Epidemiological Approaches in Prevention and Control of Infectious Disease of Fish in Aquaculture

Epidemiological Approaches in Prevention and Control of Infectious Disease of Fish in Aquaculture; With Emphasis on Fish Vaccines: A Review

 

1. Introduction

 

Fisheries play a great role in food security, livelihood, source of income and social development g developing countries. Recently the sector attracted a great attention and it is growing rapidly much more than any other food producing animal sectors (Diei-Ouadi & Mgawe, 2011). During the last three decades, involvement of fisheries in the global food production for domestic use has increased considerably. This progress has been achieved through the development of aquaculture. Beside this development, some issues have been raised regarding the global distribution, utilization and trade of the fisheries resources as well as quality, safety and future sustainability (Cka & Boukal, 2016).

Even though the sector is backbone of poor livelihood, due to the high prevalence of disease as well as the emergence of new pathogens, hindered its enhanced growth and sustainability (Subramani & Michael, 2017). To overcome these challenges, it is necessary to act up on every health constraint based on scientifically proven and recommended ways. These ways may include Both observational and theoretical epidemiological approaches to investigate the processes driving emergence and spread of infectious disease in aquaculture (Faisal, Samaha, & Loch, 2017). The need for epidemiological approaches to protect aquatic animal health will inevitably increase in the face of the combined challenges of climate change, increasing anthropogenic pressures, limited water sources and the growth in aquaculture (Peeler & Taylor, 2011).

Integrated pathogen management (IPM) is a holistic approach that gathers the best available preventative, treatment, and control strategies to minimize the impact of pathogens in fish production, while striving to increase sustainability. The development of IPM strategies starts by gathering knowledge on pathogen, host, and environmental risk factors. This is followed by the development, evaluation of feasibility, and implementation of the best strategies. Monitoring of the disease (pathogen surveillance, host performance, and impact on environment) is crucial to review the effectiveness of the applied strategies and revise them, based on the knowledge gained (Sitjà-Bobadilla & Oidtmann, 2017).

As always ‘prevention is better than cure’, it is highly recommended to give emphasis on preventing the occurrence of disease rather than treating it. The development of effective disease control strategies is one aspect of fish disease research that has resulted in substantial progress from new approaches to vaccination to the use of probiotics, prebiotics, nonspecific immune stimulants, plant products, antimicrobial compounds, water disinfection, and prevention of/restriction in the movement of infected stock (Adams, 2016). These newer approaches have been matched by a downturn of interest in the use of antimicrobial compounds insofar as there is increasing concern about the development and spread of resistance and thus a reduced efficacy against human pathogens, and tissue residues. (Bruno, 2011; Dhar & Manna, 2014; Woo, 2010). In response to the banning of chemicals and residues in imported fish many governments have imposed restrictions on the utilization of antimicrobials in aquaculture. Application of chemicals to treat viral and bacterial diseases in aquaculture is being discouraged because of the negative impacts they pose to the aquatic environments (Harikrishnan, Balasundaram, & Heo, 2011).

In response to reduced antibiotic use in fish production vaccines has been playing a key role in infectious disease control in aquaculture for decades. Prevention and control of infectious disease by fish vaccination is becoming increasingly important as a part of aquaculture biosecurity. Vaccinated animals have a reduced risk of disease development and even non-vaccinated animals may be protected due to herd immunity (Gudding, 2014a).

As part of prevention and control of disease in fisheries, in addition to vaccination there have been developments in veterinary approved chemotherapy, biological methods of controlling disease, quarantine and import restrictions, and the use of immune stimulants are some of highly recommended approaches in modern aquaculture technologies. In order to apply these strategies meticulously, implementation of effective surveillance, monitoring and availability of appropriate diagnostic tests with a high sensitivity and specificity is mandatory. By bearing this idea in mind this extensive literature review has the objective of pointing out some of the paramount approaches in infectious disease of fish in aquaculture.

2. Literature Review

 

1. The Role of Vaccines in Control and Prevention of Infectious Disease of Fish in Aquaculture

 

1.  Historical Overview of Fish Vaccines

The history of fish vaccination dates back as early as 1942. By that year a scientist named Duff demonstrated vaccination against Aeromonas salmonicida infection in cutthroat trout by oral immunization strategies. (Gudding, 2014a; Vallejos-Vidal, Reyes-López, & MacKenzie, 2017). Many of the pioneers in the fields of fish vaccination were people with a scientific background that combined good theoretical knowledge with excellent understanding of practical fish farming. The second vaccine for fish was licensed by a subsidiary of Johnson & Johnson (Tavolek Inc.). Biomed Inc. was the first company to launch an oil-based vaccine against furunculosis in Norway in 1992/3 (Adams, 2016; Thompson, 2017).

2.    Current Status

 

The majority of fish vaccines intended for injection are inactivated, whole virus/bacteria vaccines, often prepared with an adjuvant like mineral or vegetable oil. Immersion vaccines are usually non adjuvanted and the majority of these vaccines are bacterin-based, while live vaccines, for injection or immersion, are used to a very limited extent. In the years to come we will see that the current vaccine formulations are refined to give reduced side-effects, local and systemic. It is also foreseen that the vaccine formulations and immunomo dulating compounds can direct and improve the immune response, particularly against intracellular pathogens(Magnadottir, 2010).

The majority of vaccines currently available for farmed fish are prepared by conventional methods, i.e. typically a suspension-based fermentation of bacteria or virus harvested from cell culture. Inactivation methods typically include the use of formalin or alkylating compounds, and downstream methods can include filtration, concentration of antigens or purification of antigen preparations. Vaccines available are oil adjuvanted, injectable vaccines.  In addition to these currently there are some modern types of vaccines like DNA vaccine for use in fisheries (Gudding, Lillehaug, & Evensen, 2014).

3.    Future Directions

 

The salmon genome is soon to become publicly available, and several model fish species have been fully sequenced. This will be a new era in fish vaccinology, particularly with the detailed fingerprinting of the protective immune response following immunization or natural infection. With new tools like high throughput sequencing and powerful analysis of transcriptome and proteome levels that likely will become available over the next decade, it is foreseen that the understanding of underlying fish responses to vaccination will also take a major step forward (Gudding et al., 2014).

DNA vaccines will also likely be part of the future vaccine portfolio. Immunization through the mucosal surfaces will be refined and improved in coming years, probably with a focus on improved local responses that can evoke an immune protection at the site of entry of the pathogen or the primary replication site. Vaccines against intracellular bacterial and viral pathogens will be one of the big challenges for the next 10 years of fish vaccine research. Use of DNA vaccine technology will likely play a role here, and so will new and improved formulations, oral boosting regimes, and possibly live vaccine preparations (Gudding et al., 2014).

4.    Types of Fish Vaccines

 

Depending on their designing strategy, vaccines can be classified as non-replicating vaccines, replicating vaccines, and DNA vaccines which intern have their own sub classifications(Gudding, 2014b). It is difficult to identify any particular type of preparation which excels in terms of protection. Generally, the simplest approach of using inactivated whole cells has received greatest attention. whole cell vaccines gave superior results to other more complex forms of vaccines. However, even the best vaccines do not completely prevent the occurrence of disease, necessitating the use of costly drugs to combat low levels of infection. Some of the approaches of designing of vaccines are discussed in the next session.

1. Inactivated vaccines (Non Replicating Vaccines)

These types of vaccines are prepared by killing the infectious agent and using it as an antigen to induce an immune response. The whole process can be classified into four basic steps, namely: isolation of the pathogen; propagation; inactivation; followed by administering the inactivated microorganism into the host. Inactivated vaccines depend upon the inactivation of whole pathogen, and they account for the majority of commercial vaccines currently used in aquaculture. They have the advantage of being easy to make, eco safe, and are less expensive. Inactivated bacterial vaccines account for the majority of the products on the market, followed by viral vaccines (Gudding et al., 2014).

Inactivation may target extracellular or outer surface components of microorganisms such as the cell membrane or the lipid bilayer forming the outer coat of viruses, or intracellular or inner components such as nucleic acids, with the aim of impeding the pathogen’s ability to replicate. In principle, inactivation methods fall into two broad categories, namely physical and chemical inactivation (Adams, 2016; Dhar & Manna, 2014; Gudding & Muiswinkel, 2013).

2. Subunit vaccines

Subunit vaccines are another class of vaccines that have emerged with the advent of molecular biology. Production of specific microorganism proteins using a recombinant protein expression system allowed the rapid production of focused vaccines based on a single antigen or small number of antigens. Molecular tools allowed the high expression of the most highly antigenic proteins of the target organism in bulk and subsequent delivery of these highly purified preparations as a vaccine (Harikrishnan et al., 2011; Plumb & Larry, 2011).

These vaccines rely on the expression of specific immunogenic proteins of pathogens in recombinant vectors. The recombinant proteins are then expressed in large quantities in vitro and then purified. The choice of vectors and cell systems is influenced by different factors like: the ability to facilitate the production of large quantities of heterologous proteins in vitro; ease of manipulation; and the ability to express the conformational structure of the antigenic protein. Consequently, different prokaryotic and eukaryotic cell systems have been used for the production of subunit vaccines for fish diseases (Gudding et al., 2014).

 

 

3. Synthetic peptide vaccines

Peptide vaccines use short sequences of amino acids as antigens. Because of their size, they are usually not potent enough thus require coupling to a carrier molecule. Given that there are few synthetic peptide vaccines developed for use against fish diseases, these are not used in aquaculture (Mweemba, Mutoloki, & Øystein, 2014).

4.  Attenuated vaccines (Replicating vaccines)

 

Vaccines based on live attenuated organisms have been applied extensively in humans, such as the chicken pox vaccine. Attenuated vaccines are based on live organisms that have been selected for cross reactivity (a less virulent organism that elicits an immune response to the target organisms), genetically modified to attenuate the virus, and/or cultivated under conditions that disable viral virulence. As a result, the attenuated virus replicates in the target host albeit at a much lower rate compared to the wild type organisms and has no or reduced clinical signs (Mcloughlin & Christie, 2014).

Replicating vaccines are successfully used in human, veterinary, and aquatic animal medicine to prevent disease(Craig & Klesius, 2014). Attenuation strategies used to develop live vaccines for fish include laboratory passage, antigen mimicry, physical or chemical mutagenesis and genetic modification using molecular techniques. Laboratory studies have demonstrated the effectiveness of live vaccines in fish. Replicating vaccines induce mucosal, cellular and humoral immunity(Adams, 2016)

 

5.  DNA Vaccines

Deoxyribonucleic acid (DNA) vaccination is based on the administration of the gene encoding the vaccine antigen, rather than the antigen itself(Biering & Kira, 2014). Subsequent expression of the antigen by cells in the vaccinated hosts triggers the host immune system. DNA vaccines are usually in the form of a purified bacterial plasmid, a circular molecule of DNA able to replicate in bacterial hosts. The constructs will contain regulatory elements to ensure both replication of the plasmid in the bacterial host for production purposes, and expression of the vaccine genes in the vaccinated. In some cases, the nomenclature NAV (nucleic acid vaccine) or genetic vaccine is used, and those terms also include vaccines that are based on other nucleic acids than DNA (including RNA) (Thompson, 2017).

Among the many experimental DNA vaccines tested in various animal species as well as in humans, the vaccines against rhabdovirus diseases in fish have given some of the most promising results. A single intramuscular (IM) injection of microgram amounts of DNA induces rapid and long-lasting protection in farmed salmonids against economically important viruses such as infectious haematopoietic necrosis virus (IHNV) and viral haemorrhagic septicaemia virus (VHSV) (Gudding & Muiswinkel, 2013).

As DNA vaccination is a relatively new technology, various theoretical and long-term safety issues related to the environment and the consumer remain to be fully addressed, although inherently the risks should not be any greater than with the commercial fish vaccines that are currently used. Present classification systems lack clarity in distinguishing DNA-vaccinated animals from genetically modified organisms (GMOs), which could raise issues in terms of licensing and public acceptance of the technology. The potential benefits of DNA vaccines for farmed fish include improved animal welfare, reduced environmental impacts of aquaculture activities, increased food quality and quantity, and more sustainable production. Testing under commercial production conditions has recently been initiated in Canada and Denmark (Biering & Kira, 2014).

 

5.    Methods of Administering Vaccines in Fisheries

 

Successful vaccination depends upon both the development of protective vaccines and their correct use(Lillehaug, 2014). In addition to deciding which diseases to vaccinate against, the choice of vaccination method, the timing of vaccination, and the use of revaccination must be considered. For optimal protection, vaccination should be carried out some time before exposure to the actual pathogen, in order to give immunity sufficient time to develop. On the other hand, vaccination should not be carried out too early, as the degree of immunity declines with time. Water temperature may be an important factor when deciding when to vaccinate, as well as the size of fish, being the major parameter regulating the development of immuno-competence(Vallejos-Vidal et al., 2017).

Fish offer a unique group of animals for vaccination. They can be vaccinated orally, by immersion/dipping in a vaccine or they can be injected with the vaccine. Each of these offers advantages and disadvantages and each method are suitable for aquaculture under certain conditions (Newaj-Fyzul & Austin, 2017).

1. Oral Vaccination

Oral vaccination is logistically easy to apply because fish are not confined or handled, therefore eliminating stress. Disadvantages include variable protection, a large quantity of vaccine is required, and the possible need for encapsulation of the antigen. In oral vaccination, the antigen must be incorporated into the feed at manufacturing or top coated onto the pellets and/or encapsulated(Lillehaug, 2014). When incorporated into feed the heat to which the antigen is exposed is critical because the antigen may be denatured. However, oral vaccines have very short-term stability once mixed with the feed and in many cases protection is of short duration. Although oral vaccination would appear to be the optimum way to vaccinate fish, there are few such vaccines now available for fish. Oral vaccination may best be suited for secondary or booster vaccinations. The efficacy of orally delivered fish vaccines is hindered by the potential for proteins (antigens) to be denatured in the fish’s acidic stomach before the antigen can be absorbed by the intestine and gain access to immunologically competent tissue. The problem can be circumvented by encapsulating, or coating the antigen; however, this process is expensive (Vallejos-Vidal et al., 2017).

2. Immersion Vaccination

When applying immersion vaccination methods, the surfaces of the fish (gills, skin, alimentary tract) are exposed to a dilution of the vaccine. The antigen uptake is considered to take place via the gills, the skin and the lateral line, and possibly also via the intestine. The fish may be dipped for a short period (30 seconds to one minute) in a relatively concentrated vaccine solution, or may swim for a prolonged time period in an extensively diluted mixture. The vaccine solution may also be sprayed onto the fish. Immersion vaccination is particularly convenient for small fish, fry and fingerlings, which are impractical to handle for injection. Vaccine consumption per individual is low for small fish, but increases proportionally with fish size (i.e., with total biomass of fish) It often produces an acceptable level of protection and fish are less stressed than when injected. The skin and gills have specialized cells, such as antibody-secreting cells that are activated when exposed to a specific antigen (pathogen), that will respond by producing antibody when the fish is subsequently exposed to the pathogen. Also, macrophages absorb the vaccine and transport it to the antibody producing organs. The advantage of dipping is that a solution of vaccine can be used multiple times. When fish are immunized in a diluted vaccine, the fish are usually exposed for a longer period of time. Disadvantages include a need to handle fish, it is labor intensive, and specialized equipment is required (Dalmo, Bøgwald, & Tafalla, 2016; Lillehaug, 2014).

3. Injection Vaccination

Vaccination by injection is the delivery method generally resulting in best protection, and is the only choice for adjuvated products (Lillehaug, 2014). It is the most common and widely used method of vaccinating fish. Injecting a vaccine (intraperitoneally, intramuscularly, or occasionally subcutaneously) provides the highest level of protection and requires relatively small amounts of vaccine. They are economical for use with larger and highly valuable fish, and multivalent vaccine preparations and adjuvants can be conveniently included as needed. Vaccination by injection against multiple disease organisms or different serological strains of the same species simultaneously is economically superior to other procedures and also provides a higher degree of protection. Other advantages are protection of long duration, assure correct dosage, and incorporation of multiple antigens. To facilitate handling, ideally fish that are to be injected should weigh at least 10 g each (Harikrishnan et al., 2011).

Manual injection vaccination is used extensively using a spring-loaded or air-powered syringe, but this method is more labor intensive, requires each fish to be lightly anesthetized and handled; therefore, there is risk of operator injection. Because of the need to handle each fish, some mortality, particularly of weak fish, does ensue but it is normally only about 0.25%. Evolution of automatic vaccination machines via injection has improved the cost effective- ness of vaccinating salmonids, they do not require handling individual fish, and the danger of self-injection is eliminated. These machines require fish to be a more uniform size and generally larger than for hand injection. Other automated vaccination machine advantages include a vaccination rate efficiency of 7,000–9,000 fish per hour compared to about 2,500 per hour (depending on the technician) for hand vaccination. Injection is impractical for devastating diseases of juvenile fish in their first few months of life, and there is also a potential safety hazard to persons administering the vaccine (Lillehaug, 2014).

Fig. 1. Vaccination by intraperitoneal injection. Adapted from (Lillehaug, 2014).

Fig.2 The injection needle should be short, usually a length of 3–5mm is adequate to penetrate the abdominal wall, and have a diameter of 0.5–0.7mm (Lillehaug, 2014).

Fig. 3 Maskon machine for fully automatic high-speed injection vaccination of fish (Lillehaug, 2014).

2.  Efficacy Measurement Strategies for Fish Vaccines

 

The term “efficacy” means the disease therapeutic (pharmaceuticals) or preventative (immunologicals) value that a medicinal product offers and is by default a measure of its performance in real patients its clinical effect. Any fish vaccine must therefore be “immunogenic”, i.e. able to induce clinical immunity to virulent challenge with the infectious organism it is claimed to protect against. However, in animals as in humans, there is no per se requirement that vaccine protection raised against illness must be complete nor that vaccination must provide sterile immunity (inability of the challenge organism to infect the immunized individual). This is logical, given a variety of diseases that prove notoriously difficult to prevent against and also given the emergence of new infections. The documentation of vaccine efficacy thus normally includes scientific studies to demonstrate that the clinical benefits vs. adverse effect balance is better or equal compared to existing vaccines, and evidence that at least part of the immunity elicited by the vaccine formulation is antigen specific (Midtlyng, 2016).

A successful potency test is considered proof that the vaccine has been produced according to the licensed manufacturing outline and that the amount and quality of the antigen does not fall below the licensed specifications. For fish vaccines like for many classical animal vaccines, the establishment of in vitro or antibody-based methods for batch potency testing that satisfy the methodological quality doctrines of medicinal product manufacture has proven very challenging. But irrespective of in vivo or in vitro, any potency test should be able to reveal a vaccine batch of infe- rior quality, i.e. falling below the fi nal product specifi cation irrespective if expressed as antigen amount, the ability to induce of antibody responses, or as induction of clinical protection against virulent challenge(Midtlyng, 2016)

In vitro measurement of antibody

All standard methods of titrating serum antibody are valid in fish. There can often be a wide variation in response between individual fish; so a large number should be sampled, and the geometric mean titter would be more appropriate than the arithmetic mean. It must be borne in mind that in fish serological responses rarely correlate with protection(Midtlyng, 2016).

Relatit’e percent survival

The measure commonly adopted is the Relative Percent Survival (RPS); it is derived from observations of mortality in groups of vaccinated and control (unvaccinated) fish given the same challenge.

RPS=1-% mortality in vaccinated% mortality in control*100

What appears less known is that the same article contains a comprehensive discussion about a variety of further aspects of vaccine potency and efficacy testing methods in fish and proposes detailed recommendation of test setup, challenge conditions and outcome acceptance criteria for controlled trials:

• Duplicate setup (2 × 25 fish) of vaccinate and control group

•Exposure by bath challenge in two concentrations

• Maximum 10 % non-specific mortality and 20 % within-group variation after challenge

• Control mortality ≥60 %, vaccinate mortality ≤24 % are acceptance criteria for potency equate to a standardized RPS of 60 % or above (Midtlyng, 2016).

 

3. The Economics of Fish Vaccine Production

The implementation of a vaccination strategy should aim at achieving an economically favorable solution for the fish farming industry. Disease and disease outbreaks caused by specific infectious agents generate expenses and economic losses, including reduced production due to direct losses of fish, and increased expenses for medication and extra work. In addition to the vaccine itself, vaccination costs include labor costs of the actual vaccination method, as well as losses due to side-effects. The levels of protection achieved by the respective methods should be taken into account, by including RPS into the formula (Lillehaug, 2014).

The expected savings are the product of:

1. The value of a fish (i.e. the gross profit on it at harvest)

2. The expected reduction in fish mortality= (expected mortality in unvaccinated) x RPS

A saving which was not taken into account is in the cost of disposal of dead fish. Such fish are infected irrespective of whether they have been vaccinated; they must be disposed of in a way that does not pose a hazard to survivors(Brian Austin & Austin, 2012).

 

 

 

 

4. Quarantine and Restriction of Movement in Aquaculture

Quarantine, in the strict sense, is the confinement of aquatic animals of unknown or questionable health status in secure facilities such that neither they nor any pathogens they may be carrying can escape into the external environment(Hadfield & Leigh, 2011). During the period of quarantine, the animals are observed, tested, and treatment may be applied, and a decision will be made as to whether or not they should be released to the external environment. While the concept of quarantine for aquatic animals has existed for many years, within the current framework of “national biosecurity”, quarantine is seen as one of a number of risk mitigation options that governments can apply to reduce the likelihood of serious pathogens being introduced with the importation of live aquatic animals and their products (Faisal et al., 2017).

The duration of quarantine can be anywhere from two weeks to as long as ninety days depending upon the pathogens of concern and their rate of detection, the purpose for the animals, and the established biosecurity practices at the facility. During quarantine fish should be monitored for signs of sickness (e.g., anorexia, lethargy, respiratory rate changes, equilibrium disturbances, skin lesions, changes in skin color or body confirmation, and behavioral abnormalities). When seen, sick animals should be isolated in separate tanks until diagnostic evaluation by veterinary services or fish health professional. During this time the remainder of the population should be monitored for the development of similar or different clinical signs. Treatment of fish during quarantine should be limited, unless the animals will be eventually introduced into a captive population. All treatment should be based upon appropriate diagnosis and selection of efficacious agents used for the appropriate period of time. Prophylactic treatments can inhibit the development of clinical signs and inappropriate use of antibiotics will lead to the development of antibacterial resistance (Sánchez, Tanguay, Sanders, & Jan, 2012).

 

5. The Use of Chemoprophylaxis and Antibiotics in Control of Infectious disease of fish

Antibiotics have been used extensively in aquaculture in the past but the present consensus is that this should be kept at a minimum. Excessive use of antibiotics is known to produce resistant bacteria, which are considered a major threat to animal (and human) health. With the development of immunoprohylactic control measures, the use of antibiotics in aquaculture has been greatly reduced in the past 20 years(Magnadottir, 2010). Antibiotics, many of which are important in human medicine, appear side by side with compounds used almost exclusively in fisheries. In many instances, the introduction of a compound into fisheries use has followed closely after the initial use in human medicine (Plumb & Larry, 2011).

Drugs used for food producing animals need approval prior to use due to the risk of residues and increased risk of resistance(Bowker & Trushensk, 2016). Some of the drugs approved in aquaculture including their administration strategies and dosage rates are listed below

1.    Tetracyclines

These are broad spectrum bacteriostatic drugs, two of the natural tetracyclines, oxytetracycline (OTC) and chlortetracycline, have been used in aquaculture; OTC in particular has been widely used because in most markets it is not only available but also cheaper than other broad-spectrum antibacterial drugs. OTC has been used as a first choice drug for nearly all bacterial diseases of fish (Rico et al., 2012).

Injection

Because injection is a labor intensive method of administering drugs it would normally be done only once with antibacterial agents. It is essential for fish which are not feeding and are too large for immersion treatment. Its value is in the rapid achievement of therapeutic plasma levels. The dose rate normally recommended is 20 mg/kg which is almost certainly more than necessary (Bowker & Trushensk, 2016).

Infeed medication

The normally recommended dose rate is 75 mg/kg/day; this may be appropriate for marine fish in which case it represents an overdose for freshwater fish due to the effects considered The usual regimen is daily dosage at this rate for 10 days (Bowker & Trushensk, 2016).

Immersion

Concentrations of OTC ranging from 5 to 120 mg/l have been recommended, with the appropriate concentration varying with the hardness of the water. This very wide range illustrates the absence of critical dose titration studies of this widely used drug. In fact, although OTC could be used to prevent water-borne transmission of bacterial disease it would not be therapeutic (Bowker & Trushensk, 2016).

2.    Penicillins

Most penicillins are sensitive to acids but a few are stable and therefore active by mouth. Penicillins are sensitive to hydrolysis by bacterial beta-lactamase enzymes but some can be potentiated by beta-lactamase inhibitors, notably clavulanic acid. Ampicillin and amoxycillin, are broader from the group and they are widely used in aquaculture.

In feed administration

Amoxycillin dose rates in the range 40-80 mg/kg/day have been recommended. For furunculosis the higher end of the range for 10 days is usually necessary if relapses are to be avoided. This is especially the case if twice daily feeding of half the daily dose is practised, because penicillins are rapidly excreted and low dose rates will not produce therapeutic blood levels in salmonids. For Pasteurella piscicida and streptococcal infections in yellowtail a lower dose regimen of 40 mg/kg/day for 5 days has been found satisfactory; ampicillin is usually used.

Injection

 

The recommended dose rate is 10 mg/kg/day. This is rarely used in food species however because of the labor costs involved. The route may be used in suitably large and valuable ornamental fish or brood fish of food species.

Immersion

The bacteria in aquarium filters are very sensitive to semi-synthetic penicillins so where the drugs are used it should always be by dipping with the solution being discarded afterwards. However very little drug is absorbed through the gills and this route of administration is not recommended for therapy of systemic infections.

3.    Macrolides

Macrolides are antibiotics having a large ring in the molecular structure. Microbiologically macrolides are more active in slightly alkaline conditions, and erythromycin is most active at close to the physiological pH of salmonids Macrolides are medium-spectrum antibiotics active mainly against Gram-positive bacteria, Chlamydia and Rickettsia. Because a majority of fish bacterial pathogens are Gram negative the indications for macrolides are limited and specific

4.    Sulfonamides

The sulfonamides have a broad spectrum of activity; and since they were the first modem antibacterial drugs to be developed members of the group have been used for virtually all bacterial diseases of fish. However, the doses required often leave little margin below toxicity and many bacteria develop resistance fairly easily. In consequence the group has little use now except in synergistic combination with pyrimidine potentiators.

5.    Disinfectants and Pesticides

Disinfectants are physical or chemical agents that are used to destroy microorganisms, usually on inanimate objects including hard surfaces and equipment. In aquaculture, disinfectants can also include compounds used to destroy microorganisms living on the surface of fish eggs. These agents are used in aquatic animal rearing facilities as part of biosecurity protocols  to control the spread of aquatic animal pathogens or nuisance/invasive species (Bowker & Trushensk, 2016).

In the case of compounds applied to eggs, disinfectants can be used as part of a comprehensive fish health management plan. Disinfectants are related to, but different from sanitizers, antiseptics, biocides, and sterilizers: biocides and sterilizers are agents that kill all forms of life, not just microbes; antiseptics refer to antimicrobial agents that are used to destroy microbes on living tissues; and sanitizers are compounds that clean and disinfect at the same time(Rico et al., 2012).

It is important to recognize that not all disinfectants are effective or appropriate in all circumstances. For example, iodine is appropriate for disinfecting eggs, but it is quickly neutralized by biological material and exposure to light and can stain clothing and equipment. As a result, iodine is not a good disinfectant for foot baths or net dips. Conversely, chlorine is particularly good for sanitizing nets, siphons, and other equipment, but is highly toxic to aquatic organisms unless neutralized. Disinfection can be optimized by selecting the appropriate agent for each scenario, and by following these general recommendations; Remove dirt, vegetation, or other debris before disinfecting—disinfection is unlikely to be effective unless the equipment and/or surfaces are clean and free of such material (Bowker & Trushensk, 2016).

6. Biological Control of Fish Disease

Biological control of fish disease is mainly dependent on the use of probiotics.  The concept of probiotics was introduced by Lilley and Stillwell to describe ‘substances secreted by a microorganism that stimulate the growth of another organism’, and thus ‘probiotic’, which is derived from the Greek meaning ‘for life’, is the opposite of antibiotic. Since then the description of probiotics has evolved and it is commonly used to indicate bacteria associated with beneficial effects on humans and animals (B Austin, 2014; Sommerville, 2012).

According to FAO, probiotics can be defined as live micro-organisms, which administered in adequate amounts confer a health benefit on the host. After the administration of these useful microbes into the host, they are able to colonize and multiply in the gut of the host and show numerous beneficial effects by modulating various biological systems in the host. The application of probiotics has gained special interest because of their help in promoting the indigenous microbe(s) in the intestines, and thus helps to restore the microbial balance. Probiotics can be implemented at larval and early fry stages, where vaccines cannot be administered (Mohapatra, Chakraborty, Kumar, Deboeck, & Mohanta, 2012).

1.    Mechanism of Action of Probiotics

 

One of the most important properties of probiotics is to adhere and proliferate at the specific location for the maximum usefulness to the host species. Therefore, to have maximum benefit, the probiotics should reach the specified location where it is most required.

1.  Production of inhibitory compounds

 

Probiotics play a major role in preventing the occurrence of diseases by producing certain inhibitory compounds that act antagonistically against the pathogenic microbes and hence, preventing their proliferation in the host bodies (Sharifuzzaman & Austin, 2017). The anti-pathogenic activity may be due to singular or combination of production of antibiotics, bacteriocins, siderophores, lysozymes, prote ases, hydrogen peroxide and the alteration of pH values (Mohapatra et al., 2012).

2.  Competitions for adhesion sites

 

Competition for space for adhesion and colonization on the gut and other tissue surface is another mode of action of the probiotics to fight against harmful pathogens as proper adhesion to the enteric mucus and intestinal wall surface is most important for any pathogen to cause damage to the host animal. Adhesion of the probiotics is either non-specific, based on the physicochemical factors or specific, based on the adhesion of the probiotics on the surface of the adherent bacteria and receptor molecules on the epithelial cells. Different strategies have been put forth regarding the attachment of micro-organisms to the intestinal tract, such as, passive forces, electrostatic interactions, hydrophobic, steric forces, lipoteichoic acids, adhesins and specific structures of adhesion. It has been reported that the intestinal isolates compete more effectively with Vibrio anguillarum for adhesion sites on the mucosal intestinal surface suggests that intact pilus fibers with mucus-binding capacity on the cell surface of a probiotic strain of lactic acid bacteria are helpful for competing with Escherichia coli in the human intestine(Mohapatra et al., 2012; Sharifuzzaman & Austin, 2017).

3. Modulation of Host Immune Responses

Probiotics render protection against pathogens by overcoming the adverse consequences of antibiotics and chemotherapeutic agents. Probiotics help in achieving natural resistance and high survivability of larvae and post larvae of fishes, due to the elevated immunity of the fish. Different modes of probiotics have a different stimulatory effect on the immune system of fish, namely, effect on the immune cell, anti-bodies, acid phosphatase, lysozyme and anti- microbial peptides.

4. Competition for chemicals or available energy

The basis for the existence of any microbial population depends on its ability to compete for chemicals and available energy with the other microbes residing the same ecosystem. Heterotrophs, which dominate the aquatic environment, compete for organic substrates, such as carbon and other energy sources. inoculated a bacterial strain, which had a capacity to grow actively in an organic-poor substrate, into a diatom culture and reported that it prevented the establishment of a pathogenic strain of V. alginolyticus. As the inoculated strain lacked any in vitro inhibitory effect on V. alginolyticus, so this prevention might be due to the bacterial strains ability to utilize the exudates of the diatoms and fight against the pathogen (Oliva, 2012).

5. Competition for nutrients

Probiotics make up a part of the resident micro-flora by adhering to the mucus, gastrointestinal tract, epithelial cells and other tissues, further contributing to the health or well-being of the host. The attachment ability of some bacteria has been tested in vitro and in vivo and their results suggest that the pathogen gets dis- placed by the potential probiotic based on the com- petition for essential nutrients, space, etc. Probiotics utilize the nutrients otherwise consumed by pathogenic microbes. The useful microbiota sometimes serves as a supplementary source of food and microbial activity in the digestive tract and also is a source of vita- mins or essential amino acids. It has been seen that Bacteroides and Clostridium species contribute to the host’s nutrition, especially by supplying fatty acids and vitamins(Mohapatra et al., 2012).

7. The Use of Immunostimulants

An immunostimulant (IS) is a natural or chemical substance that stimulates the immune system by specific (vaccines or antigens) or non-specific (irrespective of antigenic specificity) routes. In aquaculture, non-specific immunostimulants have been widely used (Awad & Awaad, 2017), probably due to the limited knowledge of the immune response in fish and the ease of their application. Although fish are indeed the most diverse group of vertebrates, there are well conserved mechanisms across the immune system. These systems, often innate immunity, are the most commonly used to measure the response of the fish to the dietary supplement in question (Vallejos-Vidal et al., 2017).

The ability to boost the natural defense mechanisms of cultured fish has major benefits and is the subject of a relatively new and highly active current research area. The main search has been for substances which can be incorporated in feed and delivered orally to fish but others may be injected along with vaccines. Many of the early reports of commercial benefits were not supported by investigations of the mechanism of action and evidence for involvement of the immune system could not be confirmed (Sommerville, 2012).

One of the most used substances in immunostimulation experiments in fish are various forms of β-glucans from different sources, normally introduced in the feed but also by intraperitoneal injection or as vaccine adjuvant 1,3 glucan from the cell wall of Saccharomyces cerevisiae (yeast) to yellow croaker (Pseudosciaena crocea) and demonstrated significant increase in lysozyme, phagocytic and respiratory burst activity and reduced mortality during Vibrio harveyi infection. Other types of promising immunostimulants in aquaculture include synthetic double stranded RNA, referred to as Poly I:C, which is thought to stimulate anti-viral defences through binding to TLR-3 analogue in fish (Magnadottir, 2010).

The addition of various food additives like vitamins, carotenoids and herbal remedies to the fish feed have been tested in aquaculture. Overall the effects have been beneficial, for example, reducing stress response, increasing the activity of innate parameters and improving disease resistance (Awad & Awaad, 2017; Magnadottir, 2010; Miriam, Tapissier, Pierre, & Saulnier, 2017; Oliva, 2012; Vallejos-Vidal et al., 2017).

 

8. Surveillance for Diseases of Fish

The purposes of surveillance in aquatic animals are in principle the same as for terrestrial animals (Peeler & Taylor, 2011). However, special challenges for surveillance planning do occur due to the fact that the animals are kept in water, are kept in often complex rearing system (hatchery, freshwater or marine site), the size of the fish population on farm, and accessibility for inspecting and sampling animals. Furthermore, some basic information relevant to planning, such as expected prevalence in infected populations and diagnostic test performance, is often limited available (Plumb & Larry, 2011).

Surveillance activity is important in reducing the risk of the international spread of aquatic animal pathogens as well as control of important endemic diseases (Oidtmann et al., 2013). These issues have continued to increase in importance since the formation of the World Trade Organization and the subsequent implementation of various multilateral agreements on trade. Disease surveillance should be an integral and key component of all government aquatic animal health services. It is important for risk analysis, early detection of diseases, planning and monitoring of disease control programs, provision of sound aquatic animal health advice to farmers, certification of exports, international reporting and verification of freedom from diseases, and assurance of pathogen status (e.g., Specific Pathogen-Free stocks). It is particularly important for aquatic animal disease emergency preparedness (Hadfield & Leigh, 2011).

1.    Passive surveillance

 

The main method of collecting information on aquatic animal diseases currently used in most countries is through a passive disease reporting system. When disease is noticed in the aquatic animals, the producer may contact the authorities, who may then either submit a disease report, or send a specimen to a diagnostic laboratory. These reports and/or the results of examination of the specimens provide information on what diseases are present in the country. Information is not collected about all diseases, or all cases of disease. Many countries have compulsory disease notification regulations to encourage the reporting of priority diseases, but other diseases are not reported. This system is called a passive reporting system because the main users of the information (the aquaculture or fisheries service) take no action to initiate the collection of the information. The producer initiates the report, and the central authorities wait (passively) for the report to arrive. Passive surveillance also includes the use of information that was collected for a different purpose, such as disease diagnosis (Bruno, 2011).

2.    Active surveillance

Active surveillance differs from a passive reporting system in that it uses surveys of a relatively small, representative sample of the population to gather specific information about that population. The key advantages of active surveillance are that the quality of information collected is usually better, the information reflects the true situation in the entire population, and it is often faster and cheaper to collect than with passive methods. Despite their problems, passive reporting systems are an important source of disease information. Passive disease-reporting systems in one form or other are in place in virtually all countries, but relatively few countries make regular use of active surveillance, in spite of its advantages. This is partly due to the fact that appropriate techniques have not previously been available, and aquaculture and fisheries staff may not have been trained in the skills necessary. This book describes how to implement appropriate, active surveillance techniques and provides the skills needed to do so (Bruno, 2011; Landman & Ling, 2011).

9. Diagnostic Tests for Infectious Disease of Fisheries and Their Reliability

 

Fish are susceptible to diverse pathogenic microorganisms, including bacteria, viruses, parasites and fungi, some of which pose a major threat to the aquaculture industry. Unlike terrestrial animals, aquatic animals live in complex and dynamic environments and they are not readily visible. This makes control of fish and shellfish disease problematic so monitoring of the health status of aquatic animals is vital. Rapid and accurate diagnostic methods are useful, as early detection and diagnosis are critical for the management and control of infectious disease, especially in aquatic animals. Many diagnostic techniques have been developed, some of which are widely used for detection of pathogens. The diagnostic approaches used in aquaculture comprise conventional microbiological, immuno serological and molecular methods (Kim, Nguyen, & Kim, 2017).

Development of immunodiagnostic methods has revolutionized aquaculture diagnostics as they are more sensitive and specific than traditional approaches, and can be used at the farm level without the aid of instruments. Also, these methods can detect non culturable micro-organisms. However, antibody selection is critical for acceptable results. Many monoclonal or polyclonal antibodies against fish pathogens are now available commercially, which are different in sensitivity and specificity. However, insufficient availability of antibodies against fish and shellfish pathogens still hampers fish immunodiagnostics. Western blotting and immunoblotting techniques are not used routinely as diagnostic methods, but can be useful for confirmation of certain pathogens, such as viral pathogens (e.g.white spot syndrome virus and yellow head virus in shrimp), that cannot be cultured in cell lines. (Kim et al., 2017; Weidmann, 2017).

 

Lethal sampling is commonly used for routine diagnostics in aquatic animals and diagnostic tests are usually based on the direct detection of the pathogen in a tissue sample rather than non-lethal methods to test for presence of antibodies against a specific pathogen (an appropriate diagnostic target in clinically normal animals). For fish with high individual economic values, non-lethal sampling may be performed. Reasons for the different developments in diagnostic testing are mainly based on the value of the individual animal, which is usually low for aquatic animals, but may be substantial in terrestrial animals, making lethal sampling inappropriate. The diagnostic test sensitivity of many diagnostic tests for the notifiable aquatic animal diseases is unknown. The tests are suitable to diagnose clinical outbreaks (where the pathogen load in samples tissues is high); however, the diagnostic test sensitivity for screening of clinically little or unaffected populations is likely to be low (Harikrishnan et al., 2011).

Sensitivity of screening tests for pathogens can be further reduced by pooling samples. The OIE manual of diagnostic tests for aquatic animals generally permits pooling of samples for diagnosis of many listed diseases (e.g. ten fish can be pooled for viral hemorrhagic septicemia testing). Pooling allows the number of fish sampled in a population to increase for the same cost as individual sampling. However, pooling negative fish with positive ones may dilute the concentration of a pathogen below the minimum detection level of the diagnostic test, negating the potential benefits of testing more fish. To determine whether pooling is worthwhile, the minimum detection limit of the diagnostic test, the average and range of con- centration of the agent in the tissue sampled is needed but generally not known (Oidtmann et al., 2013).

 

2.10. Challenges in Prevention and Control of Fish Disease

 

The nature of the aquatic environment, and of the aquatic animals themselves, poses some challenges for trying to understand and respond to disease problems. The key implications of the differences from terrestrial animal disease prevention and control are:

• They are more difficult to catch.

• They are more difficult to see.

• Disease agents spread quickly and easily.

• They often gather or are cultured in large numbers.

• They are difficult to isolate.

• Disease is often difficult to detect and characterise.

To understand disease, we must be able to identify disease in an individual animal, and to examine the patterns of disease in populations or groups of animals

 

That fact that aquatic animals live in water, and that there are often large numbers in relatively small areas means that they have a much closer connection to their environment than terrestrial animals. In fact, it is really not possible to consider either the animals or the environment in isolation. Anything that affects one will inevitably affect the other. The interactions that take place within the aquatic environment are often much more complex than on land. For instance, in a fish pond, there are not just fish and water. There is an entire ecological system made up of millions of algae, protozoa, and bacteria, all in a dynamic balance. These microorganisms may provide the fish with food, and contribute to maintaining an appropriate environment, for instance by buffering the pH of the water. However, they may also be parasites or otherwise threaten the health of the fish (Crumlish, 2017).

To maintain the health of the fish, and achieve good production, it is not enough to understand about the fish, or to understand about the microorganisms, nutrients, minerals, toxins and other factors present in the water. We must understand how these different elements interact, and the effect they all have on each other. Because of this, it is often misleading to think about ‘aquatic animal health’ because it places the focus on the individual fish. Instead, it is more realistic to think about ‘aquatic system health’ that is, the state of health of the entire system. If any aspect of a pond’s ecosystem is ‘diseased’, for instance a crash in the algal bloom, it will inevitably result in disease in other organisms that share that environment, including the fish that are being cultured (Kim et al., 2017).

Traditionally, when trying to diagnose the cause of a disease, we examine diseased animals. The individual animal is the unit of interest, the thing that we study to make our conclusions. When we think in terms of aquatic system health, we are no longer trying to diagnose the cause of disease in an individual animal, but the cause of disease in an aquatic system. The unit of interest is no longer a sick fish, but a ‘sick’ pond. The tests we use on individual animals, such as skin scrapings or histology, must be combined with tests that we use on the pond, such as measuring pH, soil bottom conditions and turbidity. In many situations, being able to step back from the individual and consider the system is essential to understanding disease in aquatic animals (Newaj-Fyzul & Austin, 2017).

 

 

 

 

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