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<p>Aim Because of the major problems that certain Vibrio specie, especially Vibrio harveyi, can cause the aquaculture industries a rapid method to identify Vibrio isolates is required. Early diagnosis of a V. harveyi infection could facilitate disease surveillance, treatment and prevention in cultured marine animals. Therefore, the use of PCR to aid in the identification of Vibrio is increasing and a way of extracting DNA in a cheap, fast and easy way is also of an important requirement to facilitate rapid diagnosis.</p><p>Methods This report comprises biochemical profiling and PCR methods in the characterisation of four isolates of V. harveyi and single isolates of V. tubiashii, V. alginolyticus, V. anguillarum, V. splendidus, V. tapetis and V. parahaemolyticus.</p><p>Strains were examined for adherence to a Hep-2 cell line. Four different DNA extraction methods were evaluated and compared. The detection limits and the analytical limits of two PCR methods for Vibrio were determined.</p><p>Results The overall findings were that the use of a greater range of biochemical substrates than are in the API 20E is necessary to identify Vibrio strains, and that none of the strains tested adhered to Hep-2 cells. All extraction methods successfully produced DNA with the kit method giving the purest samples. RNA was a contaminant of the other techniques but this could be overcome by treating extracts with RNase. The rapid microwave extraction method gave appropriate PCR amplicons when tested.</p><p>Conclusion PCR determination of the VH-sequence in combination with VHA and a distinguishable colonial morphology may be a good choice for the identifying of Vibrio harveyi.</p>

La résistance bactérienne induite par l’utilisation massive d’antibiotiques en aquaculture, conduit à la recherche de nouveaux modes de traitement, des traitements anti-virulents et non toxiques. Différentes stratégies sont étudiées parmi lesquelles l’inhibition d’un système de régulation : le quorum sensing. Vibrio harveyi est une bactérie pathogène en aquaculture qui régule sa bioluminescence et la production de protéines de virulence par l’intermédiaire du quorum sensing. L’étude des éponges Leucetta chagosensis, Dysidea sp. 2669 et Cacospongia sp. 2110 collectées en Polynésie française, a permis l’isolement de huit métabolites secondaires dont quatre nouveaux : l’isonaamine D, le bis-isonaamidine A-Zn, la leucettamine D et le tuamotuolol. Leurs effets et leurs modes d’action ont été évalués sur deux phénotypes contrôlés par le quorum sensing chez V. harveyi: la bioluminescence et la production de métalloprotéases. Trois molécules ont ainsi montré une activité inhibitrice du quorum sensing de V. harveyi : l’isonaamine D, l’isonaamidine A et l’ilimaquinone. / The bacterial resistance induced by the massive use of antibiotics in aquaculture, leads to the search for new treatment methods, anti-virulent and non-toxic treatments. Different strategies are studied among which the inhibition of a system of regulation: the quorum sensing. Vibrio harveyi is a pathogenic bacterium in aquaculture that regulates its bioluminescence and the production of virulence proteins through quorum sensing. The study of sponges Leucetta chagosensis, Dysidea sp. 2669 and Cacospongia sp. 2110 collected in French Polynesia, allowed the isolation of eight secondary metabolites including four new ones: isonaamine D, bis-isonaamidine A-Zn, leucettamine D and tuamotuolol. Their effects and their modes of action were evaluated on two phenotypes controlled by quorum sensing in V. harveyi: bioluminescence and production of metalloproteases. Three molecules have thus shown an inhibitory activity of the quorum sensing of V. harveyi: isonaamine D, isonaamidine A and ilimaquinone.

Tilapia culture in Indonesia was started with the Mozambique Tilapia (Oreochromis mossambicus) in the 1930’s, and the Nile Tilapia (Oreochromis niloticus) the 1960’s. The genetic improvement program of the Nile Tilapia, has led Indonesia to be one of the main tilapia producers in the world. On the other hand, shrimp aquaculture in the country was not started until the 1960’s, it became more popular after the eye ablation technology for broodstock maturation was developed in the early 1980’s. The first experimental study was conducted to investigate the feasibility of low salinity shrimp farming in a polyculture system with tilapia. Polyculture increased the survival for shrimp (77% compared to 62%), but at the same time decreased the survival of tilapia (87% compared to 97%). Together, the data on survival, specific growth rates, and feed conversion ratios showed that the shrimp performed well at low salinity. The second experimental study investigated the feasibility of brackishwater shrimp farming in a polyculture system with tilapia. Polyculture increased the survival for shrimp (82% compared to 65%), and had higher survival for the tilapia (60% compared to 43%). The Red hybrid Tilapia strain used in the study experienced mortalities after one month, suggesting the need for a salt tolerant strain. The presence of tilapia stimulated the growth of microalgae (Chlorella dominance), promoted higher numbers of heterotrophic bacteria in the water, and had lower presumptive vibrios on TCBS agar. A challenge study was conducted by mixing pathogenic luminescent Vibrio harveyi UAZ-651 into shrimp and tilapia feed. The survival of shrimp in monoculture were significantly lower (20%) compared to in polyculture systems (75 - 95%). Mortality was not found in tilapia. Based on 16S rRNA gene sequence, shrimp monoculture water was dominated by marine Vibrio spp., while the polyculture system had Bacillus spp. and Vibrio spp. with high homology to V. cholerae. The presence of Bacillus spp. which produce a lactonase enzyme AiiA, seems to inhibit vibrio growth. While providing advantages, shrimp-tilapia polyculture might also contribute to streptococcosis transmission. Injecting shrimp with Streptococcus iniae and S. agalactiae resulted in mortalities. S. iniae caused higher mortality in the shrimp cultured in 20 ppt (40%) compared to 10 ppt (20%), and no mortality in 5 ppt. S. agalactiae caused higher mortality in 5 ppt (40%) compared to 10 ppt (20%) and 20 ppt (20%). Quorum sensing (QS) is a density dependent cell to cell communication process in bacteria. Based on challenge studies in shrimp, the luminescent Vibrio harveyi BB120 wild-type strain caused 75-90 % mortality through injection of 106 CFU/shrimp. The mortality patterns in the QS mutants suggest that QS defined, when specific virulence genes were expressed or repressed. As QS in V. harveyi consists of three different circuits, further experiments deployed six mutants lacking either a synthase or a receptor for each circuit. The highest survival in the CqsS (a receptor for CAI-1 circuit) mutant group indicates that the CAI-1 circuit is the most crucial for virulence, followed by the AI-2 and HAI-1 cascades. Chitin acquisition and oxygen scavenging may be two reasons for luminescence in V. harveyi evolution and why they infect shrimp.

Carbohydrates are known to play important roles in a large number of physiological and pathological processes. Conceivably, “binders” of carbohydrates of biological importance could be used as diagnostic and therapeutic agents. Currently, lectins are the major available tools in research for carbohydrate recognition. However, the available lectins often have cross-reactivity issues, along with the high costs and stability issues. Therefore, there is a critical need to develop alternatives (lectin mimics). In this regard, there have been very active efforts in developing different “binders”, such as small molecule lectinmimics and aptamers. Among all the small molecule lectinbmimics developments, boronic acid stands out as the most important building blocks of the sensors design for carbohydrates biomarkers due to its intrinsic binding affinities with diols. To address a fundamental question that whether boronic acid also binds to six-membered ring sugars, with very limited precedents, we provided a concrete experimental evidence of the binding. Specifically, a series of isoquinolinylboronic acids were found to have remarkably high binding affinities with fluorescence change upon binding to representative sugars. Most importantly, these isoquinolinylboronic aicds showed weak but very encouraging bindings with six-membered sugar model. All these promising results paves the way of using boronic acids, especially isoquinolinylboronic acid as building blocks for chemosensors design for biological carbohydrates biomarkers, which universally contain six-membered ring and liner diols. Aptamer provides another alternative way for sensors development for carbohydrates biomarkers as lectin mimics. Compared to lectins, they are normally cheaper and more stable. However, there is much less options. Another challenging area for aptamer-based lectin mimics development is the difficulty to differentiate changes in glycosylation patterns of a glycoprotein, which affect the function of a glycoprotein and thus recognized as biomarkers. To address this major challenge, our group first demonstrated that the incorporation of a boronic acid into DNA would allow for the aptamer selection process to gravitate towards the glycosylation site. To examine the generality of boronic acid incorporation, increase the structural diversity, and broaden the application of boronic acid-modified DNA, a series of B-TTP analogues with simplified structures were designed, synthesized, and successfully incorporated into DNA. A simple route was also developed using 1,7-octadiyne as a linker for both Sonogashira coupling with thymidine and CuAAC tethering of a boronic acid moiety. This paves the way for the preparation of a large number of B-TTPs with different structural features for aptamer selection or array analysis. Finally, bacterial quorum sensing has received much attention in recent years because of its relevance to pathological events such as biofilm formation. As one of the very first groups that developed a series of antagonists for AI-2 mediated quorum sensing, we herein designed and synthesized a series of analogues based on the structures of two lead inhibitors identified through virtual screening. Besides, we also examined their inhibitory activities, twelve of which showed equal or better inhibitory activities compared with the lead inhibitors. The best compound showed an IC50 of about 6 mM in a whole cell assay using Vibrio harveyi as the model organism. This encouraging results and SAR discuss also paves the way for the finding of more potent compound through further structure optimization.

Aim Because of the major problems that certain Vibrio specie, especially Vibrio harveyi, can cause the aquaculture industries a rapid method to identify Vibrio isolates is required. Early diagnosis of a V. harveyi infection could facilitate disease surveillance, treatment and prevention in cultured marine animals. Therefore, the use of PCR to aid in the identification of Vibrio is increasing and a way of extracting DNA in a cheap, fast and easy way is also of an important requirement to facilitate rapid diagnosis. Methods This report comprises biochemical profiling and PCR methods in the characterisation of four isolates of V. harveyi and single isolates of V. tubiashii, V. alginolyticus, V. anguillarum, V. splendidus, V. tapetis and V. parahaemolyticus. Strains were examined for adherence to a Hep-2 cell line. Four different DNA extraction methods were evaluated and compared. The detection limits and the analytical limits of two PCR methods for Vibrio were determined. Results The overall findings were that the use of a greater range of biochemical substrates than are in the API 20E is necessary to identify Vibrio strains, and that none of the strains tested adhered to Hep-2 cells. All extraction methods successfully produced DNA with the kit method giving the purest samples. RNA was a contaminant of the other techniques but this could be overcome by treating extracts with RNase. The rapid microwave extraction method gave appropriate PCR amplicons when tested. Conclusion PCR determination of the VH-sequence in combination with VHA and a distinguishable colonial morphology may be a good choice for the identifying of Vibrio harveyi.

Vibrio harveyi is a bioluminescent bacterium and the organism in which quorum sensing was discovered. It was recently found that a class of molecules, cyclic dipeptides, may be a new kind of quorum sensing signal that may affect other species in the genus. The purpose of this study was to determine if V. harveyi produced one of these molecules, cyclo(Phe-Pro) or cFP, and the effects it has on bioluminescence, growth and gene expression. Electrospray Mass Spectrometry was used to detect cFP, and it was found. While growth and gene expression were not significantly affected by cFP, bioluminescence was slightly induced at low concentrations. It appears that V. harveyi does not produce cFP and it does not significantly affect the luminescence quorum sensing controlled genes, and is most likely not a true signal, in V. harveyi.

β-Glucans have long been used as an immunostimulant in aquaculture. However, the relationship of its structure to its immunomodulatory properties are poorly understood. In this study, the particle size and chemical structure of β-glucans extracted from wild-type strain of baker's yeast (Saccharomyces cerevisiae) and its null-mutant yeasts Gas1 were characterised. Using Sigma β-glucan as a reference, the immunomodulatory properties of these polysaccharides in the germ-free Artemia franciscana model system in the presence of Vibrio harveyi bacterial challenge were investigated. The survival of the A. franciscana nauplii, upon challenge with V. harveyi, was significantly higher in all three glucan-treated groups compared to the control. The glucan Gas1 with a lower degree of branching and shorter side chain length had the most prominent V. harveyi-protective effects. The particle size did not affect the nauplii survival when challenged with V. harveyi. Results also showed that the salutary effect of the tested glucans was associated with the upregulation of innate immune genes such as lipopolysaccharide and β-1,3-glucan-binding protein (lgbp), high mobility group box protein (hmgb), and prophenoloxidase (proPO). Interestingly, the up-regulation of superoxidase dismutase (sod) and glutathione-s-transferase (gst) was only observed in Gas1 treated group, indicating that Gas1 could function to induce higher reactive oxygen species and stronger immunomodulatory function in A. franciscana, and therefore higher survival rate. The expression of heat shock protein 70 (hsp70), peroxinectin (pxn), and down syndrome cell adhesion molecule (dscam) remain unaltered in response to glucan treatment. Taken together, this study provides insights into the structure-function relationship of β-glucan and the results confirmed that β-glucan can be an effective immunostimulant in aquaculture, especially the Gas1 glucan.

Depuis une quinzaine d’années, des mortalités estivales d’ormeaux européens, Haliotis tuberculata, surviennent sur le littoral breton et normand, et en structures aquacoles. Ces mortalités sont attribuées à l’espèce bactérienne Vibrio harveyi, et se produisent chez des ormeaux sexuellement matures lorsque la température de l’eau dépasse 17°C.Ce travail de thèse visait en une approche multifactorielle de l’étude de cette interaction hôte-parasite, afin de spécifier les conditions intrinsèques aux ormeaux dans le déclenchement de cette vibriose, le cycle infectieux de V. harveyi chez l’ormeau européen et le rôle de la température dans l’accomplissement de ce cycle infectieux, et enfin la réponse physiologique de l’ormeau lors d’une exposition à V. harveyi.Les principaux résultats montrent un différentiel d’expression génomique entre des ormeaux résistants et des ormeaux sensibles au cours d’une exposition à V. harveyi, attestant ainsi l’importance du statut physiologique de l’hôte dans la survie à la vibriose chez l’ormeau européen. Ce constat est supplémenté de la mise en évidence de sensibilité à cette maladie chez des ormeaux sexuellement immatures, habituellement résistants, acclimatés à 19°C et exposés à des conditions contraignantes de type manipulation. Par ailleurs, l’étude de la voie d’entrée et de la dynamique d’infection de V. harveyi chez l’ormeau européen a révélé un tropisme particulier de ce vibrion pathogène vers les tissus branchiaux dès les premières heures de contact, et son invasion dans le système circulatoire dès 24h de contact. L’étude de la réponse hémocytaire des ormeaux et du métabolisme branchial, à l’échelle moléculaire et cellulaire, lors des premières heures de contact, démontre 1/ la genèse d’un stress oxydatif au niveau des branchies d’ormeaux sensibles à la vibriose et 2/ une altération du fonctionnement des hémocytes, ce qui présume de l’une des stratégies majeures de virulence de V. harveyi. / For fifteen years, summer mortalities have been observed in wild and farmed populations of European abalone, Haliotis tuberculata, along the north French coast. These mortalities are attributed to the bacterial species Vibrio harveyi and occur in sexually mature animals, when the seawater temperature exceeds 17°C.A multifactorial approach to the study of this host-parasite interaction was done during this thesis, in order to specify the intrinsic abalone conditions in vibriosis mortalities, the infectious cycle of V. harveyi in European abalone and the role of temperature in the fulfillment of this infectious cycle, and finally the physiological response of abalone, at cellular and molecular level, when exposed to V. harveyi.The main results showed a differential gene expression between resistant and susceptible abalone during exposure to V. harveyi, indicating the importance of the physiological status of the host, in survival to vibriosis. This hypothesis is supplemented by the susceptibility of sexually immature abalone at 19°C to this disease, usually resistant, and exposed to manipulation stressor. Moreover, the study of the portal of entry and the dynamics of infection by V. harveyi in European abalone revealed a particular tropism of this vibrio pathogen for gill tissues, in the earlier hours of contact, and its invasion into the circulatory system from the first 24 hours of contact. The study of the response of abalone hemocyte and gill metabolism, at the cellular and molecular level, in the earlier hours of contact, shows 1/ a genesis of oxidative stress in gills of susceptible abalone, and 2/ an alteration of hemocyte functions, which may constitute one of the major strategies of virulence in V. harveyi.