Improvements in Host Resistance - biological methods of disease prevention and control in Aquaculture

Improvements in Host Resistance

Dietary Enhancement

The resistance of fish to diseases involves a complex array of mechanisms that include maintenance of epithelial integrity and mucus coat, non-specific cellular factors such as phagocytosis by leukocytes, non-specific humoral factors such as lysozyme, complement, and transferrin, and specific humoral and cellular immunity. A variety of nutritional components can influence the inci-dence and severity of a number of infectious diseases. Some micronutrients that are known to enhance disease resistance include vitamins C, B6, E, and A, and the minerals iron and fluoride. The role of the macronutrients (protein, lipid, and carbohydrate) in disease resistance has not yet been clearly defined. There are evidences that certain fatty acids may have essential roles in disease resistance.

Artificial diets that produce the best growth performance may not necessarily produce the optimal immune status. In marginal deficiencies, the fish continue to grow, appear healthy, and show no gross or histopathological signs of dis-ease, yet significant depression of disease resistance is present and disease out-breaks become evident only when the fish are subjected to the slightest stress.

Increased levels of certain macro and micronutrients may be beneficial before and during the exposure to certain disease agents, but may be detrimental in other instances. Nutritional enhancement of disease resistance is a new field of research yet despite the need for more studies; the potential for dietary en-hancement of disease resistance certainly exists.

Improving Genetic Resistance to Disease

Disease resistance can also be achieved by genetic improvement of cultured stocks. Research to improve genetic resistance to disease in farm animals has been in progress for some years now. Loses due to diseases in farm animal production are estimated at 10 to 20% of total production values. In aquacul-ture this figure could reach 100% during disease outbreaks. Thus breeding pro-grams that improve disease resistance may significantly increase production values.

Selective breeding for disease resistance in fish and shellfish was given empha-sis only recently. Most of the work done in fish were at first focused on im-proved growth rates, control of maturation, maintenance of genetic vigor and other characteristics like color (e.g. Red Tilapia), later studies then included selection for disease resistance. There are three main strategies that can be used for the improvement of disease resistance in farm and aquatic animals, namely: conventional selective breeding programs based on morphological traits; marker-assisted selection utilizing associated DNA polymorphism; and transgenic approaches.

Genetic selection for pathogen resistance in cultured shrimp species is given more attention by shrimp farmers because shrimp, unlike fish, cannot be effi-ciently vaccinated due to their lack of a lymphoid system that produces anti-bodies to the antigens of disease agents. The international shrimp breeding program has come up with a list of quantifiable immune traits for individual selection, namely: hemograms (or blood profile), hemocyte respiratory burst (related to killing ability of hemocytes), plasma antibacterial activity, levels of plasma coagulogen, and immune index (total of these quantifiable immune traits). These indexes can easily be determined by getting a sample of blood from the shrimps. Individuals scoring high in many or all these quantifiable traits can then be selected as the breeders in a breeding program.

Vaccination

Cells with the same morphology as the lymphocytes in warm-blooded animals are also present in the spleen, thymus, kidney, and blood of fishes. It is also possible to distinguish between B and T cells in fish as they are identified in higher vertebrates. Vaccination in fish is therefore possible and specific vac-cines can be developed along the same principles as for warm-blooded ani-mals. It appears that the thymus organ in young fish and the head kidney are the primary organs of lymphocyte differentiation in fish, whereas the spleen is a secondary lymphoid organ that harbors both B and T cells. The hemocytes of crustaceans do not include any cell types with properties comparable to B and T cells. Although “vaccines” for shrimps in aquaculture are being developed and marketed, such products do not fall within the current definition of a vac-cine. If they reduce disease, it may be the result of a non-specific stimulation of the hemocytes by the cell wall fragments from the bacteria used in the “vac-cine,” or by the adjuvants it contains and not due to a specific antibody devel-opment against the disease,

Reports on the capability of fish to produce antibodies against bacterial patho-gens first came out in 1935. Then in 1942, it was demonstrated that this anti- body response in fish translated into a protective immune response. What fol-lowed, however, was a long period of disinterest in vaccines due to the fascina-tion of the new antimicrobial compounds (antibiotics) that came on the market immediately after the Second World War. It was only in the mid to late 1970s that attention was again given to vaccination as a means of preventing and controlling fish disease and the development of commercially available vac-cines. The reasons for these turn of events were varied: the high cost of using chemotherapy, the short-term nature of the protection obtained with antibiot-ics, the increasing incidence of antibiotic resistant fish pathogens, and environ-mental concerns on the use of antibiotics.

1. Vaccine Development

Bacterial Vaccines.A number of vaccines are currently being developedagainst bacterial fish pathogens. Most of these works are focused on salmo-nid pathogens and a few are being directed against bacterial pathogens in carp and catfish. Japanese researchers are focusing their work on their cul-tured fish especially the yellowtail for the development of vaccines against streptococcal and Pasteurella infections. To date, there are still no commer-cially available vaccines for bacterial diseases in warm water fish.

Viral Vaccines.For some years, the only commercially available vaccineagainst a fish virus was the spring viraemia of carp (SVC), caused by Rhab-dovirus carpio. It was administered by injection since the disease affectscarp at a size and age (9-12 months) when they are easy to handle. How-ever, the same is not applicable for the other important fish viruses such as infectious pancreatic necrosis virus (IPN), viral hemorrhagic septicemia (VHS) virus, infectious hematopoetic necrosis (IHN) virus, and channel cat-fish (CC) virus. These cause severe mortalities in fish during the fry stage in which injection is not practical. Immersion in a suspension of inactivated virus has given unsatisfactory results. Another approach had been tried us-ing live attenuated virus or avirulent forms of the virus. Although reason-able protection has been achieved using this approach, it has been aban-doned due to concerns on residual virulence in target species, virulence in non-target species, and persistence in the treated fish leading to the fear that the virus might back-mutate to virulence.

Parasite Vaccines.Parasitologists have only recently exploited the immunesystem to protect fish against parasitic disease. Vaccination techniques are being developed against parasitic protozoans by intraperitoneal injection of live attenuated parasites. Some evidence shows the passive transfer of pro-tective immunity against these parasites from immune to naïve fish, and to egg. Studies are also in progress in the development of vaccines against helminthic and copepod parasites.

2. Types of Vaccines

Inactivated.The pathogenic organism is culturedin vitrousually in broththen killed using heat or formalin.

Live Attenuated.When the approach of using inactivated pathogens failsto elicit an immune reaction then live attenuated pathogens are used. Re-moving some genes from the pathogen, which is usually, a virus, making it non-virulent develops attenuated strains. There is always the danger of back-mutation to the virulent wild type.

Recombinant.The antigens in pathogenic organisms are just minor por-tions of their structures, such as cell wall proteins or part of the protein coat of viruses. The genes that code for these antigenic structures can be isolated and inserted into yeast or bacterial DNA (e.g. E. coli) where they become incorporated and expressed in large amount. The products, usually pro-teins, are then harvested from the broth of cultured recombinant yeast or bacteria and used as vaccines.

DNA.The most recent approach to vaccine design is by genetic immuniza-tion or the injection of naked DNA of a pathogen into the muscle of the host. The DNA, which usually encodes a single gene of the pathogen, is expressed extrachromosomaly in the muscle cells. The newly synthesized antigen can then stimulate the immune defense of the host conferring the host with life long immunity.

3. Modes of Vaccination Delivery

The route vaccine administration depend largely on the species of fish, the size, the husbandry, the disease, the stage of the life cycle of the fish.

Direct Immersion. Vaccination by immersion is commonly practiced withvery small fish where it is a convenient and highly cost-effective method of vaccine administration. Antigen uptake takes place mostly through the gills, although some may be taken up through the skin, and the lateral line and some are swallowed.

Spray Administration. Spraying a solution of a vaccine is a variation of theimmersion method suitable for larger fish than those given immersion treat-ment. Fish are run through a conveyer belt under two or more jets contain-ing the vaccine at 1:10 dilution for not less than 10 seconds. Antigen, like the immersion technique, is taken up through the gills.

Peroral Administration.The oral route of administration of vaccine as feedadditive should have many clear advantages like the absence of handling stress, no scars on the fish to lower its value, the freedom to choose vaccina-tion dates, no safety risk to the operator, and no risk of spreading infection through needles. However, to date, there are no cost-effective vaccines that can be administered orally.

Injection.This is the route of administration commonly used throughoutthe salmon industry. A single injection can provide a high degree of protec-tion through the whole length of the culture period or production cycle. The added advantage of this method is that the process allows fish to be graded, counted, and monitored for abnormalities and signs of disease.

Immunostimulation

Immunostimulants are chemical compounds that activate the immune system of animals and increase their resistance to infectious diseases. It has been known for many years that cell wall fragments when introduced into animals will render them more resistant to pathogenic diseases. The ability of the im- mune system to respond to microbial surface components is the result of an evolutionary process whereby animals have developed mechanisms to detect common and highly conserved chemical components of pathogenic organisms and to use these chemical components as “alarm signals” to switch on the defense mechanisms against infection. The immune system will therefore re-spond to an immunostimulant as if challenged by a pathogenic organism.

Immunostimulants offer many advantages when used in fish farming: 1) they may be used alone, inducing elevated activities of the non-specific defense mechanisms; 2) they promote a more effective immune response to pathogens; 3) they enhance the level and duration of the specific immune response, both cell-mediated and humoral, following vaccination; 4) they overcome the im-munosuppressive effects of stress and of those pathogens that damage or inter-fere with the cells of the immune system.

Immunostimulants may be used prior to situations known to result in stress (handling and transfer, crowding, poor water quality, etc.) or during develop-ment stages when the animals are more susceptible to infectious diseases (lar-val phases, maturation and spawning, etc.). Larval or very young fish and crus-taceans at all stages of their life cycle do not posses the specific (or adaptive) immune system and largely rely on nonspecific cellular defense functions to resist infections. The use of immunostimulants could improve growth and sur-vival of juvenile fish and crustaceans. Another field where immunostimulants might be of use is in the application of antibiotics to combat infectious dis-eases. Most antibiotics have been proven to be immunosuppressants. A com-bined administration of immunostimulants and antibiotics may counteract this suppressive effect.

Substances with Immunostimulatory Effect

1.  Bacterial products - these are usually cell wall components of bacteria (e.g. glycoproteins and lipopolysaccharides)

2.  Products from mycelial fungi - the immunostimulants derived from mycelial fungi are all glucose polymers (e.g. lentinan, schizophyllan and scleroglucan)

3.  Yeast cell wall products - these are structural components of yeast cell wall (e.g. zymosan, ß-1, 3-glucans)

4.  Soluble and particle bound ß-glucans - these are animated ß-1, 3-glucans or soluble ß-1, 3-glucans bound to microbeads

5. Glycans - polysaccharides also containing sugars other than glucose

6. Chitosan – extracted from the exoskeleton of shrimps and other crustaceans

7.  Peptides from animal extracts - examples of these are peptones and protein concentrates from fish, peptides extracted from the thymus of animals, the compound EF-203 from chicken eggs

8.  Unspecified extracts - examples under this category are extracts from a tuni-cate, a peptidoglucan extract from Bifidobacterium thermophilum, extracts from the seeds of “malunggay” (Moringa oleifera)

9.  Synthetic compounds - these are usually dipeptides or lipopeptides ex-tracted from microorganisms as well as other chemical compounds that are incidentally found to have immunostimulating properties (e.g. the antihelminthic drug levamisole)

10.      Cytokines - these are the molecules involved in the transmission of signals between leukocytes, such as interleukins, interferons, tumor necrosis fac-tors, colony-stimulating factor and monocyte chemotactic factor and have dominated research in immunotherapy in humans