonas shigelloides, Escherichia coli, and Alteromonas hanedai, a luminous strict aerobe. The agreement between these and previous studies of evolution of informational molecules in Vibrio and Photobacterium is best explained by vertical evolution (involving no genetic exchange between species) rather than by its opposite — horizontal evolution.Non-Standard Abbreviations Anti-Plei, anti-Valg, anti-Vcho, anti-Vfis, anti-Vspl antisera to the Fe-containing superoxide dismutases from Photobacterium leiognathi strain 480, Vibrio alginolyticus strain 90, V. cholerae strain M 13, V. fischeri strain 61, and v. splendidus biotype II strain 2, respectively - AP alkaline phosphatase - ATCC American Type Culture Collection - GS glutamine synthetase - ImD immunological distance - NCMB National Colleciion of Marine Bacteria - NCTC National Collection of Type Cultures - NRC National Research Council of Canada Culture Collection - SDS sodium dodecyl sulfate - SOD superoxide dismutase