The use of immunostimulant from phycocyanin of Spirulina platensis to control motile aeromonad septicaemia (MAS) disease in common carp Cyprinus carpio Pemanfaatan imunostimulan fikosianin dari Spirulina platensis untuk mengatasi penyakit motile aeromonad septicaemia (MAS) pada ikan mas Cyprinus car

Motile aeromonad septicaemia (MAS) is a major disease in common carp Cyprinus carpio caused by Aeromonas hydrophila. This study aimed to evaluate the performance of phycocyanin imunostimulant extracted from Spirulina platensis to control MAS disease in common carp. This study was conducted into two phases. First phase was conducted by adding 150 mg/kg, 250 mg/kg, 350 mg/kg feed phycocyanin dose, and 0 mg/kg feed phycocyanin dose as control treatment. Fish was challenged with pathogenic A. hydrophila after 14 days rearing. Second phase was conducted by applying the best dose obtained from the first phase added in the feed for feeding the fish in one week/month, two weeks/month, three weeks /month, and two weeks/month with one week interval. Fish was challenged with pathogenic A. hydrophila after 28 days rearing. First phase study result showed that the relative percent survival (RPS) for fish fed 150 mg/kg, 250 mg/kg, and 350 mg/kg phycocyanin dose were 87.50%, 81.25%, and 75.00% respectively. Total erythrocytes, hemoglobin, total leucocytes, phagocytic activity, and respiratory burst showed higher results than control treatment on all treated fish. The second phase study showed that fish fed one week/month, two weeks/month, three weeks/month, and two weeks/month with one week interval had RPS value 65.38%, 69.23%, 76.92%, and 69.23% respectively. The immune responses of treated fish were higher than control treatment, as well as the number of pathogenic A. hydrophila in the liver, kidney, and intestine. Fish fed with phycoyanin dose 150 mg/kg feed and three weeks/month administration had the highest RPS value.


INTRODUCTION
Aeromonas hydrophila is one of the pathogenic bacteria that attacks fish both in the culture environment and common water (Shayo et al., 2012). These bacteria are opportunistic and can be deadly when environmental conditions are in a deteriorated state. The infection type of A. hydrophila is acute, chronic, until latent by forming a septic infection, which is better known as hemorrhagic septicaemia disease or motile aeromonad septicaemia (MAS) (Ismail et al., 2010). The clinical symptoms of acute infection are the occurrence of systemic inflammation, resulting in fish death after 24 to 48 hours of infection. A. hydrophila bacterial infection causes tissue swelling, dropsy, necrosis, ulcer, bleeding (hemorrhagic), then massive mortality reaching 90-100% in one to two weeks of the infection period (Lukistyowati & Kurniasih, 2011). A. hydrophila infection invades a variety of freshwater fish culture, including the common carp, Cyprinus carpio, which is one of the important fish species in fish culture industries (Jeney et al., 2009).
One of the preventive efforts in A. hydrophila infection that is safe, effective, and environmental friendly is immunostimulant utilization obtained from the natural ingredient. Immunostimulant is a chemical compound, drug, or other substances that is capable of enhancing non-specific immune responses. The use of immunostimulant from natural materials currently has demonstrated its ability to increase the disease resistance of fish through non-specific immune system mechanisms enhancement (Fauziah et al., 2015). One of the natural ingredients that have an immunostimulatory effect is Spirulina platensis (Satyantini et al., 2016).
Spirulina platensis is a filamentous unicellular cyanobacterium, which belongs to the prokaryote algal group containing chlorophyll-A. Cyanophyceae is also called as blue-green algae due to the presence of phycocyanin and phycoerithrin, which gives the chlorophyll color (Usharani et al., 2012). Spirulina has also contained high protein content, which is 50-70%, besides antioxidant, anti-inflammatory, and antibacterial properties, which can improve the immune function (Wu et al., 2016). Phycocyanin extract of S. platensis is an antibacterial agent against Gram-negative and Gram-positive bacteria, besides affecting the stem cells in the spinal cord (Sarada et al., 2011). Based on several studies, the administration of S. platensis successfully stimulated the non-specific immune to tilapia (Ragap et al., 2012), Rainbow trout (Yegfreak et al., 2015), and growth improvement of juvenile great sturgeon (Adel et al., 2016).
A study conducted by Satyantini (2014) showed that the administration of phycocyanin extract from S. platensis with the dose 250 mg/ kg feed for 14 days increased growth, nonspecific immune response, and the survival rate of juvenile humpback grouper against Vibrio alginolyticus infection. Further studies dealing with phycocyanin bioactive compound of S. platensis as fish immune system enhancement agent needs to be done. Applied administration of phycocyanin in feed for common carp culture is expected to improve health, immune system, and resistance to disease infections. This study was conducted to testify the immunostimulatory performance of phycocyanin obtained from S. platensis in overcoming MAS disease on common carp, C. carpio. This study will obtain the optimal administration dose and duration that is able to improve common carp immune system.

Fish sample
Fish samples used in this study were common carp C. carpio, obtained from fish culturist in Ciseeng, Bogor, West Java, with average weight 8.28±0.19 g for the first phase and 8.72±0.22 g for the second phase. Fish were reared using aquarium sized 60×30×30 cm 3 in 20 cm water height with 10 fish/aquarium stocking density. Fish were acclimatized in the aquarium until showing good feeding response.

S. platensis phycocyanin extraction
The extraction of phycocyanin S. platensis was conducted based on Boussiba and Richmond (1979) with the modification of Hayashi et al. (2006). Dried S. platensis was dissolved with 0.1 M Na-phosphate pH as a solvent with 4% concentration. S. platensis was sonicated for 30 minutes and shaken for 24 hours with 140 rpm speed. The shaken solution was centrifuged on 12000 rpm speed at 4°C for 15 minutes. The supernatant was harvested and inserted into the test tube for subsequent precipitated in (NH4)2SO4 50%. The pellet was once again centrifuged on 12000 rpm speed at 4°C for 10 minutes, thus obtained the blue precipitate (blue pellets) after clearing the supernatant. Phycocyanin pellet was dissolved with 0.025 M Na-phosphate pH 7 as buffer solution and analyzed using Snakeskin Dialysis Tubing 3500 MWCO (molecular weight Ccutoff) in 0.025 M Na-phosphate pH 7 buffer solution at 4-5°C for 24 hours. The analyzed phycocyanin was frozen in the freezer under -80°C and subsequently deployed in a freeze dryer for 24 hours.

Fish feed
The feed given to the sample fish was commercial floating pellet with 39% protein content. Feed in this study was produced by the coating technique. Four feed treatments were prepared, comprising the addition of phycocyanin with the dose 150 mg/kg feed, 250 mg/kg feed, 350 mg/kg feed, and 0 mg/kg feed as a control treatment. Feed treatments and control material were mixed using egg white binder as much as 2% (v/w) and water as a solvent with 6% concentration (v/w) on the first phase study. Mixed feed was dried on the room temperature. The feed used for the second phase study was obtained from the best feed treatment on the first phase study for inducing common carp immune system.

A. hydrophila pathogenic bacteria
Bacteria used in this study was A. hydrophila (ATCC 49140) pathogenic bacteria. A. hydrophila bacteria was characterized using API 20E kit and grown on Rimmler-Shotts media. One bacterial colony from Ose needle was grown on Trypticase Soy Broth (TSB) with 5 mL volume and incubated in the waterbath shaker at 29°C with 140 rpm speed for 24 hours. A. hydrophila density obtained was 10 9 CFU/mL. This bacterial stock was serially diluted three times to obtain 10 6 CFU/mL (LD50 dose) for the challenge test.

Fish rearing
This study used six aquariums for each treatment, containing three aquariums for survival rate parameter, while other aquariums for blood sampling to obtain immune system and total plate count (TPC) parameter. Fish was fed using at satiation method with feed treatment three times at 08.00, 12.00, and 17.00 (GMT+7). Water quality condition was kept by 50% water exchange once every two days. Water quality value during the rearing period consisted of temperature 28-31°C, pH 7.2-7.6, DO 4.5-6.4 mg/L, and TAN 0.03-0.11 mg/L.

In vivo test
This study consisted of two phases, Phase one was conducted to determine the appropriate phycocyanin dose in feed, containing four treatments and three replications, namely control, 150 mg/kg feed (PF1), 250 mg/kg feed (PF2), and 350 mg/kg feed (PF3) phycocyanin dose. Fish were reared for 14 days, then intramuscularly injected (IM) with 10 6 CFU/mL A. hydrophila (ATCC 49140) on the 15 th day of rearing as much as 0.1 mL/fish. The test fish were fed commercially and observed for seven days. Phase two study was conducted to analyze the period of proper phycocyanin administration, consisting five treatments and three replications, namely control (without phycocyanin administration), one week/month (F1), two weeks/month (F2), three weeks/months (F3), and two weeks/month treatment at one week interval (F4) treatment. Fish were reared for 28 days and challenged on the day 29 of rearing using the same procedure with phase one study.

Parameter
Parameters obtained were mortality rate (MR) and relative percent survival (RPS) (Choudhury et al., 2008), which were observed after seven days of challenge test. Blood profiles observation included total erythrocytes and leucocytes (Blaxhall & Daisley, 1973), hemoglobin levels (Wedemeyer & Yasutake, 1977), phagocytic activity (Anderson & Siwicki, 1993), and respiratory burst (Singh et al., 2013). First phase study observations were conducted on the 14 th (prior to the challenge), 17 th , and 21 st day (after the challenging test) of rearing. The second phase study observations were conducted on the 28 th (before the challenge test), 31 st , and 35 th day of rearing. The abundance observation of A. hydrophila was focused on the target organs, namely liver, kidney, and intestines (Madigan et al., 2014), which was performed two days after the challenge test. Here is the calculation formula of A. hydrophila bacterial abundance.

Statistical analysis
Study design for the first and second phase used completely randomized design (CRD) with three replications on each treatment. Blood profiles, A. hydrophila abundance in the liver, kidney, and intestine obtained were analyzed with ANOVA test using SPSS.22 software program on 95% degree of confidence level (P<0.05). Significant result data was continually analyzed using Duncan's multiple range test. RPS value obtained was observed using a descriptive method.

Phase one study result Survival rate after the challenge test
The mortality rate on all phycocyanin treatments were lower than the control treatment at the end of the observation period. Relative percent survival (RPS) value after challenged with A. hydrophila indicated that phycocyanin administration with the dose 150 mg/kg feed (PF1) had the highest value with 87.50%, followed with the dose 250 and 350 mg/kg feed, which were 81.25% and 75.00%, respectively. Mortality rate (MR) and relative percent survival (RPS) of common carp after challenged with A. hydrophila are presented in Table 1. then decreased on the 21 st day on PF1, PF2, and PF3 treatment. The highest phagocytic activity on the 14 th and 17 th day was demonstrated by PF1 with 26.00 ± 0.82% and 36.00 ± 0.82% respectively, having a significant difference with control treatment (P<0.05). The highest phagocytic activity on the 21 st day was shown by PF3 with 25.33 ± 1.25%.
Respiratory burst on the 14 th day indicated that the administration of phycocyanin with the dose 150 and 250 mg/kg feed gave no significant difference at 0.31 ± 0.01 and 0.30 ± 0.01 absorbance level, contradictory with control and the dose 350 mg/kg feed ( Table 2). The highest respiratory burst value was also shown on PF1 treatment, after A. hydrophila bacterial infection on the 17th day with 0.40 ± 0.01 absorbance level, significantly different from all treatments (P<0.05). The results of total erythrocytes, hemoglobin level, total leucocytes, phagocytic activity, and respiratory burst level are presented in Table 2.

A. hydrophila abundance in target organs
The total abundance of A. hydrophila bacteria in the liver, kidney, and intestine of common carp indicated that each phycocyanin dose treatment was capable of suppressing A. hydrophila growth significantly compared to control treatment (P<0.05). A. hydrophila abundance after the challenge test is presented in Table 3.

Phase two study result Survival rate after the challenge test
The mortality rate on F3 treatment was lower than other treatments. The highest relative percent survival (RPS) value after challenged with A. hydrophila was shown on F3 with 76.92%. mortality rate and RPS value of common carp are presented in Table 4.

Immune response
Total erythrocytes, hemoglobin level, and total leucocytes after 14 days of phycocyanin administration showed a significant difference among all treatments and control (P<0.05). The highest total erythrocytes, hemoglobin level, and total leucocytes were presented on PF1 treatment with 2.80 ± 0.06 (× 10 6 cells/mm 3 ); 9.00 ± 0.00 g%, and 8.30 ± 0.26 (× 10 5 cells/mm 3 ) respectively, showing a significant difference on each treatment and control. The total erythrocytes and hemoglobin levels decreased in all treatments on two days after the challenge test, however, phycocyanin administration in feed showed a higher value than control treatment (P<0.05). Total leucocytes increased in all phycocyanin treatments significantly than control treatment (Table 2).
Phagocytic activity of common carp administered with phycocyanin in feed showed increased level on the 14 th and 17 th day of rearing,

Immune response
The observation result on the 28 th day of rearing indicated that F3 had the highest total erythrocytes, hemoglobin level, total leucocytes, and respiratory burst among other treatments (P<0.05). F3 also showed significant difference with control treatment (P<0.05). Total erythrocytes, hemoglobin level, total leucocytes, and respiratory burst observation result are presented in Table 5.

Discussion
The survival rate observation result of the first phase study after challenge test showed that the administration of phycocyanin with the dose 150 mg/kg feed (PF1) had the highest relative percent survival (RPS) compared to other treatments with 87.5% (Table 1). This condition indicates that the feed containing phycocyanin is able to be optimally absorbed by common carp, stimulating the immune system to perform the body defense process in inhibiting the pathogenic bacterial proliferation. The administration of Spirulina sp. in feed was able to increase the non-specific immune response of carp as the initial defence against antigen attack before the specific immune systems formed (Bai et al., 2014). Increased fish immune system happened as phycocyanin stimulates cells in the spinal cord, affecting the spleen cells, granulocyte macrophage-colony stimulating factor (GM-CSF), and interleukin-3 (IL-3) to form erythrocytes and immune cells system modulated by erythropoietin for erythrocytes and cytokines for immune cells (Hayashi et al., 2006).
The optimization of phycocyanin with proper dose should be followed by the efficient duration and effective administration to provide the best results on improving the fish health status. Phase two study result showed the highest RP) after the challenge test was observed in F3 with 76.92% (Table 4). Feed containing phycocyanin at the dose 150 mg/kg feed with longtime administration duration (three weeks/month) gives the best results. Dosage and administration duration are essential to produce an optimal immunity response, because excessive immunostimulatory administration can suppress fish resistance to disease and growth (Mastan, 2015). Another study result illustrated the introduction of S. platensis on tilapia at the dose 10 mg/fish for four weeks and challenged with A. hydrophila could improve the immune system and produce 80% survival rate (Ragap et al., 2012). This result was in line with Satyantini (2014), who reported that the administration of phycocyanin at the dose 250 mg/kg feed with 14 days duration in juvenile humpback grouper demonstrated better growth, non-specific cellular and humoral immune response, and resistance capability against pathogenic bacteria V. alginolyticus with 81.83% RPS value.
Blood profiles describe the fish health status. Total erythrocytes, hemoglobin level, total leucocytes, phagocytic activity, and respiratory burst can be used as indicators of the immune response. Total erythrocytes, hemoglobin level, total leucocytes, phagocytic activity, and respiratory burst showed higher value after phycocyanin administration treatments than control treatment in phase one study on the 14 th day and phase two study on the 28 th day before challenge test (Table 2; Table 5). This indicates that phycocyanin is effective as an immunostimulant material, the same as reported by Satyantini (2014). Immunostimulant has specific receptors against phagocytic cells (neutrophils, monocytes, and macrophages), which binds to receptor molecules on the circulatory surface and phagocytic tissue. This binding can increase the phagocytic activity for immune cell adaptation, attack, and digestion against pathogenic bacteria (Elala et al., 2013). Phagocytic cells have an important function in the fish body defence. Along with these activities, there is a release of molecular signals (cytokines) that can stimulate the formation of phagocytic cells. This causes elevated leucocytes, although there has not been any infection. This condition is in accordance with Kozenko and Henson (2010), who mentioned that phycocyanin affected cells in the spinal cord to produce erythrocytes and leucocytes. Satyantini et al. (2014) also added that the administration of phycocyanin with the dose 250 mg/kg feed enhances the number of leucocytes and phagocytic activity in juvenile humpback grouper. Reduced total erythrocytes and hemoglobin level were observed on the 17 th day in phase one study and 31 st day in phase two study after the challenge test (Table 2; Table 5). Kumar and Ramulu (2013) stated that A. hydrophila bacteria has aerolysine and β-hemolysine toxin which can suppress total erythrocytes and hemoglobin levels, resulting in anemia and hematopoetic organ disorders. There was a strong correlation between erythrocytes and hemoglobin level, whether decreased erythrocyte number would result decreased hemoglobin level. However, the phycocyanin treatment in both phases showed significant better value than control treatment (P<0.05). Enhanced total erythrocytes and hemoglobin level occurred again on the 21 st day in phase one study and day 35 th in phase two study. This indicates the occurrence of the recovery process after bacterial infection in fish with the help of phycocyanin compound.
Phase one and two studies after the challenge test occurred increased level of total leucocytes, phagocytic activity, and respiratory burst ( Table  2; Table 5). This was due to the resistance mechanism from the fish body against A. hydrophila infection by suppressing the bacterial proliferation. This was demonstrated in the phase one study which showed lower abundance level of bacteria in the liver, kidney, and intestine observed on all treatments compared to control treatment (Table 3). Phase two study also showed reduced bacterial abundance observed on all duration treatments compared to control (Table  6). According to Janda and Abbot (2010), A. hydrophila systemic infection that causes acute death occurs at 24-48 hours after bacterial exposure, showing clinical symptoms, such as septicaemia, ascites, ulcer, followed with the liver, spleen, and kidney damages in postmortem phase. Abdel-Tawwab and Ahmad (2009) implied that there was a decreased number of A. hydrophila in the liver and kidney organ of tilapia, after the administration of S. platensis.
Increased total leucocytes after the challenge test is an attempt to phagocytize pathogenic bacterial cells entering the fish body of fish, thus reducing bacterial growth and development (Kurniawan et al., 2014). Control treatment showed lower total leucocytes compared to phycocyanin administration treatments. This shows that the administration of phycocyanin in common carp provides non-specific immune system enhancement characterized by elevated leucocyte production to attack pathogens along with increased phagocytic activity and respiratory burst. Increased respiratory burst value indicated that there is a lot of pathogen exposure in the fish body, triggering the phagocytic activity to attack the pathogenic microbes. Phagocytic cells that perform phagocytosis activity produce H2O2 anions and superoxide (O2 -) that are highly toxic to bacteria, thereby increasing the ability of phagocytes to destroy pathogenic bacteria (Rawling et al., 2012).

CONCLUSION
Phycocyanin administration with the dose 150 mg/kg feed and three weeks/month duration provides the best results for improving survival rate and non-specific immune response of common carp against A. hydrophila bacterial infection.