The effect of Sea urchin ( Diadema setosum ) gonad extract on IgM and IgG antibodies production in BALB / c mice infected by Salmonella typhi

Background: Salmonella typhi infection decreases of the immune system and infl uences the adaptive antibodies among malnourished children. The gonad of Diadema setosum (D. setosum) is one of food sources from marine biota that contains high-quality nutrients and potentially can be used as a dietary supplement for typhoid fever condition. Objective: This study aimed to determine the effect of gonad D. setosum extract on the production of antibody IgM and IgG in an animal model. Method: This experimental study was used BALB/c mice before and after infected Salmonella typhi through intraperitoneally at 0.2 mL x the unit 103 CFU/mL. The level of IgM and IgG production was measured by Enzyme Linked Immune Sorbent Assay (ELISA). Experimental animals were divided into 2 groups. The control group was only fed with standard diets, while at the intervention group received the extract of D. setosum gonad in two doses (100 and 200 mg/kg body weight). Results: Production of IgM antibodies in the control group signifi cantly increased twofold (p=0.001) whereas the intervention group received the extracts of D. setosum gonad (200 mg/kg body weight) could suppress the increase in IgM antibody production and indicate the highest increase of IgG antibody signifi cantly (p<0.05) at day 7. Conclusion: The gonad of Diadema setosum extracts (200 mg/kg body weight) could suppress the increase in IgM antibody productions and indicate the highest increase of IgG antibody titers in mice infected with Salmonella typhi. The role of anti-microbial substances of the gonad of Diadema setosum, is potential to be utilized as dietary supplement to increase body immune system among patients infected by Salmonella typhi.

to determine the effect of gonad of D. setosum extract to levels of IgM and IgG antibodies production. This study using animal models of BALB/c mice stimulated by Salmonella typhi bacteria.

The collection of gonad of D. setosum
The gonads of D. setosum were collected from Wakatobi National Marine Park, Wakatobi District Southeast Province, Indonesia. The extraction process of D. setosum gonads was conducted in Biopharmacy and Phytopharmacy Laboratories, Faculty of Pharmacy, University of Hasanuddin, Makassar, Indonesia. The extraction process was done by using a suitable solvent acetone as described previously (11) and by making two doses (100 and 200 mg/kg body weight) (13).

Preparation of experimental animals
Male BALB/c mice of 10 to 12 weeks old, weighing 30-40 g free of pathogens were obtained from Molecular Biology and Immunology Laboratory for Diseases Infection, Faculty of Medicine, Hasanuddin, Makassar, Indonesia. Mice were reared in a room of proper air circulation and they were maintained in a temperature-controlled facility at 23°C and 50% humidity with 12 h light/dark cycle and were maintained in cages with care and were cleaned routinely. During rearing, mice were fed with standard natural pellets [the food contained protein (17.5-19.5%); lipid (3%); fi ber (8%); ash (7%); calcium (0.9%); and phosphor (0.6%) based on the previous study (13)] and were given aquadest ad libitum. Before of treatment, treated BALB/c mice were preconditioned for two weeks to adapt to the laboratory condition to keep physical and physiological conditions of mice were in a stable state. All experimental procedures for the treatment and maintenance of any laboratory animal were reviewed and approved by the Research Ethics Committee of Medicine Faculty of Hasanuddin

INTRODUCTION
Typhoid fever is caused by Salmonella typhi, and this infection is still being a health concern in developing countries (1). Indonesia is one of the fi ve countries in Asia that experiences the endemic of typhoid fever (2). The prevalence of this disease is common during the dry season that generally infects children (3)(4)(5). Deficiency of nutrient and infectious diseases will suppress immune function (6) by infl uencing the physical barrier, cellular and antibody production (7). The IgG antibodies production have role in activating other cells to form body immune against viruses and bacteria (8). Acute typhoid infection showed a strong IgM antibody response compared with healthy patients (9). IgM levels were higher in malnutrition than well-nourished childrens (10).
The gonad of Diadema setosum (D. setosum) is one source of food from marine biota that contains high -quality nutrients and is abundance in Indonesia. The amount of vitamin A and E are higher compared to the ones found in salmon, sardines, mackerel, cork fi sh (Chana striata) and eggs, indicate that the gonad of D. setosum is suffi cient to become a new source of nutrient to boost the immune response (11). In addition, the gonad of D. setosum contains protein, high quality polyunsaturated fatty acids (PUFA) and antioxidant substances (12).
As confirmed in our previous study, BALB/c mice were stimulated by LPS of Salmonella typhi at the control group noticeably shows the signifi cant reduction of body weight at 2,24 g (p=0.001), whereas, mice group of intervention with the gonad of D. setosum extracts (200 mg/kg body weight) clearly show the signifi cant increase of 1,7 g of body weight at days 7 (13). Concerning to this fi nding, the gonad of D. setosum delivers a prospect to be used as a potential food source widely for the reason that it improves nutritional status. It is important that tangible scientific research is conducted to discover the possible health benefi ts the gonad of D. setosum to adaptive antibodies immune response. This study aims tikus yang terinfeksi Salmonella typhi. Aktivitas anti-mikroba dari gonad D.setosum berpotensi dimanfaatkan sebagai suplemen makanan untuk meningkatkan status kekebalan tubuh pada pasien yang terinfeksi Salmonella typhi.

Experimental procedure and treatment
After the adaptation period, twelve tail mice (n=12) were then divided into three groups, i.e. group I (control), group II and III (intervention), respectively the fi rst blood sampling of 0.2 mL was done at tail of each mice at day 0 (baseline), and then all mice were let to rest for 2 hours. After that, the intervention group II and III received the extract of D. setosum gonad in two doses (100 and 200 mg/kg body weight), whereas those the control group were only fed with standard diets. The second blood respectively sampling of mice was done at day 10 and they were let to rest for 2 hours, and then the three mice groups were infected by Salmonella typhi through intraperitoneally at (0.2 mL × the unit 10 3 CFU/mL) based on the previous study (13). Blood of mice were collected at day 5 and day 7 after infected by Salmonella typhi.

Measuring the levels of IgM and IgG serum
Blood of mice was collected and centrifuged to obtain blood serums. The collected blood serums were kept under the room temperature of 20 °C before performing the examination of IgM and IgG antibodies titers using Enzyme Linked Immune Sorbent Assay (ELISA) technique. Plasma IgM and IgG antibodies levels were measured using the Rat ELISA Kits (Rat IgM E-25 and Rat IgG E-25G) respectively; ICL, Gentaur, the Netherlands). The use direction of laboratory instruments referred to the information sheets asserted in the KIT. The absorption values of antibody titers were automatically counted by the ELISA reader at a 450 nm wavelength.

Data analysis
The investigational results acquired are expressed as the mean ± standard deviation (SD). One-way ANOVA test was used to determine the statistically signifi cant differences of measurement results of IgM antibodies whereas IgG antibodies production using dependent t-test. Signifi cance was accepted at p<0.05. Figure 1, IgM antibody production at baseline and day 10 were not statistically different before infected by Salmonella typhi (p>0.05) however IgM antibodies production were different after infected by Salmonella typhi (p>0.05) between the control group and the intervention group. IgM production for the control group signifi cantly increased twofold at days 7 (p=0.001) compared to the intervention group (100 and 200 mg/kg body weight). Figure 2, the production of antibody IgG was statistically higher (p=0.004) for the intervention group III (200 mg/kg body weight) compared to the intervention group II (100 mg/kg body weight) and control group.

RESULTS
Our study result indicated at the mice control group, IgM production increased twofold whereas the level of IgG antibodies lower at days 7 compared to that of the intervention group, this shows that failed to  protect them from infection. Conversely the intervention group the gonad of D. setosum extracts (200 mg/kg body weight) could suppress the increase in IgM antibody production and indicate the highest increase of IgG antibody signifi cantly at days 7. The nutrient of gonad of D. setosum is likely to have an antimicrobial effect through its role as an antioxidant so it can suppress the bacteria Salmonella typhi.

DISCUSSION
Antioxidants treatment has the effects and important implications for humoral immunity during infection (14). The high vitamin A and E composition in foods have an important role in inducing signifi cant adaptive antibodies and providing protection against infection diseases (15). Vitamin A has an important role in the immune system and sustain the balance of cell function and prevent longer infl ammation (16), increased level of IgM and IgG production in mice infected with tetanus toxoid (17). Vitamin E restores humoral immune function and intracellular cell mediation (18), facilitate in the switching from IgM of IgG antibody production (19).
The level of vitamin E contained in the gonad of D. setosum was 23.47 mg in 100 g of the gonad and was highest compared to salmon, mackerel, anchovies, sneak head and eggs (11). Interestingly, around 80% of PUFA was contained in the gonad of D. setosum (12), specifi cally eicosapentaenoic (EPA) and decoshaexaenoic (DHA) acid (20). Research on human showed that EPA and DHA possess complementary effect to prevent over-infl ammation and oxidative stress (21), and can be effective to treat memory disturbance, maintain cognitive function and immune system (22). However, PUFA also has negative effects when it is over-consumed. PUFAs are easily oxidized and combined with free-radicals that can disturb the function of cell membrane (23). There have been much evidence that vitamin E is capable of protecting easily oxidized chemical substances and maintaining the stability of PUFA in lypo-protein and cell membrane. On the other hand, PUFA is required for transporting vitamin E to body tissues (24). Defi ciency in vitamin E may have detrimental effect on imuno humoral and cell mediation across intra-cellular (18).
The level of vitamin A found in the gonad of D. setosum was 1.79 mg for 100 g of the gonad, which is equal to 2983.93 IU. This level was highest compared to the level of vitamin A found in Salmon, mackerel, anchovies, sneak head and eggs. Moreover, the level of protein reached 1.24 g for 100 g of the gonad (11). Intervention of biscuit (60 g) extracted from the protein of sneak head given to 28 pre-school children (4-5 years old) for 56 days is known to enhance immune respond of IgG antibody (25). Protein is required for transporting vitamin A to the body tissues (26). Defi ciency in vitamin A affects humoral respond and may reduce cellular activities and lymphoid organ experiences bad development (27).
The interaction between micro and macro substances plays an important role in protecting and maintaining immune system. It was expected that nutritional interactions contained in the gonad affected immune respond of antibody IgM and IgG. Overall, the nutrients contained in gonad were sufficiently complete to fulfi ll the requirements as an alternative food resource.
Salmonella typhi can invade wider areas and lead to acute and chronic infections since this microorganism can replicate and protect itself from phagocytosis by epithelial and dendritic cells as well as macrophages in the immune system (28). Low inflammatory responses due to the infection of Salmonella strain lead to persistent infection and facilitate longer survival of pathogens (29). This is affi rmed by the proportion of the total typhoid fever patients (193 of 237 patients) noticeably have high value differences between the highest IgM titer (3200 pg/mL) and the lowest IgG titer (200 pg/mL) compared to the IgG antibody value (400 pg/mL) of normal individuals, as confi rmed in a study at a hospital in Kathmandu, Nepal (30).
Higher serum IgG levels but lower IgM antibodies suggesting the late class switching from IgM to IgG in response to the infection well after elimination of pathogen (9). The lag phase IgM has a half-life of about 5 days and to decline before IgG levels highest in the blood and has a half-life of about 23 days (31). Interestingly, in our study, in BALB/c mice after infected by Salmonella typhi, IgM (lag phase) has a half-life of about 7 days at the intervention group (200 mg/kg body weight). Not yet known exactly when the late class switching occurs but the highest increase of IgG productions compared with the intervention group (100 mg/kg body weight) and control groups indicate possibility activation adaptive antibodies immune response after exposure to infected with Salmonella typhi occur at days 7. It is estimated that IgG levels in the intervention group (200 mg/kg body weight) will increase again after 7 days.
The activation lymphocytes (antibody production and effector T cell lymphocytes) occur at days 7 and days 14 by the elimination of antigen (8). The cytokine interferon-gamma (IFN-fl ) binds to Fc-R results in the switching of IgG subclass that (32). The IgG antibodies have a relatively high affi nity and persist in the circulation for a long time (33). The important role of IgG antibodies can also bind to and neutralize bacterial toxins (34). Our study provides new insight on the immune nutrient of the gonad of D.setosum could increase the IgG antibody productions through its role as anti-microbial substances in mice infected with Salmonella typhi.

CONCLUSION
The gonad of Diadema setosum extracts (200 mg/kg body weight) could suppress the increase in IgM antibody productions and indicate the highest increase of IgG antibody titers in mice infected with Salmonella typhi. The role of anti-microbial substances of the gonad of Diadema setosum, is potential to be utilized as dietary supplement to increase body immune status among patients infected by Salmonella typhi.