Penaeus monodon larvae can be protected from Vibrio harveyi infection by pre-emptive treatment of a rearing system with antagonistic or non-antagonistic bacterial probiotics

This study shows that the disease resistance and survival rate of Penaeus monodon in a larval rearing systems can be enhanced by supplementing with antagonistic or non-antagonistic probiotics. The antagonistic mode of action of Pseudomonas MCCB 102 and MCCB 103 against vibrios was demonstrated in larval mesocosm with cultures having su⁄cient concentration of antagonistic compounds in their culture supernatant. Investigations on the antagonistic properties of Bacillus MCCB 101, Pseudomonas MCCB 102 and MCCB 103 and Arthrobacter MCCB 104 against Vibrio harveyi MCCB111under in vitro conditions revealed that Pseudomonas MCCB 102 and MCCB 103 were inhibitory to the pathogen.These inhibitory propertieswere further con¢rmed in the larval rearing systems of P. monodon. All these four probionts signi¢cantly improved larval survival in long-term treatments as well as when challengedwith a pathogenic strain ofV. harveyiMCCB111. We could demonstrate that Pseudomonas MCCB 102 andMCCB103 accorded disease resistance and a higher survival rate in P. monodon larval rearing systems throughactive antagonism of vibrios,whereas Bacillus MCCB 101 and Arthrobacter MCCB 104 functioned as probiotics through immunostimulatory and digestive enzyme-supporting modes of action.

Vibrio harveyi phages: Isolation, characterization and evaluation of their potential as phage therapeutics on Vibrio harveyi in shrimp hatcheries

Aquaculture is a global industry providing food and employment thereby contributing to the economy. For the sustenance of aquaculture, disease management is a major requirement. Among the bacterial pathogens Vibrio harveyi remains to be the major one especially in shrimp culture systems. Rapid and mass mortality of shrimp larvae due to Vibrio harveyi infection is well known, and the pathogen causes serious economic losses in grow out systems as well. It suggests that a well defined management strategy has to be built up to protect the crop from Vibrio harveyi infection in aquaculture systems. Antibiotics have been the choice for quite some times which led to residues in meat and development of multidrug resistant bacteria which invited ban on their application. In this context several alternate options have been thought off such as probiotics, immunostimulants and vaccines. Phage therapy is yet another option. Phages being natural parasites of bacteria and are abundant in aquatic environments their application to control bacterial pathogens in aquaculture has commendable potential in lieu of antibiotics. For that matter the therapeutic effect of phages has been proven in several antibiotic resistant pathogens inclusive of Vibrio harveyi.

Biocontrol of Vibrio harveyi in Penaeus monodon larval rearing systems employing probiotics and vibriophages

National Centre for Aquatic Animal Health, School of Environmental Studies, Cochin University of Science and Technology.

Molecular Approaches For Characterization And Determination Of Pathogenicity Of Vibrios With Special Reference To Vibrio Harveyi From Penaeus Monodon Larval Production Systems

The present study focuses on vibrios especially Vibrio harveyi isolated from shrimp (P. monodon) larval production systems from both east and west coasts during times of mortality. A comprehensive approach has been made to work out their systematics through numerical taxonomy and group them based on RAPD profiling and to segregate the virulent from non- virulent isolates based on the presence of virulent genes as well as their phenotypic expression. The information gathered has helped to develop a simple scheme of identification based on phenotypic characters and segregate the virulent from non virulent strains of V. harveyi.

An inhibitory compound produced by Pseudomonas with effectiveness on Vibrio harveyi

Persistence of the antivibrio property of the potential antagonistic probiotics, Pseudomonas MCCB 102 and 103, at di¡erent temperatures, pH and in organic solvents was studied. The antivibrio compound was extracted, puri¢ed and characterized using thin-layer chromatography, high-pressure liquid chromatography, liquid chromatography-mass spectroscopy, UV^ Vis and nuclear magnetic resonance spectroscopy and identi¢ed as N-methyl-1-hydroxyphenazine, a phenazine antibiotic. The toxicity of the compound was tested in Penaeus monodon haemocyte culture and the IC50 valuewas found to be1.4 0.31mg L 1. The compound was found to be bacteriostatic at 0.5mg L 1. Its stability to varying temperature, pH, organic solvents, prolonged shelf-life and vibriostatic nature point to its suitability for prophylatic aquaculture application.

Isolation and characterization of broad spectrum bacteriophages lytic to Vibrio harveyi from shrimp farms of Kerala, India

Of 33 phages isolated from various shrimp farms in Kerala, India, six were segregated to have broad spectrum lytic efficiency towards 87 isolates of Vibrio harveyi with cross-infecting potential to a few other important aquaculture pathogens. They were further tested on beneficial aquaculture micro-organisms such as probiotics and nitrifying bacterial consortia and proved to be noninfective. Morphological characterization by transmission electron microscopy (TEM) and molecular characterization by RAPD and SDS-PAGE proved them distinct and positioned under Caudovirales belonging to Myoviridae and Siphoviridae

Identificación de las proteínas implicadas en la respuesta al estrés en Escherichia coli y Vibrio harveyi.

187 p.

Determination of Extracellular Molecules Produced by Vibrio Harveyi Using MS/MS

Quorum sensing (QS) is a process that allows bacteria to sense the population density of cells around them by communicating with each other via autoinducer molecules. This cross-communication is crucial in the regulation of bacterial processes such as bioluminescence, virulence, and biofilm formation. Previous research by Milburn and Makemson on Vibrio harveyi suggested that in addition of the known biosynthesis of three well-characterized autoinducers, dozens of unknown molecules are also produced and released to the environment by V. harveyi. This study was performed using electrospray tandem mass spectrometry with the purpose of detection and characterization of the extracellular molecules produced by V. harveyi, and assessment of their relationship to QS. A total of 11 molecules were characterized, from which three could be related to QS. These findings provide a glimpse of the nature of novel secondary metabolites produced by V. harveyi and provide the groundwork for further research.

Haematological responses of Penaeus monodon to environmental alterations and pathogenic invasion

This thesis Entitled Haematological responses of penaeus monodon to environmental alterations and pathogenic invasion. Thesis concluded from the present study that stress is accompanied by alterations in haemolymph metabolic variables and immune responses that influences the susceptibility of P. monodon to infection. Acute salinity variations were proved to be a stress condition that enhances the susceptibility of P. monodon to V. harveyi and WSSV infection. Ambient Cu at 0.1 mg 1" and ambient Zn at 1.0 mg 1" proved immunostimulatory in increasing the immunocompetence of P. monodon to WSSV infection and higher concentrations of Cu and Zn proved immunosuppressive. Haemolymph total protein, total carbohydrates and total lipids showed the highest relation with immune responses. THC, PO, ACP and ALP that greatly correlated with the survival rate proposed as reliable biomarkers of health in P. monodon. The study highlights the need for proper management practices and regular health monitoring to be adopted to avoid mass mortality in shrimp culture ponds.

Quorum-sensing regulates biofilm formation in Vibrio scophthalmi

Background: In a previous study, we demonstrated that Vibrio scophthalmi, the most abundant Vibrio species among the marine aerobic or facultatively anaerobic bacteria inhabiting the intestinal tract of healthy cultured turbot (Scophthalmus maximus), contains at least two quorum-sensing circuits involving two types of signal molecules (a 3-hydroxy-dodecanoyl-homoserine lactone and the universal autoinducer 2 encoded by luxS). The purpose of this study was to investigate the functions regulated by these quorum sensing circuits in this vibrio by constructing mutants for the genes involved in these circuits. Results. The presence of a homologue to the Vibrio harveyi luxR gene encoding a main transcriptional regulator, whose expression is modulated by quorumsensing signal molecules in other vibrios, was detected and sequenced. The V. scophthalmi LuxR protein displayed a maximum amino acid identity of 82% with SmcR, the LuxR homologue found in Vibrio vulnificus. luxR and luxS null mutants were constructed and their phenotype analysed. Both mutants displayed reduced biofilm formation in vitro as well as differences in membrane protein expression by mass-spectrometry analysis. Additionally, a recombinant strain of V. scophthalmi carrying the lactonase AiiA from Bacillus cereus, which causes hydrolysis of acyl homoserine lactones, was included in the study. Conclusions: V. scophthalmi shares two quorum sensing circuits, including the main transcriptional regulator luxR, with some pathogenic vibrios such as V. harveyi and V. anguillarum. However, contrary to these pathogenic vibrios no virulence factors (such as protease production) were found to be quorum sensing regulated in this bacterium. Noteworthy, biofilm formation was altered in luxS and luxR mutants. In these mutants a different expression profile of membrane proteins were observed with respect to the wild type strain suggesting that quorum sensing could play a role in the regulation of the adhesion mechanisms of this bacterium.

Reducing Vibrio load in Artemia nauplii usingantimicrobial photodynamic therapy: a promising strategy to reduce antibiotic application in shrimp larviculture

We propose antimicrobial photodynamic therapy (aPDT) as an alternative strategy to reduce the use of antibiotics in shrimp larviculture systems. The growth of a multiple antibiotic resistant Vibrio harveyi strain was effectively controlled by treating the cells with Rose Bengal and photosensitizing for 30 min using a halogen lamp. This resulted in the death of > 50% of the cells within the first 10 min of exposure and the 50% reduction in the cell wall integrity after 30 min could be attributed to the destruction of outer membrane protein of V. harveyi by reactive oxygen intermediates produced during the photosensitization. Further, mesocosm experiments with V. harveyi and Artemia nauplii demonstrated that in 30 min, the aPDT could kill 78.9% and 91.2% of heterotrophic bacterial and Vibrio population respectively. In conclusion, the study demonstrated that aPDT with its rapid action and as yet unreported resistance development possibilities could be a propitious strategy to reduce the use of antibiotics in shrimp larviculture systems and thereby, avoid their hazardous effects on human health and the ecosystem at large.

The 4-aza-S-ribosyl-L-homocysteine derivatives and the related gamma-lactam and azahemiacetal analogs: Synthesis, inhibition and quorum sensing activity

Quorum sensing (QS) is a population-dependent signaling process bacteria use to control multiple processes including virulence, critical for establishing infection. There are two major pathways of QS systems. Type 1 is species specific or intra-species communication in which N-acylhomoserine lactones (Gram-negative bacteria) or oligopeptides (Gram-positive bacteria) are employed as signaling molecules (autoinducer one). Type 2 is inter-species communication in which S-4,5-dihydroxy-2,3-pentanedione (DPD) or its borate esters are used as signaling molecules. The DPD is biosynthesized by LuxS enzyme from S-ribosylhomocysteine (SRH). Recent increase in prevalence of bacterial strains resistant to antibiotics emphasizes the need for the development of new generation of antibacterial agents. Interruption of QS by small molecules is one of the viable options as it does not affect bacterial growth but only virulence, leading to less incidence of microbial resistance. Thus, in this work, inhibitors of both N-acylhomoserine lactone (AHL) mediated intra-species and LuxS enzyme, involved in inter-species QS are targeted. The γ-lactam and their reduced cyclic azahemiacetal analogs, bearing the additional alkylthiomethyl substituent, were designed and synthesized targeting AHL mediated QS systems in P. aeruginosa and Vibrio harveyi. The γ-lactams with nonylthio or dodecylthio chains acted as inhibitors of las signaling in P. aeruginosa with moderate potency. The cyclic azahemiacetal with shorter propylthio or hexylthio substituent were found to strongly inhibit both las and rhl signaling in P. aeruginosa at higher concentrations. However, lactam and their azahemiacetal analogs were found to be inactive in V. harveyi QS systems. The 4-aza-S-ribosyl-L-homocysteine (4-aza-SRH) analogs and 2-deoxy-2-substituted-S-ribosyl-L-homocysteine analogs were designed and synthesized targeting Bacillus subtilis LuxS enzyme. The 4-aza-SRH analogs in which oxygen in ribose ring is replaced by nitrogen were further modified at anomeric position to produce pyrrolidine, lactam, nitrone, imine and hemiaminal analogs. Pyrrolidine and lactam analogs which lack anomeric hydroxyl, acted as competitive inhibitors of LuxS enzyme with KI value of 49 and 37 µM respectively. The 2,3-dideoxy lactam analogs were devoid of activity. Such findings attested the significance of hydroxyl groups for LuxS binding and activity. Hemiaminal analog of SRH was found to be a time-dependent inhibitor with IC50 value of 60 µM.

Small RNAs in bacterialcell-cell communication

When all three separate quorum-sensing signals act in concert in Vibrio harveyi, they maximize bioluminescence and fully repress type III secretion. V. harveyi has five qrr loci encoding small RNA regulatory molecules, each consisting of about 100 nucleotides; several of them are involved in repressing bioluminescence. Small RNAs also play roles in population density-dependent activities, including regulation of virulence factors, for bacterial pathogens such as Pseudomonas fluorescens, V. cholerae, Salmonella enterica, Pseudomonas aeruginosa, and Erwinia spp. Although some bacteria appear to carry redundant copies of small RNA genes with which to finely tune expression

Time-related action of Lactobacillus plantarum in the bacterial microbiota of shrimp digestive tract and its action as immunostimulant

The objective of this work was to assess the time-related action of probiotic Lactobacillus plantarum in the bacterial microbiota of the digestive tract of Litopenaeus vannamei, and the relation of total haemocyte count and serum phenol oxidase activity of shrimp challenged with Vibrio harveyi. Shrimps were fed with a probiotic-supplemented diet, for eight days, then shifted to a commercial diet. Shrimps fed only with the commercial diet served as control. Evaluations were made on the 8th day of experiment and repeated two, four, six and eight days later. Total lactic bacteria in the digestive tract was higher until the 4th day of evaluation in the probiotic-supplemented group. Vibrio spp. counts were higher in the control at days zero and two. Until the 4th day of evaluation, the total haemocyte counts in shrimps after challenge with V. harveyi were higher in probiotic-supplemented group than in control group. Significant difference was not observed in phenol oxidase activity. On the 6th day after shifting from supplemented to control diet, all parameters were equal in both groups, suggesting that the time-related action of L. plantarum in shrimp is short.

Haematology of Fenneropenaeus indicus in response to environmental alterations and microbial infections

Aquaculture has developed to become one of the fastest growing food producing sectors in the world.Today India is one among the major shrimp producing countries in the world.There are extensive and intensive shrimp culture practices. In extensive shrimp culture, shrimps are stocked at low densities (< 25 PLs m'2)in large ponds or tidal enclosures in which little or no management is exercised or possible. Farmers depend almost entirely on natural conditions in extensive cultures. Intensive shrimp culture is carried out in high densities (>200 PLs m'2). Much of the world shrimp production still comes from extensive culture.There is a growing demand for fish and marine products for human and animal consumption. This demand has led to rapid growth of aquaculture, which some times has been accompanied by ecological impacts and economic loss due to diseases. The expansion of shrimp culture always accompanies local environmental degradation and occurrence of diseases.Disease out breaks is recognised as a significant constraint to aquaculture production. Environmental factors, water quality, pollution due to effluent discharge and pathogenic invasion due to vertical and horizontal transmission are the main causes of shrimp disease out breaks. Nutritional imbalance, toxicant and other pollutants also account for the onset of diseases. pathogens include viruses, bacteria, fungi and parasites.Viruses are the most economically significant pathogens of the cultured shrimps world wide. Disease control in shrimp aquaculture should focus first on preventive measures for eliminating disease promoting factors.ln order to design prophylactic and proactive measures against shrimp diseases, it is mandatory to understand the immune make up of the cultivable species, its optimum culture conditions and the physico chemical parameters of the rearing environment. It has been proven beyond doubt that disease is an end result of complex interaction of environment, pathogen and the host animal. The aquatic environment is abounded with infectious microbes.The transmission of disease in this environment is extremely easy, especially under dense, culture conditions. Therefore, a better understanding of the immune responses of the cultured animal in relation to its environmental alterations and microbial invasions is essential indevising strategic measures against aquaculture loss due to diseases. This study accentuate the importance of proper and regular health monitoring in shrimps employing the most appropriate haematological biomarkers for application of suitable prophylactic measures in order to avoid serious health hazards in shrimp culture systems.