Immunostimulants are chemical, drug, stressor or actions that enhance the innate or non-specific immune response in animals by activating immune cells. They include chemical agents, bacterial preparations, polysaccharides, animal or plant extracts, and cytokines. Common immunostimulants used in aquaculture include glucan, levamisole, chitin, and chitosan. Glucan is derived from yeast cell walls and plants and has excellent immuno-stimulatory properties. Levamisole is a synthetic anthelmintic that can also enhance the non-specific immune response in fish. Chitin and chitosan are derived from shrimp and crab shells and have been shown to increase disease resistance

1. Immunostimulants

2. What are immunostimulants?
An immunostimulant is defined as a chemical, drug, stressor or action
that enhances the innate or non-specific immune response by interacting
directly with cells of the system activating them.
Immunostimulants can be grouped under chemical agents, bacterial
preparations, polysaccharides, animal or plant extracts, nutritional factors
and cytokines.
Immunostimulants are dietary additives that enhance the innate (non-
specific) defense mechanisms and increase resistance to specific
pathogens.
biological and synthetic compounds that enhance the cellular
Immunostimulants and immuno-modulators comprise a group of
and
humoral defense mechanism in animals

4. Types of immunostimulants
Natural Synthetic
Microbial derivates: B-glucans, LPS,
bacterins.
Natural factors: Vit-C and Vit-E
Animal and plant extracts
Polysaccharides (Chitin, Chitosan,
Lentinan)
Hormones (GH, Thyroid hormone,
prolactin), Cytokines and others (ds
RNA, recombinant proteins)
•Macrogard
•Immersion grade
•Aquasalor
•Ergason (rich in
polysaccharides)
•Lomal

5. Most commonly used immunostimulants in aquaculture:
Glucan:
Most popular immunostimulants.
Derived from yeast cell wall and from certain higher plants.
It has excellent immuno-stimulatory properties and
work well when injected or fed to the fish.
Most common brands are MacroGard, Vetregard and EcoActiva.
ß-1,6, branched ß-1,3 Glucans were effective in stimulating the non-
specific immune response in carp (Yano et al., 1991).
Studies of glucan-activated macrophages in trout revealed an increased
ability to kill salmonid pathogen Aeromonas salmonicida (Jorgensen et
al., 1993).

6. Levamisole:
A synthetic Imidazothiazole, extensively used in both humans and
veterinary medicine as an anti-helminthic agent .
Levamisole is an effective treatment for Camallanus roundworm
infestations in freshwater tropical fish.
In fish, levamisole has been used in a few studies with the aim of
enhancing the non-specific immune response (Baba et al. 1993) or as
an adjuvant with a vaccine (Jeney and Anderson 1993).

7. Chitin:
Linear Beta-1,4-linked polymer of N-acetyl-D-glucosamine, a
common constituent of insect and crustacean exoskeleton and fungal
cell wall.
Commercially manufactured from the shrimp and crab shells.
As chitin is a non-toxic biodegradable and biocompatible
substance, its derivatives are used in medical practice (Shibata et al.
1997).
Increased protection against Aeromonas salmonicida has been
observed in brook trout when injected with chitin. Injection of
abalone extract and chitin increase phagocytic response and natural
killer cell activity in fish (Stickney, 2000).

8. Chitosan:
Chitosan is a linear homopolymer of ß-(1,4)-2-amino-deoxy-
Dglucose and is prepared by the alkaline deacetylation of chitin
obtained from shrimp and crab shell.
Chitosan is used as an immunostimulant in aquaculture to protect
salmonids and carps against bacterial diseases (Anderson and
Siwicki 1994; Siwicki et al. 1994).

9. Use of herbal and medicinal plants as
immunostimulants in fish:
Natural plant products have been reported as antistress, growth
promotion, appetite stimulation, tonic and immunostimulation, and
to have aphrodisiac and antimicrobial properties in finfish and
shrimp larviculture due to the presence of active principle
components such as alkaloids, flavanoids, pigments, phenolics,
terpenoids, steroids, and essential oils (Citarasu et al. 2002;
Sivaram et al. 2004).

11. Probiotics

12. Definition of Probiotics:
Probiotics 1st used to describe a microbial feed/food supplement was
by Parker in 1974. Probiotics as “organisms and substances that
contribute to intestinal microbial balance.”
Fuller defined as “live microbial food supplement that benefits the host
(human or animal) by improving the microbial balance of the body” &
said that it would be effective in arange of extreme temperatures and
salinity variations.
In 1998, Guarner & Schaafsm aassumed that probiotics are live
microorganisms which, when consumed in adequate amounts, confer
health benefits to the host.
Gatesoupe in 1999, Probiotics as “microbial cells administered in a
certain way, which reaches the gastro-intestinal tract & remain alive with
the aim of improving health”.

13. Flow chart for selection of probiotics

14. Characteristics of good probiotics:
It should be a strain, capable of exerting a beneficial effect on the
host animal. e.g. increased growth or resistance to disease.
Non pathogenic and non-toxic.
Present as viable cells preferable in large numbers.
Capable of surviving & metabolizing in the gut environment. e.g.
resistance to low pH & organic acid.
Stable & capable of remaining viable for periods under storage and
field conditions.

15. Function of probiotics:
a)Growth promotion:
Lactobacillus (LAB) act on complex carbohydrates (starch, cellulose &
synthesize many B–complex & vitamin K. Increase appetite & good health in
host. Infection control & from raised digestibility of nutrients.
b)Immunostimulation / Immunomodulation:
Eg; Lactobacillus casei YIT9018 (BPL) shown to act as immuno-modulators
altering anti-tumor responses.
c)Antagonism effect of probiotics:
Lactobacillus effective by producing certain weak organic acids as lactic acid &
formic acids as acidolin , acidophylin, lactobacillin, lactocidin effective
against many pathogenic bacteria as Escherichia, Salmonella, Streptococcus,
Shigella, Proteus, Klebsiella, Pseudomonas, Bacillus and Vibrio thus helps to
fight disease. Lactobacillus balgaricus are potent toxin neutralizer.

16. d) Antimicrobioses for host benefits:
Intestinal microbiota of higher animals contributes to resistance against invading
pathogens. P. pentosaceous 43200 most promising strain for further research on
bacteriocins mediated protection against C. botulinum hazardous spores.
e) Probiotic feeding:
Probiotics once colonized in the gastro-intestinal (GI) tract can effect the florain 3
ways as:
1)By providing preformed metabolites.
2)By continuous provision of viable cells which do not colonize but which survive
& metabolize in-situ.
3)By provision of viable cells which colonize the gut & produce useful metabolites
in-situ.

17. Modes of action of probiotics:
1) Production of inhibitory compounds / substances
2) Competion for chemicals or available energy
3) Competion for iron & production of Siderophores
4) Competion forAdhesionsites
5) Enhancement of immune response.
6) Improvement of water quality
7) Interaction with phytoplankton
8) Production of enzymes
9) Synthesis of vitamins & absorption of minerals
10) Improvement of stress tolerance
11) Antimicrobial effects
12) Digestive simulation effect
13) Cholesterol – lowering effect

20. Benefits of probiotics in aquaculture:
1)Production of inhibitory compounds: (bacteriocins, sideropheres,
lysozymes, proteases, hydrogen peroxides)
2)Competion for adhesion sites (probiotics on gut or external mucous
epithelial surface against pathogens as Vibrio anguillarium, Aeromonas
hydrophila)
3)Competion for nutrients.
4)Source of nutrients & enzymatic contribution to digestion: (bacteriodes &
Clostridium sp. Supplying fatty acids & vitamins to
host. Probiotics produce enzymes as proteases, lipases in bivalves)
5)Enhancement of immune response: (probiotics can stimulate on specific
immune system eg: Clostridium butyricum in rainbow trout enhanced
phagocytic {leucocytes} activity & resistance to vibriosis.

21. 6)Influence on water quality: (Nitrosomonas {convert ammonia to nitrite},
Nitrobacter {convert nitrite to nitrate}, methane reducing bacteria use
CO2 as source of molecular O2)
7)Interaction with phytoplankton: (eg: probiotics
algicidal effect microalgae red tide plankton)
8)Anti-viral activity: (Pseudomonas sp., Vibrio sp.,
Aeromonas sp., isolated from salmonid hatcheryhad
anti-viral activity against IHNV
{infectous hematopoitic necrosis virus} with >50%
plaque reduction. Moraxella showed Anti-viral capacity high
specificity for polio virus.

22. Conclusion:
FAO should recommend the sustainable use of Immunostimulants and
Probiotics & their complex (Biofloc) in replacing the Antibiotics & excess
artificial feed in Aquaculture to discourage aquatic habitat pollution.
prophylactic treatment of farmed fish and shrimp. It is safer
 Immunostimulants appear to be most promising and useful tools for
than
chemotherapeutics and their range of efficacy is wider than vaccination.
Investigated immunostimulants, probiotics, and prebiotics have numerous
beneficial effects in aquaculture including improved disease resistance and
nutrient availability, leading to increased sustainability and profitability of fish
and crustacean production.

23. References:
Anderson DP. 1992. Immunostimulants, adjuvants and vaccine carrier in fish;
application to Aquaculture. An Rev Fish Dis 2: 281-307.
Selvaraj V, Sampath K, Sekar V. Administration of yeast glucan enhances survival
and some non-specific and specific immune parameters in carp (Cyprinus carpio)
infected with Aeromonas hydrophila. Fish Shellfish Immunol. 2005;9:293–306.
Pionnier N, Falco A, Miest J, Frost P, Imazarow I, Shrive A, et al. Dietary β-glucan
stimulates complement and C-reactive protein acute phase responses in common carp
(Cyprinus carpio) during an Aeromonas salmonidica infection.
Fish Shellfish Immunol. 2013;34:819–31
Jorgensen JB, Sharp GJ, Secombes CJ, Robertsen B. Effects of a yeast-cell-wall
glucan on the bactericidal activity of rainbow trout macropages. Fish Shellfish
Immunol. 1993;3:267–77.