Optimization of carbon and nitrogen sources and growth factors for the production of an aquaculture probiotic (Pseudomonas MCCB 103) using response surface methodology

Aim: To develop a new medium for enhanced production of biomass of an aquaculture probiotic Pseudomonas MCCB 103 and its antagonistic phenazine compound, pyocyanin. Methods and Results: Carbon and nitrogen sources and growth factors, such as amino acids and vitamins, were screened initially in a mineral medium for the biomass and antagonistic compound of Pseudomonas MCCB 103. The selected ingredients were further optimized using a full-factorial central composite design of the response surface methodology. The medium optimized as per the model for biomass contained mannitol (20 g l)1), glycerol (20 g l)1), sodium chloride (5 g l)1), urea (3Æ3 g l)1) and mineral salts solution (20 ml l)1), and the one optimized for the antagonistic compound contained mannitol (2 g l)1), glycerol (20 g l)1), sodium chloride (5Æ1 g l)1), urea (3Æ6 g l)1) and mineral salts solution (20 ml l)1). Subsequently, the model was validated experimentally with a biomass increase by 19% and fivefold increase of the antagonistic compound. Conclusion: Significant increase in the biomass and antagonistic compound production could be obtained in the new media. Significance and Impact of the Study: Media formulation and optimization are the primary steps involved in bioprocess technology, an attempt not made so far in the production of aquaculture probiotics.

Studies on isolation and molecular characterization of plasmids from Vibrios

While the seriousness of the problem of antibiotic resistance is now recognized, the complex web of resistance linking humans, animals, and the environment is getting realized. More often, antibiotics are used as a preventive measure against diseases. Antibiotic use for agriculture leads to the increased resistance in the environment since antibiotics are inevitable element during agriculture/aquaculture and antibiotic residues are excreted as waste that is frequently spread onto farmland as organic fertilizer. Fecal bacteria survive long periods in the environment and spread through runoff into groundwater, rivers, and marine ecosystems.However, horizontal gene transfer occurs in the animals and guts of humans and in a variety of ecosystems, creating a pool of resistance in the rice fields and open waters. Even if people are not in direct contact with resistant disease through food animals, there are chances of contact with resistant fecal pathogens from the environment. Additionally, pathogens that are autochthonous to the environment can acquire resistance genes from the environment. Our study revealed that autochthonous , bacteria Vibrio spp gained antibiotic resistance in the environment. Further, it was evident that horizontal gene transfer occurs in Vibrio by means of plasmids, which further augments the gravity of the problem. Non-pathogenic bacteria may also acquire resistance genes and serve as a continuing source of resistance for other bacteria, both in the environment, and in the human gut. As the effectiveness of antibiotics for medical applications decline, the indiscriminate use of in aquaculture and in humans can have disastrous conditions in future due to horizontal gene transfer and the spread of resistant organisms: We must recognize and deal with the threat posed by overuse of antibiotics.

Purification and characterisation of Vibriophage isolated from Mangalavanam mangrove

The temperate, filamentous phage ФMV -5 isolated from Mangalavanam mangrove of Kochi, using the environmental strain of Vibrio sp. MV-5 shares many similar properties with other marine phage isolates, while also remaining unique. The study has revealed that the interaction of temperate phages and the microbial population in the marine environment may contribute significantly to microbial genetic diversity and composition by conversion and transduction and which requires greater study.Prophages contribute a substantial share of the mobile DNA of their bacterial hosts and seem to influence the short-term evolution of pathogenic bacteria. Automated methods for systematic investigation of prophages and other mobile DNA elements in the available bacterial genome sequences will be necessary to understand their role in bacterial genome evolution. In the past, phages were mainly investigated as the simplest model systems in molecular biology. Now it is increasingly realized that phage research will be instrumental in the understanding of bacterial abundance in the environment. One can predict that phage research will impact diverse areas such as geochemistry and medicine. Success will largely depend on integrative multidisciplinary approaches in this field. Clearly, further studies are required to understand how vibriophages interact with Vibrios to promote this organism's acquisition of the critical genes which alter its virulence or adaptation to its environmental niche.It is evident from this study and comparison with those reports cited above that vibriophage ФMV-5 is a previously unreported bacteriophage. It is recommended that the minimum requirement for reporting a new phage should be novel morphological markers and a description of host range, both of which have been achieved in this study.

lncidence of enterotoxigenic Staphylococcus aureus in relation to the microbial safety of seafood

A detailed study was made on the microbial quality, with special reference to food safety, of the fish and fishery products in the retail trade in Cochin and around. Also, farmed molluscan shellfishes like mussels and oysters were investigated for the microbial quality including the presence of pathogenic bacteria. Special stress has been given to monitor the incidence of coagulase positive as well as coagulase negative Staphylococcus in these products and their relative incidence have been recorded.In the next part, the investigation was centered mainly on toxigenic S.aureus. This is because among the Gram positive toxigenic bacteria, the Saureus with potential to produce thermostable enterotoxins are more relavent in food safety conceming seafoods in comparison with the Gram-negative pathogens like Salmonella and V.cholerae.The incidence, toxigenic potential and conditions of toxin production by S.aureus have been investigated in detail. An attempt has also been made to relate the toxigenisis with the presence of the concerned toxigenic genes in the genomes of S. aureus strains.

Ecophysiology of nontuberculous Mycobacteria in marine aquaculture ponds

The world demand for fish and fishery products is increasing steadily and it is generally accepted that it will not be possible to meet the heavy demand with resources exploited from capture fishery alone. Now aquaculture is well established and fastdeveloping industry in many countries and is a major focus sector for development. During recent decades, aquaculture has gained momentum, throughout the world especially in developing countries. According to Food and Agricultural Oganisation (FAO, 2000), global aquaculture production was 26.38 tones in 1996 have reached 32.9 million tonnes during 1999. Only marine aquaculture sector has contributed 13.1 million tonnes during 1999.India is a major fish producing country. About one half of lndia’s brackish water lands are currently being utilized for farming in order to reduce the gap between supply and demand for fish. Aquaculture has become a major source of livelihood for people and its role in integrated rural development, generation of employment and earning foreign exchange, thereby alleviating poverty is being greatly appreciated around the world.Among the infectious agents, bacteria are becoming the prime causal organisms for diseases in food fishes and other marine animals. Sindermann, (1970) reported that bacterial fish pathogen most commonly found among marine fishes is species of Pseudomonas, Vibrio and Mycobacterium. These can be categorized into primary pathogens; secondary invaders that may cause systemic disease in immunocompromised hosts; and normal marine flora which are not pathogenic but may occur on body surfaces or even within the tissues of the host. I-Iigh density of animals in hatchery tanks and ponds is conducive to the spread of pathogen and the aquatic environment with regular application of protein rich feed, is ideal for culturing bacteria. Bacteria, which are normally present in seawater or on the surface of fish, can invade and cause pathological effects in fishes, which are injured or subjected to other environmental stresses.Mycobacteria except parasites are known as nontuberculosis mycobacteria (NTM), atypical mycobacteria or mycobacteria other than tuberculosis(MO'l'l"). This group of mycobacteria includes opportunistic pathogens and saprophytes. Environmental mycobacteria are ubiquitous in distribution and the sources may include soil, water, warm-blooded as well as cold-blooded animals. Disease caused by environmental mycobacterial strains in susceptible humans (Goslee & Wolinsky, 1976; Grange, 1987), animals and fishes are increasingly attracting attention. Greatest importance of environmental mycobacteria is believed to be their role in immunological priming of humans and animals, thereby modifying their immune responses to subsequent exposure to pathogenic species.

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.

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.

Modification of Linear Low Density Polyethylene for Improved Photo and Biodegradation

LLDPE was blended with poly (vinyl alcohol) and mechanical, thermal, spectroscopic properties and biodegradability were investigated. The biodegradability of LLDPE/PVA blends has been studied in two environments, viz. (1) a culture medium containing Vibrio sp. and (2) a soil environment over a period of 15 weeks. Nanoanatase having photo catalytic activity was synthesized by hydrothermal method using titanium-iso-propoxide. The synthesized TiO2 was characterized by X-Ray diffraction (XRD), BET studies, FTIR studies and scanning electron microscopy (SEM). The crystallite size of titania was calculated to be ≈ 6nm from the XRD results and the surface area was found to be about 310m2/g by BET method. SEM shows that nanoanatase particles prepared by this method are spherical in shape. Linear low density polyethylene films containing polyvinyl alcohol and a pro-oxidant (TiO2 or cobalt stearate with or without vegetable oil) were prepared. The films were then subjected to natural weathering and UV exposure followed by biodegradation in culture medium as well as in soil environment. The degradation was monitored by mechanical property measurements, thermal studies, rate of weight loss, FTIR and SEM studies. Higher weight loss, texture change and greater increments in carbonyl index values were observed in samples containing cobalt stearate and vegetable oil. The present study demonstrates that the combination of LLDPE/PVA blends with (I) nanoanatase/vegetable oil and (ii) cobalt stearate/vegetable oil leads to extensive photodegradation. These samples show substantial degradation when subsequent exposure to Vibrio sp. is made. Thus a combined photodegradation and biodegradation process is a promising step towards obtaining a biodegradable grade of LLDPE.

Studies on the pathobiology of penaeid larvae and postlarvae

The thesis presents the results of the studies carried out on certain diseases encountered in the larvae and postlarvae of penaeid prawns raised in the hatcheries at Cochin, Madras and Mandapam Camp during September 1985- April 1988. In the preliminary survey carried out to understand the common diseases occurring in the penaeid larvae and postlarvae, seven cases of diseases and abnormalities were encountered. These included ciliate infestation, Nit_zschia closteriurn infestation, parasitic protozoaninfection, parasitic dinoflagellate infection, appendage necrosis, heteromorphic eye and abnormal eggs and deformed nauplii .The clinical signs, seasonal occurrence and incidence of each of the above cases were provided along with the information on environmental factors such as salinity, dissolved oxygen, temperature and pH of the rearing medium. The Thésis is presented in nine chapters. Chapter 1 surveys the literature on the diseases of penaeid larvae, postlarvae and adult prawns from India and abroad. This is followed by a chapter on the material and methods employed during the present investigation. In the third chapter, seven cases of diseases and abnormalities encountered in the larvae and postlarvae of Penaeus indicus and p. semisulcatus during the survey carried out in the hatcheries located at different centres of Central‘ Marine Fisheries Research Institute are presented and discussed .A bacterium responsible for appendage necrosis was isolated and its taxonomy was studied. It was Gram-negative, fermentative and motile rod. It was sensitive to vibriostatic compound, 0/129. This bacterium was found to be a new isolate of vibrio on the basis of its morphological, biological, physiological and biochemical characters and comparison of these characters with those described for other related vibrios. This new isolate of vibrio was deposited in vibro Referrence Laboratory, Centres for Disease Control, Georgia, U.S.A. and coded as vibrio sp. 2448-88.

Pyocyanin (5-methyl-1-hydroxyphenazine) produced by Pseudomonas aeruginosa as antagonist to vibrios in aquaculture: overexpression, downstream process and toxicity

Pyocyanin is a versatile and multifunctional phenazine, widely used as a bio-control agent. Besides its toxicity in higher concentration, it has been applied as bio-control agents against many pathogens including the Vibrio spp. in aquaculture systems. The exact mechanism of the production of pyocyanin in Pseudomonas aeruginosa is well known, but the genetic modification of pyocyanin biosynthetic pathways in P. aeruginosa is not yet experimented to improve the yield of pyocyanin production. In this context, one of the aims of this work was to improve the yield of pyocyanin production in P. aeruginosa by way of increasing the copy number of pyocyanin pathway genes and their over expression. The specific aims of this work encompasses firstly, the identification of probiotic effect of P. aeruginosa isolated from various ecological niches, the overexpression of pyocyanin biosynthetic genes, development of an appropriate downstream process for large scale production of pyocyanin and its application in aquaculture industries. In addition, this work intends to examine the toxicity of pyocyanin on various developmental stages of tiger shrimp (Penaeus monodon), Artemia nauplii, microbial consortia of nitrifying bioreactors (Packed Bed Bioreactor, PBBR and Stringed Bed Suspended Bioreactor, SBSBR) and in vitro cell culture systems from invertebrates and vertebrates. The present study was undertaken with a vision to manage the pathogenic vibrios in aquaculture through eco-friendly and sustainable management strategies with the following objectives: Identification of Pseudomonas isolated from various ecological niches and its antagonism to pathogenic vibrios in aquaculture.,Saline dependent production of pyocyanin in Pseudomonas aeruginosa originated from different ecological niches and their selective application in aquaculture,Cloning and overexpression of Phz genes encoding phenazine biosynthetic pathway for the enhanced production of pyocyanin in Pseudomonas aeruginosa MCCB117,Development of an appropriate downstream process for large scale production of pyocyanin from PA-pUCP-Phz++; Structural elucidation and functional analysis of the purified compoundToxicity of pyocyanin on various biological systems.

An assessment of potential public health risk associated with the extended survival of indicator and pathogenic bacteria in freshwater lake sediments

Microcosm studies were performed to evaluate the survival of Escherichia coli, Salmonella paratyphi and Vibrio parahaemolyticus in water and sediment collected from the freshwater region of Vembanad Lake (9 35◦N 76 25◦E) along the south west coast of India. All three test microorganisms showed significantly (p < 0.01) higher survival in sediment compared to overlying water. The survival in different sediment types with different particle size and organic carbon content revealed that sediment with small particle size and high organic carbon content could enhance their extended survival (p < 0.05). The results indicate that sediments of the Lake could act as a reservoir of pathogenic bacteria and exhibit a potential health hazard from possible resuspension and subsequent ingestion during recreational activities. Therefore, the assessment of bacterial concentration in freshwater Lake sediments used for contact and non contact recreation has of considerable significance for the proper assessment of microbial pollution of the overlying water, and for the management and protection of related health risk at specific recreational sites. Besides, assessment of the bacterial concentration in sediments can be used as a relatively stable indicator of long term mean bacterial concentration in the water column above

Diversity and effectiveness of tropical mangrove soil microflora on the degradation of polythene carry bags

The diversity and load of heterotrophic bacteria and fungi associated with the mangrove soil from Suva, Fiji Islands, was determined by using the plate count method. The ability of the bacterial isolates to produce various hydrolytic enzymes such as amylase, gelatinase and lipase were determined using the plate assay. The heterotrophic bacterial load was considerably higher than the fungal load. There was a predominance of the gram positive genus, Bacillus. Other genera encountered included Staphylococcus, Micrococcus, Listeria and Vibrio. Their effectiveness on the degradation of commercial polythene carry bags made of high density polyethylene (HDPE) and low density polyethylene (LDPE) was studied over a period of eight weeks in the laboratory. Biodegradation was measured in terms of mean weight loss, which was nearly 5 % after a period of eight weeks. There was a significant increase in the bacterial load of the soil attached to class 2 (HDPE) polythene. After eight weeks of submergence in mangrove soil, soil attached to class 1 and class 3 polythene mostly had Bacillus (Staphylococcus predominated in class 2 polythene). While most of the isolates were capable of producing hydrolytic enzymes such as amylase and gelatinase, lipolytic activity was low. Class 2 HDPE suffered the greatest biodegradation.

Potential Exposure Risk Associated with the High Prevalence and Survival of Indicator and Pathogenic Bacteria in the Sediment of Vembanadu Lake, India

Prevalence of faecal coliform bacteria and the survival of Escherichia coli, Vibrio parahaemolyticus and Salmonella paratyphi were studied in the water and sediment from Vembanadu Lake in the presence and absence of protozoan predators. The density of faecal coliform bacteria ranged between mean MPN value 5080–9000/100 ml in water and 110,000–988,000/1 g in sediment (p <0.01), which was 110 times greater than in overlying water. The laboratory microcosm studies revealed that E. coli, V. parahaemolyticus and S. paratyphi showed significantly higher survival (p <0.05) potential in sediment than in overlying water both in the presence and absence of protozoan predators. The results indicate that Vembanadu Lake sediment constitutes a reservoir of pathogenic bacteria and exhibits potential health hazard from possible resuspension and subsequent ingestion during recreational activities. Therefore, assessment of bacterial concentration in freshwater lake sediments used for contact and non-contact recreation is of considerable significance for the proper assessment of microbial pollution of the overlying water and the management and protection of related health risk at specific recreational sites. In addition, assessment of the bacterial concentration in sediments can be used as a relatively stable indicator of long-term mean bacterial concentration in the water column above.

A brackishwater isolate of Pseudomonas PS-102, a potential antagonistic bacterium against pathogenic vibrios in penaeid and non-penaeid rearing systems

A Pseudomonas sp PS-102 recovered from Muttukkadu brackish water lagoon, situated south of Chennai, showed significant activity against a number of shrimp pathogenic vibrios. Out of the 112 isolates of bacterial pathogens comprising Vibrio harveyi, V. vulnificus, V. parahaemolyticus, V. alginolyticus, V. fluvialis, and Aeromonas spp, 73% were inhibited in vitro by the cell-free culture supernatant of Pseudomonas sp PS-102 isolate. The organism produced yellowish fluorescent pigment on King's B medium, hydrolysed starch and protein, and produced 36.4% siderophore units by CAS assay and 32 μM of catechol siderophores as estimated by Arnow's assay. The PS-102 isolate showed wide ranging environmental tolerance with, temperatures from 25 to 40 °C, pH from 6 to 8, salinity from 0 to 36 ppt, while the antagonistic activity peaked in cultures grown at 30 °C, pH 8.0 and at 5 ppt saline conditions. The antagonistic activity of the culture supernatant was evident even at 30% v / v dilution against V. harveyi. The preliminary studies on the nature of the antibacterial action indicated that the antagonistic principle as heat stable and resistant to proteolytic, lipolytic and amylolytic enzymes. Pseudomonas sp PS 102 was found to be safe to shrimp when PL-9 stage were challenged at 107 CFU ml−1 and by intramuscular injection into of ∼5 g sub-adults shrimp at 105 to 108 CFU. Further, its safety in a mammalian system, tested by its pathogenicity to mice, was also determined and its LD50 to BALB/c mice was found to be 109 CFU. The results of this study indicated that the organism Pseudomonas sp PS 102 could be employed as a potential probiont in shrimp and prawn aquaculture systems for management and control of bacterial infections

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