Introduction

Fish farming is the principal form of aquaculture in the world and growing more rapid food animal-producing sector (FAO 2002, 2003). Increasing demands on wild fisheries by commercial fishing operations have caused widespread overfishing. Fish farming offers an alternative solution to the increasing market demand for fish and fish protein. Pisciculture has been employed to compensate the shortage of animal protein all over the world. It has the potential to create huge job opportunities, provided fish cultivation is done on a scientific basis. Disease is the single most important limiting factor in the progressive growth of aquaculture industries. Several studies conducted on the modulation of fish immune system in order to prevent the outbreak of disease were reviewed by Sakai (1999).

The immune systems of aquatic vertebrates, such as those of higher animals, are sensitive to immune challenges by environmental stresses. High-density fish farming and chemical contaminants in water, especially chronic exposure to toxic pollutants, can lead to decreased resistance to viral, bacterial and parasitic diseases. Micro-organisms and metazoans are still engaged in a never-ending arms race, which has led to the evolution of protection mechanisms in vertebrates and pathogenicity mechanisms in microbes. It has been suggested that the diverse evasive strategies of micro-organisms to their host have acted as inducers of vertebrate defence evolution (Castro and Gonzaa’lez 2001). Fishes are one of the most primitive vertebrates, which possess non-specific defence mechanisms of the invertebrates such as phagocytic mechanisms of macrophages and granular leucocytes. They were also the first animals to develop both cellular and humoral immune responses mediated by lymphocytes. The main lymphoid organs of fish are the thymus, anterior kidney and spleen. In fishes, non-specific immunity is considered as the first line of defence and represents a considerable part of the immune response (Dalmo et al. 1997). The epidermal secretions, anti-microbial proteins, lysozyme, phosphatases and trypsin, their amount and activity depend on the species (Fast et al. 2002).

In aquaculture, antibiotics have been used mainly for therapeutic purposes and also as prophylactic agents. Antibiotics are drugs of natural or synthetic origin, with the capacity to kill or inhibit the growth of micro-organisms. Aquaculture faces serious issues due to various adverse effects of antibiotics such as accumulation in the tissue and immunosuppression (Gudding et al. 1999). Chemotherapeutics and antibiotics are mainly administered in the culture practices through medicated feeds or food materials. This practice may result in antibiotics entering into the environment by leaching from uneaten feeds, unabsorbed parts in manure or aquatic animals (Robinson et al. 2007). The options adept in aquaculture for controlling and preventing diseases were vaccines, antibiotics, probiotics, genetically improved stocks, immunostimulants, husbandry practices and water treatments as given in Table 1.

Table 1 Options available for prevention, treatment and control of the incidence of diseases in aquaculture practices
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Use of antibiotics in fish production increases the prevalence of antibiotic resistance in human diseases. For reducing the usage of antibiotics, immunostimulant is one of the useful tools in aquaculture. Moreover, due to the availability of limited vaccines in few countries and their pathogen-specific protective action, much attention has been directed towards the use of immunostimulants in aquaculture to control infectious diseases. An immunostimulant is a chemical drug, stressor or action that increases the non-specific defence mechanism or the specific immune response. Sajid et al. (2011) have given an overall review about immunostimulant activity of glucan, chitosan, levamisole and herbal plant products. Various herbal products such as from Hygrophila spinosa, Withania somnifera, Zingiber officinale, Solanum trilobatum, Andrographis paniculata, Psoralea corylifolia, Eclipta erecta, Ocimum sanctum, Picrorhiza kurroa, Phyllanthus niruri, Tinospora cordifolia, purified Silajit and cod-liver oil have the characteristics of growth promotion, anti-stress, immunostimulation and anti-bacterial. These preparations had a prerogative influence on the Penaeus larviculture (Citarasu et al. 1998, 2002). Citarasu et al. (2006) used five herbal plants Cynodon dactylon, Aegle marmelos, T. cordifolia, P. kurroa and Eclipta alba extracts incorporated into basal diet formula for testing white spot syndrome virus infection in Penaeus monodon. The aim of this study is to review the positive effect of plant materials on the fish immunity for the control of microbial diseases.

Immunostimulants

Immunostimulants, also known as immunostimulators, are substances (drugs and nutrients) that stimulate the immune system by inducing activation or increasing activity of any of its components. The immunostimulants could increase the resistance of fish to infectious diseases by enhancing non-specific defence mechanism. Immunostimulants can be administered by injection, bathing or orally, with the latter appearing to be the most practicable (Jeney and Anderson 1993; Sakai 1999; Yin et al. 2006). It will boost the potency of the vaccine, thereby decreasing the dose necessary for the same effect (Jeney and Anderson 1993). The immunomodulation of larval fish has been proposed as a potential method for improving larval survival by increasing the innate responses until its adaptive immune response is sufficiently developed for effective response against the pathogen. To this end, it has been proposed that the use of immunostimulants as a dietary supplement to larval fish could be of considerable benefit in boosting the animal’s innate defences with little detriment to the developing animal (Bricknell and Dalmo 2005). Galeotti (1998) suggested that in vitro screening methods should be used to elucidate the mechanisms of immunostimulation and then in vivo methods should be used to establish whether the benefits occur in live fish. Total serum IgM levels of fish fed with the assayed immunostimulant-supplemented diets were statistically higher than those of fish fed with a non-supplemented diet (Cuesta et al. 2004). The addition of various food additives such as vitamins, carotenoids and herbal remedies to the fish feed has been tested in aquaculture. Reducing the stress response, increasing the activity of innate parameters and improving disease resistance (Amar et al. 2004; Puangkaew et al. 2004; Cerezuela et al. 2009; Yin et al. 2009) are the overall beneficial effects. In vivo and in vitro effects are the two different immune response measuring factors. There are many studies reporting a variety of substances including synthetic (Rao et al. 2006), bacterial (Goetz et al. 2004), animal and plant products (Hardie et al. 1991; Rao et al. 2006; Ardo et al. 2008) can be used as immunostimulants to enhance non-specific immune system of cultured fish species. Various types of plant active compound substances such as saponin (Ninomiya et al. 1995), glycyrrhizin (Jang et al. 1995), aloe (Kim et al. 1999) and azadirachtin (Logambal and Michael 2000, 2001; Harikrishnan et al. 2009a, b) have been reported to enhance the innate immunity in fishes. Some plants are the rich sources of compounds such as volatile oils, saponins, phenolics, tannins, alkaloids, polysaccharides and polypeptides. These natural plant products have various activities such as anti-stress, appetizer, tonic, anti-microbials and immunostimulants (Citarasu et al. 2002). Interest in the use of immunostimulants as an alternative to the drugs, chemicals and antibiotics currently being used to control fish diseases is growing, partially because immunostimulants, in contrast to vaccines, enhance the innate (or non-specific) immune response (Galeotti 1998; Sakai 1999). For the past decade, genomic organization and characterization of immune related genes and their expression through various immunostimulants (zymozan, peptidoglycan, β-glucan) were studied to understand the molecular mechanism behind the immune system of crustaceans (Rattanachaia et al. 2005; Vaseeharan et al. 2006, 2011; Lin et al. 2007; Sivakama valli and Vaseeharan 2012).

Leave extract as immunostimulant

Recently, in aquaculture, scores of plant extracts have been tested and used with good results in the control of bacterial and viral diseases. Fourteen herbs have been tested against Aeromonas hydrophila infection in Oreochromis niloticus, among them, the ethanol extract of Psidium guajava has been found to have highest anti-microbial activity (Pachanawan et al. 2008). The stimulation of specific and non-specific immunity and the protection against fish pathogen A. hydrophila in O. mossambicus by ethanol and petroleum ether extracts of T. cordifolia were observed (Sudhakaran et al. 2006). Salah and Mohamed (2008) studied the effect of Echinacea on the growth rate and disease resistance in Nile tilapia. The experiment was carried out on 1200 Nile tilapia, reared in earth ponds. A controlled infection with Pseudomonas fluorescens was also carried out by intraperitoneal inoculation. The test group showed a significant increase in body weight gain, specific growth rate, haematocrit values, lysozyme activity and total leucocytic count. The survival rate was significantly enhanced in the experimental group prior to and post-inoculation. Astragalus membranaceus extract significantly enhanced the phagocytic activity of leucocytes isolated from Nile tilapia within 1 week of being provided to the fish, and this increased activity was maintained during the entire experiment (Yin et al. 2006; Ardo et al. 2008). Carp fed with A. membranaceus extract showed enhanced survival compared with control fish, following a challenge with A. hydrophila (Yin et al. 2009). Feeding O. niloticus with two herbal extracts (A. membranaceus and Lonicera japonica) alone or in combination significantly enhanced phagocytic and respiratory burst activity of blood phagocytic cells in treatment groups compared with control group (Ardo et al. 2008). In O. mossambicus, the acetone extract of O. sanctum was found to enhance the anti-sheep red blood cell (SRBC; sheep erythrocytes) antibody response (Hemapriya 1997), while the water extract stimulated both the specific and non-specific immune mechanisms (Logambal et al. 2000). Leaves of O. sanctum contain water-soluble phenolic compounds and various other constituents, such as eugenol, methyl eugenol and caryophyllene (Chopra et al. 1956). Administration of O. sanctum leaf extract to tilapia O. mossambicus, simultaneously with or after vaccination, resulted in changes both the magnitude of antibody response and the day of peak antibody response increased protection against experimental infection with A. hydrophila (Logambal et al. 2000). The effects of water- and hexane-soluble fractions of S. trilobatum on the non-specific immune mechanisms and disease resistance of tilapia found that all doses of water-soluble fraction significantly enhanced the production of reactive oxygen and decreased the percentage mortality, following a challenge with A. hydrophila (Divyagnaneswari et al. 2008). The addition of plant extracts of four Chinese herbs (Rheum officinale, A. paniculata, Isatis indigotica and L. japonica) to the feed of crucian carp resulted in increased phagocytosis of the white blood cells (Chen et al. 2003). Dip treatment in goldfish with Azadirachta indica aqueous leaf extract exhibited a significant increase in serum glucose, cholesterol and total protein (Harikrishnan et al. 2009b). Dip treatment of C. carpio with aqueous leaf extract of A. indica significantly increased serum protein levels and protected the fish from A. hydrophila infection (Harikrishnan et al. 2003). Methanolic extracts of the herbals O. sanctum, W. somnifera and Myristica fragrans herbs significantly improved the immune parameters such as phagocytic activity, serum bactericidal activity, albumin–globulin (A/G) ratio and leucocrit against Vibrio harveyi challenge in juvenile grouper, Epinephalus tauvina larviculture (Sivaram et al. 2004). The acetone extracts of four plants C. dactylon, A. marmelos, W. somnifera and Z. officinale were screened for their inhibitory activity against seven fish Vibrio pathogens V. alginolyticus, V. parahaemolyticus, V. mimicus, V. campbelli, V. vulnificus, V. harveyi and P. damselae, and these extracts were mixed with fish feed in proper ratio; as a result, there was an enhancement in leucocrit, phagocytic and lysozyme activities in the blood of O. mossambicus fed with experimental diet compared with the control diet-fed fish (Immanuel et al. 2009). Five herbs Acalypha indica, H. spinosa, P. kurooa, T. cordifolia and Z. officinale were selected to screen for the in vitro immunostimulant activity against the shrimp pathogen Vibrio harveyi, and the herbal extract improved the total haemocyte count (THC), phagocytosis, phenol oxidase (PO) haemagglutinin activity and bacterial clearance activity in Fenneropenaeus indicus (Raja Rajeswari et al. 2012). Yin et al. (2009) reported that the complementary vaccine with Ganoderma and Astragalus to carp showed a significantly enhanced respiratory burst activity of phagocytic cells as well as enhanced phagocytosis and lysozyme activities in plasma. The specific immune response was also increased, although there were no significant differences between the vaccinated group not fed with herb extracts and the vaccinated fish fed with herb extracts. The herbal plant extracts of A. indica, C. dactylon, W. somnifera, Z. officinalis and P. kurooa having anti-viral and immunostimulant characteristics, which offer better growth and immunostimulation and act as anti-viral during the dual administration against the WSSV infection in Penaeus monodon (Yogeeswaran et al. 2012). Fish fed with both herbs and vaccine showed best survival against infection with A. hydrophila. The herbal immunostimulants Emblica officinalis, C. dactylon and Adhathoda vasica improved the immune system and reduced microbial infection in the goldfish Carassius auratus (Minomol 2005). Punitha et al. (2008) screened the herbal plant extracts of C. dactylon, Piper longum P. niruri, Tridax procumbens and Z. officinalis testing immunostimulant activity in grouper E. tauvina against V. harveyi infection, and the petroleum ether extract is very effective against vibrio pathogens in in vitro screening. Herbal extracts have a potential application as an immunostimulant in fish culture, primarily because they can be easily prepared, are inexpensive and act against a broad spectrum of pathogens. Most of the herbs and herbal extracts can be given orally, which is the most convenient method of immunostimulation. However, the effect is dose dependent, and there is always a potential for overdosing (Yin et al. 2006). It has been observed that the plant extract might act directly on the immunopoietic cells as immunostimulant (Jeney and Anderson 1993).

Plant parts as immunostimulant

Many parts of the plant materials possess medicinal properties. Numerous plant materials are widely used in aquaculture for preventing diseases by controlling the pathogenic microbes and enhancing the immunity. A. hydrophila infection in rainbow trout (Oncorhyncus mykiss) was controlled by garlic (Nya and Austin 2007). At an inclusion level of 0.5 and 1.0 mg/g feed, a 4 % reduction in mortality was observed than the control group. Garlic can help in the control of bacteria and fungi and increase the welfare of fish (Corzo-Martinez et al. 2007). Nya and Austin (2009) used ginger to control an experimental infection of A. hydrophila in rainbow trout, and mortality was reduced to zero compared with the control group. They have also recorded the enhancement of growth rate, feed conversion and protein efficiency in the rainbow trouts fed with ginger. Several medicinal-plant-based materials were administered as immunostimulants in various fish species against pathogens and are given in Table 2. Hemapriya (1997) reported that the acetone extract of P. emblica enhanced the anti-SRBC antibody response in tilapia, while Balasubramani and Michael (2002) found that both crude extracts and a water-soluble fraction of P. emblica fruit had a stimulatory effect on the immune response of tilapia. P. emblica fruit pulp contains large proportion of vitamin C, which has also been identified as an immunostimulant (Li and Lovell 1985). Achyranthes aspera seed was incorporated into the diet of Labeo rohita, rohu fingerlings, and the results indicated that A. aspera seed stimulated immunity and increased resistance to A. hydrophila infection in fish (Rao et al. 2006). Dorucu et al. (2009) reported that black cumin seed extract enhances the total immunoglobulin level in Oncorhynchus mykiss after 3 weeks feeding period. Natural immunostimulants are biocompatible, biodegradable and safe for both the environment and human health. Moreover, they possess an added nutritional value (Ortuno et al. 2002). Rainbow trout fed with Z. officinale (ginger) extract had significantly higher extracellular activity of phagocytic cells in blood and in trout fed with nettle, and mistletoe extracts increased the production of extracellular superoxide anion (Dugenci et al. 2003).

Table 2 Medicinal-plant-based materials used as immunostimulants in fish against pathogens
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Essential oil as immunostimulant

In aquaculture practice, pathogenic microbes are controlled by essential oils, and they also act as one of the immunostimulants. The carbonyl group of cinnamaldehyde is thought to be responsible for anti-microbial action by binding to cellular proteins and preventing them from functioning properly (Wendakoon and Sakaguchi 1995). Pongsak and Parichat (2010) have studied the cinnamon oil potential to control Streptococcus iniae infection on Nile tilapia. They studied and compared inhibitory capacity of four essential oils: Citrus hystrix (leech lime), Cymbopogon citratus (lemon grass stems), Curcuma longa (turmeric) and Cinnamomum verum (cinnamon). The highest inhibitory activity was observed in cinnamon oil. As for the growth parameters, there was no significant difference observed between the control group and the experimental groups. Diet supplemented with Zataria multiflora essential oil enhanced common carp immunity to some extent even though fish cannot express its potential immunity during low temperature. Recently, Vaseeharan et al. (2012) reported the inhibitory activity of essential oils from medicinal plants against Pseudomonas spp. isolated from aquatic environments, and its control efficiency was visualized under confocal laser scanning microscopy. The Z. multiflora essential oil had the potential for enhancing innate immune system of carp under temperature stress (Soltani et al. 2010). Oral administration of Quil A saponin increased leucocyte migration in yellowtail fish (Ninomiya et al. 1995). Some effective components such as thymol and terpinene present in the oils have the properties in stimulating the fish immunity.

Herbal drugs as immunostimulators

Resveratrol (RESV; trans-3,5,40-trihydroxystilbene), a natural polyphenol, was first isolated in 1940 as a constituent of the roots of white hellebore, but since then, it has been found in various plants, including grapes, berries and peanuts (Khanna et al. 2007). It was found that RESV strongly inhibited intracellular and extracellular myeloperoxidase (MPO) activity, behaving as a non-competitive and reversible inhibitor, and also induced a decrease in MPO mRNA levels in Turbot Psetta maxima neutrophils (Castro et al. 2008). Papaya leaf meal contains an enzyme, namely papain, which increases the protein digestion, food conversion ratio, specific growth rate and weight gain in 16 % unsoaked papaya meal diet fed to P. monodon post-larvae (Penaflorida 1995). Edahiro et al. (1991) reported that yellowtail fish treated orally with glycyrrhizin showed increased protection against Edwardsiella seriola infection, although lysozyme activity of blood and phagocytic activities of macrophages were not enhanced. Glycyrrhizin is a glycosylated saponin, containing one molecule of glycyrrhetinic acid, which has anti-inflammatory and anti-tumour activities, mediated by its immunomodulatory activities (Wada et al. 1987; Zhang et al. 1990). Livol (IHF-1000) is a herbal growth promoter containing different plant ingredients such as Boerhavia diffusa, Solanum nigrum, Terminalia arjuna, Colosynth and black salt and has been found to significantly improve digestion, thereby leading to better growth, production and health in cultivable fishes (Shadakshari 1993; Unnikrishnan 1995; De Bolle et al. 1996; Jayaprakas and Euphrasia 1996). The herbal extracts from Astragalus membranaceus, Portulaca oleracea, Flavescent sophora and A. paniculata act as an anti-stressor and induce the immunological parameters such as serum lysozyme activity, SOD, NOS and levels of total serum protein, globulin and albumin in Cyprinus carpio (Wu et al. 2007). Azadirachtin, a triterpenoid derived from A. indica, enhanced respiratory burst activities, the leucocyte count and the primary and secondary antibody response against SRBC in tilapia (Logambal and Michael 2000, 2001).

Conclusion

Antibiotics, chemotherapeutants and vaccines are expensive and lead to many adverse effects such as bioaccumulation and multi-resistance species development in the environment. Plant materials have a potential application as an immunostimulant in fish culture, primarily because they are not expensive and act against a broad spectrum of pathogens. The preparation of plant extract is much easier and inexpensive. Many plant products are used as anti-bacterial and anti-viral materials. The use of plant products as immunostimulants in fish culture systems may also be of environmental value due to its biodegradability. The enhancement compounds present in the leaf extracts such as phenolics, polyphenols, alkaloids, quinones, terpenoids, lectines and polypeptides have shown to be very effective alternatives to antibiotics and other synthetic compounds. Plant phenolics, polysaccharides, proteoglycans and flavonoids play a major role in preventing or controlling infectious microbes. In many studies, the usage of plant products as immunostimulant has revealed that they increase the immune responses, survival and growth rate of the fish. Due to the beneficial effect of plant material as immunostimulants, it can be used in fish farming as alternatives to vaccines, antibiotics and chemical drugs. For getting better growth and diseases management in aquaculture, attention has to be focused around many medicinal plants and drugs derived from medicinal plants, and these can be used easily as feed supplementation. In future, purification and experimental evaluation of active compounds from the herbal plants are needed for the effective control of disease-causing agents in aquaculture. Further, aquaculture practice with medicinal plant derivatives can get healthy natural protein-rich food for consumers and will be profitable for aquaculture farmers.