A marine actinomycete nocardiopsis mccb 110 as source of novel drugs to manage vibriosis

Aquaculture is a form of agriculture that involves the propagation, cultivation and marketing of aquatic plants and animals in a controlled environment (Swann, 1992). After growing steadily, particularly in the last four decades, aquaculture is for the first time set to contribute half of the fish consumed by the human population worldwide. Given the projected population growth over the next two decades, it is estimated that at least an additional 40 million tonnes of aquatic food will be required by 2030 to maintain the current per capita consumption (FAO, 2006). Capture fisheries and aquaculture supplied the world with about 110 million tonnes of food fish in 2006. Of this total, aquaculture accounted for 47 percent (FAO, 2009). Globally, penaeid shrimp culture ranks sixth in terms of quantity and second in terms of value amongst all taxonomic groups of aquatic animals cultivated (FAO, 2006). In places where warm-water aquaculture was possible black tiger shrimp, Penaeus monodon became the preferred variety of shrimp cultivar owing to its fast growth, seed availability and importantly due to high prices it fetches (Pechmanee, 1997). World shrimp production is dominated by P.monodon, which accounted for more than 50 % of the production in 1999 (FAO, 2000). In the last few years the whiteleg shrimp, Litopenaeus vannamei, has replaced P.monodon in many countries. Indian shrimp culture is dominated by P.monodon with the East Coast accounting for 70% of the production (Hein, 2002). Intensive culture, apart from other problems, results in enhanced susceptibility of the cultured species to diseases (Jory, 1997), which in fact have become the biggest constraint in shrimp aquaculture (FAO, 2003).

Application of bacterins and yeast Acremonium dyosporii to protect the larvae of Macrobrachium rosenbergii from vibriosis

The fresh water prawn, Macrobrachium rosenbergii, has proven potential for use as an aquaculture species (Hanson & Goodwin, 1997; Kurup, 1984). In India alone, culture of this species of prawn in low saline areas requires about 200 million seed per year (Kurup, 1984). In hatcheries poor survival rate has been associated with vibriosis at di#erent stages of the larval cycle. Members of the family Vibrionaceae associated with the larvae of M. rosenbergii were shown to be pathogenic under laboratory conditions (Bhat et al., 2000, in press). Vibrios have been associated with mortality of penaeid prawns by several workers (Aquacop, 1977; Hameed, 1993; Karunasagar et al., 1994). Two methods have been suggested to protect both the larvae and juveniles from vibriosis; one is the administration of bacterins prepared from pathogenic strains (Itami et al., 1989, 1991; Adams, 1991; Song & Sung, 1990; Sung et al., 1991) and the other is the utilization of yeast 1-3 and 1-6 glucans as immunostimulants for enhancing the non-specific defense system (Sung et al., 1994; Song et al., 1997). In the light of these observations it was hypothesised that bacterins and yeast glucans may also be e#ective in protecting the larvae of M. rosenbergii from vibriosis as has been achieved in the case of penaeids. To examine this hypothesis, the ability of bacterins and an extracellular glucan-producing yeast to increase the overall survival and metamorphosis of larvae in a hatchery, as well as to protect against an experimental challenge under laboratory conditions, was evaluated

Diagnosis of vibriosis in the era of genomics: lessons from invertebrates

Global changes linked to increases in temperature and ocean acidification, but also to more direct anthropogenic influences such as aquaculture, have caused a worldwide increase in the reports of Vibrio-associated illnesses affecting humans and also animals such as shrimp and molluscs. Investigation of the emergence of Vibrio pathogenesis events requires the analysis of microbial evolution at the gene, genome and population levels, in order to identify genomic modifications linked to increased virulence, resistance and/or prevalence, or to recent host shift. From a more applied point of view, the elucidation of virulence mechanisms is a prerequisite to devising prophylactic methods to fight infectious agents. In comparison with human pathogens, fairly little is known about the requirements for virulence in vibrios pathogenic to animals. However, the advent of genome sequencing, especially next-generation technologies,the possibility of genetically manipulating most of the Vibrio strains, and the recent availability of standardised animals for experimental infections have now compensated for the considerable delay in advancement of the knowledge of non-model pathogens such as Vibrio and have led to new scientific questions.

Antibody Based Diagnostic for Detection Of Vibrios and their Biological Control using Antagonistic Bacteria in Macrobrachium Rosenbergii Larval Rearing Systems

The main objective of the work undertaken here was to develop an appropriate microbial technology to protect the larvae of M.rosenbergii in hatchery from vibriosis. This technology precisely is consisted of a rapid detection system of vibrios and effective antagonistic probiotics for the management of vibrios. The present work was undertaken with the realizations that to stabilize the production process of commercial hatcheries an appropriate, comprehensive and fool proof technology is required primarily for the rapid detection of Vibrio and subsequently for its management. Nine species of Vibrio have been found to be associated with larvae of M. rosenbergii in hatchery. Haemolytic assay of the Vibrio and Aeromonas on prawn blood agar showed that all isolates of V. alginolyticus and Aeromonas sp., from moribund, necrotized larve were haemolytic and the isolates of V.cholerae, V.splendidus II, V.proteolyticus and V.fluvialis from the larvae obtained from apparently healthy larval rearing systems were non-haemolytic. Hydrolytic enzymes such as lipase, chitinase and gelatinase were widespread amongst the Vibrio and Aeromonas isolates. Dominance of V.alginolyticus among the isolates from necrotic larvae and the failure in isolating them from rearing water strongly suggest that they infect larvae and multiply in the larval body and cause mortality in the hatchery. The observation suggested that the isolate V. alginolyticus was a pathogen to the larvae of M.rosenbergii. To sum up, through this work, nine species of Vibrio and genus Aeromonas associated with M.rosenbergii larval rearing systems could be isolated and segregated based on the haemolytic activity and the antibodies (PA bs) for use in diagnosis or epidemiological studies could be produced, based on a virulent culture of V.alginolyticus. This could possibly replace the conventional biochemical tests for identification. As prophylaxis to vibriosis, four isolates of Micrococcus spp. and an isolate of Pseudomonas sp. could be obtained which could possibly be used as antagonistic probiotics in the larval rearing system of M.rosenbergii.

Biochemical and molecular characterization of pathogenic vibrios from hatcheries and aquaculture farms

Vibrio are important during hatchery rearing. aquaculture phase and post-harvest quality of shrimps. Vibrio spp are of concern to shrimp farmers and hatchery operators because certain species can cause Vibriosis. Vibrio species are of concern to humans because certain species cause serious diseases.With the progress in aquaculture, intensive systems used for shrimp aquaculture create an artificial environment that increases bacterial growth. To maintain the productivity of such an intensive aquaculture, high inputs of fish protein have to be employed for feeding together with high levels of water exchange and the massive use of antibiotics/ probiotics / chemicals. It seems that the combination of these conditions favours the proliferation of vibrios and enhances their virulence and disease prevalence. The risk of a microbial infection is high, mainly at larval stages. The effect and severity are related to Vibrio species and dose, water, feed, shrimp quality and aquaculture management.Consumption of seafood can occasionally result in food-bome illnesses due to the proliferation of indigenous pathogens like Vibrio.Of the l2 pathogenic Vibrio species, 8 species are known to be directly food associated. Strict quality guidelines have been laid by the importing nations, for the food products that enter their markets. The microbiological quality requirement for export of frozen shrimp products is that V.cholerae, V.parahaemolyticus and V. vulnificus should be absent in 25g of the processed shrimp (Export Inspection Council of India, 1995). The mere presence of these pathogenic Vibrios is sufficient for the rejection of the exported product.The export rejections cause serious economic loss to the shrimp industry and might harm the brand image of the shrimp products from the countiy.There is a need for an independent study on the incidence of different pathogenic vibrios in shrimp aquaculture and investigate their biochemical characteristics to have a better understanding about the growth and survival of these organisms in the shrimp aquaculture niche. PCR based methods (conventional PCR, duplex PCR, multiplex-PCR and Real Time PCR) for the detection of the pathogenic Vibrios is important for rapid post-harvest quality assessment. Studies on the genetic heterogeneity among the specific pathogenic vibrio species isolated from shrimp aquaculture system provide; valuable information on the extent of genetic diversity of the pathogenic vibrios, the shrimp aquaculture system.So the present study was undertaken to study the incidence of pathogenic Vibrio spp. in Penaeus monodon shrimp hatcheries and aquaculture farms, to carry out biochemical investigations of the pathogenic Vibrio spp isolated from P. monodon hatchery and. aquaculture environments, to assess the effect of salt (NaCl) on the growth and enzymatic activities of pathogenic Vibrio spp., to study the effect of preservatives, and chemicals on the growth of pathogenic Vibrio spp. and to employ polymerase chain reaction (PCR) methods for the detection of pathogenic V ibrio spp.Samples of water (n=7) and post-larvae (n=7) were obtained from seven Penaeus monodon hatcheries and samples of water (n=5), sediment (n=5) and shrimp (n=5) were obtained from five P. monodon aquaculture farms located on the East Coast of lndia. The microbiological examination of water, sediment, post-larvae and shrimp samples was carried out employing standard methods and by using standard media.The higher bacterial loads were obtained in pond sediments which can be attributed to the accumulation of organic matter at the pond bottom which stimulated bacterial growth.Shrimp head. (4.78 x 105 +/- 3.0 x 104 cfu/g) had relatively higher bacterial load when compared to shrimp muscle 2.7 x 105 +/- 1.95 x 104 cfu/g). ln shrimp hatchery samples, the post-larvae (2.2 x 106 +/- 1.9 x 106 cfu/g) had higher bacterial load than water (5.6 x 103 +/- 3890 cfu/ml).The mean E.coli counts were higher in aquaculture pond sediment (204+/-13 cfu/g) and pond water (124+/-88 cfu/ml). Relatively lower Escherichia coli counts were obtained from shrimp samples (12+/-11 to 16+/-16.7 cfu/g). The presence of E.coli in aquaculture environment might have been from the source water. E.coli was not detected in hatchery waters and post-larvae.

Studies on vibrio spp. in juveniles of penaeus indicus in culture systems

The present study is carried out to understand (i) the incidence and occurrence of species of Vibrio in different culture systems in and around Cochin, (ii) characteristics of vibrios isolates, their ecology including growth response to various hydrological parameters, sensitivity to about 40 antibiotics, and (iii) role of physico-chemical parameters in pathogenicity of vibrios, etc. and the results emerged from the investigations are important and encouraging for better understanding of the 'vibriosis' in the culture systems to develop remedial measures to control diseases. The Thesis begins with an “Introduction” followed by “A review of literature” on diseases of penaeid shrimps with particular reference to 'vibriosis' and “Material and methods” which details with the methods and procedures followed in the experiments and analyses of data. This Thesis consists of three chapters. Chapter Ideals with the incidence and ecology of Vibrio spp. in water, sediment and in juveniles of the Indian white prawn Penaeus indicus in the culture systems. In Chapter 2, characteristics of vibrio isolates including growth response to various levels oftemperature, salinity and pl 1, sensitivity to 40 antibiotics and minimal inhibitory concentration tests are detailed.e out-breaks. The Chapter 3 discusses the role of physico-chemical parameters in the incidence, seasonal abundance of Vibrio spp. and in 'vibriosis'. A summary of the whole work and list of references are also included at the end. This study gives a detailed information regarding the incidence and ecology of vibrios in the culture systems. their characteristics and pathogenicity

‘Systematics and pathogenicity of Vibrionaceae associated T ¢ S with the larvae of M. rosenbergii in hatchery

Even though Bergey '5 Manual has been recognized globally as the guide to bacterial systematics, it has to be emphasized that descriptions given to a large extent are based on studies made with temperate isolates This leads one to conclude that any attempt to identify the tropical isolates with identification keys and tables generated from this information may lead to erroneous conclusions. And there is every possibility of the existence of genotypic and phenotypic variants or even nev. species in this part ofthe aquatic ecosystem. Applications ofa polythetic scheme of classification based on the principles of Numerical Taxonomy opens up exciting avenues for bringing to light, this possibility which otherwise would have been masked by the unidirectional approach as in monothetic schemes. Another added advantage of clustering a ‘natural’ bacterial population by numerical taxonomy, is the ease by which genotypic characterization could be performed on the clusters by selecting a representative from each cluster This helps overcome the practical impossibility of analyzing all the isolates in a pani:'_lar cluster. The genotypic characteizarion would either be mole °/o G-'rC. DNA-D.\_-X hybridization, DNA-RNA hybridization or DNA fingerprinting. Considering the requirement creating a broad base in the understanding of the family Vibrionaceae associated with the larvae ofM rosenbergii, the present work was undertaken to channelize every new information generated for developing appropriate managerial measures to protect the larvae from vibriosis during the unusually prolonged larval phase.

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.

Isolation of a pathogenic strain of Vibrio alginolyticus from necrotic larvae of Macrobrachium rosenbergii (de Man)

Giant freshwater prawn, Macrobrachium rosenbergii (de Man), is an important commercial species with considerable export value, ideal for cultivation under low saline conditions and in freshwater zones (Kurup 1994). However, despite more than a decade of research on its larval production systems, vibriosis still hampers seed production resulting in high mortality rates. Among the different species of vibrios, Vibrio alginolyticus has been isolated frequently from diseased shrimp as the aetiological agent of vibriosis and has been described as a principal pathogen of both penaeids and nonpenaeids (Lightner 1988; Baticados, Cruz-Lacierda, de la Cruz, Duremdez-Fernandez, Gacutan, Lavilla- Pitogo & Lio-Po 1990; Mohney, Lightner & Bell 1994; Lee, Yu, Chen, Yang & Liu 1996). Vibrio fluvialis, V. alginolyticus, V. cholerae non-O1 (Fujioka & Greco 1984), Aeromonas liquifaciens and V. anguillarum (Colorni 1985) have been isolated from the larvae of M. rosenbergii. A profound relationship between the abundance of members of the family Vibrionaceae and larval mortality (Singh 1990) and the predominance of Vibrio in eggs, larvae and post-larvae of M. rosenbergii (Hameed, Rahaman, Alagan & Yoganandhan 2003) was reported. The present paper reports the isolation, characterization, pathogenicity and antibiotic sensitivity of V. alginolyticus associated with M. rosenbergii larvae during an occurrence of severe mass mortality at the ninth larval stage.

Synechocystis MCCB 114 and 115 as putative probionts for Penaeus monodon post-larvae

Synechocystis MCCB 114 and 115 were segregated as putative probionts for shrimp larvae from a collection of 54 cyanobacterial cultures enriched from seawater. On feeding Penaeus monodon post-larvae with the cyanobacteria, the generic diversity of the intestinal bacterial flora could be enhanced with substantial reduction or total absence of Vibrio spp. A significant difference (p < 0.001) in the percent survival of batches of post-larvae fed on the cyanobacterial cultures was observed and, on repeated challenge with V. harveyi, the relative percent survival of those batches of larvae fed on Synechocystis MCCB 114 and 115 was significantly higher. The Synechocystis MCCB 114 and 115 cultures were found to contain high levels of protein (34 to 43%), in addition to carotenoids

Antimicrobial activity of chitosan against vibrios from freshwater prawn Macrobrachium rosenbergii larval rearing systems

Chitosan is a biocompatible and biodegradable natural polymer with established antimicrobial properties against specific microorganisms. The present study demonstrates its antibacterial activity against 48 isolates of Vibrio species from prawn larval rearing systems. The antibacterial activity had a positive correlation with the concentration of chitosan. This work opens up avenues for using chitosan as a prophylactic biopolymer for protecting prawn larvae from vibriosis.

Investigation on possibility of survival and growth enhancing by selected seaweeds from Persian Gulf in post larvae shrimp Litopenaeus vannamei

The use of antibiotics in aquaculture has been limited. Scientifics seeking for natural substitutes to prevent of aquatic animals diseases. Considering seaweeds are rich of nutritions and bioactive compounds, the purpose of this study is: investigation the potential and use possibility of native seaweeds from Persian Gulf in shrimp aquculture industry to improve growth, survival of postlarvae and to resistance against pathogens such as vibriosis. For this propose 7 macroalgae species from Bushehr province coast, inclouding: green algae (C. iyengarii), brown algae (S. angutifolium and S. ilicifolium) and red algae (L. snyderiae, K. alvarezii and G. corticata) were collected and identified. Then seaweed extracts abtained by Water, Ethanol, Methanol and Chloroform solvents by soaking method. In vitro antibacterial activity of extracts against Gr+ bacteria (S. aureus and B. subtilis) and Gr- bacteria (V. harveyi, V. alginolyticus and E. coli) was conducted by Agar diffusion, MIC and MBC methods. Antioxidant activity also by DPPH and EC50 methods was investigated. According to results of these two tests four seaweeds species (S. angutifolium, L. snyderiae, K. alvarezii and G. corticata) were selected for use in shrimp postlarvae (PL22) diets by Bio-Encapsulation (Artemia enrichment). Before of enrichment, toxicity effect of extracts to Artemia nauplii were evaluated by determination of LC50 24 h method. From results of this section Ethanol extracts were selected to bioencapsulation. After encapsulation shrimp postlarvae divided to 12 groups in triplicate, namely: C-, C+, S (200), S (400), S (600), L(200), L(400), L(600), G(300), G(600), K(300) and K(600). During 30 days of reared period C- and C+ use of basal diet and unenriched Artemia, but the other groups use of basal diet and enriched Artemia. Except C-, the shrimps in first day of culture put in 107 cfu/ml v. harveyi suspension for 30 minutes, and after water exchange 10 ml of this dose was added to reared aquaria. After 30 days survival percentage, obtained weight and SGR% were investigated. To evaluate vibrio loading, every 10 days 5 postlarvae were sampled randomly for vibrio count. Results showed that vibrio count in C- was less than the others and in C+ was more than the others. In treatments vibrio count in L(200) was the most and L(600) was the less. Survival rate in C- was the most and after that G(600) with 79.4±6.6% and then S(300) and K(600) were 73.3±7.3% and 70.6±6.6% respectively that were significantly compare the other (P < 0.01). Also the C+ was the less with 33.3±6.6% that difference was significant (P< 0.01). In this study growth parameters of all groups that fed by enriched Artemia were better than C+ (P<0.05). After cultre period 10 shrimp of every aquarium disinfected and reared for 10 days like before treatment. After 10 days the shrimps were challenged by 3×108 cfu/ml V. harveyi and mortality was recorded for 7 days. The all of animals in C- were survive but more than 90% of C+ were dead. And survival in all of treatments were better the C+ (P<0.05). The study showed the ethanol extracts of selected seaweed from Persian Gulf is a good source for growth, Survival and disease control in shrimp larviculture.

Antiadhesive activity of poly-hydroxy butyrate biopolymer from a marine Brevibacterium casei MSI04 against shrimp pathogenic vibrios

Vibrio pathogens are causative agents of mid-culture outbreaks, and early mortality syndrome and secondary aetiology of most dreadful viral outbreaks in shrimp aquaculture. Among the pathogenic vibrios group, Vibrio alginolyticus and V. harveyi are considered as the most significant ones in the grow-out ponds of giant black tiger shrimp Penaeus monodon in India. Use of antibiotics was banned in many countries due to the emergence of antibiotic-resistant strains and accumulation of residual antibiotics in harvested shrimp. There is an urgent need to consider the use of alternative antibiotics for the control of vibriosis in shrimp aquaculture. Biofilm formation is a pathogenic and/or establishment mechanism of Vibrio spp. This study aims to develop novel safe antibiofilm and/ or antiadhesive process using PHB to contain vibrios outbreaks in shrimp aquaculture.