The Application of Asam Sunti as Feed Additives for Bacterial Infection Control of Edwardsiella

Cultivating catfish is one of the businesses that are of interest in Indonesia. The success of cultivation begins to be determined by the provision of seeds. Healthy seeds lead to a high survival rate, and unhealthy seeds due to disease will cause a high mortality rate, causing losses in the cultivation business. Bacterial infectious diseases Edwardsiella tarda can cause a low survival rate of catfish seeds, and even the death rate can reach 100%. This study aimed to evaluate and determine the best dosage of asam sunti in feed to control the pathogenic bacteria E. tarda infection in catfish. Asam sunti was given through feed with different treatment doses, with concentrate 0% (K), 0.5% (P1), 1% (P2), 2% (P3), and 4% (P4) for 14 days after being infected with pathogenic bacteria. The effect of giving asam sunti was measured by observing the survival of fish. The results showed that the administration of asam sunti could control the bacterial diseases of E. tarda bacteria sequentially control (K) is 0%, (P1) is 16.67%, (P2) 40%, (P3) 66.67%, and (P4) 90%. From these results, it can be concluded that the 4% asam sunti dose is the best dose for the survival of catfish seeds, with the survival rate for pathogenic bacteria E. tarda at 90%. Keywords


Background
Catfish (Clarias gariepinus) is one of the fisheries in Indonesia which is in great demand by fish cultivators. Disease attacks often constrain the increase in fish production from microorganisms, one of which is a group of bacteria. This condition impacts decreased production, so it is insufficient for market demand and increases in selling prices and affects consumer consumption (Ryan et al., 2012;Mathew et al., 2014;Tran et al., 2017). Clarias gariepinus (C. gariepinus) inhabits almost all zones of freshwater bodies, including rivers, lakes, floodplains, large and shallow rivers. In addition, C. gariepinus is a basic omnivorous eater, eating almost anything it comes into contact with (Ogueji et al., 2019).
Bacterial infectious diseases can cause low fish survival, even the death rate can reach 100% (Sri, 2022). Increasing catfish production is often done by increasing feed efficiency and adding antibiotics. However, in recent years, strains of pathogenic microbes have emerged that are resistant to certain antibiotics (Widodo, 2019). The use of antibiotics for a long time can cause resistance to pathogenic bacteria in the body, both in fish and in environments polluted by antibiotics which result in nontarget organisms being killed (Scientific, 2012). Natural ingredients that can potentially control bacterial infectious diseases in catfish seeds include the star fruit plant (Averrhoa bilimbi). Wijayanti and Safitri (2018) said that starfruit leaves had antibacterial activity at concentrations of 2.5%, 5%, and 10% against the growth of Staphylococcus aureus. Research results from Maryam et al., (2015) showed that the ethanol extract of star fruit which are called 'belimbing wuluh' at a concentration of 0.4% had antibacterial activity for Shigella dysenteriae, Salmonella typhi, Pseudomonas aeruginosa, Vibrio cholera, Escherichia coli, and S. aures. Thus, a way of handling it is needed by looking for natural products that have antimicrobial properties (Chotiah, 2013) and can be added to feed (feed additives), as a substitute for synthetic antibiotics to control disease in catfish farming.

Materials and Method Experimental Design
This study used a completely randomized design (CRD) with one treatment factor (sunti acid administration) with treatment levels of 0%, 0.5%, 1%, 2%, and 4%, which were mixed with commercial feed.

Preparation of Containers and Test Fish Seeds
The place or container used to maintain catfish seeds is an aquarium with a size of 30 x 30 x 20 cm totaling 5 pieces. The initial stage is to clean the aquarium and arrange it on the shelf where the aquarium is, then fill it with 10 liters of fresh water and add airase. The aquarium was filled with 10 test catfish seeds before treatment, followed by acclimatization for three days. During the research, the water quality conditions with a pH between 7-8.5, water temperature between 23-30 C, and dissolved oxygen (DO) of 3.5 mg/L-6 mg/L (Nikhlani et al., 2022). Fish were fed using the at satiation method with a frequency of twice a day during rearing, around 08.00 and 17.00 WIB. During the treatment, every two days siphoning was carried out, and 50% water replacement of the total volume was carried out to maintain water quality.

Preparation of Edwardsiella tarda Pathogenic Bacterial Cultures
Pathogenic bacterial cultures were obtained from the Aceh KIPM Station. One ose of bacteria was cultured in 10 mL of Tryptic Soy Broth (TSB) medium and incubated for 24 hours. After incubation, 1 mL of culture medium was re-cultured on Tryptic Soy Agar (TSA) medium and then incubated for 24 hours.

Pathogenicity Test
The pathogen test is to see or confirm that pathogenic bacterial isolates can infect healthy catfish fry with the same clinical symptoms (Ariani et al., 2009). Target bacterial isolates were cultured into 10 mL of Tryptone Soya Broth (TSB) and adjusted or equalized to 0.5 McFarland standard (10 8 CFU/mL). As much as 0.05 mL of a bacterial suspension with a density of 10 8 CFU/mL was injected intraperitoneally using a syringe into 10 catfish seeds, and previously the catfish seeds were inactivated in water with a temperature of 25 C for 30 seconds by dipping (Wibowo et al., 2010).
After injection, the catfish seeds were then put back into an aquarium containing 10 liters of water and observed for seven days or until clinical symptoms and death appeared in the catfish seeds. The presence of pathogenic bacteria was then isolated from the liver and kidneys on Tryptic Soy Agar (TSA) media and incubated for 24 hours at 30 C, and reidentified by biochemical tests (Wibowo et al., 2010). Pathogenic bacteria that can be deadly or cause clinical symptoms in catfish fry are used again in the next test stage.

Preparation of Suspension of Pathogenic Bacteria and LC50 Test
Target bacterial colonies growing on TSA media suspension, equivalent to the McFarland 0.5 standard (Ariyanti et al., 2012). The way to test LC50 in this method is to inactivate catfish seeds at 25 C for 30 seconds by dipping (Wibowo et al., 2010), then as much as 0.05 mL of pathogenic bacteria (bacterial suspension) with a dilution of 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 were injected intraperitoneally into 50 catfish fry. After the injection, the catfish seeds were transferred to the maintenance aquarium. The number of catfish seeds was observed from one day after the injection of pathogenic bacteria until the seventh day. Probit analysis using Microsoft Excel to calculate the LC50 determination.

Preparation of Feed with Asam Sunti
This study used commercial feed in crushed granules containing 30% protein, 3% crude fat, 12% moisture, and 4% fiber. Then the feed is added with commercial asam sunti at a predetermined dose and adjusted according to the treatment (Widanarni et al., 2012).

Asam Sunti Test in Controlling The Pathogenic Bacterial Infection
In each aquarium, 10 fish/aquarium of catfish seeds were kept for seven days to control pathogenic bacteria. For feeding with a frequency of twice a day at satiation, namely at 08.30 and 17.00 WIB according to the treatment dose, namely treatment 0% (K), 0.5% (P1), 1% (P2), 2% (P3), and 4% (P4).
Catfish fry (test fish), inactivated by immersion in water at 25 C for 30 seconds, were infected intraperitoneally with pathogenic bacteria (density according to LC50 test). Then the test fish were placed in a maintenance aquarium by providing a feed without asam sunti two times a day at satiation, namely at 08.30 and 17.00 WIB. Fish maintenance and treatment for 14 days (Dinamella et al., 2013).

Testing The Number of Pathogenic Bacteria in The Liver and Kidneys of Fish
The determine of total plate count (TPC) of each pathogenic bacteria were determined to determine the number of pathogenic bacteria present in catfish seeds (test fish). The method is that 1 g of liver and kidney of the test fish was taken aseptically and put into 10 mL of sterile butterfield's phosphate buffered (BFP), then homogenized and serial dilutions were carried out up to 10 -6 . As much as 1 mL was inoculated from each dilution into a cup containing TSA media. At 30 C, incubation was carried out for 48 hours. After 48 hours of incubation, the number of colonies was read using a colony counter.

Observation of Survival Rate
According to Agung et al. (2022), the stages of reducing and preventing pathogenic bacterial infection on survival using a formula to know the effect of treatment. The formula is: With : SR = Survival Rate (%); No = Initial population (tail) Nt = Final population (tail)

Research Parameters
The parameters of this study included the level of pathogenicity of pathogenic bacteria, LC50 concentration of bacterial infections, control of pathogenic bacterial infections, TPC of pathogenic bacteria, absolute weight gain of fish, and environmental parameters, including temperature, and pH.

Data analysis
Data from observations of the pathogenicity of pathogenic bacteria, environmental parameters were analyzed descriptively presented in tabular form, LC50 observations of the concentration of bacterial infections were analyzed with probit. For controlling bacterial infection, TPC of pathogenic bacteria and absolute weight gain were analyzed using analysis of variance (ANOVA) with a 95% degree of confidence (P<0.05) using Statistical Package for the Social Science (SPSS) version of IBM 25 if there are significant differences significant, further tested by Duncan's Test 95% (P<0.05).

Results and Discussion Pathogenicity of Pathogenic Bacteria in Catfish Seeds
Pathogenicity of pathogenic bacteria in catfish seeds was seen in the death of Edwardsiella tarda treatment. The results showed that catfish infected with E. tarda had a mortality rate of 50% each, while in the control treatment, there was a mortality rate of 10%. Table 1.
The death of catfish fry due to infection with E. tarda showed that the bacterium was indeed a disease that attacked catfish fry. Nur (2019) said that E. tarda bacteria are pathogenic, which can cause death in catfish fry. According to Firma et al. (2012) E. tarda bacteria can cause disease in catfish fry, these two types of bacteria can infect the liver and kidneys so that their function is disrupted. Wahidi et al. (2022) explained that impaired liver function in Clarias gariepinus causes fish to become susceptible to disease because the liver is unable to carry out the detoxification process and disrupts the phagocyticity of foreign bodies that enter the body, as a result of this disturbance, the catfish seeds become weak accompanied by reduced appetite until it ends with death. Meanwhile, the death of control catfish fry was thought to be due to stress conditions and environmental factors (Hendri, 2022). Mortality of over 50% of catfish seeds in a very short time was caused by E. tarda where the bacteria have a very high ferocity, major losses in catfish seed cultivation can be caused by E. tarda (Qosimah, D., & Khotimah, H, 2020).

LC50 Concentration of Bacterial Infection in Catfish Seeds
The higher number of dead catfish fry caused by the higher density of isolates of pathogenic bacteria was shown from the LC50 test results (Table 2.). These results are consistent with previous studies where isolates of the pathogenic bacteria E. tarda with higher densities increased fish mortality in a relatively short time of death (Narwiyani, 2011;Wahidi, B. R., 2022). Probit analysis using Microsoft Excel generated data where the LC50 value generated for E. tarda was 10 -8 , this result is to the findings of Mufidah et al. (2022).

Controllingn Pathogenic Bacterial Infections with Sunti Acid Treatment
Control of bacterial infections in catfish seeds can be done in various ways, including by improving feed quality. The results showed that fish infected with E. tarda bacteria could survive, which was marked by increased survival in the feed treatment with the addition of sunti acid. Table 3 shows a significant difference between the treatments of adding asam sunti in feed, with the best treatment being 4%. Table 3 shows that increasing the concentration of asam sunti increased the average survival rate of catfish fry in the presence of both pathogenic bacteria E. tarda. The survival of catfish fry in the presence of E. tarda showed a significant difference between the control treatment (K) and other treatments (P1, P2; P3 and P4). From this test, it can also be concluded that the highest concentration results were in the treatment of sunti acid with a concentration of 4% (P4), with the survival of catfish seeds reaching 90% in the presence of E. tarda. These results also show that the addition of asam sunti at a concentration of 4% is more effective in controlling pathogenic bacterial infections by the treatment of adding asam sunti to feed for catfish seeds, these findings are also by the research of Misrahanum (2022) and Pertiwi et al. (2020) where the administration of asam sunti was able to prevent the occurrence of pathogenic bacterial infections. According to Amelia (2020), the content of asam sunti is useful for strengthening immunity in fish where the accumulation of primary metabolites (acetic acid, ethanol, carbon dioxide, and lactic acid) present in asam sunti can inhibit bacteria.

Total Plate Count of Pathogen Bacteria
At the end of the infection reduction test, the number of pathogenic bacteria in catfish seeds was known by calculating the total plate count (TPC) of each pathogenic bacteria Table 4. Table 4. TPC value of pathogenic bacteria in the tested catfish seeds.

Bacteria
ALT (CFU/g) 0% (K) 0.5% (P1) 1% (P2) 2% (P3) 4% (P4) E. tarda 1.8 x 10 6 0.1 x 10 5 8.6 x 10 4 3.6 x 10 4 9.0 x 10 3 Note: In Control E. tarda showed significantly different in each treatment (P1; P2; P3 and P4) After treatment, pathogenic bacteria E. tarda were still found in the liver and kidneys of live catfish fry. In general, it was found that there was a decrease in the number of pathogenic bacteria in the treatment (4%, 2%, 1%) compared to the treatment (0.5% and 0%). The data in (Table  4.) shows that sunti acid can inhibit or prevent the growth of bacterial pathogens. In accordance with the opinion of Pertiwi et al. (2020), Misrahanum et al. (2022) found that sunti acid produces flavonoids, terpenoids and saponins which have the potential as antimicrobials and according to Misrahanum et al. (2022) asam sunti contains organic acids that can prevent bacterial growth.

Conclusion
The pathogenicity of Edwardsiella tarda in the pathogenicity test showed survival in catfish seeds with a mortality rate of up to 50%. Adding asam sunti to feed effectively controls pathogenic bacteria E. tarda in catfish fry. The concentration of 4% asam sunti in the feed shows the highest effectiveness in controlling pathogenic bacterial infections, whereas the E. tarda bacteria shows a figure of 90%.