Process optimization for fermented siwu decoction by multi-index-response surface method and exploration of the effects of fermented siwu decoction on the growth, immune response and resistance to Vibrio harveyi of Pacific white shrimp (Litopenaeus vannamei).

The purpose of this study was to explore the optimal fermentation technology of Chinese herbal medicine formula-Siwu Decoction and the effects of fermented Siwu Decoction (FSW) on the growth performance, immune response, intestinal microflora and anti microbial ability of Litopenaeus vannamei. Response to surface methodology (RSM) was used to optimize the fermentation process of Siwu Decoction. The optimal fermentation conditions were obtained as follows: inoculation amount of mixed strains was 4.5%, fermentation time was 36 h, and the ratio of material to liquid was 20%. A total of 1260 shrimps were selected and divided into seven groups, three in parallel in each group. The dietary level of each group was as follows: Control （No additions）, USW1 (0.2% unfermented herbal medicine), USW2 (0.5% unfermented herbal medicine), USW3 (0.8% unfermented herbal medicine), FSW1 (0.2% fermented herbal medicine), FSW2 (0.5% fermented herbal medicine), FSW3 (0.8% fermented herbal medicine). The immune response and antioxidant defense ability of hemocytes and intestine were measured at 21 and 42 days of feeding and the intestinal flora and growth performance were measured at 42 days of feeding, after that, a 7-day challenge test against Vibrio harveyi was conducted. The results showed that fermented Siwu Decoction significantly improved the growth performance and body composition of Litopenaeus vannamei; significantly increased the total number of hemocytes, phagocytic activity, antibacterial activity and bacteriolytic activity of Litopenaeus vannamei, and improved the antioxidant activity of Litopenaeus vannamei; the addition of fermented Siwu Decoction significantly increased the gene expression level of hemocytes and intestinal tract of Litopenaeus vannamei, and improved the antioxidant activity of Litopenaeus vannamei. The abundance of Bacillus increased, while the abundance of Vibrio decreased. After Vibrio harveyi challenge, the cumulative mortality of FSW group was significantly lower than that of control group. Fermented Siwu Decoction may be a potential physiological enhancer in aquaculture, and can be widely used in aquaculture.

Effects of reducing dietary fishmeal with yeast supplementations on Litopenaeus vannamei growth, immune response and disease resistance against Vibrio harveyi.

The aim of this experiment was to investigate the effects of reducing dietary fishmeal (FM) with yeast culture (SYC) supplementation on growth, immune response, intestinal microbiota, intestinal morphology, and disease resistance of Litopenaeus vannamei. A total of 480 shrimps with an average initial body weight of 0.35 ± 0.002 g were randomly distributed into twelve tanks. Three isonitrogenous (40.00 crude protein) and isolipidic (8.00 crude lipids) diets with yeast culture supplementing fishmeal were formulated. The groups were divided into two (2) namely control group and experimental groups. The formulations of the groups were control (0 %, without yeast culture) and the experiment groups (SYC) [(1 % of yeast culture), and (2 % of yeast culture)]. Each diet was delivered in four replicate per treatment group. The results indicate significant improvement on the growth indices (specific growth rate, weight gain rate, survival rate and lower feed conversion ratio) with yeast culture treatment group after 56 days feeding trials (P < 0.05). Total hemolymph protein, superoxide dismutase, catalase, alkaline phosphatase, acid phosphatase, lysozyme and phenoxidase were enhanced but low aspartate aminotransferase, alanine aminotransferase, and glucose were observed in shrimp fed yeast culture diets (P < 0.05). The SYC groups showed insignificant differences in hemolymph cholesterol and triglyceride. Proteobacteria, Bacteroidetes, and Actinobacteria were the dominant bacteria found in all the SYC groups. At the genus level, Vibrio was significantly decreased (P < 0.05) in 2 % yeast culture diets supplemented group whereas the beneficial bacteria Pseudoalteromonas was significantly enhanced. Moreover, intestinal villus length and width in shrimps fed yeast culture diets were improved (P < 0.05). Dietary yeast culture supplementation can improve growth, intestinal health, immune response, and resistance against Vibrio harveyi infections in L. vannamei.

Transcriptome analysis of Catarina scallop (Argopecten ventricosus) juveniles treated with highly-diluted immunomodulatory compounds reveals activation of non-self-recognition system.

Marine bivalve hatchery productivity is continuously challenged by apparition and propagation of new diseases, mainly those related to vibriosis. Disinfectants and antibiotics are frequently overused to prevent pathogen presence, generating a potential negative impact on the environment. Recently, the use of highly diluted compounds with immunostimulant properties in marine organisms has been trailed successfully to activate the self-protection mechanisms of marine bivalves. Despite their potential as immunostimulants, little is known about their way of action. To understand their effect, a comparative transcriptomic analysis was performed with Argopecten ventricosus juveniles. The experimental design consisted of four treatments formulated from pathogenic Vibrio lysates at two dilutions: [(T1) Vibrio parahaemolyticus and Vibrio alginolyticus 1D; (T2) V. parahaemolyticus and V. alginolyticus 7C]; minerals [(T3) PhA+SiT 7C], scorpion venom [(T4) ViT 31C]; and one control (C1) hydro-alcoholic solution (ethanol 1%). The RNA sequencing (RNAseq) analysis showed a higher modulation of differentially expressed genes (DEG) in mantle tissue compared to gill tissue. The scallops that showed a higher number of DEG related to immune response in mantle tissue corresponded to T1 (V. parahaemolyticus and V. alginolyticus lysate) and T3 (Silicea terra® - Phosphoric acid®). The transcriptome analysis allowed understanding some interactions between A. ventricosus juveniles and highly-diluted treatments.

Evaluation of Homeopathic Phosphoric Acid, Silica and Pathogenic Vibrio on Digestive Enzyme Activity of Longfin Yellowtail Fish (Seriola rivoliana).

BACKGROUND: This research aimed to observe the effect of homeopathically prepared Vibrio parahaemolyticus (ViP) and V. alginolyticus (ViA) and the commercial homeopathic compound Similia (Phosphoricum acidum and Silicea terra) on the digestive enzyme activities of Seriola rivoliana juveniles under usual culture conditions. MATERIALS AND METHODS: Biochemical analysis was used to study the effect of highly diluted substances (7C potency) prepared from ViP and ViA (Treatment 1: T1) and the homeopathic compound Phosphoricum acidum and Silicea terra (Treatment 2: T2) on changes in the main digestive enzymes on weaning-state fish (WS; 30 days post-hatching [DPH]) and early juveniles (EJ; 62 DPH) versus a reference control group that received no homeopathic medicines. RESULTS: Treatment T2 significantly increased the activity of trypsin and lipase and decreased the activity of amylase, whereas treatment T1 increased the activity of chymotrypsin and reduced the activity of aminopeptidase-N in WS fish. Except for alkaline phosphatase, which was significantly reduced in the intestine, no significant differences in enzymatic activity were found between treated EJ fish and controls. The fish of the WS group had a higher growth rate with the T2 treatment. CONCLUSIONS: T1 treatment stimulated chymotrypsin in EJ fish and T2 promoted intestinal maturation of WS fish. Higher growth rate with the T2 treatment may be associated with the stimulation of trypsin activity. Thus, T2 may be applied, under hatchery conditions, during larval stages with an aim to enhance digestion and assimilation of inert food.

Dietary phytogenics and galactomannan oligosaccharides in low fish meal and fish oil-based diets for European sea bass (Dicentrarchus labrax) juveniles: Effects on gut health and implications on in vivo gut bacterial translocation.

European sea bass were fed four low FM/FO (10%/6%) diets containing galactomannan oligosaccharides (GMOS), a mixture of garlic oil and labiatae plants oils (PHYTO), or a combination of both functional products (GMOSPHYTO) for 63 days before exposing the fish to an intestinal Vibrio anguillarum infection combined with crowding stress. In order to evaluate functional diets efficacy in terms of gut health maintenance, structural, cellular, and immune intestinal status were evaluated by optical and electron microscopy and gene expression analyses. A semi-automated software was adapted to determine variations in goblet cell area and mucosal mucus coverage during the challenge test. Feeding with functional diets did not affect growth performance; however, PHYTO and GMOS dietary inclusion reduced European sea bass susceptibility to V. anguillarum after 7 days of challenge testing. Rectum (post-ileorectal valve) showed longer (p = 0.001) folds than posterior gut (pre-ileorectal valve), whereas posterior gut had thicker submucosa (p = 0.001) and higher mucus coverage as a result of an increased cell density than rectum. Functional diets did not affect mucosal fold length or the grade of granulocytes and lymphocytes infiltration in either intestinal segment. However, the posterior gut fold area covered by goblet cells was smaller in fish fed GMOS (F = 14.53; p = 0.001) and PHYTO (F = 5.52; p = 0.019) than for the other diets. PHYTO (F = 3.95; p = 0.049) reduced posterior gut goblet cell size and increased rodlet cell density (F = 3.604; p = 0.068). Dietary GMOS reduced submucosal thickness (F = 51.31; p = 0.001) and increased rodlet cell density (F = 3.604; p = 0.068) in rectum. Structural TEM analyses revealed a normal intestinal morphological pattern, but the use of GMOS increased rectum microvilli length, whereas the use of PHYTO increased (p≤0.10) Ocln, N-Cad and Cad-17 posterior gut gene expression. After bacterial intestinal inoculation, posterior gut of fish fed PHYTO responded in a more controlled and belated way in terms of goblet cell size and mucus coverage in comparison to other treatments. For rectum, the pattern of response was similar for all dietary treatments, however fish fed GMOS maintained goblet cell size along the challenge test.

Stenotrophomonas maltophilia AHL-Degrading Strains Isolated from Marine Invertebrate Microbiota Attenuate the Virulence of Pectobacterium carotovorum and Vibrio coralliilyticus.

Many Gram-negative aquacultural and agricultural pathogens control virulence factor expression through a quorum-sensing (QS) mechanism involving the production of N-acylhomoserine (AHL) signalling molecules. Thus, the interruption of QS systems by the enzymatic degradation of signalling molecules, known as quorum quenching (QQ), has been proposed as a novel strategy to combat these infections. Given that the symbiotic bacteria of marine invertebrates are considered to be an important source of new bioactive molecules, this study explores the presence of AHL-degrading bacteria among 827 strains previously isolated from the microbiota of anemones and holothurians. Four of these strains (M3-1, M1-14, M3-13 and M9-54-2), belonging to the species Stenotrophomonas maltophilia, were selected on the basis of their ability to degrade a broad range of AHLs, and the enzymes involved in their activity were identified. Strain M9-54-2, which showed the strongest AHL-degrading activity, was selected for further study. High-performance liquid chromatography-mass-spectrometry confirmed that the QQ enzyme is not a lactonase. Strain M9-54-2 degraded AHL accumulation and reduced the production of enzymatic activity in Pectobacterium carotovorum CECT 225T and Vibrio coralliilyticus VibC-Oc-193 in in vitro co-cultivation experiments. The effect of AHL inactivation was confirmed by a reduction in potato tuber maceration and brine shrimp (Artemia salina) mortality caused by P. carotovorum and Vibrio coralliilyticus, respectively. This study strengthens the evidence of marine organisms as an underexplored and promising source of QQ enzymes, useful to prevent infections in aquaculture and agriculture. To our knowledge, this is the first time that anemones and holothurians have been studied for this purpose.

Indole reduces the expression of virulence related genes in Vibrio splendidus pathogenic to sea cucumber Apostichopus japonicus.

Indole is a metabolite of tryptophan that can be synthesized by various bacteria. In the present study, production of indole by Vibrio splendidus Vs was determined using Kovac's reagent, and m/z was further determined by HPLC-MS. Extracellular indole reached a maximum concentration of 160 µM, when OD600 of V. splendidus Vs was approximately 0.9. In addition, glucose could reduce indole level, and 1% (m/v) glucose could reduce the mRNA level of tnaA, the gene encoding tryptophanase, down to 0.2%. To investigate the effects of indole on the mRNA levels of virulence related genes of V. splendidus Vs, mRNA levels of vsm, vsh and ABC respectively related to protease activity, haemolytic activity and ABC transporter ATP-binding protein were determined. Exogenous indole supplemented at a concentration of 125 µΜ could respectively down regulate the mRNA level of vsm, vsh and ABC to 16%, 13% and 11%. Meanwhile, indole could alter the expressions of immune related gene in Apostichopus japonicus. When coelomocytes were co-cultured with exogenous indole at a concentration of 125 µΜ, the mRNA level of Ajp105 and AjLBP/BPI1, were up regulated by 1.6-fold and 2.1-fold, respectively. Combined all the results in our study suggested that indole could alter the expressions of the virulence related genes in pathogenic V. splendidus Vs as well as the immune related genes in A. japonicus.

A novel biomarker for marine environmental pollution of HSP90 from Mytilus coruscus.

Heat shock protein 90 (HSP90) is a conserved molecular chaperone contributing to cell cycle control, organism development and the proper regulation of cytosolic proteins. The full-length HSP90 cDNA of Mytilus coruscus (McHSP90, KT946644) was 2420bp, including an ORF of 2169bp encoding a polypeptide of 722 amino acids with predicted pI/MW 4.89/83.22kDa. BLASTp analysis and phylogenetic relationship strongly suggested McHSP90 was a member of HSP90 family, and it was highly conserved with other known HSP90, especially in the HSP90 family signatures, ATP/GTP-Binding sites and 'EEVD' motif. The mRNA of McHSP90 in haemolymph was upregulated in all treatments including Vibrio alginolyticus and Vibrio harveyi challenge, metals stresses (copper and cadmium) and 180 CST fuel exposure. All the results implied the expression of McHSP90 could be affected by Vibrio challenge and environmental stress, which might help us gain more insight into the molecular mechanism of HSP against adverse stresses in mollusca.

Phage therapy treatment of the coral pathogen Vibrio coralliilyticus.

Vibrio coralliilyticus is an important coral pathogen demonstrated to cause disease outbreaks worldwide. This study investigated the feasibility of applying bacteriophage therapy to treat the coral pathogen V. coralliilyticus. A specific bacteriophage for V. coralliilyticus strain P1 (LMG23696), referred to here as bacteriophage YC, was isolated from the seawater above corals at Nelly Bay, Magnetic Island, central Great Barrier Reef (GBR), the same location where the bacterium was first isolated. Bacteriophage YC was shown to be a lytic phage belonging to the Myoviridae family, with a rapid replication rate, high burst size, and high affinity to its host. By infecting its host bacterium, bacteriophage YC was able to prevent bacterial-induced photosystem inhibition in pure cultures of Symbiodinium, the photosymbiont partner of coral and a target for virulence factors produced by the bacterial pathogen. Phage therapy experiments using coral juveniles in microtiter plates as a model system revealed that bacteriophage YC was able to prevent V. coralliilyticus-induced photoinactivation and tissue lysis. These results demonstrate that bacteriophage YC has the potential to treat coral disease outbreaks caused by the bacterial pathogen V. coralliilyticus, making it a good candidate for phage therapy treatment of coral disease.

Nutrient and microbial dynamics in eutrophying shrimp ponds affected or unaffected by vibriosis.

A field survey was conducted on two intensive shrimp farms using similar technical practices: one (DF) historically affected by a vibriosis, the other (HC) in which the pathogen has been observed although no mortality event has occurred. Because historical data suggest that eutrophication process may directly or indirectly play a role in the disease outbreak, we focussed our research on its dynamics. A higher variability of the phytoplanktonic compartment linked to an imbalance in the molar N:P ratio was observed in farm DF compared to farm HC, implying a modification on the linkage between the bacteria and phytoplankton compartments at DF. The beginning of the mortality outbreak at DF followed a shift from pico- to nanophytoplankton. The organic matter mineralization process at the water-sediment interface may explain the disturbance observed in the water column during eutrophication. The consequences of this disturbance on shrimps' health status and pathogen ecology are discussed.

A selenium-dependent glutathione peroxidase (Se-GPx) and two glutathione S-transferases (GSTs) from Chinese shrimp (Fenneropenaeus chinensis).

Glutathione S-transferases (GSTs) and glutathione peroxidases (GPxs) are essential components of cellular detoxification systems that defend cells against reactive oxygen species (ROSs). Two GSTgenes have recently been cloned from Fenneropenaeus chinensis and BLAST P analysis shows that one GST, designated FcMuGST, is similar to members of MuGST while the other has similarities to ThetaGST (FcThetaGST). A selenium-dependent glutathione peroxidase (Se-GPx) has also been cloned from F. chinensis. The alignment of the deduced GST and GPx amino acid sequences with those from other species showed that the residues essential for enzymatic function of these three proteins are highly conserved. Tissue distribution and response to pathogens for the three genes was investigated by RT-PCR analysis, which showed that the transcript of FcMuGST and FcGPx increased in response to Vibrio anguillarum infection, while FcThetaGST showed little change at the transcript level. GPx activity in gill tissues quickly increased at 6 h after V. anguillarum challenge and maintained at a relatively high level from 6 h to 24 h. Total GST activity in hepatopancreas and intestines of the bacterial challenged shrimp was increased at 6 h, and gradually recovered from 12 and 24 h to the normal level. These three genes were all predicted to play an important role in detoxification defense reactions. FcMuGST primarily scavenges excess ROS produced after bacterial infection, while clearance of endogenous hydrophobic electrophile molecules was mainly dependent on activities of FcThetaGST.

A prophenoloxidase from the Chinese mitten crab Eriocheir sinensis: gene cloning, expression and activity analysis.

Prophenoloxidase (proPO) is a conserved copper-containing enzyme that plays important roles in immune response of crustaceans and insects. In the present study, the full-length cDNA of a prophenoloxidase (designated EsproPO) was cloned from haemocytes of Chinese mitten crab Eriocheir sinensis by expressed sequence tag (EST) and PCR techniques. The isolated 3549bp full-length cDNA of EsproPO contained a 2040bp open reading frame (ORF) encoding a putative proPO protein of 679 amino acids, a 5'-untranslated region (UTR) of 68bp, and a long 3'-UTR of 1441bp. Two putative copper-binding sites, a proteolytic activation site, and a complement-like motif (GCGWPQHM) were identified in the deduced amino acid sequence of EsproPO. Homology analysis revealed that EsproPO was highly similar to other proPOs from crustaceans with identities from 52% to 68%. The conserved domains and motifs, and higher similarity with other proPOs suggested that EsproPO was a member of the proPO family. The mRNA expression of EsproPO and PO specific activities in the tissues of hepatopancreas, gill, gonad, muscle, heart, eye and haemocytes were measured by quantitative real-time PCR and colorimetric assay, respectively. The mRNA transcripts of EsproPO and PO specific activities could be detected in all the examined tissues with the highest level both in hepatopancreas. Three peaks of EsproPO mRNA expression were recorded at 2h, 12h and 48h in haemocytes of Chinese mitten crab post Vibrio anguillarum challenge, which was consistent with the temporal profile of PO specific activity. The mRNA expression pattern and the activity fluctuation of EsproPO post V. anguillarum stimulation indicated that it was potentially involved in the acute response against invading bacteria in Chinese mitten crab.

The effect of oral immuno-stimulation in juvenile carp (Cyprinus carpio L.).

The effect of a 2-week period of oral immuno-stimulation from the age of 2 or 6 weeks post-fertilisation (wpf; before and after reaching the ability to produce antibodies) onwards was investigated on various immune functions of the common carp, Cyprinus carpio. The immuno-stimulants Aeromonas salmonicida lipopolysaccharide, Yeast DNA (containing unmethylated CpG motifs) or high-M alginate (an extract of algae containing poly-mannuronic acid) were used. The effect of this treatment was studied on the kinetics of B cells in head kidney and peripheral blood leucocytes using flow cytometry, on the total plasma IgM level using ELISA, on cytokine and inducible nitric oxide synthase (iNOS) expression in the intestine, and acute phase protein expression in the liver, using real time quantitative PCR, and on exposure to Vibrio anguillarum. Oral administration of immuno-stimulants from 6 wpf resulted in decreased WCI12(+) (B) cell percentages in PBL (only after administration of LPS) and head kidney (all test groups), and a decreased total IgM level in plasma, suggesting that suppressive effects are strongly indicative of oral or juvenile tolerance. After administration from 2 wpf, the effects on WCI12(+) (B) cell percentages were less pronounced: the group fed with Yeast DNA showed higher percentages compared to the control group at 6 wpf, but lower percentages at 8 wpf. No changes were observed in the cytokine or iNOS expression levels in the intestine or acute phase protein expression in the liver. A challenge with V. anguillarum resulted in an initially higher cumulative mortality in the group fed with LPS, but lower mortality in the groups fed with Yeast DNA or high-M alginate compared to the control group, providing a provisional warning especially for the use of pathogen-derived immuno-stimulants, such as A. salmonicida LPS, in larval and juvenile fish.

Characterization of Vibrio fluvialis-like strains implicated in limp lobster disease.

Studies were undertaken to characterize and determine the pathogenic mechanisms involved in a newly described systemic disease in Homarus americanus (American lobster) caused by a Vibrio fluvialis-like microorganism. Nineteen isolates were obtained from eight of nine lobsters sampled. Biochemically, the isolates resembled V. fluvialis, and the isolates grew optimally at 20 degrees C; none could grow at temperatures above 23 degrees C. The type strain (1AMA) displayed a thermal reduction time (D value) of 5.77 min at 37 degrees C. All of the isolates required at least 1% NaCl for growth. Collectively, the data suggest that these isolates may embody a new biotype. Pulsed-field gel electrophoresis (PFGE) analysis of the isolates revealed five closely related subgroups. Some isolates produced a sheep hemagglutinin that was neither an outer membrane protein nor a metalloprotease. Several isolates possessed capsules. The isolates were highly susceptible to a variety of antibiotics tested. However, six isolates were resistant to erythromycin. Seventeen isolates harbored plasmids. Lobster challenge studies revealed that the 50% lethal dose of a plasmid-positive strain was 100-fold lower than that of a plasmid-negative strain, suggesting that the plasmid may enhance the pathogenicity of these microorganisms in lobsters. Microorganisms that were recovered from experimentally infected lobsters exhibited biochemical and PFGE profiles that were indistinguishable from those of the challenge strain. Tissue affinity studies demonstrated that the challenge microorganisms accumulated in heart and midgut tissues as well as in the hemolymph. Culture supernatants and polymyxin B lysates of the strains caused elongation of CHO cells in tissue culture, suggesting the presence of a hitherto unknown enterotoxin. Both plasmid-positive and plasmid-negative strains caused significant dose-related intestinal fluid accumulations in suckling mice. Absence of viable organisms in the intestinal contents of mice suggests that these microorganisms cause diarrhea in mice by intoxication rather than by an infectious process. Further, these results support the thermal reduction data at 37 degrees C and suggest that the mechanism(s) that led to fluid accumulation in mice differs from the disease process observed in lobsters by requiring neither the persistence of viable microorganisms nor the presence of plasmids. In summary, results of lobster studies satisfy Koch's postulates at the organismal and molecular levels; the findings support the hypothesis that these V. fluvialis-like organisms were responsible for the originally described systemic disease, which is now called limp lobster disease.

Elucidation of the Vibrio anguillarum genetic response to the potential fish probiont Pseudomonas fluorescens AH2, using RNA-arbitrarily primed PCR.

The antagonistic interaction between a potential fish probiont, Pseudomonas fluorescens strain AH2, and its target organism, Vibrio anguillarum, was investigated by studying the genetic response of the target organism when it was exposed to the antagonist. We compared the differential display of arbitrarily PCR-amplified gene transcripts in V. anguillarum serotype O1 when it was exposed to AH2 supernatant with the display of transcripts in nonexposed control cultures. Growth of V. anguillarum was immediately arrested when the organism was exposed to 50% (vol/vol) AH2 supernatant. A total of 10 potentially differentially expressed transcripts were identified. Among these we identified a gene homologous to rpoS that was induced in a dose-dependent manner when V. anguillarum was cultured in media supplemented with sterile filtered supernatant from AH2. rpoS was also induced when growth was arrested with the iron chelator 2,2-dipyridyl. A chromosomal transcript homologous to vibE that participates in vibriobactin synthesis in Vibrio cholerae was also upregulated during AH2 exposure. This transcript could represent a functionally active gene in V. anguillarum involved in biosynthesis of anguibactin or another V. anguillarum siderophore. On the pJM1 plasmid of V. anguillarum serotype O1, a pseudogene designated open reading frame E (ORF E) that contains a frameshift mutation was previously identified. The gene homologous to vibE identified in this study, interestingly, also has significant homology to ORF E on the amino acid level and does not possess the frameshift mutation. Thus, the chromosomally encoded vibE homologue could fulfil the role of the inactive plasmid-encoded ORF E pseudogene. Addition of Fe(3+) to the system eliminated the growth arrest, and the genes homologous to rpoS and vibE were not induced. To our knowledge, this is the first study linking rpoS induction to iron starvation. Taken together, the results of this study suggest that a major part of the antagonistic property exhibited by strain AH2 is caused by the ability of siderophores in the supernatant to efficiently chelate iron, which results in instant iron deprivation of the pathogen V. anguillarum and complete growth arrest.

Inhibition of luminescence and virulence in the black tiger prawn (Penaeus monodon) pathogen Vibrio harveyi by intercellular signal antagonists.

Expression of luminescence in the Penaeus monodon pathogen Vibrio harveyi is regulated by an intercellular quorum sensing mechanism involving the synthesis and detection of two signaling molecules, one of which is N-hydroxy butanoyl-L-homoserine lactone and the other of which is uncharacterized. Indirect evidence has suggested that virulence, associated with a toxic extracellular protein, and luminescence in V. harveyi are coregulated. In this study the effects of an acylated homoserine lactone antagonist produced by the marine alga Delisea pulchra on luminescence and toxin production in a virulent strain of V. harveyi were analyzed. Luminescence and toxin production were both inhibited by the signal antagonist at concentrations that had no impact on growth. Toxin production was found to be prematurely induced in V. harveyi cultures incubated in a 10% conditioned medium. Additionally, a significant reduction in the toxicity of concentrated supernatant extracts from V. harveyi cultures incubated in the presence of the signal antagonist, as measured by in vivo toxicity assays in mice and prawns, was observed. These results suggest that intercellular signaling antagonists have potential utility in the control of V. harveyi prawn infections.

Utilization of hemin and hemoglobin by Vibrio vulnificus biotype 2.

The eel pathogen Vibrio vulnificus biotype 2 is able to use hemoglobin (Hb) and hemin (Hm) to reverse iron limitation. In this stud, the adjuvant effect of both compounds on eel pathogenicity has been evaluated and confirmed. Further, we have studied the heme-iron acquisition mechanism displayed by this bacterium. Whole cells were capable of binding Hb and Hm, independently of (i) iron levels in growth medium and (ii) the presence of polysaccharide capsules on bacterial surface. The Hb- and Hm-binding capacity was retained by the outer membrane protein (OMP) fraction and was abolished after proteolytic digestion of OMP samples. Western blotting (immunoblotting) of denatured OMPs revealed that two major protein bands of 36 and 32 kDa were involved in both Hm and Hb binding. The expression of these proteins was not affected by iron levels. In addition, V. vulnificus biotype 2 produced extracellular proteases, not regulated by iron, that were active against native Hb. In conclusion, the overall data suggest that the eel pathogen V. vulnificus biotype 2 can obtain iron by means of a mechanism which involves a direct interaction between the heme moiety and constitutive OMPs.

Analysis of starvation conditions that allow for prolonged culturability of Vibrio vulnificus at low temperature.

The response of the estuarine human pathogen Vibrio vulnificus to starvation for carbon, nitrogen or phosphorus, or all three nutrients simultaneously (multiple-nutrient), was examined with respect to the maintenance of culturability during incubation at low temperature. V. vulnificus showed similar survival patterns during starvation for the individual nutrients when kept at 24 degrees C. On the other hand, cultures prestarved at 24 degrees C and then shifted to 5 degrees C maintained culturability at low temperature in a starvation-condition-dependent manner. Carbon and multiple-nutrient starvation were indistinguishable in their ability to mediate maintenance of culturability in the cold. Prolonged starvation for phosphorus had a similar effect, but nitrogen starvation did not allow for maintenance of culturability. Extracellular factors produced during starvation were not observed to have an effect on the culturability of cells incubated at low temperature. Protein synthesis during starvation for individual nutrients was analysed by two-dimensional PAGE of pulse-labelled proteins. Carbon and multiple-nutrient starvation gave nearly identical protein induction patterns involving at least 34 proteins, indicating that carbon starvation determines both responses. Nitrogen starvation for 1 h induced 24 proteins, while phosphorus starvation induced a set of 10 proteins after 1 h and about 40 proteins after 18 h. It is suggested that starvation for carbon or phosphorus induces maintenance of culturability of V. vulnificus incubated at low temperature via the synthesis of distinct sets of starvation-specific proteins.

Presence of a capsule in Vibrio vulnificus biotype 2 and its relationship to virulence for eels.

Strains of Vibrio vulnificus biotype 2, isolated from internal organs of diseased European eels as pure cultures of opaque cells, together with some reference strains from Japanese eels, were used in this study. Spontaneous translucent-phase variants were obtained from the corresponding parent strains and compared for a variety of phenotypic traits related to virulence for eels. The rate of colony dissociation from opaque to translucent cells was higher (around 10(-2)) than that observed for translucent to opaque cells (10(-3) to 10(-4)). Electron microscopy with ruthenium red revealed the presence of a capsule of variable thickness on opaque cells, whereas translucent-type colonies had no observable capsular materials. No differences in plasmid profiles were detected between the two cell types so that plasmids do not seem to be implicated in the mechanism of phase shift of biotype 2 strains. No apparent difference in outer membrane protein and lipopolysaccharide patterns could be observed between the cell types. Both isogenic morphotypes were able to grow in eel serum and minimal medium supplemented with ethylenediamine di(O-hydroxyphenyl-acetic acid) or transferrin. Therefore, the presence of capsule was not required for the acquisition of iron from iron chelators or for resistance to serum bactericidal action. Both morphotypes were highly virulent for elvers, although the 50% lethal dose for translucent cells was higher than that for the corresponding opaque cells. The latter observation, together with the overall data, suggests that the production of capsular materials by biotype 2 of V. vulnificus is not essential for the development of vibriosis in eels, at least when cells are injected intraperitoneally.

Collagenolytic activity of Vibrio vulnificus: potential contribution to its invasiveness.

Vibrio vulnificus (lactose-positive vibrio) produced collagenase when grown in 2% synthetic sea salts supplemented with hydrolyzed casein. The addition of collagen or peptone to the medium increased the level of collagenase production. Collagenase activity was inhibited by EDTA but not by fetal calf serum.