The comparative studies of Borneo plant extracts to increases vaccine efficacy in tilapia , Oreochromis niloticus

This study investigated the adjuvant effect of Boesenbergia pandurata (BP), Zingiber zerumbet (ZZ), Solanum ferox (SF) on protection of tilapia with injection Pseudomonas sp. (Pseumulvacc) vaccination. The extract concentrations of BP (600 mg/L), ZZ (200 mg/L), and SF (900 mg/L) were combined with the vaccine, ratio between vaccine and extract was 1:1. Tilapia fish were injected with vaccine mix the extract and challenged at day 7 (d7), 14 (d14), and 21 (d21) post vaccination through injection with A. hydrophila and P. fluorescens (105 CFU/ mL each bacteria). The results shown that the fish with BP+V were found in fin rot at d14 days challenge. The same symptom was found in ZZ+V at d14 challenge as much 11.11% and 42.86% while, in the vaccine groups (V), after the challenge, tilapia were found fin rot and darkness color until the last experiment. The BP+V and SF+ZZ+V groups shown reducing the number of bacteria in the fish body after challenge test on d7, d14, and d21. The efficacy of Pseumulvacc vaccine has increased after its administration with BP (BP+V) on day 7 and day 14 after challenge (90%) and 100% at the time of challenge test d21. The conclusion is B. pandurata extract was a promising adjuvant candidate, and the extract is the best plants as an adjuvant that mixed with the vaccine to against A. hydrophila and P. fluorescens infection.


INTRODUCTION
The cultivation of tilapia grows rapidly, followed by the used for its rich source of protein (Wang et al., 2016), excellent gelatin source taken from the bone (Alfaro et al., 2013), or skin and scales (Jamilah & Harvinder, 2002).It triggers the increase of intensive aquaculture production, which leads to the increasing number of mortality caused by pathogens, disruption of cultivated land sustainability, and the slowing growth (Kibenge et al., 2012).
Vaccination is a more environmental friendly in pathogen prevention technology compared to the application of antibiotics and other drugs (Sommerset et al., 2005;Rodger, 2016;Sukenda et al., 2018).In general, there are three methods for immunostimulant or vaccine application in fish include injection, immersion, and feed.Pasnik et al. (2005); Hardi et al. (2013);and Evensen (2016) stated that vaccination through intraperitoneal injection (IP) has a better efficacy rate than two other methods.
Giving the vaccine in combination with oilbased adjuvants would potentially improve vaccine efficacy in fish.Some of the adjuvant materials used in fish include Freund's adjuvants used with fish vaccine (Bøgwald & Dalmo, 2012;Dalmo et al., 2016), EseD a Putative T3SS (Edwardsiella tarda has a type III secretion system) (Wang et al., 2010;Jiao et al., 2010b); Streptococcus agalactiae and Aeromonas hydrophila (Pasaribu et al., 2018).In addition, the use of other adjuvant mineral oils reported being effective in increasing the performance of Moritella viscosa vaccine and Aeromonas salmonicida in salmon (Mutoloki et al., 2010).Furthermore, non-oil material which also acts as adjuvant is aluminum salts (Jiao et al., 2010a), β-Glucans (Dalmo & Bøgwald, 2008;Mizel & Bates 2010); plant extract Quillaja saponaria saponin (Wang et al., 2016); a combination of Ocimum sanctum plant extract (Tulsi), Withania somnifera (Ashwagandha), Tinospora cordifolia (Guduchi), and Emblica offcianalis s (Amlaki) (Priyadarshini et al., 2012).Saponin is steroids and terpenoid glycosides produced by several species of plants (Song, 2009;Sun et al., 2009;Tafalla et al., 2013) and could serve as specific adjuvants (Dalmo et al., 2016).This material has immunostimulant capabilities for animals including fish; is able to increase macrophage cell phagocytosis activity, antibody production, and produce cytotoxic T-lymphocytes (CTLs) which can inhibit exogenous antigen (Zhang et al., 2007;Xie et al., 2008;Tam & Roner, 2011;).According to Freitas et al. (2006) and Wang et al. (2016), the saponins from the Q. saponaria plant are non-toxic and capable of enhancing the specific immune system and tend to be immune protective in mixing with the Leishmania donovani vaccine.Saponin testing as an adjuvant has also been performed by Wang et al. (2016), the result indicated that the Vibrio anguillarum vaccine combined with Q. saponaria could enhance humoral antibody responses and increases the protective level of turbot (Scophthalmus maximus) fish after bacterial infection.
The Borneo plant extract of Boesenbergia pandurata (BP), Solanum ferox (SF), and Zingiber zerumbet (ZZ) are plants which easily grown in the yard and used as herbs plants by communities in east Kalimantan (Borneo).Based on previous research, that plant extracts containing saponins could serve as immunostimulants for tilapia (Hardi et al., 2016a,b andHardi et al., 2017a,b).This study aimed to obtain information and test the associated potential adjuvant of B. pandurata extract, S. ferox, and Z. zerumbet in improving the efficacy of Pseumulvacc vaccine in tilapia.

Experimental fish
The experimental fish sized about 15 g was tilapia originated from Seed Fishery Seedling Center Sebulu, Kutai Kartanegara District, which previously quarantined and isolated to ensure A. hydrohila and P. fluorescens free.The experimental fish was checked with A. hydrophila and P. fluorescens through isolating the gill and kidney in GSP media then incubated at 30°C for 18-21 hours.If the bacteria was not growth, the fish was ready to be used for this study, otherwise, if the bacteria was growth, the fish was immersed with formaline solutions 3% for five minutes (Kent et al., 2009).

Plant material extraction
Boesenbergia pandurata, Z. zerumbet, and S. ferox herbal materials were collected from the traditional market in Samarinda.The extraction process carried out using Limsuwan and Voravuthikunchai et al. (2008) and Hardi et al. (2016a) methods.The first step in extraction methods was cutting the plants into smaller pieces, oven-dried for four days or until dry and the air-dried plant samples were mashed using a blender.The dried samples were soaked in ethanol (96%) at room temperature with the ratio 1:1 for 48 hours.The extract solution was filtered with Whatman ® filtration paper and the filtered sample was centrifuged for 24 hours at 50 rpm to obtain a crude extract.The last step was to keep the crude extract in the oven (30-40 o C) until the ethanol lost from the extract, then the extract were kept in the refrigerator at -4 o C until used.The extract concentrations used were 600 mg/L B. pandurata, 900 mg/L S. ferox and 200 mg/L Z. zerumbet (Sun et al., 2016a,b andHardi et al., 2017a,b).The dose was achieved by diluting it using a sterile distilled water.

Bacteria test
Aeromonas hydrophila (EA-01) and P. fluorescens (EP-01) used as a bacterial test challenge, and Pseudomonas sp. as a bacterial vaccine came from the Microbiology Laboratory, Faculty of Fisheries and Marine Science, Mulawarman University, Indonesia.Bacteria were grown in the media brain heart infusion broth (BHIB DIFCO ® ) and brain heart infusion agar (BHIA, DIFCO ® ) for 24 hours at 30 o C.
The vaccine (Pseumulvacc) for Pseudomonas sp.bacteria was produced by inactivating using 3% formalin for 24 hours.The density of bacteria vaccine used was 10 4 CFU / mL (Hardi et al., 2014b).
The bacteria for the challenge test used were a combination of A. hydrophila and P. fluorescens 1:1 ratio with the density of each bacterium 10 5 CFU/mL (Hardi et al., 2017a).The administration was intramuscularly injected of 0.1 mL/fish (Hardi et al., 2014a).

Effectiveness test of B. pandurata, S. ferox, and Z. zerumbet as an adjuvant of Pseumulvacc vaccine
Experiment test was conducted by mixing each extract with a vaccine ratio 1:1.This experiment consisted of 6 groups: (1) extract of B. pandurata mix with vaccine (BP + V); (2) Solanum ferox extract mix with vaccine (SF + V); (3) Zingiber zerumbet extract mix with vaccine (ZZ + V); (4) concoction S. ferox and Z. zerumbet mix with vaccine (SF + ZZ + V); (5) Vaccine without extract (V); (6) control group.This extract that using as an adjuvant in this research based on the preresearch, the concoction S. ferox, and Z. zerumbet was the best extract as an immunomodulatory extract than other concoction (B.pandurata and S. ferox or B. pandurata and Z. zerumbet).
Vaccine plus extract was injected to fish by intraperitoneal injection as many of 0.1 mL/fish.Challenge test was conducted on days 7, 14, and 21 post vaccination.The observations parameters were the percentage of fish undergoing changes in the anatomy of the external organs and internal organs, the total number of bacteria in the body of the tilapia fish, the total leucocyte, antibody titers, phagocytic index, and cumulative of mortality and RPS (relative percent survival).

Fish pathology anatomy
This parameter was done to evaluate the percentage of fish pathology anatomy post vaccination and challenge test.The observed fish pathology anatomy were fin rot, body darkness, and exophthalmia.The percentage of fish pathology anatomy was calculated according to Hardi et al. (2017 a,b) formula.

Total bacteria
Record the results for the total bacteria in the fish body after challenges test, calculate the mean colony count of five sample.As much 1 g of sample organ was crushed, then put into 9 ml of sterile distilled water and continued by dilution 7, 8, 9, and 1 ml of suspense were grown on TSA agar medium, incubated at 30°C for 24 hours.The number of bacterial cells is calculated using colony number as Hardi et

Total leucocyte
Total leucocyte describes the number of leucocyte cells in the fish body after vaccine and challenge test with pathogenic bacteria (Blaxhall, 1972).The first step of total leucocyte examination has collected the blood from the fish, put into microtube and then the blood sample was sucked with a leucocyte pipette up to 0.5 and added Turk's solution into 11 scales, wiggling the pipette to % Fish pathology anatomy

Fish pathology anatomy
Alive fish in the end of treatment homogeneously.Remove the first droplet, the next inserted the blood mix Turk's solution into the hemocytometer and cover with a cover glass, put on the microscope and accounted the cells.The number of leucocyte cells calculated on the five large boxes of hemocytometer and calculation by using the formula: Total leucocyte = ∑ leucocyte cells × 50 cell/ mm 3

Antibody titers
Serum preparation: fish blood was collected via caudal veins and put it in a micro tube, the next step was centrifuged at 3000 rpm for 3 minutes.After the serum was separated from the blood cells, incubated at 44°C for 20 minutes to activate the complement (Lumsden et al., 1993).The serum was stored in a refrigerator at 4°C for antibody titer observation.
The antibody titers measurements were carried out by taking 25 µL of PBS solution and inserted into the microplate at holes 1 st through 12 th , then inserting 25 µL blood serum at 1 st hole and diluting the level up to 11 th hole.As much as 25 µL of bacteria inserted into 1 st hole to 12 th , then the microplate homogenized by gently waving.Further, it stored for two hours in the incubator at 37°C, followed by storing into the overnight in refrigerator 4°C, the antibody titers were determined from the last remaining hole of the agglutination reaction.

Phagocytic index
Phagocytic index was measured using Anderson and Siwicki (1995) method as much 50 μL of blood put into the microtube, added 50 μL of a Staphylococcus aqueous suspension in PBS (10 7 cells/mL) was homogenized and incubated at room temperature for 20 min.Make a prepared on the glass object and dry it out.Next step was fixing with methanol for five minutes and airdried, stained by immersion into Giemsa for 15 minutes, washed with running water and dried with tissue, and then observed and counted the number of cells showing the phagocytic process of 100 phagocyte cells observed.

Cumulative mortality & RPS
The effectiveness of the extract as an adjuvant vaccine was measured by observing the fish mortality after challenges with A. hydrophila and P. fluorescens.Cumulative of mortality and RPS method using Ellis (1988) method.

Data analysis
All results were presented in the average and standard deviation of three independent measurements.Cumulative mortality and RPS were analyzed using nonparametric ANOVA (SPSS 16 computer program) was used to determine whether there was a significant difference (P<0.05)compared to control and vaccine groups, while the hematological and immunological parameters were analyzed with description.

Fish pathology anatomy
Bacterial infections of A. hydrophila and Pseudomonas sp.causing exophthalmia symptoms in the eyes, fin rots and bleeding (Hardi et al., 2014a).The test results show that prevention of bacterial infection of A. hydrophila and P. fluorescens using vaccine mixed with plant extract reducing the fish symptoms such as fin rot, darkness and exophthalmia average decreased on day 14 post-challenge, and the results for all treatment were significantly different from vaccine without extract (P< 0.05) on test challenges d21 (Table 1).

Total bacteria in tilapia after the challenge test
The number of bacteria in the fish body also decreased in the vaccinated without and mixed with the plant extract on day 14 after the challenge test.The highest decrease was found in the treatment of SF+ ZZ+V reached 10 3 CFU / mL and the result was significantly different from the un extracted vaccine (V) (P< 0.05).

Total leucocytes
Post vaccination leucocyte types also increased, either with extracts or with no extracts.The increase in total leucocytes occurred from day 7 to day 21 after vaccination, and the highest increase occurred in indigo fish injected with a vaccine with a mixture of S. ferox and Z. zerumbet (Table 3).

Antibody titers
This research used a simple method to examine the antibody titer; Table 4 showed the result of value -log titer antibody.The results of measurement indicated that the value of fish antibody titer has already detected since 7 days after the test.However, the highest increase occurred on the 14 th day of the 21 st day test period increased up to 6 in the treatment using a mixture of B. pandurata as well as S. ferox and Z. zerumbet treatment, but the value was not significantly different between all treatments.

Phagocytic index (%)
The results showed the number of cells that performed phagocytosis in the mixing treatment between the vaccine and the extract increased post-challenge compared with the vaccine without extract (Table 5).The highest increase occurred in the treatment with the addition of S. ferox and Z. zerumbet.The phagocytic index increased on day 14 and day 21.

Cumulative mortality and RPS
Tilapia test fish injected with bacteria A. hydrophila and P. fluorescens on day 7, 14 and 21post vaccination.Table 6 shows the cumulative mortality of tilapia.The entire tilapia fed with the mixture of extracts showed a decrease in the number of deaths compared to only vaccinated fish, ranging from seven days post-challenge test.When viewed from the post-challenge protective results, those seven days after the vaccination period has given the protection to the fish, and the better the protection as the challenge time (14 and 21 days post-vaccination) increases.Even the vaccine-administered fish plus the B. pandurata extract was able to provide 100% protection against fish post bacterial infections.
It seen from the RPS level, on the 7th day of the challenge test, the highest reached 90% in vaccine treatment plus the B. pandurata extract (P<0.05)(Table 6).It followed on the treatment of the 14th day tested vaccine with a B. pandurata mixture was able to increase the best RPS reached 91%.However, in the 21 st day of the 21 st treatment, almost all vaccine treatments added with extracts were able to increase the RPS to 100% better than only the treated vaccine without the addition of extract (Table 6).The overall RPS results were significantly different from the non-extractive vaccine with P< 0.05.It turns out that the trial of using QSS as an adjuvant of the V. anguillarum vaccine in turbot fish indicating the same increasing in post-test RPS on days 14 and 28 (Wang et al., 2016).

Discussion
The utilization of adjuvant in vaccination to human (Pasquale et al., 2015;Wang et al., 2016) and fish (Bøgwald & Dalmo, 2012) has already done for a long time, as it proved to increase the immunogenicity of the vaccine.Some ingredients which known as an adjuvant were aluminum, water-in-oil emulsions (Freund›s adjuvants), part of microorganism cell and components of plant extracts (Rajput et al., 2007;Pasquale, 2015).
Plant extracts containing saponins, flavonoids, carbohydrates have the ability to immunomodulate fish such as B. pandurata, S. ferox and Z. zerumbet (Hardi et al., 2017a,b).Furthermore, Azadirachta indica extract, Ocimum sanctum, and Curcuma longa were able to increase the activity of phagocytosis, respiratory burst, and alternative complement activity and lysozyme goldfish (Carassius auratus) (Harikrishnan et al., 2009).Stratev et al. (2018) research showed that the treatment of fish pathogen infection using medicinal plants (secondary metabolites, fractions, or plant extracts) would be the best choice for sustainable aquaculture.
There are many of the phytocomponents of plant extracts, one of them is saponin which already proven to boost the fish's specific immune system when mixed with vaccines (Milgate & Roberts, 1995;Song & Hu, 2009;Bagherwal, 2011).The saponin component of O. aponaria extract was able to prevent rotavirus infection by inhibiting viral attachment of host cells, through the destruction of cell membrane proteins virus receptors (Tam et al., 2011).Saponin also stimulates growth and mucosal immune response that can prevent viral infections in humans (Wang et al., 2016;Tafalla et al., 2017).The results of the experiment by Wang et al. (2016) showed that utilization of Q. saponaria saponin (QSS) (45 mg/L) mixed with Vibrio anguillarum formalin vaccine increased the antibody production of Scophthalmus maximus on the 28 th day post vaccination.This indicated that QSS was capable as an adjuvant vaccine through immersion.QSS was able to increase the effectiveness of immune cells by increasing complement activity and macrophage cell phagocytosis capable of consequent antigen presenting activity, thereby initiating the downstream humoral adaptive immune responses of the immunized fish.
The results also showed that the administration of B. pandurata extract was able to increase antibody titer production, in accordance with the increasing of post-trial RPS with combined bacteria A. hydrophila and P. fluorescens extracts act as permeabilizing agents by which allowed the molecules penetrated into the cell, and have not been adequately addressed (Secombes & Belmonte, 2016).
The addition of plant extracts increased the ability of antibodies to react with antigenic epitopes so that antigens were unable to recognize host cell receptors which would lead to failure of the antigen attachment process on the host cell surface (antibodies act as an inhibitor).In addition, the extract was also able to accelerate the elimination of antigen by the opsonizing process (antibody as opsonin).Formalin-killed E. tarda vaccine mixed with the Quil-A saponin increasing the survival rates after E. tarda infection and showed better survival.Adjuvant utilization mix with the fish vaccine was strongly capable to induce a specific immune response and long duration time protection, therefore the impact increased the vaccine efficiency (Tafalla et al., 2013).

CONCLUSION
The conclusion of this research was the B. pandurata extract was a potential adjuvant in the application of vaccine in freshwater fish.It was shown by the RPS after challenges was significantly higher than other treatment.The fish protection to bacterial infection was faster increase with B. pandurata administration (D7 and D14) than control (vaccine without extract).

Table 1 .
The anatomical pathology of the outer organ of tilapia observation in day 14 th after post challenge test with bacteria A. hydrophila and P. fluorescens

Table 2 .
Total bacteria in tilapia organ observation in day 14 th after post challenge test with bacteria A. hydrophila and P. fluorescens

Table 4 .
Tilapia's antibody titers (-log 2 ) at postvaccination observation in day 14 th after post challenge with bacteria A. hydrophila and P. fluorescens

Table 5 .
Phagocytic index (%) observation in day 14 th after post challenge test with bacteria A. hydrophila and P. fluorescens

Table 6 .
Number of mortality and RPS of post-test indigo fish on day 7, 14 and 21 with bacteria A. hydrophila and P. fluorescens