The compounds that modulate the by-host immune system called immunostimulants are naturally present and commonly used in aquaculture (Robertsen, 1999). One of the most capable approaches in aquaculture for managing diseases is developing the fish defence system during prophylactic immunostimulant administration (Ringo et al., 2010; Selvaraj, Sampath, & Sekar, 2009). Due to the emergence of bacterial resistance and environmental and habitat safety issues, conventional disease prevention methods using Chemical disinfectants and antibiotics will be no longer required. While vaccination is an efficient prophylactic approach for preventing infection in fish, there are few clinical illnesses associated with high costs and stress (Ellis, 1999). With remarkable results, the use of immunomodulators as an even more eco-friendly method of deterring disease control has already been achieved (Peddie, Zou, & Secombes, 2002). Immunostimulants improve the adaptive immune system and therefore eliminate infectious diseases (Sakai, 1999).

Microalgae contain many bioactive compounds that can satisfy people's food and energy needs, encourage health conditions, and avoid chronic illness disease (Morais, Vaz, Morais, & Costa, 2014). The capacity of these bacteria and fungi in feeding and foodstuffs, cosmetics manufacturing and die industry and additive production have been defined by various processes. The ability of these micro-organisms in the food, animal feed, cosmetics, and die and additive manufacturing industries has been defined in various processes (Morais et al., 2014). These nutrients contribute to the development of these microorganisms by different enzymatic reactions for the biosynthesis of specific compounds (Brennan & Owende, 2010; Karemore, Pal, & Sen, 2013; Wang, Li, Wu, & Lan, 2008).

Microalgae cultivation provides an economic outlet for humans and considerably helps decrease the greenhouse effect appropriate to carbon dioxide (CO2) fixation potential of microalgae (Brennan et al., 2010; Wang et al., 2008). Microalgae have been recognized as bioactive substances, such as Botryococcus, Chlorella, Spirulina, Dunaliella, Haematococcus, and Nostoc. The natural bioactive compound is essential for algae metabolism and has many advantageous biological activities, including certain features many favourable biological activities such as anti-carcinogenic, anti-obesity, anti-angiogenic, antioxidant, anti-inflammatory and neuroprotective activities (Guedes, Barbosa, Amaro, Pereira, & Malcata, 2011; Pangestuti & Kim, 2011). Microalgae, including carotenoids and phycobiliproteins, were present at the primary pigments. Carotenoids are commonly used mainly for dietary supplement diets for animals and human beings, food colouring dyes, animal feed, fortified foods and pharmaceuticals, and few cosmetic goods productions products (Vílchez et al., 2011).

Enzymes are ideal sources of proteins for human nutrition since they are cheaply further processed by the gastrointestinal tract relative to intact amino acids (Lisboa, Pereira, Ferreira, & Jorge, 2014). Hydrolysates are an ideal source of protein for human nutrition while they are quickly processed additionally by the gastrointestinal tract relative to intact amino acids (Hoseini, Khosravi-Darani, & Mozafari, 2013). Spirulina's high 50-70 percent weight basis protein content provides all the required amino acids, particularly valine, leucine and isoleucine and others (83-90 percent) (Becker, 2007). Studies in nutrition and toxicology have already shown that microalgal biomass is helpful as a replacement food or substitute for conventional rich sources of protein (Christaki, Florou-Paneri, & Bonos, 2011; Spolaore, Joannis-Cassan, Duran, & Isambert, 2006). They comprise water-soluble vitamins and can be used to supplement nutrients and diets. Microalgae are considered a pool of essential vitamins, including ascorbic acid, tocopherols and several B vitamins complexes. These microbes comprise water-soluble vitamins it can be used as food supplements and diets. Microalgae have the primary source of essential vitamins such as ascorbic acid and multi-complex B tocopherols (Ambrosi, Reinehr, Bertolin, Costa, & Colla, 2008). Spirulina is a concentrated supplement to vitamin A (β-carotene) and B12 (Ambrosi et al., 2008; Raa, 1996). The level of immune tolerance may be improved by adding immune stimulants by enriched feed or vaccines while preventing the use of antibiotics and chemotherapy agents (Raa, 1996). Many products of seaweed materials such as Chaetomorpha aerea (Sattanathan et al., 2020), Eugena viridis (Das, Pradhan, Pattnaik, Samantaray, & Samal, 2005), Euglena gracilis (Kondo et al., 1992) fish immunity has been reported to be improved the immune mechanism of fish. There is no known study of mixed algae against A. hydrophila on innate immune functions in C. carpio. Therefore, the present studies aimed to assess the effectiveness of the mixed algal extract against A. hydrophila on the immunity point of C. carpio.

Material and methods

Experimental fish and their maintenance

C. carpio wet weight: 8.50 ± 0.50 g; length: 11 ± 0.50 cm was bought from a nearby fish farm, Kumbakonam, Tamil Nadu, India, and cultivated with dechlorinated water. Before treatment, all experimental fish were acclimatized at pH 7.0 ± 0.2 at a steady temperature of 22 ± 1 °C and commercial fish food was fed once a day.

Collection and culture techniques

Spirulina platensis

S. platensis was grown in a modified Zarrouk medium and sterilized at 121 °C for 20 min with 100 ml of the ideal combination. Each conical flask was inserted with a 10 ml culture usually containing a minimum level of 107–108 colony forming units/ml incubated for seven days and transferred to a 20 L white, clear polyethylene vessel incubated for 21 days. S. platensis samples were cultivated under laboratory conditions of 30 ± 2 °C, and continuous illumination was 5500-6500 Lux. During incubation, the sterilised air was used to spread and mix the culture. The culture mixture was filtered with a sterile cotton cloth and washed with distilled water. The cells were air dried for 24 hours at room temperature before being baked at 70°C (Ravelonandro, Ratianarivo, Joannis-Cassan, Isambert, & Raherimandim, 2008).

Euglena viridis

The algal blooms were gathered from a local fish farm (0.2 to 2.5 ha) at Kumbakonam, Tamilnadu. Under the circumstances, the water samples were observed under a microscope. The water samples were examined under the microscope, under the circumstances. The E. viridis organisms have been flexibly enlarged, spindle-shaped, single-celled mobile, and greenish colored, and were observed (Das et al., 2005). For triplet methods in sterilized water to remove the suspended particles, the collected water samples were washed in bolting and silk cloth and then centrifuged at 1000 g using a macro rotor (Sorvall CE, UK). The mixed pellet, which can be prepared, was harvested at room temperature and dried for 2 or 3 days.

Chlorella vulgaris

C. vulgaris was cultivated in a 20 L tank of glass procured from marine water. Seawater 30 ppt filtered from the pollution-free zone (0, 5 lm pore-size Millipore filters), sterilized for 30 min at 120 °C, and accompanied by the Erd-Schreiber medium (Vijayavel, Anbuselvam, & Balasubramanian, 2007). The cultivations of C. vulgaris were maintained at 28 ± 1 °C with sufficient aeration, and continuous flow centrifugation (10 l / h) harvested the algae for 40 min at 2,000 g at 4 °C. Weighed and air-dried the ensuing whole-cell pellet to remove moisture and refrigerated until it was used.

Preparation of mixed algal extract

Different shade-dried from the three different microalgae with seven days until weight constancy was achieved. The E. viridis, C. vulgaris and S. platensis algae powders were gently mixed with a ratio of 1:1:1 for preparation of the mixture, and the algae sample was lightly crushed using an electric blender. The mixed algal extract was obtained by dissolving 100 g of the mixture with 1000 ml (sterile) distilled water and transferred to 2000 ml conical flasks densely covered with aluminium foil coating, kept at room temperature agitated daily for seven days. Mixed algae methanol solvent extract was prepared and applied using the standard methods followed by slight adjustments (Harikrishnan et al., 2009). Using a cold maceration process previously reported, coarse powdered dry algae were gradually extracted using solvents such as petroleum ether, CH3Cl3, EtOAc, MeOH and 0.25 percent CH3Cl3:H2O (v/v). The extracts were then dried and stored at -20 °C until utilised using a rotating vacuum evaporator (Buchi, Switcherl).

Experimental design

Fifteen tanks were distributed to the C. carpio 180 fish, with each tank containing twelve fish being maintained with triplicate. The fish were injected intraperitoneally with 0.2 ml of reconstituted methanol extract at tenfold increases in body weight of 0; T1, 0.1; T2, 1; T3, 10; T4-100 mg/kg. It received 0.2 ml of sterile water from the control groups. Seven days before treatment, the fish were bled, and 7, 14, 21 and 28 days after treatment

Collecting blood and separating serums

   The blood samples are taken using an approximately 1ml tuberculin syringe, placed with a 24 gauge needle (Michael, Srinivas, & Sailendri, 1994). The serum was prepared within 1 min after collection of blood by collecting 200μl of blood. The blood in the serological tubes was store overnight in the refrigerator. On the clot, the centrifugation was performed at 400 g for 10 min. (Michael et al., 1994). For the use of assays, the obtained serum was placed in the storage vials with the highest sterility at -20 °C.

Immunological assays

Nitroblue tetrazolium assay

The neutrophil activity was assayed using Nitro blue tetrazolium (NBT, Sigma) assay using theChung and Secombes (1988) method with modifications previously described (Sahu, Das, Mishra, Pradhan, & Sarangi, 2007). Then at 620 nm was observed the OD value for the microplate reader (Systronics, India). They used KOH / DMSO as blank.

Serum lysozyme activity

Serum lysozymes were measured using the technique described by Parry et al. (1965), slightly modified to (Hutchinson & Manning, 1996) . One unit of lysozyme activity was defined as the total amount of enzyme that produces a decrease of 0.001 min-1 and mL in serum absorbance.

Bactericidal activity

According to standard serum bactericidal activity procedure Kajita, Sakai, Atsuta, and Kobayash (1990). An adequate amount of 100 ml serum and bacterial suspension (105 CFU / ml) at room temperature was already mixed and incubated for one h. They prepared the blank control, too, by replacing serum with sterile PBS. The solution was diluted again with Buffered saline phosphate (PBS) at a ratio of 1:10. The diluted 100 mL suspension was poured on nutrient agar plates at 37 °C with 24 h incubation. The number of bacterial cells was determined by the new colonies cultivated in agar plates counted with nutrients.

Myeloperoxidase activity

The total amount of myeloperoxidase activity was determined using the described method (Quade & Roth, 1997). In brief, 10 mL of serum has been diluted with 90 mL of HBSS, adding 35 mL of 20 mM 3, 3, 5, 5 tetramethyl benzidine hydrochloride (TMB) (Genei, India) and 5 mM of H2O2 at 1:20. The reaction was stopped by adding 35 mL of 4 M sulfuric acid ( H2SO4) to it after 2 minutes of exposure, and the optical density was examined at 450 nm using a micro plate reader.

Serum antiprotease activity

The antiprotease activity was determined by following the previous method Bowden, Butler, Bricknell, and Ellis (1997), and 2 mN BAPNA was used as the substrate. 10mL of serum was incubated with 20 ml of trypsin solution, then 0.1 M Tris HCL pH 8.2 was placed and incubated at 22 ° C for 25 min from the 500 ml volume substratum. The reaction was halted in a plate reader with 150 ml 30% acetic acid, and the OD was read at 415 nm.

Percentage of trypsin inhibition (%) = (Trypsin blank OD-Sample OD)/(Trypsin blank OD) X 100

Culture of A. hydrophila and the Challenge study

A. hydrophila microbial strain (MTCC 1739) was obtained from IMTECH, Chandigarh, India, where it was grown at 37 ° C in (TSB) tryptone soy broth (HiMedia, India) for 24 hours; TSB broth culture was centrifuged at 3000 g for 10 min. The supernatant was filtered, and the phosphate-buffered saline pellets (PBS, pH 7.4) were suspended while the solution's OD values were adjusted to 0.5 at 456 nm, equivalent to 1 x 107cfu / ml. Defined by intraperitoneal injection of A. hydrophila evaluated doses (106, 107, 108 and 109 CFU per fish), the seven-day lethal dose 50 (LD50) in 20 fish was 107cfu / ml. Nine fish from each group completely at random treated with selected fish have injected i.p at the end of the feeding trial. A. hydrophila with 1 x 107 live, containing 0.2 ml PBS. Fourteen days after infection, all groups recorded total mortality. Furthermore, the relative survival percentage (RPS) was illustrated according to the formula below after the challenge study.

Relative percentage survival (RPS) = (Number of surviving fishes after challenge)/ (Number of fishes infected with bacteria) ×100

Statistical analysis

The quantitative data was evaluated using one-way variance analysis (ANOVA). All data were analyzed statistically using the SPSS (version 21) software. The mean level was p<0.05, and the results are analyzed as mean (S.E.M) values.

Results and Discussion

Figure 1 to Figure 6 shows the effect of mixed algae extract i.p (intraperitoneal) injection at different doses like (0, 0, 1, 10 and 100 mg/kg) on immunity response at weekly intervals of 28 days, respectively. The C. carpio neutrophils' experimental group respiratory burst activities (NBT reduction) are shown in Figure 1.

Figure 1

Neutrophil activity in C. carpio after varied treatments with mixed algal extract (results are mean±SE).

In comparison with the control group, respiratory burst activity increased statistically significantly during the experiment after treatment with mixed algae extract. The highest value of respiratory burst activity was found in the T2 group, and the least amount of respiratory burst activity in the control group was found. The lysozyme activity revealed an increasing trend in exposure to mixed algal groups during the 7-day to 28-day sampling period and differed significantly ( p<0.05) between treatment groups. Furthermore, the highest lysozyme activity was found in group T2, followed by control of sampling on T3, T4 and 14 days (Figure 2).

Figure 2

Various doses of mixed algal extract were used to treat C. carpio serum lysozyme activity (values are mean±SE).

Due to the administration of different dosages of a mixed algal diet during the 28-day sampling period, serum myeloperoxidase activity increased significantly ( p<0.05) compared to control (Figure 3).

Figure 3

During post-treatment days, C. carpio serum bactericidal activity was supplied with varied amounts of mixed algal extract (results are mean±SE).

During all sampling days, serum bactericidal activity improved significantly ( p<0.05) during the experimental period (Figure 4).

Figure 4

During post-treatment days, serum myeloperoxidase activity was measured in C. carpio with varying amounts of mixed algal extract (results are mean±SE).

Statistically significant ( p<0.05) increases in serum antiprotease activity were noted up to 28 days after sampling in fish from different treatment groups (Figure 5).

Figure 5

Ceruloplasmin activity was measured in C. carpio after several treatments with mixed algal extract (results are mean±SE).

The relative percentage (percent) survival of C. carpio in mixed algal treatment conditions is illustrated in Figure 6 .

Figure 6

Post-treatment serum antiprotease activity in C. carpio treated with varied doses of mixed algal extract (results are mean±SE).

The highest post-challenge survival rate (p<0.05) was observed in the fish group with 1 and 10 mg/kg mixed algal exposure (95 and 90 per cent). The control group was shown at 50 per cent. Microalgae are microscopic unicellular organisms that can describe food and food metabolites, including certain carbohydrates, proteins, vitamins, fat, and organic minerals. Environmental pollution occurs when environmental degradation crosses limits and leas lethal to living organisms (Usha, Pugazhendy, Tamizhazhagan, Sakthidasan, & Jayanthi, 2017). Adversely human activities are directly or indirectly affect the environment (Tamizhazhagan et al., 2017). Mainly, the indiscriminate use of pesticides resulted in the contamination of the aquatic system has become a global problem and is being extensively researched worldwide (Tamizhazhaganv, Pugazhendy, & Sakthidasan, 2016). Microalgae are microscopic unicellular that can make a wide range of metabolites for food and feeding stuffs, such as carbohydrates , proteins, vitamins, lipids, and organic and inorganic minerals (Becker, 1994) .

The present study shows that mixed algal extract enhanced concentrate upgraded the safe reaction in C. carpio against A. hydrophila. In Figure 1 to Figure 6 , respectively, the impact of mixed algal growth removes i.p (intraperitoneal) infusion at different portions (0, 0.1, 1, 10 and 100 mg/kg) on non-specific immune mechanisms parameters at weekly intervals for 28 days 1 month. The experimental group's respiratory burst activities (NBT decrease) of C. carpio neutrophils appear in Figure 1. In the research analysis, various agents, including bacteria and bacterial products, have found the increase in NBT levels beneficial for fish in protecting them from invading pathogens (Khan, Bhadouria, & Bisen, 2005).

Figure 7

Effect of mixed algal extract on C. carpio survival when challenged with virulent A. hydrophila (mean±SE).

In this current study, the maximum higher neutrophil activity was noted compared to control in all treatment groups. In this study, higher respiratory burst activity was recorded as compared to control in all treatment groups. Similar results were reported with varying levels in E. viridis fed by Labeo rohita. Similar results have been reported in Labeo rohita fed E. viridis with varying levels (Lamas & Ellis, 1994; Solem, Jorgensen, & Robertsen, 1995), levamisole (Wijendra & Pathiratne, 2007) . T1 and T2 were found to have a high maximum value of respiratory burst activity, and decreased respiratory burst activity was defined in the un-treated control group. During sampling periods from 7 to 28 days, lysozyme activity (Figure 2 ) showed an increasing trend in exposed mixed algal groups and significantly increased (p<0.05) between treated groups. Additionally, the highest lysozyme activity was found in group T2, followed on the 14th day by T3, T4 and untreated control. In addition, the maximum activity of lysozyme was recorded in group T2, followed by T3, T4 and untreated control on the 14th day.

Serum bactericidal activity increased statistically during all sampling days (p<0.05) during the experimental period. Figure 6 shows significant increases (p<0.05) in serum antiprotease activity from the treatment group compared to the control groups up to a sampling day of 28 were noted in fish. The neutrophils are considered an indicator of lysozyme, and the enzyme seems to be much more bactericidal than the higher lysozyme of the vertebrate (Barsanti, Vismara, Passarelli, & Gualtieri, 2001; Ellis, 1999). In this research, serum lysozyme activity increased considerably at different concentrations in treatment groups fed with a mixed algal diet. It was found that E. viridis is microalgae may improve the activity of antiprotease in fish as recognized in L. rohita (Amar, Kiron, Satoh, & Watanabe, 2004; Das et al., 2005). Similar results with Chinese herbal medicine have been observed for lysozyme activity on 20 to 30 days after feeding with Pseudosciaenas crocea (Guzmán, Gato, & Calleja, 2001; Janczyk, Halle, & Souffrant, 2009; Kotrbacek, Halouzka, Jurajda, Knotkova, & Filka, 1994). The serum myeloperoxidase levels were increased significantly ( p<0.05) compared with the control group (Figure 3 ) due to the administration during the 28-day sampling period of different dosages of mixed algal diet.

In the current study of this investigation, a maximum higher of myeloperoxidase activity was observed compared to the control group in the mixed algal dietary fed groups. The relative percentage of C. carpio survival (percentage) under mixed algal treatment conditions are shown in (Figure 6). The maximum relative percent survival was significantly (p<0.05) increased in experimental group exposure with 1 and 10 mg/kg mixed algal (95 and 90 percent), and the control group was observed at 50 percent. In L. rohita, a similar diet study supplemented E. viridis showed significantly higher relative percentage survival after challenge with A. hydrophila after immunization (Das et al., 2005). The results provide strong evidence that the mixed algal extract displayed a more robust immune response and sustainability in common carp, C. carpio. The promising effects of the challenge trial indicate that due to the ubiquitous presence and opportunistic pathogenesis of Aeromonas, a wide-spectrum disease resistance develops.

Conflicts of interest

The authors declared that there is no conflict of interest.

Author contributions

TV - Research concept and design, SG - Collection and/or assembly of data, SG - Data analysis and interpretation, SG - Writing the article, TV - Critical revision of the article, TV - Final approval of the article .