Efficacy of Streptococcus agalactiae vaccine strains N 3 M and N 4 M in fry tilapia infected by different strains of S . agalactiae

Streptococcus agalactiae is a major bacterial streptococcosis disease that infects tilapia. This study aimed to analyze a specific and nonspecific immune system in fry tilapia that has been given with S. agalactiae vaccine from N3M and N4M strain and examine the protective immunity against S. agalactiae N3M, N4M, N17O, NK1, and N14G strains infection. Fry tilapia used in this study has the weight of 7.086±0.948 g and length of 7.443±0.353 cm. The S. agalactiae strains that used were N3M, N4M, N17O, NK1, and N14G. Fry tilapia was vaccinated through intraperitoneal injection method with 0.1 mL per fish of N3M and N4M vaccines. Fish reared in aquarium sizing of 60×30×50 cm3 with a density of 10 fishes/aquarium. Two weeks after vaccination, fry tilapia was tested with 0.1 mL/fish of each N3M, N4M, N17O, NK1, and N14G strains through intraperitoneal injection method. Antibody level measured with indirect enzym-linked immunosorbent assay (ELISA) method. The result of antibody level in vaccinated fish after vaccination of N3M, N4M vaccine, N3M and N4M control were 0.767; 0.743; 0.587; and 0.544, respectively. Relative percent survival in N3M vaccinated fish after challenged with N3M and N4M was 87.50% dan 64.70%, respectively, otherwise in N4M vaccinated fish was 62.50% dan 76.47%, respectively. N3M and N4M vaccine strain have better protection as only if it tested with similar bacteria strain.


INTRODUCTION
Development of intensive tilapia fish farming caused disease problems, such as the viral, bacterial, and parasitic disease which needed to get more attention. Disease outbreak in fish was caused by a low fish defense, endurance, feed quality, and water quality (Giordano et al., 2010). One of the bacterial diseases which often attacked tilapia was streptococcosis (Sheehan et al., 2009). Streptococcosis was a disease caused by bacterial pathogen Streptococcus sp. (Abuseliana et al., 2011). Indonesia became one of the countries exposed mass mortality on tilapia farming as the result of streptococcosis outbreaks (Taukhid & Purwaningsih, 2011). Tilapia intensive culture had a high risk of great loss as streptococcosis disease caused more than 90% mortality from the population (Ye et al., 2012).
Tilapia fish which was infected by Streptococcus agalactiae suffered exophthalmia, opacity, purulent, whirling, discoloration, abdominal abscess, and clear operculum (Hardi et al., 2011). Isolation of S. agalactiae from some regions in Indonesia which were discovered from streptococcosis disease outbreak obtained some isolation strains, such as N3M (eyes), N4M (eyes), N17O (brain), N14G (kidney) came from Cirata and NK1 (brain) came from Klaten (Hardi et al., 2013). S. agalactiae was the main bacteria which attacked various freshwater, brackishwater, and marine fish in the tropical and subtropical region, infecting fry until adult stadia (Bowater et al., 2012). Some steps that have been made to control this disease outbreak were the use of antibiotics and other chemical products. However, these treatments had an impact on the environment and human health in a long period (Giordano et al., 2010). One way to prevent a disease outbreak is vaccination (Chen et al., 2012). This activity would help form specific and non-specific immune system. Vaccination also formed a longterm memory for the immune system, therefore it only took one or two times for the treatment of fish (Barman et al., 2013).
Vaccination on tilapia fish had been tested to be capable to enhance specific antibody formation and to protect from S. agalactiae infection (Giordano et al., 2010;Chen et al., 2012;Hardi et al., 2013). However, S. agalactiae from the isolation result contains too many strains, producing various type of vaccine. Every vaccine from S. agalactiae strain has a different protection mechanism. Hardi et al. (2013) reported that fish which was given S. agalactiae N14G strain after challenged with N14G and NK1 strain produced relative percent survival as much as 79% and 75% respectively, while fish vaccinated with NK1 strain, then challenged with N14G and NK1 strain produced relative percent survival as much as 62.5% and 75%, respectively.
Fish that had been vaccinated by the different strain of S. agalactiae showed crossed protection in the sampled tilapia (Sukenda et al., 2014). Various vaccine type made the vaccine was difficult to choose for protecting the fish from S. agalactiae exposed strain. Information about the effectivity of a vaccine for protecting the fish from S. agalactiae strain infections is necessary to be discovered. This study aimed to analyze the specific and nonspecific immune system on the tilapia fry which was given S. agalactiae strain N3M and N4M, as well as investigating the protective immunity against S. agalactiae N3M, N4M, N17O, NK1, and N14G strain infection.

Streptococcus agalactiae bacteria
Bacteria of S. agalactiae from N3M, N4M, N17O, N14G isolates were obtained from the Center for Research and Development of Freshwater Aquaculture, Bogor, West Java, Indonesia. Five strains of S. agalactiae bacterial virulence was recovered with Koch's postulate test. Bacteria were cultured in brain heart infusion broth (BHIB) liquid media for 72 hours on 140 rpm shaker and injected on fish as much as 0.1 mL in the intraperitoneal body part. Bacteria were isolated from the fish suffered the clinical signs of S. agalactiae. Isolation result was purified and characterized with Kit API 20 Strep.

Vaccine production
Vaccine production from S. agalactiae N3M and N4M strain were cultured in the liquid media for 72 hours (10 9 CFU/mL bacterial density), added with 38% neutral formalin buffer (NBF) as much as 3% of the liquid culture volume, and incubated for 24 hours. Bacteria were harvested using 7000 rpm centrifugation for 30 minutes and washed with phosphate buffered saline (PBS) twice, then added with PBS based on the initial volume. The vaccine produced was tested its viability level on brain heart infusion agar (BHIA) media.

Tank preparation and experimental fish rearing
As many as 48 pieces of aquarium tanks sized (60×30×50) cm 3 with set aeration on each tank were prepared. Aquarium was washed and filled with water reaching 25 cm height, then disinfected along with the media using 30 mg/L of chlorine (Ca(ClO)2) for 24 hours and neutralized with sodium thiosulfate (Na2S2O3) as much as 15 mg/L. As many as 10 fish on each aquarium was maintained and given commercial feed three times a day with satiation. Water media was replaced as much as 75% of the volume every three days. Water quality maintained during the rearing process was 25.8-26.6°C of temperature, 6.7-7.1 of pH, 5.26-6.23 of dissolved oxygen, and 0.0019-0.0038 of TAN.

Parameter
Parameters observed in this study were mortality rate, relative percent survival, antibody level (ELISA), blood characterization (erythrocyte total, leucocyte total, hematocrit, hemoglobin, phagocytic activity, and respiratory burst), lysozyme activity, and clinical signs.
Blood characterization, antibody levels, and lysozyme activity parameter were observed before vaccination, two weeks after vaccination, and a week after the challenge test. Mortality range and relative percent survival were observed two weeks after challenge.

Relative percent survival
Relative percent survival (RPS) was calculated to measure the vaccine protection level against S. agalactiae infection on the fry sample. The relative percent survival was calculated on the following formula.

Antibody level
Antibody level was measured using an indirect method of enzyme-linked immunosorbent assay (ELISA), based on Sumiati et al. (2015). S. agalactiae antigen was obtained from the sonication result. Protein concentration on antigen was measured with Bradford spectrophotometry method and diluted with carbonate-bicarbonate buffer (pH 9.6) until reaching 10 µg/mL concentration. As much as 100 µL diluted antigen was put into the microtiter plate wells and incubated for 12 hours at 4°C. The microtiter plate was washed with PBS-T (PBS, pH 7.4 + 0.05% Tween-20). Bovine serum albumin (BSA) 3% in aquabidest w/v was added 100 µL on the well and incubated at 25°C for an hour, then washed with PBS-T. Tilapia serum samples were diluted with 1:50 PBS-T, added to the microtiter plate wells in accordance with 100 µL antigen. The microtiter plate was incubated at 25°C for an hour and washed with PBS-T. A hundred µL long  chain immunoglobulin of anti-tilapia for a specific polyclonal antibody which had been diluted 1:200 (v/v) with PBS-T was added on the microtiter plate well and incubated 25°C for an hour, then washed with PBS-T. Peroxidase-conjugated goat anti-rabbit diluted with PBS-T 1:15,000 was added 100 µL onto the microtiter plate wells and incubated at 25°C for 1 hour. The microtiter plate was washed with PBS-T and added with 100 µL of One Step Ultra-TMB-ELISA (TMB 5 mg + 10 µL 38% H2O2 in 5 mL of acetate buffer pH 5), then incubated at 25°C for 15 minutes. ELISA reaction was stopped with the addition of 100 µL H2SO4 3 M into microtiter plate wells. Optical density on the microtiter plate was read using a Microplate Reader (Kayto RT-2100C) with 450 nm wavelength. The cut off value (CV) was determined as the antibody formation standard. The optical density with a higher value than CV would represent antibody existence. CV value was determined with the following formula:

Erythrocyte total
Blood which was taken from the fish sample was absorbed using red pulpy Sahli pipette until scale 0.5 and diluted using Hayem's solution until reaching scale 101. Blood solution was homogenized by shaking the pipette forming number eight for 3-5 minutes. One drop of blood was thrown to eliminate the unmixed blood part and closed with cover glass. Total erythrocyte was calculated using :

Hemoglobin
Hemoglobin was measured based on Sahli method using haemometer. Hemoglobin tube was filled with 0.1 N HCl until reaching scale 10 on the red scale. Blood was taken using Sahli pipette until scale 20 and put into the hemoglobin tube, then steadied for 5 minutes. The solution was added with aquadest until the solution color was the same as the standard color. Hemoglobin level was read on the yellow scale (g/dL).

Hematocrit
Hematocrit was measured to determine the blood cell percentage existed in the fish blood. Fish blood was taken in the microhematocrit tube until ¾ part of the tube, then the tube tip was closed using crytoceal. Tube filled with fish blood was centrifuged using 3000 rpm for 5 minutes. Hematocrit content was determined with the following formula:

Leucocyte total
Blood was absorbed using white pulpy Sahli pipette until scale 0.5 and added with Turk's solution until scale 11, then homogenized for 3-5 minutes by shaking the pipette to form a number eight. One to two solution drops from the pipette was thrown and another one was dropped on the haemocytometer. Leucocyte total was calculated with :

Phagocytic activity (PA)
Staphylococcus aureus bacteria with 10 7 CFU/ mL was added on the blood in the sterile microtube with 1:1 ratio and incubated for 20 minutes at the room temperature. The blood sample was spread thinly on the object glass and dried at the room temperature. The blood sample was fixated using methanol 95% for 5 minutes. The blood sample was dried and colored with Giemsa 5% for 15 minutes, then cleaned with aquadest. The phagocytic activity on blood sample was observed using a microscope and counted 100 phagocytic cells (monocytes and neutrophils). Observation result was calculated with the following formula:

Respiratory burst (RB)
Respiratory burst measurement was based on the nitroblue tetrazolium (NBT) reduction existence as the anion superoxide. The blood sample was put into the microtiter plate as much as 50 µL and incubated at 37°C for 1 hour. Blood inside the microtiter plate was thrown and cleaned with PBS pH 7.4 three times. 100 µL NBT (0.2% in PBS pH 7.4) was added in the microtiter plate and incubated at 37°C for 1 hour, then cleaned using PBS. Methanol absolute was added as much as 100 µL and kept steady for 10 minutes, then thrown and cleaned with methanol 30% three times. A sample in the microtiter plate was added with 60 µL KOH 1 M and 70 µL dimethyl sulfoxide (DMSO). Respiratory burst value was determined from the optical density read using Microplate Reader (Kayto RT-2100C) on 630 nm wavelength.

Lysozyme activity
A hundred µL blood serum was put in a microtiter plate and added with a liquid suspension of Micrococcus lysodeikticus (Sigma) bacteria as much as 100 µL (0.4 mg/mL in 0.1 M PBS pH 6.2) at 25°C. Absorbance was read using Microplate Reader (Kayto RT-2100C) with 450 nm wavelength for 30 seconds and 30 minutes mixing. Lysozyme activity measurement unit was based on the decreased 0.0001 per minute of optical density (OD) on the following formula:

Clinical signs
Clinical signs on the fry were observed after the challenge test conducted by comparing the external organ of the fry infected with and without S. agalactiae. Clinical signs observed were exopthalmus, dropsy, whirling, opacity, purulent, and discoloration.

Data analysis
Measured parameter data result was tabulated in Microsoft Office Excel 2007 and analyzed using analysis of variance (ANOVA) on Minitab 16 application with 95% confidence level (P<0.05), as well as continued Tukey test.

Mortality and relative percent survival
The mortality rate of fry vaccinated by vaccine A and B were lower significantly (P<0.05; Table  2), compared with the positive control, except vaccine B treatment with d challenge test. The best mortality range was observed at vaccine A on a challenge test with 6.67 ± 5.77%. The highest relative percent survival on every vaccine type treatment was at vaccine A on a challenge test (87.50 ± 10.82%) and B on b challenge test (76.47 ± 10.18%) that both were significantly different (P<0.05) with d challenge test.

Antibody level
Antibody level after vaccination increased significantly (P<0.05; Table 3) for two vaccine types compared with control treatment, however, showing no significant difference among vaccine

Clinical signs
Based on the observation result, the clinical signs occurred on fry tilapia were blackish body color, purulent, dropsy, opacity, whirling, and exophthalmia.

Discussion
Vaccination used formalin-killed cells vaccine of bacteria S. agalactiae strains N3M (A) and N4M (B) given was able to lower the mortality rate of tilapia fry from Streptococcosis. The mortality rate of fish fry vaccinated was significantly lower (P<0.05) compared with the positive control, except vaccine B vaccine on d challenge test treatment. The lowest mortality rates from vaccine treatments were vaccine A on a challenge test (6.67±5.77%) and vaccine B on b challenge test (13.33 ± 5.77%). The mortality rate of tilapia fry infected with the same bacteria as the vaccine was lower than the different bacteria infection, which showed that the vaccine worked specifically against homologous bacteria. Giordano et al. (2010) reported that tilapia fish mortality rate which injected with inactive bacteria of S. agalactiae was 10.74%, based on the challenge test with homologous bacteria. This study reports that vaccine challenged with different bacteria had 20-36.67% mortality rate. Shoemaker et al. (2010) reported that fish mortality rate observed from the S. iniae vaccination using four strains were ranged from 0-7.5% after the challenge test. The mortality rate of tilapia vaccinated with one type of vaccine and tested using 10 combined strains of S. agalactiae was ranged 40-80% (Chen et al., 2012). All treatments on d challenge test (NK1) had the highest mortality rate from control treatment with 60%, vaccine A treatment with 33.33%, and vaccine B with 36.67%. According to Hardi et al. (2011), the bacteria strain NK1 used to challenge test had virulence which was higher than the other. Vaccine A treatment had an averagely low mortality rate after challenged with different bacteria. The increased protection level of tilapia against streptococcosis indicated the relative percent survival. The relative percent survival of both vaccines with different challenge tests showed different results. Tilapia relative percent survival on vaccine A after a, b, c, d and e challenge test were 62.50%, 76.47%, 47.05%, 38.89%, and 52.94%, respectively. Vaccine A was capable of protecting more than 50% infection of S. agalactiae strains a, b, c, and e, while vaccine B was capable of protecting from the bacterial infection from a, b, and e strains. The vaccine produced that was homologous with bacteria gives higher protection than the different bacteria after the challenge test. Homologous antigen comes from other individuals in one species. Meanwhile the heterologous antigen comes from other individuals in other species. The difference source of antigen will usually produce different immune response compared to other (Munang'andu et al., 2016). Evans et al. (2004) showed tilapia vaccinated and challenged with the heterologous bacteria generated 25% of relative percent survival, whereas with the bacterial homologous was 70%. Vaccine A and B for homologous bacterial protection also provided heterologous infected bacterial protection, called cross-protection. Chen et al. (2012) showed three among ten vaccine types were capable of protecting from different strains and genotypes of S. agalactaie with relative percent survival ranged 53.57-100%. Vaccine protection from S. iniae infection in tilapia with four different strains showed different relative percent survival, ranging 79-100% (Shoemaker et al., 2010). Based on the result obtained in this study, vaccine A gave a lot more protection from different S. agalactiae strains.
Parameters of the mortality and relative percent survival on vaccination treatment were influenced by the specific immune system followed with a non-immune system. Vaccination performed stimulated the formation of the specific immune system shown with increased antibody level in fish. Increased antibody level was supported by the activity of T cell (

T helper cells, cytotoxic T cells, and memory T cells) and B cells (B cells and plasma B cell memory). The interaction between antigen and lymphocytes (B cells and T cells) caused an increased level of antibodies through vaccination. The vaccine contained antigens which induced the proliferation of B cells into memory B cells and plasma B cells. The role of memory B cells stored information characteristics
of the antigen into the body, while the plasma B cells played a role in the production of specific antibodies against these antigens. Incoming antigen was phagocytized by macrophages, the process of antigen phagocytosis would transfer helper T cells via major histocompatibility complex (MHC) class II. Macrophage would activate helper T cells through cytokine molecules. Helper T cells produced cytokines that stimulated B cells, memory T cells, and cytotoxic T cells. The same antigen that penetrated the body for the second time would stimulate the role of memory T cells and memory B cells to conduct proliferation into the plasma cells as well as producing antibodies (Mashoof et al., 2016).
Antibody levels after vaccination on both vaccine treatments happened to conduct an increased level of specific antibodies against S. agalactiae strain A and B. This was shown on the value of vaccine A (0.767 ± 0.026) and B (0.743 ± 0.026) optical density (OD) which were higher than any vaccine the cut of value (CV). Both vaccine treatments showed a significant increase (P<0.05) compared with the control treatment. Martins et al. (2011) also reported increased antibody level in tilapia serum compared with control treatment, after vaccinated with vaccines S. iniae. The antibody level after challenge test on vaccine A and B treatment was significantly higher (P<0.05) than positive and negative control treatment as well as higher than CV. OD value on vaccine A after a,b,d challenge test treatment and vaccine B after all challenged tests were increased compared with OD value after vaccination. Increased OD value after challenge test also occurred on Sukenda et al. (2015), who reported that measurable antibodies were correlated with the relative percent survival. Increased OD treatment on vaccine A after all challenge tests were not significantly different (P>0.05) with vaccine B after a, c, d and e challenge test. Measurement of antibody level after challenge test using the same vaccine antigens, making the measured OD value was obtained from the antibodies formed from the same antigen and vaccines. This caused the OD value after challenge test was not much different to the value OD after vaccination. This result was on the contrary from Shomaker et al. (2010), who used the same antigen as a vaccine to testify the antibody level of fish vaccinated with S. iniae vaccine strain ARS-98-60 which was challenged with four different strains showed an increased antibody level. This was suspected to happen as of the protein profile of the five strains of S. agalactiae bacteria were used. Shomaker et al. (2010) stated that bacterial strains used had protein profile similarities inducing the antibody formation that allowed cross-protection. Based on antibody level measurement in this study, vaccine A had OD value that was higher after the challenge test treatments. The nonspecific immune system also had a role in mortality and relative percent survival which could be shown from hereditary immune system and hematology parameter (Sukenda et al., 2017). Leucocyte total is one of the hematology parameters on the nonspecific immune system. Leucocyte total measured on vaccine A and B (2.23-2.47×10 4 cell mm ˗3 ) treatment after vaccination were significantly higher (P<0.05) than the control treatment, nevertheless showing no significant difference (P>0.05) on the vaccination treatments. Suanyuk and Itsaro (2011) also reported that the leucocyte total which had no significant difference between vaccination treatments. Leucocyte total on vaccine A and B treatment after all challenge tests (6.33-10.47×10 4 cell/mm 3 ) were significantly higher (P<0.05) than the negative control. The vaccine was suggested as an antigen penetrating the fish body which was responded by producing leucocyte and forming an antibody, though it was less effective during the initial infection, or in this case was vaccination period (Sukenda et al., 2014). Increased leucocyte total after the challenge test showed that the fish body still defended against the infection. The highest leucocyte total was observed at vaccine A on c challenge test and vaccine B on b challenge test treatment. According to Hardi et al. (2013), vaccination was not yet completely capable of helping eliminate the pathogen existed in the body, which was marked as high leucocyte total after the challenge test.
Leucocyte total is related to the phagocytic activity. Phagocytic activity on vaccine treatment A and B (32.33% and 34%) was significantly higher (P<0.05) than the control treatment after vaccination. Sukenda et al. (2018) showed that tilapia fish which were given FKC S. agalactiae vaccine differed significantly against the phagocytic activity. Improvement of phagocytic activity was in line with the significant increased of total leucocytes compared with control treatment. Phagocytic activity experienced an increased immune system, showing bacterial infection resistance after the challenge test. This was also supported by the leucocyte total and antibody level which were quite high after the challenge test. Formed antibodies after vaccination would accelerate the antigen elimination under opsonization process. Opsonized antigen will be more recognizable for macrophages as well as more effective to be destructed (Hardi et al., 2013).
The activity of phagocyte in destroying pathogen was indicated with the respiratory burst parameter. According to Uribe et al. (2011), the respiratory burst was produced by neutrophils and macrophages in the phagocytosis process to attack pathogens by producing reactive oxygen species (ROS). Phagocyte cells that successfully ingested pathogens would expose ROS as well as enzymes for pathogen digestion (Holmstrom & Finkel, 2014). The number of phagocyte cells exposed ROS was measured by NBT reduction method to become formazan in the form of optical density (OD). In this study, the value of the respiratory burst after vaccination vaccine treatment A and B did not differ significantly (P>0.05) with the control. Aly et al. (2015) also reported similar results with FKC vaccination on tilapia with Aeromonas hydrophila on respiratory burst parameter after vaccination. The value of the respiratory burst was significantly higher (P<0.05) on the vaccine treatment A and B after all challenge test treatments test challenge than control treatment after on b, c and e challenge test treatments. Pridgeon and Klesius (2013) showed an increased production of ROS by macrophages of fish vaccinated against different bacteria after two and five minutes incubation compared with controls measured using the chemiluminescence assay method. The value of respiratory burst increased along with the phagocytic activity on the same treatment.
Nonspecific immune response associated with the lysozyme activity is leucocytes. Lysozyme is an important component in the immune system that separates a peptidoglycan layer cell wall of Gram-positive bacteria for preventing from infection. The measurement of lysozyme was commonly used as an indicator for fish vaccination evaluation (Yi et al., 2014). This study discovered increased lysozyme activity after vaccinations on treatment A and B, which were significantly different (P < 0.05) with control treatment. Pasaribu et al. (2018) reported an increased lysozyme activity in the tilapia broodstock after vaccination was followed by its descendants. The lysozyme activity on vaccine A and B treatment was higher than the positive and negative control treatment. The increased activity of lysozyme would enhance the antibacterial ability in tilapia (Huang et al., 2013).
Nonspecific immune response parameter that affected the fish health status is the erythrocyte total. Measured erythrocyte total after vaccination showed a significantly lower decline on vaccine B treatment (P<0.05) than control treatment, while vaccine A treatment was not significant difference compared with the control treatment (P>0.05). Martins et al. (2011) reported that the erythrocyte total of tilapia fish after vaccinated using S. iniae vaccine had declined result with no significant difference against the control treatment. Total erythrocyte decline was suspected because the vaccine given was suggested as an antigen, making the fish become more stressful along with the declined erythrocyte production and increased leucocyte production. Every treatment showed higher erythrocyte total compared with the control treatment. Different bacteria used for challenge test did not influence the erythrocyte total. According to Evans et al. (2005), vaccination was able to minimalize fish stress infected by the bacteria, making the energy needs to protect from S. agalactiae was less than the vaccinated fish.
Hemoglobin parameter was related with the erythrocyte total which functioned as oxygen binder that was able to bring in all body parts. Hemoglobin level on vaccine B treatment had no significant difference (P>0.05) with control treatment after vaccination. Martins et al. (2011) reported that the hemoglobin level was not significantly differencing with the control treatment, which had a correlation with erythrocyte total and hematocrit. Hemoglobin level on vaccine A treatment after a,c,e challenge test and vaccine B treatment after e challenge test were significantly different (P<0.05) with the control treatment after a challenge test. Normal hemoglobin level of fish was 5.8±1.04% (Lourenco et al., 2012).
Fish health status parameter associated with erythrocytes is hematocrit. Hematocrit shows the percentage of the total volume of blood cells in fish. Hematocrit on vaccine A and B after vaccination did not differ significantly (P<0.05) with the control. Suanyuk and Itsaro (2011) reported that hematocrit level on tilapia vaccinated with S. iniae vaccine was not significantly different with the control treatment. Vaccine treatment had higher hematocrit level compared with positive control. Martin et al. (2011) also reported that the vaccinated fish showed higher hematocrit level during 18 days after challenge test compared with the control treatment.
Clinical signs due to the infection of S. agalactiae strains N3M, N4M, N17O, NK1, and N14G were black skin color change, opacity, dropsy, purulent, whirling and exophthalmia (Figure 1). Clinical signs generally appeared on the fifth day after the challenge test. Based on the research of Hardi et al. (2011), clinical signs appeared after infection were shrunk pupil, opacity, purulent, exophthalmia, followed with bleeding, blackened discoloration, stomach ulcer, abscess, whirling and gasping. These signs appeared after 24 hours after the challenge test.

CONCLUSION
Based on the result obtained, it concluded that tilapia fry after vaccinated with S. agalactiae N3M dan N4M strain showed the increased level of antibodies, leucocyte total, phagocytic activity, respiratory burst, and lysozyme activity. Vaccine S. agalactiae N3M strain was capable of protecting the fry from N3M, N4M, N17O, and N14G strain, while N4M strain vaccine was only capable of protecting the fry from N3M, N4M, and N14G strain.

ACKNOWLEDGMENTS
We would like to thank the Directorate General of Higher Education who has helped fund this study, laboratory of Aquatic Organisms Health of FPIK IPB for the facilities assistance provided during the research and Center for Research and Development of Freshwater Aquaculture, Bogor, West Java, Indonesia for providing Streptococcus agalactiae bacterial isolates.