Harveyi Strain Research Articles

Detection of Luminous Vibrio harveyi in Penaeid Shrimp Through Nested PCR Using Haemolysin Gene Primer

Whiteleg shrimp ( Litopenaeus vannamei ) is one of the most important aquaculture commodity in Indonesia. However, the luminous disease primarily caused by Vibrio harveyi bacteria still becomes an obstacle in penaeid shrimp farming, especially in shrimp hatchery. This study was aimed to identify the presence of V. harveyi in L. vannamei through nested PCR using haemolysin gene primer. First, initial primers were designed using V. harveyi VIB 391 haemolysin gene sequence (accession number: DQ640264), flanking the position 133 to 756. This primer pairs were used to identify haemolysin gene in both V. harveyi MR5339 and V. harveyi 275 strain. Sequencing results from each sample showed 99% similarity with haemolysin gene sequence in Genebank. Furthermore, the sequence of V. harveyi MR5339 haemolysin gene was used to design the nested PCR primers. The first primer pairs of nested PCR have successfully amplified the haemolysin gene fragment of all V. harveyi strains samples from position 52 to 405. The second primer pairs of nested PCR have amplified position 204 to 405 where it can detect all of V. harveyi strains used as sample sources in this study. The application of nested PCR technique in this study was able to identify V. harveyi strains at serial dilution of cells density as low as 10 0 cfu/mL, which is equal to a single cell or at DNA concentration up to 10 1 fg/µL.

Seasonal variation in the antivibrio activity of two organic extracts from two red seaweed:Palmaria palmataand the introducedGrateloupia turuturuagainst the abalone pathogenVibrio harveyi

The wide polarity range and highly polar compounds of two selected red seaweed, Grateloupia turuturu and Palmaria palmata were extracted using two different types of solvent, dichloromethane/methanol and methanol/water. Monthly in vitro antibacterial activities were studied using the microplate method against the marine bacteria Vibrio harveyi strain ORM4, known to infect abalone. Inhibition, slowdown and delay of Vibrio harveyi growth were investigated. Polar compounds of seaweed showed an activity against the abalone pathogen. The best activity was recorded from P. palmata collected in spring, with an inhibition of 7.9% of the bacterial growth. Preliminary 1H NMR profiles identified the differences between the extracts.

Simulated hatchery system to assess bacteriophage efficacy against Vibrio harveyi.

Vibriosis caused by luminous Vibrio harveyi commonly contributes to poor survival in shrimp hatcheries and aquaculture ponds. Lytic bacteriophages pathogenic for V. harveyi are currently being investigated as an alternative to antibiotics to prevent vibriosis. Here, 8 bacteriophages were isolated from oysters and clams using V. harveyi strains as baiting hosts. Among these bacteriophages, 1 strain (VHP6b) identified as broadly pathogenic for 27 V. harveyi strains examined was further characterized by electron microscopy and genome sequence analysis. Phage VHP6b possessed a tail and morphology consistent with it being a member of the family Siphoviridae, and its genome and proteome were most closely related to the Vibrio phages SSP02 and MAR10. An integrase gene essential for lysogeny was not evident. The ability of bacteriophage VHP6b to protect shrimp postlarvae against vibriosis caused by V. harveyi strain VH6 was demonstrated in a model system designed to simulate typical hatchery conditions. Bacteriophage treatment improved survival of postlarvae by 40 to 60% under these conditions, so therapies based on this or other bacteriophages may be useful in shrimp hatcheries.

Assessment of mutagenic, genotoxic, and cytotoxic potential of water samples of Harike wetland: a Ramsar site in India using different ex vivo biological systems.

Harike is a wetland of international importance under the Ramsar Convention. The present study entails the investigation of mutagenic, genotoxic and cytotoxic effect of surface water samples collected from five different areas of the Harike wetland by using the histidine reversion point mutation assay in Salmonella typhimurium (TA98 and TA100) strain with or without S9, bioluminescence mutagenicity assay using Vibrio harveyi (A16) strain, plasmid-nicking assay using pBR322 and 3(4,5-dimethyl-thiazol-2-yl)2,5-diphenyl-tetrazolium bromide assay as well as confocal imaging studies using Chinese hamster ovarian cell line, respectively. It was observed that although, the water sample of all the areas of wetland demonstrated mutagenic, genotoxic as well as cytotoxic activity, the effect was quite significant with the water samples from River Satluj and Khatan area (i.e. reservoir mainly contains Satluj water). The metal analysis of water samples was also conducted with atomic absorption spectrophotometer. The mutagenicity, genotoxicity and cytotoxicity of water samples emerged to be correlated with metal concentration. The source of toxic components seems to be associated with various industrial effluents and agricultural run-off. The results of the present study carry great importance in documenting the water quality monitoring data of the wetland.

Preliminary assessment of mutagenic and anti-mutagenic potential of some aminoalkanolic derivatives of xanthone by use of the Vibrio harveyi assay

The Vibrio harveyi assay was used to evaluate mutagenic and anti-mutagenic effects of four new aminoalkanolic derivatives of xanthone with anticonvulsant activity, to select the potentially safe compounds for further in vivo studies in animal models.The study showed that at a concentration of 40ng/ml the test compounds were not mutagenic. Additionally, two of the investigated compounds, namely the (R,S)-N-methyl-1-amino-2-propanol derivative of 6-methoxyxanthone (compound III) and the (R)-N-methyl-2-amino-1-butanol derivative of 7-chloroxanthone (compound IV) were strong inhibitors of the mutagenicity induced by 4-nitroquinoline-N-oxide (4-NQO) in V. harveyi strains BB7M and BB7XM. The inhibition percentages for compound IV were 49 (in BB7M) and 69 (in BB7XM), whereas for compound III these percentages were 47 (in BB7M) and 42 (in BB7XM), respectively.The present study demonstrates that four bioactive derivatives of xanthone display no mutagenic activity in the V. harveyi assay. In addition, compounds III and IV demonstrated considerable anti-mutagenic activity in this test. Based on the results obtained here, these compounds could be selected for further studies in animal models, while compounds III and IV should be tested further for their anti-mutagenic properties.

Seasonal antibacterial activity of two red seaweeds,PalmariapalmataandGrateloupia turuturu, on European abalone pathogenVibrio harveyi

Vibrio harveyi is the main pathogen of the European abalone Haliotis tuberculata, and recently caused important mortalities at the production sites of this marine gastropod in France. In the present work, the monthly an- tibacterial activity of two red seaweed species from the French Atlantic coast, the native Palmaria palmata and the intro- duced Grateloupia turuturu, were investigated against the abalone pathogen Vibrio harveyi strain ORM4. Water-soluble extracts were screened using the microplate method. Grateloupia turuturu showed an antibacterial activity with a max- imal growth inhibition in spring of around 16%. In contrast, Palmaria palmata was inactive, as further growth of the bacteria was observed. Preliminary one-dimensional proton nuclear magnetic-resonance ( 1 H NMR) profiles identified the differences between the two water-soluble extracts.

In vitro biofilm forming capacity on abiotic contact surfaces by outbreak-associated Vibrio harveyi strains

Objective: To evaluate the in vitro biofilm forming capacity on abiotic food contact surfaces by Vibrio harveyi (V. harveyi) strains. Methods: Thirty six Gram-negative V. harveyi strains were isolated from various street vended seafood outlets in a food processing line and evaluated for their ability to produce mucoid biofilms on food contact surfaces using a microplate assay. Phenotypic characterization of mucoid biofilm producing V. harveyi strains were screened on Congo red agar, thiosulfate-citrate-bile salts-sucrose agar and tryptic soy agar, respectively. Results: Only five V. harveyi strains (14%) were mucoid biofilm producers characterized by formation of black colonies, whereas the remaining 31 strains (86%) were not capable of producing biofilm characterized by formation of red colonies or pinkish-red colonies with darkening at the centre. The morphological, physiological and biochemical characteristics of these isolates were studied using standard protocols. Strain identification was confirmed by polymerase chain reaction targeted to species-specific polymerase chain reaction primers VH-1 and VH-2 corresponding to variable regions of V. harveyi 16S rRNA sequence. All the biofilm-forming strains showed resistance to at least three antimicrobial compounds tested. V . harveyi strains isolated from various seafood were able to form biofilms of different capacity, and the strains VB267, VB238 and VB166 isolated from cat fish, shrimp and eel fish exhibited significantly greater biofilm forming ability compared to other isolates. Conclusions: It can be concluded from the present study that the strain VB166 was able to better attach and form subsequent biofilms on glass and stainless steel compared to high density polyethylene. These properties allow these bacteria to survive, proliferate and persist in street vended seafood outlets.

Single or combined effects of Lactobacillus sakei and inulin on growth, non-specific immunity and IgM expression in leopard grouper (Mycteroperca rosacea)

The aim of this study was to evaluate the single or combined effects of Lactobacillus sakei with inulin suitable for immunological in vivo studies in farmed fish. By in vitro assays, L. sakei strain 5-4 showed antibacterial activities against all assayed fish pathogens (except the Vibrio harveyi strain CAIM-1793). L. sakei was able to survive at high fish bile concentrations. Fermentation of the agave inulin resulted in a large increase in number of lactobacilli. For the in vivo study, fish were fed for 8 weeks four practical diets: control diet (control), L. sakei 5-4 (10(7) CFU/g), inulin (1% or 10 g/kg) and L. sakei + inulin (10(7) CFU/g + 10 g/kg). The weight gain showed clearly the synergistic effect of L. sakei 5-4 and inulin at 6 and 8 weeks of treatments. Leopard grouper fed with L. sakei alone or combined with inulin have significantly increased the assayed physiological and humoral immune parameters. By real-time PCR assays, the mRNA transcripts of immunoglobulin M (IgM) were found to be higher expressed in intestine, head kidney, mucus, gill, spleen and skin. Moreover, mRNA expression levels of IgM in head kidney and anterior intestine were measured by real-time PCR. L. sakei 5-4 and L. sakei + inulin supplemented diet up-regulated the expression of IgM at week 4 and 8 in intestine and head kidney, respectively. These results support the idea that the L. sakei 5-4 alone or combined with agave inulin improved growth performance and stimulates the immune system of leopard grouper.

Vibrio harveyi Hsp70: Immunogenicity and application in the development of an experimental vaccine against V. harveyi and Streptococcus iniae

Vibrio harveyi and Streptococcus iniae are severe bacterial pathogens of farmed fish. In a previous study, we have identified a secretory antigen, Sia10, from S. iniae. In this study, we cloned and analyzed an hsp70 gene from a pathogenic V. harveyi strain. We found that purified recombinant Hsp70 possesses ATPase activity and, when used as a subunit vaccine, could induce protection in Japanese flounder (Paralichthys olivaceus) against V. harveyi infection. Based on this observation, we fused Hsp70 to Sia10 and thus obtained a chimeric antigen Sia10-Hsp70, which was expressed from a plasmid pTSH contained in Escherichia coli DH5α. Western blot analysis showed that DH5α/pTSH was able to secret Sia10-Hsp70 into the extracellular milieu. The potential of DH5α/pTSH as a live vector vaccine was examined in a flounder model, which showed that DH5α/pTSH induced effective protection against not only V. harveyi infection but also S. iniae infection. ELISA analysis showed that fish vaccinated with DH5α/pTSH produced specific serum antibodies against both Hsp70 and Sia10. These results indicate that (i) V. harveyi Hsp70 is a biologically active protein with immunoprotective property; (ii) the chimeric antigen Sia10-Hsp70 delivered by a live bacterial host is an effective vaccine candidate against V. harveyi and S. iniae infection in aquaculture; (iii) using recombinant bacteria as vaccine hosts not only enables easier vaccine preparation but also easy construction of vaccines with cross protective potentials.

Deciphering Bacterial Universal Language by Detecting the Quorum Sensing Signal, Autoinducer-2, with a Whole-Cell Sensing System

Bacteria communicate with neighboring bacteria of the same species or of other species by means of chemical signaling molecules. The concentration of such signaling molecules is proportional to the bacterial population size; upon reaching a threshold concentration, corresponding to a threshold cell density, certain specialized genes are expressed. This system of communication among bacteria is known as quorum sensing (QS). QS regulates diverse behaviors, such as formation of biofilms and production of pathogenic factors. Autoinducer-2 (AI-2) is a QS signaling molecule that is used for interspecies communication by both Gram-positive and Gram-negative bacteria. Bacteria are known to play an important role in many diseases, from infections to chronic inflammation. Therefore, QS is involved in a variety of disorders of bacterial origin or where bacteria play a crucial pathogenic role. One such condition is inflammatory bowel disease (IBD), a chronic inflammation of the gastrointestinal (GI) tract that includes debilitating diseases, such as ulcerative colitis (UC) and Crohn's disease (CD). To date, noninvasive methods are unavailable for the diagnosis and monitoring of IBD. We hypothesized that detection of QS molecules in physiological samples, specifically saliva and stool specimens, would provide with a method for the noninvasive, early diagnosis and monitoring of IBD conditions. To that end, we developed and optimized a whole-cell sensing system for AI-2, which is based on Vibrio harveyi strain BB170. Furthermore, we standardized and applied the biosensing system for the quantitative detection of AI-2 in saliva, stool, and intestinal samples from IBD patients.

Evaluation of mutagenic and antimutagenic properties of new derivatives of pyrrolidine-2,5-dione with anti-epileptic activity, by use of the Vibrio harveyi mutagenicity test

The Vibrio harveyi test was used to evaluate mutagenic and antimutagenic properties of nineteen new derivatives of pyrrolidine-2,5-dione (compounds 1–19) with antiepileptic activity. Four V. harveyi strains were used: BB7 (wild type) and the genetically modified strains BB7M, BB7X and BB7XM (i.e. strains with additional mucA and mucB genes, UV hypersensitivity, and UV hypersensitivity with plasmid pAB91273, respectively). None of the derivatives of 2-ethyl-2-methylsuccinic acid (compounds 1–7) had mutagenic activity against the tester strains of V. harveyi, but this set had strong or moderate antimutagenic activity against 4-nitroquinoline-N-oxide (NQNO) in the tester strains BB7, BB7X, and BB7M. This antimutagenic activity ranged from 51% to 67%, through 51–66% to 71–83% for V. harveyi BB7, BB7X and BB7M strains, respectively. Mutagenic activities in the group of 2,2-diphenyl-succinic acid derivatives (compounds 8–19) were variable and depended on the tester strain used. Compounds 8–19 were devoid of mutagenic properties against BB7 (wild-type strain). Among this group only compound 9, with the fluorine substituent in position 2 of the aromatic system, was devoid of mutagenic potential against all tester strains. The compounds in this group (8–19) demonstrated strong antimutagenic activity only against strain BB7 (inhibition ranging from 51% to 71%). We conclude that there are various mutagenic and antimutagenic activities of derivatives of pyrrolidine-2,5-dione. Moreover, our studies have proven that the V. harveyi test can be applied for primary mutagenicity and antimutagenicity assessment of these new compounds.

Immunological study of the outer membrane proteins of Vibrio harveyi: Insights that link immunoprotectivity to interference with bacterial infection

Vibrio harveyi is a bacterial pathogen that affects marine vertebrates and invertebrates. In this study, we identified 13 outer membrane proteins (OMPs) from a pathogenic V. harveyi strain and analyzed their immunological properties. In vivo immunogenicity analysis showed that antibodies specific to recombinant proteins of the 13 OMPs were detected in the antiserum of V. harveyi-infected rat. When used as subunit vaccines to immunize Japanese flounder (Paralichthys olivaceus), all OMPs were able to elicit specific serum antibody production in the vaccinated fish; however, only two OMPs (OMP173 and OMP214) induced high levels (>70%) of relative percent survival. Pre-incubation of V. harveyi with the antisera of protective OMPs significantly impaired bacterial infectivity against peripheral blood leukocytes (PBL), whereas the antisera of non-protective OMPs had no apparent effect on infection. OMP173 antibodies could bind whole V. harveyi cells and exhibit bactericidal effect in a complement-dependent manner. Passive immunization showed that fish received OMP173 antiserum before being infected with V. harveyi exhibited significantly reduced mortality rate and lower bacterial loads in liver, spleen, and kidney. Finally, treatment of FG cells with OMP173 prior to V. harveyi infection protected the cells from bacterial invasion to a significant extent. Take together, these results indicate that two of the examined OMPs induce protective immunity through production of specific antibodies that block bacterial invasion, and that one OMP is likely to be involved in host cell interaction during the infection process. Thus, the immunoprotectivity of the OMPs is probably associated with functional participations of the OMPs in bacterial infection.

English

Vibrio harveyi strains were isolated from the water samples of low salinity.  Isolates were confirmed by both the presence of vhh gene using polymerase chain reaction and bio-chemical tests.  V. harveyi was grown in LB medium and its spent culture was treated with ethyl acetate. The organic layer was pooled and dried by Roto - Vapour with vacuum at 30ËšC. The dried residues were dissolved with 50 % acetonitrile and water. The presence of N-acyl homoserine lactones (AHL) compounds from the residues of crude extract and the standard “3 - Oxo hexanoyl homoserine lactone” were analyzed separately using Liquid chromatography and mass spectrometry (LC-MS). Different peaks were detected and peaks corresponding to 214.4 m/z were identified as N- (3-oxohexanoyl)-L-homoserine lactones in the sample. Different unknown peaks were also identified.  Aqueous extracts of 47 terrestrial plants were evaluated against luminescence disease causing V. harveyi, through “Agar well diffusion assay”. Ten plant extracts showed zone of inhibition (5 to 7 mm) on V. harveyi. Extracts from Phyllanthus niruri and Cynodon dactylon showed the highest zone of inhibition (8.3 mm) against V. harveyi followed byCalotropis gigantean and Costus speciosus (7.3 mm). Plant extracts were reported for the presence of alkanes, alkenes, aromatics, primary and secondary amines etc, byFourier transform infra red spectroscopy (FT-IR). The antagonistic activity may be due to by the presence of these functional compounds in the extracts. Therefore, terrestrial plants could be used to control aquatic bacterial pathogens, which can also be extended to control human bacterial pathogens.   Key words: V. harveyi, extraction of N-acyl homoserine lactones (AHL), analysis by Liquid chromatography and mass spectrometry (LC-MS), terrestrial plants, antagonism,Fourier transform infra red spectroscopy.

Characterization of abalone Haliotis tuberculata–Vibrio harveyi interactions in gill primary cultures

The decline of European abalone Haliotis tuberculata populations has been associated with various pathogens including bacteria of the genus Vibrio. Following the summer mortality outbreaks reported in France between 1998 and 2000, Vibrio harveyi strains were isolated from moribund abalones, allowing in vivo and in vitro studies on the interactions between abalone H. tuberculata and V. harveyi. This work reports the development of primary cell cultures from abalone gill tissue, a target tissue for bacterial colonisation, and their use for in vitro study of host cell-V. harveyi interactions. Gill cells originated from four-day-old explant primary cultures were successfully sub-cultured in multi-well plates and maintained in vitro for up to 24days. Cytological parameters, cell morphology and viability were monitored over time using flow cytometry analysis and semi-quantitative assay (XTT). Then, gill cell cultures were used to investigate in vitro the interactions with V. harveyi. The effects of two bacterial strains were evaluated on gill cells: a pathogenic bacterial strain ORM4 which is responsible for abalone mortalities and LMG7890 which is a non-pathogenic strain. Cellular responses of gill cells exposed to increasing concentrations of bacteria were evaluated by measuring mitochondrial activity (XTT assay) and phenoloxidase activity, an enzyme which is strongly involved in immune response. The ability of gill cells to phagocyte GFP-tagged V. harveyi was evaluated by flow cytometry and gill cells-V. harveyi interactions were characterized using fluorescence microscopy and transmission electron microscopy. During phagocytosis process we evidenced that V. harveyi bacteria induced significant changes in gill cells metabolism and immune response. Together, the results showed that primary cell cultures from abalone gills are suitable for in vitro study of host-pathogen interactions, providing complementary assays to in vivo experiments.

Proteomic Analysis of Protein Expression in the Induction of the Viable But Nonculturable State of Vibrio harveyi SF1

Vibrio harveyi has been reported to enter into a viable but nonculturable (VBNC) state. One marine V. harveyi strain, SF1 became nonculturable when incubated in seawater microcosm at 4°C within 60days. We investigated protein expression in the exponential phase of V. harveyi SF1 and compared it to the VBNC state. Cytosolic proteins were resolved by two-dimensional polyacrylamide gel electrophoresis using pH 4-7 linear gradients. Among these proteins, sixteen proteins which were strongly downregulated or upregulated in the VBNC cells were identified by MALDI-TOF-TOF mass spectrometry. The results indicated that the differentially expressed proteins were mainly focused on stress response proteins and key components of central and intermediary metabolism, like carbohydrate metabolism, transport, and translation. This study provided clues for understanding the mechanism of adaptation to the VBNC state.

Pathogenicity of halophilicVibrio harveyiin giant freshwater prawns (Macrobrachium rosenbergiiDe Man)

In this study, the phenotypic and pathogenic properties in freshwater host were characterized in 14 strains of halophilic Vibrio harveyi isolated from infected marine black tiger shrimp, Penaeus monodon. The lysogenic phenotype was assayed via prophage excision and mitomycin C induction. The bacteria were grouped into two types, corresponding to lysogenic and non-lysogenic strains. The pathogenicity was determined via direct injection of bacterial cultures into post-larval juvenile giant freshwater prawns, Macrobrachium rosenbergii De Man. All of the infected prawns showed similar symptoms and inflamed hepatopancreas. The V. harveyi isolates derived from the first-injected infected prawns were re-isolated and re-injected into healthy giant freshwater prawns, in which they retained similar infectivity. Both lysogenic and non-lysogenic Vibrio spp. showed identical virulence associated with 100% mortality within one day post-injection. TEM micrographs showed hepatopancreatic nuclear deformation and lipid breakdown caused by lysogenic γ-hemolytic VL19 and non-lysogenic β-hemolytic V33. However, the V33 strain was associated with severely disrupted mitochondria. None of the V. harveyi strains was able to produce a biofilm. Together, our findings indicate that the lysogenic and non-lysogenic halophilic V. harveyi isolated from marine shrimps may use different virulence factors that are responsible for their pathogenicity in freshwater prawns.

Development of TaqMan real-time PCR assays for monitoring Vibrio harveyi infection and a plasmid harbored by virulent strains in European abalone Haliotis tuberculata aquaculture

The Gram-negative bacterium Vibrio harveyi is known to be highly pathogenic for the European abalone Haliotis tuberculata, which is a gastronomically important marine gastropod with a high commercial value. Since 1998, some particular bacterial strains are described as implicated in recurrent mortality outbreaks in French farm and field stocks of abalone. Recently, a 9.6kb plasmid named pVCR1, was shown to be harbored by one highly V. harveyi virulent ORM4 strain suggesting its involvement in virulence phenotype. Thus, we have developed in the present study two TaqMan real-time PCR assays allowing to (i) rapidly and specifically detect, by a duplex procedure and in less than 2h, both V. harveyi and the presence of plasmid pVCR1 from unidentified bacterial colony and to (ii) quantify both V. harveyi and the plasmid pVCR1 in the hemolymph of abalone or its surrounding seawater. Quantification curves of V. harveyi or ORM4 strain seeded in hemolymph or artificial sea water samples were equivalent showing excellent qPCR efficacies and detection level as low as 18V. harveyi cell-equivalent genomic DNA in a PCR reaction well. This qPCR allowed us to monitor V. harveyi ORM4 strain in experimentally infected H. tuberculata. These diagnosis assays could provide powerful and useful tools to better understand the epidemiology of vibriosis caused by V. harveyi in different cultured marine species including H. tuberculata.

Autoinducers Act as Biological Timers in Vibrio harveyi

Quorum sensing regulates cell density-dependent phenotypes and involves the synthesis, excretion and detection of so-called autoinducers. Vibrio harveyi strain ATCC BAA-1116 (recently reclassified as Vibrio campbellii), one of the best-characterized model organisms for the study of quorum sensing, produces and responds to three autoinducers. HAI-1, AI-2 and CAI-1 are recognized by different receptors, but all information is channeled into the same signaling cascade, which controls a specific set of genes. Here we examine temporal variations of availability and concentration of the three autoinducers in V. harveyi, and monitor the phenotypes they regulate, from the early exponential to the stationary growth phase in liquid culture. Specifically, the exponential growth phase is characterized by an increase in AI-2 and the induction of bioluminescence, while HAI-1 and CAI-1 are undetectable prior to the late exponential growth phase. CAI-1 activity reaches its maximum upon entry into stationary phase, while molar concentrations of AI-2 and HAI-1 become approximately equal. Similarly, autoinducer-dependent exoproteolytic activity increases at the transition into stationary phase. These findings are reflected in temporal alterations in expression of the luxR gene that encodes the master regulator LuxR, and of four autoinducer-regulated genes during growth. Moreover, in vitro phosphorylation assays reveal a tight correlation between the HAI-1/AI-2 ratio as input and levels of receptor-mediated phosphorylation of LuxU as output. Our study supports a model in which the combinations of autoinducers available, rather than cell density per se, determine the timing of various processes in V. harveyi populations.

Characterization and Experimental Infection of Vibrio harveyi Isolated from Diseased Asian Seabass (Lates calcarifer)

Aims: Vibrio harveyi causes vibriosis to Asian seabass ( Lates calcarifer ). The disease spreads rapidly among fish stocked in the same cage. It causes high mortality especially in weak and small sized fish stocked at high density in poorly managed net cage. Study to determine the virulence levels of the bacterial pathogen in various aquaculture animals is a key to prevent vibriosis in marine aquaculture. Methodology and Result: Isolation of bacteria from diseased Asian seabass was done using tryptic soy agar (TSA) and thiosulphate citrate bile sucrose agar (TCBS) plates. Virulence of two strains of Vibrio harveyi (VHJR4 and VHJR7) was tested against clinically healthy aquaculture animals. The analysis revealed that the two bacterial strains differ in pathogenicity. The V. harveyi strain VHJR7 was virulent to Asian seabass at 1.40 x 10 4 c.f.u. g �1 , humpback grouper (Cromileptis altivelis ) at LD 50 8.33 x 10 3 c.f.u. g �1 and black tiger shrimp ( Penaeus monodon ) at LD 50 3.26 x 10 4 c.f.u. g �1 , respectively. The V. harveyi strain VHJR4 was not virulent to Asian seabass and humpback grouper but it caused mortality to black tiger shrimp at LD 50 1.32 x 10 6 c.f.u. g �1 . Phenotypically, the two strains shared most of the biochemical features except that the V. harveyi strain VHJR7 was a urease positive and grew at 8.5 % NaCl, and at 10 °C. The percentage similarity of nucleotide sequences of 16S rDNA in V. harveyi VHJR4 and V. harveyi VHJR7 was higher (99 %) but reduced at 95 % in hemolysin gene. Conclusion, significance and impact of study: Pathogenic strain of V. harveyi causes mortality and affects aquaculture production of Asian seabass. Hence, vaccine development against the bacterial pathogen is urgently needed for sustainability of Asian seabass aquaculture in Malaysia.

Development and efficacy of an attenuated Vibrio harveyi vaccine candidate with cross protectivity against Vibrio alginolyticus

Vibrio harveyi is a Gram-negative bacterial pathogen that can infect a wide range of marine animals. In previous studies, we have reported a virulent V. harveyi strain, T4D. In the present study, an attenuated mutant of T4D, T4DM, was obtained by selection of rifampicin resistance. Compared to the wild type, T4DM was different in whole-cell protein profile and much slower in growth rate when cultured in stress conditions caused by iron depletion. Virulence analysis showed that compared to T4D, T4DM exhibited a dramatically increased median lethal dose, impaired tissue dissemination capacity, defective hemolytic activity, and significantly reduced resistance against the killing effect of host serum. To examine the potential of T4DM as a live attenuated vaccine, Japanese flounder (Paralichthys olivaceus) were vaccinated with T4DM via intraperitoneal injection or immersion. The results showed that at one and two months post-vaccination, fish administered with T4DM via both approaches, in particular that of immersion, were effectively protected against not only V. harveyi but also Vibrio alginolyticus, another important fish pathogen. Microbiological analysis showed that following immersion vaccination, T4DM was recovered from the internal organs of the vaccinated fish in a time-dependent manner within the first 6 days post-vaccination. Serum antibodies against V. harveyi and V. alginolyticus were detected in T4DM-vaccinated fish, and, compared to serum from control fish, serum from T4DM-vaccinated fish was significantly enhanced in bactericidal activity. These results indicate that T4DM is an attenuated strain with residual infectivity and that T4DM can induce effective cross-species protection against both V. harveyi and V. alginolyticus when used as a live immersion vaccine.