THE POTENTIAL OF ADJUVANT AGAINST PRODUCTION OF ANTISTREPTOCOCCAL IMMUNOGLOBULIN Y (IGY) IN AQUACULTURE

This study was conducted to explore the potential of adjuvant for the production of immunoglobulin Y (IgY) as antistreptococcosis in layer chicken with mass production orientation. Enterococcus faecalis which causes streptococcosis in the red tilapia was selected as a candidate antigen. The production of immunoglobulin Y (IgY) was carried out on Isa Brown layer chickens and aged around 20 weeks. Furthermore, the chickens were grouped into four groups (A, B, C, and D groups), each consisting of three chickens based on the type of adjuvant, while two chickens were used as a control group. Each group was treated by giving MONTANIDETM ISA 71R VG adjuvant (A), Freund's adjuvant (B), aluminum potassium sulphate adjuvant (KAl(SO4)2∙12H2O) concentration of 50 ppm in pH 7 (C), and only antigens without adjuvant (D). Chickens were kept for 35 days and each week was checked for presence the IgY antigen in the serum and egg yolk. Booster was conducted on 14 and 28 days of maintenance. The results showed that IgY in treatment group A was detected on day 28 in the serum and day 35 in the yolk. Whereas the treatment group B could be detected on day 35 in the serum. However, the IgY was not detected in the serum and yolk in C, D, and control groups until the end of the maintenance. Based on the results, it can be concluded that the appearance of IgY in serum and yolk in a relatively fast time is obtained in the combination of Enterococcus faecalis antigen with the emulsion of water-in-oil adjuvant (SEPPIC MONTANIDETM ISA 71R VG) compared to the other types of adjuvant that use in this study. ____________________________________________________________________________________________________________________


INTRODUCTION
Adjuvant is a substance that mixed with antigens which is more able to increase the antibody titer due to an increase in the immune system response compared to a single antigen application (Awate et al., 2013). One of the most ingredients that widely used as an adjuvant is an emulsion material, such as water-in-oil or W/O emulsion and a water-in-oil-in-water or W/O/W emulsion. In addition, mineral salts such as aluminum potassium sulfate, aluminum phosphate, and aluminum hydroxide; microorganism products such as Complete Freund's Adjuvant (CFA) containing dried preparations of inactive bacteria Mycobacterium sp.; saponin; type of cytokine; polymer; microparticles; and liposomes can also be used as adjuvants (Guy, 2007).
In terms of safety, aluminum mineral adjuvants have the best safety history of their use for 70 years (HogenEsch, 2013). Therefore, this type of adjuvant is widely used in studies of immunomodulatory substances. The workings of mineral salt adjuvants such as aluminum adjuvants are largely determined by the optimization of the absorption of antigens by the aluminum mineral surfaces used as adjuvants. According to Hem and HogenEsch (2007), absorption of antigens with aluminum adjuvants is influenced by hydrophobicity, van der Waals forces, electrostatic mechanisms between antigens and adjuvants, and the presence of ligand changes. Therefore, the effectiveness of aluminum adjuvant is largely determined by the chemical and physical stability of the antigen used (HogenEsch, 2013).
The mechanism of emulsion-type adjuvants in increasing antibody titers begins with the formation of antigen depots at the injection site in muscle tissue. The depot will release antigens slowly into the tissue. The presence of antigen depots can also trigger the secretion of cytokines and chemokines that invite immune cells that play a role in non-specific immune systems, such as macrophages and other cells that are phagocytosis. Adjuvants also play a role in increasing antigen phagocytosis and macrophage activity which acts as antigen presenting cells (APCs). In addition, adjuvants also enable the activation and maturation of APCs, the increase of Major Histocompatibility Complex (MHC) class II, increased migration of the matured and activated APCs into lymph node flow, and ultimately increased activation of antibody-producing B cells in the process of humoral immune systems and cells cytotoxic T lymphocytes (CD8 + cells) in the process of the cellular immune system. Thus, it can be said that adjuvants are also an immunomodulatory material to improve the performance of the non-specific and specific immune system (Awate et al., 2013).
The Freund adjuvant works by three mechanisms, namely prolonging the presence of antigens in the injection area, facilitating antigens to be quickly distributed to the lymphatic system and the lungs as the place where adjuvants trigger the accumulation of cells that work in the immune system, and allowing the third mechanism that has not yet been identified but suspected to be related to how antibodies form and how sensitization develops (Dienes, 1929). Complete Freund's adjuvant containing Mycobacterium tuberculosis can induce the production of some chemokines, tumor necroting factor α (TNF α), interleukin (IL) 12, IL-6, and interferon γ (IFN γ). The Mycobacterium sp. component will be phagocytosed by the dendritic cells and macrophages which are the target of these cells and directly cause stress that allows natural killer (NK) cells to secrete IFN-γ as cytokines that will be captured by the dendritic cells and macrophages as chemical signals in the phagocytic process. The dendritic cells and macrophages will then secrete IL-12 which can activate the helper T cells and humoral immune systems as in the mechanism of action of emulsion adjuvants (Billiau and Matthys, 2001). Shah et al. (2019) states that the droplet size of an adjuvant emulsion material can also affect its immunomodulatory potential due to differences in activation and direction of immune cells that lead to antigenic depots. The smaller emulsion droplet size (20 nm) is less effective compared to the larger droplet size (~ 160 nm). Adjuvants with a 160 nm droplet allow better movement of immune cells to the depot where the antigen is injected, more antigen uptake by phagocytic cells, and faster APC translocation into lymph nodes, thereby increasing humoral and cellular immune responses.
Today chicken eggs are widely used in research as a biological factory that produces immunoglobulin Y (IgY) which is specific to infectious diseases in aquaculture commodities. Koi Herpes Virus (KHV) in carp and koi (Pasaribu et al., 2012), White Spot Syndrome Virus (WSSV) that causes white spot disease in shrimp (Soejoedono, 2012), Aeromonas hydrophila bacterial infection in chef carp (Carassius auratus Gibelio) (Xiao-liang et al., 2006), and vibriosis in vanamei shrimp (Litopenaeus vannamei) (Gao et al., 2016) are several diseases that have been studied in the development of IgY as a therapeutic basis. This was done as a new therapeutic effort that is more environmentally friendly in fighting infectious diseases by reducing the use of antibiotics in aquaculture activities. The purpose of this study was to explore the potency of various adjuvants for the production of antistreptococcal IgY using field isolates as a prospect for mass pellet production for the treatment of streptococcosis in fish.

Preparation of Inactive Antigen from Enterococcus faecalis Field Isolate
The Enterococcous faecalis field isolate strain 7INB derived from the liver of red tilapia (accession number GenBank MT105346) which has been confirmed morphologically, biochemically, and molecularly was used as an antigen candidate in the production of antistreptococcal IgY. The isolate was inoculated on BHI broth medium with a volume of 300 mL which was then incubated at 35 C for 24 hours. The media was centrifuged for 10 minutes (10000 rpm) and the pellets were collected. The obtained pellets were then washed three times with the addition of 10 mL sterile physiological sodium chloride (NaCl) and then re-centrifuged for 10 minutes (10000 rpm) before being collected again. The pellets were then suspended using physiologically sterile NaCl and compared with McFarland 4 standard tubes to obtain an estimate of the desired bacterial cell concentration (10 9 CFU/mL). The inactive bacteria were then confirmed by being regrown on the agar medium made of 5% sheep blood. If it did not grow, then the bacterial suspension could be injected as an antigen in the production of IgY in experimental chickens (Modified from Wibawan et al., 2010).

Production Antistreptococcosis Immunoglobulin Y (IgY) in Chicken
The production of IgY was carried out on Isa Brown layer chicken aged around 20 weeks for 35 days of maintenance. The chickens were grouped into four groups (A, B, C, and D groups) where each group consisting of three chickens based on the type of adjuvant, while two chickens were used as a control group. The treatment that given in each group as follows: group A was treated using inactive bacterial antigens injection with the addition of water-in-oil emulsion adjuvants (MONTANIDE ™ ISA 71R VG) with the formulation of 60% adjuvant: 40% antigens (w/w); group B was injected with inactive bacterial antigens with the addition of CFA at the beginning of vaccination and Incomplete Freund's Adjuvant (IFA) at the first and second revaccinations, each with the formulation of 50% adjuvant: 50% antigen (v/v); group C was treated by inactive bacterial antigens injection with the addition of an adjuvant solution of aluminum potassium sulfate (KAl (SO4) 2 •12H 2 O) with a concentration of 50 ppm at pH 7; and group D was injected with inactive bacterial antigens without adjuvants; whereas the control was a group of chickens that were not injected with inactive bacterial antigens.
The treatment groups (A, B, C, and D) were vaccinated intramuscularly with inactivated bacterial antigens with a total volume of mixture (antigen and adjuvant) of 1 mL/chicken on the chest muscles. Vaccination was repeated on 14 th and 28 th days of the maintenance period, each with a total volume of mixture (antigen and adjuvant) of 1 mL/chicken. Serum and egg yolk were collected and the antibody titers were calculated using the Agar Gel Precipitation Test (AGPT) method with dilution on 0, 7, 14, 21, 28, and 35 days after vaccination (Modified from Wibawan et al. 2010). During production, feed and drinking water were given ad libitum. The method of using experimental animals had been approved by the Animal Ethics Commission of the Institute for Research and Community Service of the Bogor Agricultural Institute (LPPM IPB) with ethical approval number: 171-2019 IPB.

Preparation of Dissolved Antigen
Pure bacterial isolates that had been through a series of bacteriological examinations were inoculated on a 150 mL BHI broth medium and then incubated at 28 C for 24 hours. The media was centrifuged for 10 minutes (10000 rpm) and the pellets were collected. The pellets were then washed three times with a sterile 0.9% NaCl solution. The pellets obtained were then given 350 µL of 0.2 N HCl solution and homogenized with vortex. The suspension was evaporated for ± 2 hours at 60 C. The suspension that had been evaporated was then given 1-3 drops of phenol red and a few drops of 1N NaOH solution until it turned pink so that the pH remained 7. The suspension that had changed in color was centrifuged again for 10 minutes (10000 rpm). The pellets were removed and the supernatant was collected. The supernatant was collected as dissolved antigen in the test using the AGPT method with dilution (Arnafia et al., 2016).

Preparation of The Egg Yolk Extract
Preparation of egg yolk extract was referred to Wibawan et al. (2018). The procedure was divided into two stages, namely the preparation of egg yolk Water Soluble Fraction (WSF) and the IgY precipitation. The preparation of the egg yolk began with carefully breaking the egg and separating the egg whites and yolk using filter paper. The vitelin membrane was torn with a tweezers until the egg yolk came out. The volume of the yolk was measured and then mixed with distillate water with pH 5.0 with the addition of 0.5 M HCl seven times the volume of yolk obtained. The mixture was frozen at -20 C and thawed at 4 C. The egg yolk aggregate granules were precipitated by centrifugation at 13500 g for 15 minutes at 4 C, and the supernatant was collected and filtered with filter paper to obtain clear WSF filtrate. The IgY precipitation step was carried out using solid NaCl which was then added to WSF up to 8.8% of the total WSF volume. The mixture was stirred with magnetic stirrer for 2 hours at room temperature, and the pH of the solution was adjusted to the pH point 4 by adding 0.5 M HCl. The mixture was then centrifuged at 3700 g for 20 minutes at 4 C. The supernatant was removed and pellets were dissolved in Phosphate Buffer Saline (PBS).

Agar Gel Precipitation Test (AGPT) Method with Dilution
The AGPT method with dilution to test the presence of IgY was carried out in the following stages: 0.502 g agarose gel, 1.2 g PEG6000, 0.04 g Na-azide were mixed into 20 mL PBS with pH 7.2 and 20 mL distilled water in the Erlenmeyer tube. The Erlenmeyer was heated in the microwave until the gel solution became clear. The gel solution was removed and immediately molded on the object glass by taking 4 mL of the gel solution using a scale pipette which was then poured slowly until the gel solution covered the entire surface of the object glass and bulged. The gel solution was allowed to stand until it hardened. It was then perforated with a special AGPT puncher to make a well.
Each serum sample or egg yolk extract was diluted before being put into an AGPT well using a sterile PBS solution. A total of 100 µL of serum sample or egg yolk extract was put into the microplate of well 1. Every 50 µL PBS solution was added to the microplate in eell 2 to well 12. A total of 50 µL of serum sample or egg yolk extract from well 1 was then put into well 2 and a stratified dilution was performed up to well 12.
The filling of gel wells was carried out by the following steps. The middle well was filled with 50 μL of dissolved bacterial antigen, and the other wells were filled with 50 μL of serum sample or egg yolk extract. All wells were then incubated for ± 24 hours in a moist container covered with wet tissue at room temperature. Positive results were shown by the presence of whitish precipitation lines between wells containing dissolved antigens and wells containing serum samples or egg yolk extracts suspected of containing IgY (Oudin, 1946).

RESULTS AND DISCUSSION
Serum examination showed that IgY was only present in the treatment groups which were given antigens with a combination of adjuvant emulsions in water oil (MONTANIDE ™ ISA 71R VG) and antigens with a combination of Freund adjuvants. The IgY was formed during 35 days maintenance period. In two chickens in the water-oil emulsion adjuvants group (MONTANIDE ™ ISA 71R VG), IgY began to be detected in the serum on day 28 after the initial vaccination and the first revaccination on day 14. Whereas in one chicken in the Freund adjuvant group, IgY was only detected on day 35 after initial vaccination and the first and second revaccinations on day 14 and day 28. Negative results were found in all chickens in the group C which was treated with aluminum potassium sulfate mineral salt adjuvant and the group D that were given antigens without the adjuvant combination.
The IgY was detected in the yolk extract of two chickens from group A which were given the antigen with a combination of water-in-oil emulsion adjuvant (MONTANIDE ™ ISA 71R VG) on day 35. The two individuals chicken also showed positive results on serum examination. Furthermore, negative results were observed in all egg yolk extracts from the Freund adjuvant group (group B), aluminum potassium sulfate adjuvant group (group C), and chicken groups given antigens without adjuvant combinations (group D). The results of IgY titer examination are presented in Table  1, Table 2, Table 3, and Table 4.
Results of studies on the potential of adjuvants as an immunopotentiation material in the production process of antistreptococcal IgY showed that the vaccine formulations using antigens from the field isolate Enterococcus faecalis with the addition of aluminum potassium sulfate adjuvant concentrations of 50 ppm at pH 7 did not cause a significant immune response until the end of the maintenance period. This is indicated by the negative AGPT test results or the absence of precipitation lines in all three chickens from this adjuvant group. According to Hem and HogenEsch    (2007), absorption of antigens with aluminum adjuvants is influenced by hydrophobicity, van der Waals forces, electrostatic mechanisms between antigens and adjuvants, and the presence of ligand changes. Electrostatic mechanism as one of the factors that influence the success of vaccination using adjuvants containing aluminum minerals is strongly influenced by three things, namely the pH during the vaccine formulation process, point-of-zero-charge (PZC) of the aluminum adjuvant used, and isoelectric points of antigen protein used. Aluminum Adjuvants have different PZCs. Likewise, isoelectric points on proteins that become antigens. For example, the aluminum phosphate adjuvant has a PZC point at pH 5, where the adjuvant is neutral. If aluminum phosphate is in an environment of pH <5, the surface of the adjuvant will be positively charged. Meanwhile, if the aluminum phosphate is in an environment with a pH>5, the adjuvant surface will change to be negatively charged. An antigen in the form of an ovalbumin protein (OVA) has an isoelectric point at pH 4.6, where the protein is neutral. If OVA is in an environment with a pH <4.6, the protein will be positively charged. Meanwhile, if the OVA protein is in an environment with a pH>4.6, the protein will be negatively charged. Vaccine formulations that have a pH of 7 (neutral) will cause both aluminum phosphate and OVA proteins to be negatively charged, so they have no affinity or bonds. This can cause a failure in the adsorption process to stimulate the immune system because antigens are not bound by adjuvants. However, the opposite will occur if the OVA protein is combined with an aluminum hydroxide adjuvant that has a PZC point at pH 11.4. If the aluminum hydroxide is in an environment of pH <11.4, then the surface of the adjuvant will be positively charged. Meanwhile, if the aluminum hydroxide is in an environment with a pH>11.4, then the adjuvant surface will change to be negatively charged. If the vaccine formulation is maintained at pH 7, then the surface of the aluminum hydroxide adjuvant will be positively charged and the OVA protein will be negatively charged. This will cause an electrostatic   Chicken 3 (-) (2 0 ) (-)= Negative (no precipitation line), n = Dilution, A = Inactive bacteria + water-in-oil emulsion (MONTANIDE ™ ISA 71R VG) adjuvant mechanism, in which the antigen will be absorbed by the adjuvant and cause an immunostimulatory effect (HogenEsch, 2013;Lindblad and Duroux, 2017). The negative results on the immune response of the formation of antistreptococcal IgY in the aluminum potassium sulfate adjuvant group is thought to be related to the fact that the electrostatic mechanism between the surface of the aluminum potassium sulfate adjuvant and the Enterococcus faecalis antigen used during the vaccination process did not occur optimally. The vaccine formulation used in this study, which was a combination of intact cell antigens from Enterococcus faecalis bacterium and aluminum potassium sulfate adjuvant at a concentration of 50 ppm at pH 7, is thought to have the same effect, namely analogy between aluminum phosphate antigen formulations and antigens in the form of proteins ovalbumin is maintained at pH 7, electrostatic mechanisms did not occur because they both had the same charge. As a result, the immune system in chicken was not stimulated until the end of the maintenance period because antigens were not bound and absorbed by adjuvants. However, further studies need to be carried out on the PZC points held by aluminum potassium sulfate adjuvants and the isoelectric points of each protein contained in Enterococcus faecalis bacterial cells used as antigens, so that an optimal pH environment is obtained during the vaccine formulation.
The IFA adjuvants contain an essential paraffin oil emulsion where mannide monooleate is used as a surfactant. The adjuvants will form emulsions of water in oil (water phase) when interacting with aqueous solution or antigen suspension. Meanwhile, CFA adjuvants containing Mycobacterium tuberculosis and inactivated by heating work by pathogenesis of lesio tubercles in the injected tissue. Exposure to the Mycobacterium component causes an immune response that is delayed-type hypersensitivity (DTH) (Billiau and Mattys, 2001). The DTH is also called hypersensitivity type IV which requires time to produce an excessive immune response. The process of DTH can be divided into three phases. The first phase is the sensitization phase, in which the process of forming and exposing antigens to immunocompetent cells occurs the first time. The second phase is the phase of development, in which sensitive lymphocytes are produced specifically. The third phase begins after the administration of new antigens for the second time to make lymphocytes recognize the antigen more effectively. The process that occurs in this last phase will later develop into a typical inflammatory reaction. Furthermore, there are two models in the occurrence of DTH. The first is the permanent type in which the hypersensitivity reaction lasts for several months or years after the sensitization stage is complete, and the inflammatory reaction can still be seen up to 72-96 hours after intradermal injection exposure. The second is a temporary type in which the hypersensitivity reaction disappears one to two weeks after the sensitization process, and the peak of inflammation occurs 24 hours after injection with lesions that will disappear on days 48-72 (de Weck, 1998). This DTH condition is thought to cause IgY titers in the Freund adjuvant group to take longer than the adjuvant type of water-in-oil emulsion (MONTANIDE ™ ISA 71R VG) used in this study. This condition also requires revaccination a second time to produce IgY titers in the serum so that the lymphocytes responsible for the immune process can further recognize the antigen being exposed.
Adjuvants of water-in-mineral-oil or W/O emulsion type are a formulation that is very often used in inactivated vaccines for poultry commodities. This adjuvant can induce antigens in the long run because it contains a mixture of specific mineral oil and a surfactant system that can stabilize the emulsion and vaccine depot in the tissue (Heegaard et al., 2011). MONTANIDE ™ ISA 71R VG adjuvant is an example of a commercial type of water-in-oil emulsion that has been widely used in poultry vaccination programs. A special surfactant system that can stabilize commercial adjuvants for 7-30 days at 4 C, 20 C, and 37 C after vaccine formulation is thought to be the reason for the success rate in making antigen depots in the tissues high (Arous et al., 2013). This triggers the slow release of antigens and forms the immune system in gradual but stable and strong way, so that IgY is formed in the serum with only one revaccination.

CONCLUSION
Based on the results of comparative study of the three types of adjuvants used in the IgY production, it can be concluded that the water-in-oil emulsion (MONTANIDE ™ ISA 71R VG) as adjuvant of can help increase the production of IgY in serum and egg yolk in a relatively faster time than without the use adjuvants or the use of two other types of adjuvant.