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I have edited and also contributed 16 chapters to this book on "Fish and shellfish health management". My colleagues in universities in India and abroad and the Indian Council of Agricultural Research(ICAR), New Delhi have contributed rest of the chapters. This book has my experience of 40 years long career in research, teaching and extension in Aquaculture and Fish and shellfish health management at the Karnataka Veterinary, Animal and Fisheries Sciences University (KVAFSU), India. My six year long exposure and experience in North America during doctoral programme in microbiology / fish virology has further helped in writing the book. Experience in Teaching UG and PG programmes in aquatic health management, contributed immensely to carve chapters to this book. I had an opportunity to serve as Chairman (Fisheries), 5th Deans Committee ICAR, New Delhi 2014 for revising syllabus for the four years fisheries programme (BFSc) in the ICAR and the State Agricultural Universities(SAU). Experience gained through interaction and discussion with the colleagues for introduction of Pharmacology, Aquatic medicine, Immunology and Biotechnology for the first time in India has been helpful in writing this book. My research experience in important key areas of fish health management- Production and application of monoclonal antibodies for pathology, development of diagnostics and vaccine, Development of a novel biofilm oral vaccine model for fishes, Artificial substrates for boosting fish health and production through microbial biofilm, besides work on general fish and shellfish diseases has been useful for completion of this book. National and international grants with research collaboration at national /international level helped me to a great extent in quality research with publications and technology developments. Experiences of extensive field visits to carp, freshwater prawn, shrimp and aquarium fish breeding farms in India over the four decades, interacting with farmers, demonstrating farmer level diagnostics are worth mentioning which also contributed to this book.

An Overview of Fish and Shellfish Health Management in Aquaculture in India Aquaculture in India in particular freshwater fish culture comprised mainly of Indian major carp cultivation has a long history. Since 1980s commercial carp culture expanded, particularly in Andhra Pradesh and in few years became an Asia model. Then on, it is slowly and steadily expanding to different parts of the country particularly Punjab, Haryana, West Bengal, Orissa and Tamil Nadu and Karnataka. Of late, culture of Pungasius (Basa) has come up on large scale in AP. In addition, there is also growing small scale commercial cultivation of catfish, murrels, tilapia and freshwater prawn(Macrobrachium rosenbergii). Aquarium fish breeding is also expanding to establish itself as an industry in India. Brackish water culture has been practiced traditionally in India in Pokkali fields of Kerala, Bheries of West Bengal and Ghaznis of Karnataka since hundreds of years. From 1990 onwards, commercial shrimp culture came into existence along the coasts of India. High export value, support from government and short culture period encouraged private entrepreneurs to pursue shrimp farming in a big way. There is vast potential for fish culture in brackish water and at the present efforts are on to popularize culture of Asian seabass, mullets, Eutroplus and milk fish. Mariculture is still at infancy but increasing at a slow and steady rate. Aquaculture health management has to be viewed from the incidences and outbreak of diseases due to growth and expansion of the industry. Aquaculture has expanded tremendously in the last three decades, growing at 6-7 % annually. Contribution of fish production from aquaculture in the country is around 60 % compared to 10 % it was 40 years ago. In few years to come, aquaculture contribution may go up to 80 % of total fish production. Today 2.43 m ha of freshwater and 1.15 m ha of brackish water are under culture. In addition, 8.62 m. ha of irrigated inland area mainly in Haryana, Maharastra and Karnataka which have become saline and unsuitable for agriculture are slowly being reclaimed for aquaculture. Farmers are growing acclamatized saltwater fish and shrimp spp in inland saline waters and a shift like this may favour pathogens with different virulence to cause disease. Overall, it can be seen that in addition to increased conventional areas in fresh and brackish water, aquaculture is being practiced in unconventional areas like saline soils. Performance of fish and shellfish in these non conventional water bodies in the long run needs to be studied from their health point of view.

Diseases are among the greatest deterrents to the sustained production in aquaculture. White spot disease (WSD) in penaed shrimp and epizootic ulcerative syndrome (EUS) in fresh and brackish water fishes are the well known devastating diseases with serious impact in aquaculture. Aquaculture medicine which broadly encompasses prevention and management of diseases in cultured aquatic organisms becomes a vital requirement for a sustained industry. The four K’s essential for scientific aquaculture health management are knowledge about the disease process, knowledge about the pathogen, knowledge about the host and the knowledge about the environment. Disease development process is often complicated and involves host-pathogen- environment interactions. Knowledge about the pathogen, attaching and entering the host, deriving nourishment, reproduction, transmission, overcoming host defence barriers, etc are very essential. Susceptibility of a host to a pathogen is important which depends on host species, age, size, immuno competence and stress response. Besides knowledge about how the temporal and spatial aspects of environment stress the host and favour the pathogen to cause disease is vital Disease Development Process There exist a delicate balance between the host, pathogen and the environment. When this delicate balance gets upset, disease can result. Aquaculture environments can stress the host, favour the pathogen and result in disease development. To appreciate the process of disease development understanding the pathogenicity mechanisms of the pathogens, disease resistance mechanism of the host and the role of the environment is essential. Furthermore, role of various pathogens, their adaptive modifications, the functional importance of the target tissue, interaction between pathogen and host at the target tissue level, pathogenicity mechanisms of pathogens, etc are necessary to gain insight into process of disease development. Only when a pathogen can establish on or in the host, proliferate, overcome the non-specific and/or specific defense barriers of the host, produce the pathogenic factors, cause cellular and tissue damage, produce significant pathological changes, impair the function of the target tissue cause clinical disease often with mortality. This process is very complicated which often get accelerated by stress and environmental factors. However, the sequence of disease development will to a large extent depend on the nature and load of the pathogen (parasite, bacteria, fungi, virus), its intensity per unit area or unit weight of the host, size of the host and environmental factors.

An Overview of Parasitic Diseases in Asian Aquaculture Parasitic induced diseases bring about significant economic losses in fish culture system. Estimated loss due to mortality and morbidity of fish due to parasitic infection is much more than that from microbial diseases. Protozoan ciliates (Ichthyopthirius, Trichodina) and flagellates (Ichthybodo, cryptobia) are some of the common ectoparasites of fish. Certain ectocommensal ciliates (Epistylis, Vorticella and Zoothamnium) attach to the external surface of fish leading to fouling. These protozoan ectoparasites are often associated with mortalities of younger stages of cultured fish. The situation becomes worst in water with low oxygen and high organic matter. Most extoparasitic protozoans have simple and short life cycle and reproduce by binary fission. Most protozoan ectoparasites are readily detected in direct microscopic examination of skin and gill scrapings. Histopathological changes in the integument following ectoparasitic protozoan infection are outcome of two counteracting cellular processes – hyperplasia of the epithelial cells, including mucus cells and chloride cells, versus a progressive cellular destruction. Cellular necrosis primarily occurs due to the feeding and attachment activity of the parasites. Generally, immersion therapy using suitable chemicals can control ectoparasitic protozoan infections Disease caused by endoparasitic sporozoans are a serious threat to fish farms. These sporozoan spores present in the pond soil are normally ingested by the fish. The infective element of the spore (sporoplasm) is released in the gut. The sporoplasm reaches the target tissue using the vascular route. Once inside the target tissue, the vegetative trophozoites cause massive destruction of the target tissue and produce large spore containing cysts. There are two types of sporozoan infecting fish and shell fish namely myxosporidian and microsporidian. Myxosporidian occur in fish either as histozoic (intercellular tissue parasites) or as coelozoic (inside the lumen of various ducts and tubules) parasites. Microsporidians are intracellular obligatory parasites and lead to massive hypertrophy of infected cells (xenomas). There is no effective treatment for sporozoans. However, treating pond soil with chemicals and lime is the best option.

With increasing fish culture activities several bacterial diseases causing morbidity and mortality in fish have been reported in tropical and subtropical aquaculture systems. Common and major diseases are due to motile aeromonads, Vibrios and Pseudomonas. However, bacterial diseases of minor nature have also been reported from aquacultural systems. In the last two decades several new diseases due to Streptococcus sp, Edwardsiella and Ricketsia have also emerged. Bacteria can cause disease either as primary pathogens or as secondary opportunistic invaders. Bacterial diseases in fish can be broadly classified as surface ulcerative, acute systemic and chronic granulomatous types (Table 1, Plates 4 and 5). Surface ulcerative types of diseases are characterized by hemorrhagic surface ulcers and are caused by species of Aeromonas, Pseudomonas, Vibrios, Flexibacteria and Myxobacteria. Surface ulcerative disease conditions at times develop to acute systemic disease. Acute systemic disease are characterized by the presence and proliferation of bacteria in internal organs like kidney, heart, spleen, blood and other visceral organs. These diseases produce significant necrotic changes in all affected organs and can cause mortality in a short time scale. Bacterial hemorrhagic septicemia caused by Aeromonas hydrophila is a major problem in carp farms. Chronic granulomatous type of disease conditions are characterized by the formation of granulomas or nodules with the initiating bacteria in the centre. These conditions will not produce mass mortality but can produce chronic low levels of mortality and can significantly reduce growth.

A. Bacterial Diseases in Shrimp Bacterial diseases in hatcheries and farms are being increasingly recognised as major hurdles to successful shrimp farming (Plate 6). Among the several bacterial diseases, diseases due to Vibrio sp. (vibriosis) are common. As majority of the Vibrio are secondary opportunistic pathogens, problems related to vibriosis can be traced to stress, poor water quality and bad management. It is widely accepted that the ubiquitous opportunistic vibrios are always responsible for causing mortalities in shrimp which are already stressed by poor water quality, external fouling and primary pathogens like viruses. Vibrios are gram negative, oxidase positive, motile rods. Most frequently reported species in hatchery situations include Vibrio harveyi, V. vulnificus, V. parahaemolyticus and V. alginolyticus. All species of cultured shrimp are susceptible to vibriosis under stressful condition. The common clinical signs associated with vibriosis are: 1) high mortalities, particularly in PL and young juveniles shrimp, 2) Moribund shrimp appearing hypoxic and often coming to pond surface and edges, 3) Reddening of shrimp, 4) Shell and appendage necrosis with blackening, 5) Presence of luminiscence in affected shrimp in tanks and ponds.

Viruses are submicroscopic particles made up of nucleic acid covered with a protein coat. The nucleic acid is either RNA or DNA and not both. Protein coat is often covered with an envelope and depending on the presence or absence of an envelope, virus can be an enveloped or non enveloped virus. Size of the virus vary from 20-200 nm. Shape of the virus varies from a simple icosahedra to bullet shape to complex brick shape. Virus are classified based on their nucleic acid (DNAor RNA) and protein profile. However, additional details on type of disease, host range, and geographic distribution of the virus are also used in classification. The classification of the virus is decided by the International committee on taxonomy of virus (ICTV). Viruses are obligatory intracellular parasites requiring a living cell to replicate. As the virion is lacking its own metabolic machinery, it depends fully on the living host cell for machinery and raw materials for its metabolism and replication. This complex biology of the virus has made it difficult to clearly identify a viral process to design drugs for selective killing without damaging the host cell. Any attempt to kill the intracellular virus affects the host cell. Replication of Virus in a Cell There are two kinds of entry into and exit of virus from a cell: a) receptor mediated endocytosis (RME), b) direct entry by fusion. Non-enveloped virus enter the cell by RME (Fig. 1). RME of virus is similar to regular entry of other macromolecules into the cell. With this receptor mediated specified process, virus enter a cell in a vesicle which later fuses with lysosome. Inside the lysosome, viral particles undergo partial changes in protein coat due to digestion by lysosomal enzymes and move to cytoplasm. Finally, this uncoated nucleic acid replicates followed by synthesis of proteins and assembly of viral capsid proteins and genome to full blown virus particles. The site of assembly of the virus may be in the cytoplasm or nucleus of a cell which is referred to as inclusion body. Inclusion bodies which varies in size, shape, location and staining reaction can be used as a diagnostic feature for some viruses. Non- enveloped virus exit from a cell by damaging plasma membrane and hence clearly show cytopathic effect(CPE) in cell culture.

A. Viral Diseases of Shrimp Shrimp farming which has grown to an industry in the last three decades is young compared to fish farming in the world. Yet the shrimp farming is facing serious problems with several types of viral diseases. Intensive culture in hatcheries and grow outs along with movement of stock across national and international borders of these animals with poor immune system has encouraged emergence of large number of viruses. During a short span of 30 years, 15 viruses of economic concern have been reported in shrimp culture of the Asia Pacific region. Shrimp viruses (Plate 8) can be broadly categorised into two groups: 1. Viruses infecting ectodermal (epidermis, hypodermal epithelium of fore and hindgut, nerve cord and nerve ganglia) and mesodermal (hematopoitic organs, antennal gland, gonads, lymphoid organ, connective tissue and striated muscle) origin tissues and replicating within the cytoplasm (YHV, TSV) and within the nucleus (IHHNV, WSSV), 2. viruses like MBV, HPV, BMNV, and BP infecting endodermal origin tissue and replicating within the nucleus of hepatopancreatic and mid gut epithelial cells. Lack of established shrimp cell line is the major drawback in study of shrimp viruses. Following are some of the common viruses causing diseases in shrimp hatcheries and grow out ponds in the Asia Pacific region.

Introduction Oomycete and fungal diseases are considered as the second most economically impactful diseases in aquaculture, after bacterial diseases. However, among both the diseases, oomycetes are more common. The oomycetes are members of the Kingdom Chromista, which also includes diatoms, kelps, and brown algae. These group of pathogens form free-swimming zoospores and their cell walls are composed of cellulose and glycans rather than chitin. Fish-pathogenic oomycetes (Saprolegnia, Achlya, Aphanomyces and Branchiomyces), are commonly known as water moulds, are ubiquitously distributed in a variety of aquatic environments and ecological niches, and belong to the Order Saprolegniales. These pathogens have low host specificity and infect a diverse range of fishes. On the other hand, most of the fungi (Fusarium sp., Aspergillus sp., Candida sp., Paecilomyces sp., Penicillium sp. and Cladosporium sp.) responsible for infection in fish, form non-motile spores and their cell walls are made up of chitin. In addition, Ichthyophonus sp. an important pathogen which, superficially appears like fungus and produces spores and hyphae, belongs to a distinct clade of protistan parasites. Among the oomycete diseases, infection with A. invadans is considered as one of the most serious fish disease. Therefore, the disease is discussed elaborately. Infection with Aphanomyces invadans: Infection with Aphanomyces invadans is popularly known as, Epizootic Ulcerative Syndrome (EUS). It has a complex infectious aetiology and is clinically characterised by the presence of invasive Aphanomyces infection and necrotising ulcerative lesions, typically leading to a granulomatous response (Plates 9 and 10). In an expert consultation held during the sidelines of the Fifth Symposium on Diseases in Asian Aquaculture (DAAV) at Gold Coast, Australia in November 2002, it was proposed to rename the disease as epizootic granulomatous aphanomycosis; however, the term EUS is still used popularly.

In the past several Non infectious diseases (NIDs) of fish and shellfish have been observed in natural waters. However, with expansion in fresh, brackish and marine aquaculture including aquarium keeping world wide there is a phenomenonal increase in incidences of NIDs and economic loss. Common Non infectious diseases (NIDs) in fish are 1.Gas bubble disease(GBD), 2. Body deformities(BD), 3.Nutritional diseases(ND) and 4. Disease due to harmful algal bloom(DHAB) 1. Gas Bubble Disease (GBD) Gas bubble disease (GBD), is caused by supersaturation of gases in water. The gas involved is nitrogen in most cases and rarelycarbon dioxide and oxygen can cause GBD as these two gases are used by fish tissues and readily processed.Oxygen can cause gas-bubble disease at about 350 per cent air saturation, but nitrogen can cause the disease even below 118 per cent. GBD becomes prominent whenever there is a change in temperature, pressure and turbulance in aquatic environment, leading to disturbance in metabolism of f ishes. Bubbles of gas may form in the eyes, skin, gills, fins and within the internal organs where small bubbles may coalesce into larger bubbles.Fish absorb the excess gas, which forms bubbles in the small blood vessels, due to pressure changes inside their body. Gas bubble disease (GBD), is seen both in captive and wild fish. When bubbles affect internal organs, some fish may diesometimes without obvious clear clinical signswhile other fish may be chronically affected, and have a reduced appetite, activity and buoyancy. GBD can also cause secondary bacterial, viral or protozoan infections.All fish and shelfishspecies as well as amphibians and aquatic invertebrates are susceptible to GBD.Sensitivity of fish to GBD in terms of pathology and mortality varies with species and age.

Introduction Fish is the cleanest animal, gets bath 24 hrs a day provided water is clean without any toxic material. Perhaps this was the scenario world over 5 decades ago. However, in the last few decades due to industrialisation, modernisation of agriculture and animal husbandry, human health care industry and city developments all have contributed to toxic material to water endangering the fish and the consumer, leave alone clean environment. In recent years, even intensive aquaculture also add several chemicals, pesticides, antibiotics contributing to pollution. Natural water bodies such as rivers, lakes, reservoirs, scoastal/marine waters are ultimate receivers of pollutants which also serve as the source of water for aquaculture. Although pollution control by treatment measures and awareness have improved over the years still there is a long way to achieve the clean water. Effect of toxins in aquaculture has multiple angles, differing from that of toxins on land animal. Fish depends heavily on water for respiration, physiology, reproduction, food and feeding and its very sustenance- as a supporting medium. Aquaculture is expanding world over growing at 6-8% gradually involving more areas and species contributing a major share to total fish production with gradual decline in capture fisheries from the natural water bodies. Invariably, most of the natural water bodies are polluted with one pollutant or the other in the country. In addition, several pollutants are added to the system by various aquacultural practices. Although large quantity of information is available on aquatic toxicology, toxicology in aquaculture from health point view of fish and the consumer is meager and not focused. Against this background, it is very important to study, understand various toxicants, their mode of action, accumulation and effect on fish and the consumer. Toxicology in aquaculture is incomplete unless it includes or addresses safety of the consumers. Further, poisoning of fish in farms and wild deliberately or otherwise needs forensic studies/approach to address the issues.

Epidemiology is the study of diseases in a population in its natural setting. In epidemiology, the population is the patient and hence epidemiology is population medicine. All epidemiological studies are field based. In aquatic epidemiology, aquaculture ponds serve as the laboratory. This can be a great advantage over laboratory studies, which cannot be easily extrapolated to field conditions. Diseases are among the greatest deterrents to the sustained production in aquaculture. Diseases such as Epizootic Ulcerative Syndrome (EUS) in fishes and White Spot Syndrome (WSS) in shrimps have devastated aquaculture with bad impact on productivity and profitability. Various diagnostic and health management tools are being used to devise disease control strategies. It is becoming increasingly difficult to manage pathogens that have become endemic. Epidemiological approaches which have been very successful in human and veterinary medicine are now being increasingly recognised as important weapons that can be used in aquaculture to formulate disease control programmes. Concept of Cause for a Disease Cause from an epidemiological perspective is interpreted in quite a wide sense. This is somewhat different to the more traditional view of the role of cause being restricted to etiological agents. An epidemiological definition of a cause of a disease is “an event, condition or characteristic that plays an essential role in producing an occurrence of the disease”. Epidemiologists avoid defining the word cause for any disease outbreak, but prefer to use words such as determinants, exposures and risk factors. Alternatively, they will categorise causes as direct or indirect; necessary or sufficient; and single or multiple rather than defining primary etiology.

Introduction Aquaculture and Zoonotic Diseases Diseases that can transfer from wild or domestic animals to humans are referred to zoonosis (Fig 1). Zoonosis also includes biotoxin producing agents from fish to human beings which is well documented. There are a number of pathogens which spread from fish and shellfish to human and other domestic animals causing diseases.There are also many other infectious organisms of fish origin that have not been reported but have potential to infect and harm man. Zoonotic diseases originated from fish and shellfish have been documented in the past mostly from wild caught fish and shellfish. However, in the last 30 years as the capture fisheries is declining and aquaculture is growing steadily worldwide, zoonotic incidences from the latter are increasing. Aquaculture growing at 6-7% annually world over including India is contributing to 50-60% of total fish production today. Additionally, aquarium industry is booming worldwide. Therefore,incidences and scale of zoonosis due to aquaculture are quite different from that from capture or wild fisheries. Disease outbreaks in cultured fish are often by pathogens indigenous to aquatic environment. In aquaculture, disease outbreaks are associated with quality and quantity of nutrients and high stocking density which increase pathogen load leading to easy transmission to human. Apparently healthy fishes may also harbor bacterial pathogens, especially in their kidneys and intestines. World Health Organisation (WHO) considers many diseases in aquatic animals as emerging type. Emerging diseases appear in a population for the first time or it may have been existed before, but is now increasing rapidly. Usually information on zoonotic potential of emerging diseases is limited which is essential to disseminate to human health managers. Potential biological contamination of aquaculture products can occur from bacteria, fungi, viruses, parasites and biotoxins. The location of the farm, the species being farmed, water temperature, husbandry systems, postharvest processing, and habits in food preparation and consumption are among the main factors influencing the zoonotic risk associated with aquatic animals and their products.

Disease diagnosis refers to the various procedures and techniques used to identify the nature of disease and to precisely pinpoint the primary and secondary pathogens involved. Disease diagnosis is an integral part of health management. Proper diagnosis helps to adopt accurate therapy and avoids indiscriminate use of chemotherapeutics. Information on case history and clinical signs should be carefully used while examining samples for diagnosis. By following rational and scientific sampling protocols, one can hope to arrive at accurate diagnosis. Sampling methodology is key to the success of proper disease diagnosis. Dead fish/shrimp should never be used for any diagnostic purpose. Autolytic post-mortem changes, saprophytic invaders, spoilage organisms, etc. mask the actual etiological agents and can lead to false diagnosis. The three commonly used conventional diagnostic approaches are fresh examination using microscope, microbiological methods and histopathology. With little facilities and infrastructure, fresh microscopical diagnostic approach can be done at the farm/hatchery site itself. Gross examinations along with simple skin and gill preparations examined under microscope can many a times give very good information about the disease and the etiology. All fresh preparations should be made from live or moribund fish and the preparations should be wet and moist. The fish meant for examination should be kept wet and not allowed to dry. Many of the commonly occurring external disease problems can be easily diagnosed by this method. Systemic bacterial diseases need to be positively diagnosed by adopting the microbiological approach. Samples for bacterial isolations should be taken from kidney, blood, body cavity, etc. from live or moribund fish following strict aseptic conditions. In shrimp, it is ideal to use hemolymph for bacterial isolations. Dominant bacterial isolates are identified following standard procedures and their antibacterial sensitivity tested before recommending any antibacterial therapy. This method requires a minimum of 4-5 days.

Introduction Development and standardisation of diagnostics for aquaculture health management have undergone great improvements over the last three decades. Diagnosis in aquaculture health management involves several stages starting from farm-level observations to conventional laboratory identification and histopathology to sophisticated modern molecular tools. Accordingly, diagnosis can be broadly categorised under three levels depending on the level of scientific development. 1. level-I detection system, farm site observation and record-keeping; 2. level-II system, conventional laboratory identification employing microbiology, parasitology, and histopathology and 3. level-III system, advanced and specialized immunological and biomolecular techniques. The interaction of an antibody with an antigen forms the basis of all immunochemical techniques. Antibody binds specifically to an antigen at epitope or antigenic determinant level and this property has been used for development of immunodiagnosis. Furthermore, as antibodies react with antigen at ambient conditions and do not require sophisticated equipment ideal field test kits can be developed. Biomolecular tools using nucleic acid probes although very sensitive are costly and time-consuming requiring sophisticated equipment and trained personnel. Against this background, there is always a need for developing simple, low-cost, rapid yet sensitive farm- level diagnostics. Such field-level tests facilitate screening a large number of samples by a large number of poor and medium-sized farmers who constitute a majority in developing countries. Large-scale screening besides empowering farmers is considered to be ideal for effective health management. Antibody probes provide an ideal format for developing simple field-level diagnostics as antigens ideally react with antibodies at ambient conditions without requiring sophisticated equipment. Available technology for raising large quantities of monoclonal antibodies (MAbs) against pathogens has increased the scope for developing field detection kits in human, animal and agricultural health management. Several farmer-friendly detection systems also referred to as point-of-care (POC) detection method based on antibodies have been innovated in the recent past. POCT is defined as ‘medical diagnostic testing at the time and place of patient care.

Introduction Nucleic acid based diagnosis is highly specific, sensitive and rapid method widely used in health management. The detection is mainly based on structure and sequence of bases of nucleic acid which are unique to an organism. In recent years, nucleic acid based diagnosis is becoming common and popular in fish and shell fish health management. Commonly employed nucleic acid based diagnosis are DNA hybridisation and Polymerase chain reaction (PCR). Nucleic acids are made up of nitrogen bases. The primary sequence of bases in DNA or RNA have regions which are unique for a particular organism or gene. The sequence of DNA is composed of a series of four phosphorylated bases Adenine (A), thymidine (T), guanidine (G) and cytosine (C). Pairs of bases (A=T, or G=C) can form hydrogen bonds, which cause opposing strands of DNA to form a semi-stable bonding (hybridization or annealing) to one another. If the opposing strands of DNA match exactly, then the familiar double helix form of DNA results. In the following example, the opposing strands in (1) will form a double stranded, tightly bonded structure, and the DNA strands are described as complementary. In (2) the strands are non-complementary (opposing A=T and G=C base pairs do not line up) and the two strands will not bond. DNA Hybridisation The basic principle in DNA hybridization is that, if a strand of double helix of DNA is separated from its complement (by heat) it will bind (reanneal or hybridize) only to its complementary strand (or to a duplicate of its complementary strand). Two complementary RNA structure will hybridise in the same manner. Inaccurate and mismatched bindings will be unstable and fall apart. The binding of one DNA strand to another is in some respect is analogous to binding of antigen and antibody. However, in the case of nucleic acid bindings, the precise molecular nature of binding (base pairing) is known and can be easily manipulated by choosing DNA strand of appropriate length and by incubating the strands in appropriate salt concentration, temperature. The stringency (faithfulness of the match) of hybridisation can be precisely defined and as a result coincidental cross reaction is virtually eliminated.

Introduction Scope and importance Aquaculture is breeding, rearing and husbandry of aquatic animals such as f ishes, crustaceans and molluscs for food, ornament and sports. It is a husbandry involving special animals, the cold blooded vertebrates and invertebrates, whose physiology, growth, metabolic activities and overall performance depend heavily on ambient aquatic temperature and environment. Therefore, husbandry and the health management of aquatic animals is quite different from that of the other land based warm blooded animals. Aquaculture is one of the fastest growing food production sectors in the world including India. Three decades ago, few species were cultured in India mainly in inland waters and then aquaculture was contributing less than 10 % of total f ish production in the country. In the last 25 years, there is a rapid growth in aquaculture with annual rate of 7-8% with significant contribution (above 60%)to total fish production today. The number of species cultured today in India including ornamentals is close to 40. Besides, a good number of plant and animal fish food organisms are cultured to support the hatcheries and nurseries in aquaculture. The area under aquaculture is increasing steadily in inland and coastal brackish water while marine water is also being added up for aquaculture slowly and steadily. Very interestingly, non-traditional environment such as manmade saline soils are also explored successfully for aquaculture. Intensification of culture activity with high stocking density artificial feed and fertilizers is another dimension of aquaculture noteworthy. Large scale movement of aquatic animals and their raw or processed products across national and international borders is important from aquaculture production and health point of view. In general, aquaculture package of practices alter the very water in terms of its chemistry and microbial load and has significant negative impact on the health of fish in culture system.

Farming of fish and shellfish has gained significant grounds in several parts of the world. However, the major bottleneck to successful farming is disease in farmed aquatic animals. In recent years, lot of attention is being given to health management using various forms of immunoprophylactic measures such as vaccination and immunostimulation. To rationalise immunoprophylactic applications, it is vital to have insight into the specific and non-specific defense mechanisms of farmed aquatic animals. Through disease process studies it is very well known that a pathogen can cause disease only if it can overcome the non-specific and specific defence barriers of the host and successfully establish and proliferate. Non-specific and specific immune systems are the two main branches of the defense system. The immunity which an organism derives against a pathogen is the result of a delicate interaction and cooperation between the two systems. Defense System of Fish Surface Barriers as Local Immune System The immune systems present in the surface structures of fish have their potential importance in local immune responses. Important surface barriers are skin, gills and gastrointestinal tract.

Introduction Aquaculture is given major thrust in many countries including India not only as a source of good quality food of animal origin but also because of economic gains it brings to aqua-farmers, creation of employment and export trade. It has emerged as one of the fastest growing food producing sector sowing to intensification and diversification of culture systems. However, the success of the intensive aquaculture system depends on several factors including species cultured, type of feed, water quality, management practices and strategies adopted to prevent/ control disease problems. Further, the intensive aquaculture operation results in accumulation of wastes leading to water quality deterioration, oxygen depletion, build-up of toxic metabolites such as hydrogen sulphide, methane, ammonia and nitrites that cause stress to culture animals leading to susceptibility to diseases. Among the several challenges that seriously affect the growth and sustainability of aquaculture, the incidence of microbial diseases are considered as major threat. Thus, keeping cultured aquatic animals in good health becomes major requirement for the success of aquaculture. Though application of chemicals and drugs are often considered necessary to maintain water quality and animal health, use of these, in the long run are known to cause harmful effects both to the animal and the environment. Further, accumulation of drugs and chemicals in tissues, development of resistance to drugs by pathogens, pathogen build up, destruction of useful microorganisms, deterioration of pond environment and toxicity issues have limited their use in aquaculture system. Under these circumstances several alternative strategies have been proposed as viable option for the success of aquaculture in the form of prophylactic measuressuch as use of probiotics, prebiotics, synbiotics, immunostimulants and bacterial vaccines. The term prophylaxis refers to all preventive steps taken including better aquaculture management practices that are adopted during a hatchery and farming operation to improve growth, minimize the load of pathogens and prevent occurrence of diseases.

Any technique that uses living organisms or substances from these organisms to make or modify a product or to improve plant or animal or to develop microorganism for specific uses, can be termed biotechnology. Biotechnology involves microbes or cells derived from plants and animals for efficient production of useful products for mankind. The use of microbes for production of alcohol and penicillin, to name a few, is well known. However, in the last 25 years, several novel organisms, yielding useful products have been created by genetic engineering. Today, there is a wide application of biotechnology in human medicine, agriculture and animal husbandry in the area of diagnosis prophylaxis and therapy. Aquaculture industry has undergone a sea change world over in the last three decades. The important needs of the industry are development of fast growing disease resistant fish and shellfish varieties, development of cheap and effective vaccines, disease diagnostics, cell lines sand probiotics. Efforts on these lines have already begun since 3-4 decades and results are highly promising. Some of the biotechnologies employed in aquaculture health management (Plate 15) are fish cell lines, monoclonal antibodies and nucleic acids for diagnostics sub unit and nucleic acid vaccines for prophylaxis, transgenes for disease resistance, gene editing in fishes and their pathogens and probiotics for bioremedial measures. A. Cell Lines for Health Management Cell lines are transformed cells that are adapted well to grow and multiply outside the host in in vitro culture systems. Cells lines can be developed from fast growing tissues of young fish. There are two methods of developing cell lines (1) Explant method, (2) Enzymatic method. Cell lines are grown in sterile plastic or glass containers containing appropriate medium having nutrients salts, antibiotics, buffers, serum etc. Minimum essential medium (MEM) is the common medium employed for fish cell line. Cell lines are developed, characterised and deposited in national and international repositories, which can be procured for use in research and industry.

Fish are Sentient Animals There is strong increasing evidence that finfish, like other vertebrates are sentient animals. This means that fish are self-aware, they can feel pain and distress, they have long term and short-term memory and to some extent, they can experience emotions. Fish are intelligent, sensitive creatures and like many other animals, they explore, travel, socialise, hunt and play. Some species care for their young and use tools as humans do. Fish are sentient animals capable of suffering and feeling pain. Most fish have highly developed senses with excellent taste, smell, hearing and colour vision. Until fairly recently, many people didn’t realise that fish were sentient or feel pain, and the mental abilities of fish were given limited attention by the scientific community. Now, recent discoveries open up a new world of understanding. Far more complex than we ever realised, f ish live rich social lives: communicating; hunting cooperatively; and, in some cases, developing cultural traits. Consensus arrived in the mid-2000s around science demonstrating high-level pain perception in fish, and examinations of fish behaviour are beginning to reveal a picture of fish as complex, social and emotional beings. Appreciation of these facts are not widespread in civil society, but identified in time for the principle of fish welfare to be recognised by legislation by the European Union.

 A  A invadans 117, 118, 122, 123, 124, 126, 127, 128, 129, 164, 166, 223, 347  Abalon herpres virus 116  Accepted daily intake 156, 157, 160   Acute inflammation 16, 17, 18  Aflatoxins 137, 147  AHPND 35, 65, 66, 67  Algal bloom 133, 134, 141, 148, 202  AMPs 302, 305, 306, 307  AMR 271, 272, 273, 367