This study employed probiotics Bacillus sp. NP5 from the digestive tract of tilapia (Putra & Widanarni, 2015) and made resistance to the antibiotic rifampin as a marker. Bacillus sp. NP5 was cultured on Trypticase Soy Agar (TSA) media and incubated at room temperature (27–30⁰C) for 24 h. Afterward, the bacteria was taken and inoculated on Tryptic Soy Broth (TSB) media. The inoculants were incubated in a water bath shaker at 29⁰C at 140 rpm for 24 h.
Preparation of Artemia sp.
This study employed 2 g.L− 1 Artemia sp. in the form of a system that was hatched in water with 30 g.L− 1 salinity and was given strong aeration for 24 h. After that, the nauplii of Artemia sp. were harvested by turning off the aeration. The hatching container was covered with dark plastic, and a light source was provided at the bottom. The shell of the hatched system will be on the surface while the unhatched system will settle at the bottom. Nauplii Artemia sp. will approach the light source, which was then siphoned using a hose. Meanwhile, Artemia sp. was given when the fish larvae were 2–5 days old.
Preparation of Tubifex sp. (Silkworm)
Silkworms or Tubifex sp. were kept in a rearing container and given aeration. Worms had been washed before being placed in different containers according to the treatment used. The finely chopped and washed silkworms were given to fish larvae aged 6–13 days. At this stage, the fish was not given probiotics.
Preparation of Artificial Feed
The artificial feed was prepared in the form of commercial feed with a protein content of 40%. Feeding was carried out at satiation with a rate of 11% of fish biomass. Artificial feed was given when the seeds were 14–28 days old.
Preparation of Maintenance Media Container
This study employed 12 aquariums with a size of 25 × 20 × 30 cm3. The aquarium was disinfected with 20 ppm potassium permanganate (PK) for 24 h. The aquarium was then cleaned and dried. The cleaned aquarium was placed in a fiber bath measuring 2 × 1 × 0.5 m3 and filled with 10 L of water. The fiber tub was filled with 200 L of water, and 5 heaters were installed to keep the aquarium’s temperature stable. Each aquarium was added with a hose and an aeration stone to supply oxygen.
Test Animal Preparation
This study examined catfish larvae from the Center for Development of Catfish and Catfish Cultivation (Balai Pengembangan Budidaya Ikan Patin dan Lele/BPBIPL) Cijengkol, Subang, West Java. Larvae were reared from newly hatched with an average weight and length of 1.52 ± 0.06 mg and 0.46 ± 0.007 cm. The stocking density of fish in each aquarium was 15 fish.L− 1 (150 fish.aquarium− 1).
Feed Enrichment Process
This study employed natural and artificial feed. Natural food was in the form of Artemia sp. and silkworms. Meanwhile, the artificial feed was in the form of commercial feed. The Artemia sp. and commercial feed were enriched with probiotic Bacillus sp. NP5 according to the treatment; this probiotic Bacillus sp. NP5 were 106 CFU.mL− 1, 107 CFU.mL− 1, and 108 CFU.mL− 1. The enrichment process was carried out in each different container. The process of enrichment of Artemia sp. was done by centrifuging probiotics that had been cultured in Tryptic Soy Broth (TSB) and by discarding the liquid supernatant. The precipitated pellet was added with phosphate buffer saline (PBS) as much as the volume of the removed and vortexed supernatants. The ready probiotics were added to the Artemia sp. containing 1 L of water and 30 g.L− 1 salinity and enriched with the treatment for 4 h . Afterward, Artemia sp. was harvested using a plankton net and rinsed with water. The harvested Artemia sp. were given directly to the larvae or stored in the refrigerator at 4ºC for later use that day.
The commercial feed enrichment process was carried out by centrifuging the probiotics cultured on TSB media and discarding the liquid supernatant. The precipitated pellet was added with PBS as much as the volume of the removed and vortexed supernatants. After that, probiotics were given as much as 2% of the weight of the feed; these probiotics were mixed with a binder in the form of egg whites as much as 2% of the weight of the feed . Next, the feed was sprayed with probiotics according to the treatment and air-dried. The excess feed can be stored in the refrigerator.
Maintenance and Challenge Test
Juvenile catfish received Bacillus sp. NP5 in feed with different concentrations, namely 106 CFU.mL− 1, 107 CFU.mL− 1, and 108 CFU.mL− 1; each concentration had three replicates. Meanwhile, the control was without the enrichment of Bacillus sp. NP5, but it consisted of positive control (tested on A. hydrophile) and negative control (without a test on A. hydrophila); each control has three replications. Test animals for this study follow  according to the standard guidelines of the IPB University animal ethics commission (ethical approval number 206–2021).
Probiotics were given in two stages. The first stage was giving Artemia sp. to the larvae aged 2–5 days, and the second stage was giving artificial feed to fish aged 14–28 days. Meanwhile, fish aged 6–13 days received silkworms without probiotics. Prior to treatment, catfish larvae had been sampled to determine initial weight and length. 18–24 fish− 1 larvae aged 2–5 days old were fed on the Artemia sp. every 2 h for 4 days of maintenance. Meanwhile, 0.575 g.aquarium− 1 of larvae aged 6–13 days were fed with silkworms without probiotics four times a day: at 8 a.m., 12 p.m., 4 p.m., and 8 a.m. Western Indonesian Time (WIB). Juvenile catfish aged 14–28 days were fed on artificial feed at satiation with a feeding rate (FR) of 11% of fish biomass 3 times a day: at 8 a.m., 12 p.m., and 4 p.m. WIB. The water of the rearing media was changed every other two days, starting from day 6 to 28 of rearing. The temperature (°C) ranged from 29.0-30.3, the dissolved oxygen (DO, mg.L− 1) ranged from 5.2–6.3, pH ranged from 6.19–7.12, and the Total Ammonia Nitrogen (TAN; mg.L− 1) ranged from 0.016–0.144.
After being reared with treated feed for 28 days, the fish were challenged with A. hydrophila. The challenge test process utilized a glass jar with a volume of 3 L for 15 fish. The jar was filled with water (1 L/jar) that had been deposited for 24 h. Juvenile catfish were reared at a density of 15 fish/L and adapted for 2–3 h in the container. The fish were then infected with A. hydrophila at a concentration of 107 CFU.mL− 1. The observation was conducted for seven days post-infection, and dead fish were counted as the fish survival rate data at the end of the challenge test. During the post-challenge fish rearing, the fish were fed artificial feed in the form of commercial feed without probiotic enrichment but with an FR of 11%. This artificial feed was given three times a day.
The survival rate of catfish was calculated at the end of the rearing using the following formulas .
Specific growth rate (SGR; %/day) = [ln final weight ln initial weight/days] x 100
Absolute length growth (cm) = Average length of fish at the end of rearing – the average length of fish at the beginning of rearing
Total feed consumption = Total weight of feed provided – Total weight of leftover feed
Feed conversion ratio (FCR) = (total food intake) × (weight gain) – 1
Survival rate (SR; %) = 100 × (final number of test fish)/(initial number of test fish)
Total Bacterial Count (TBC) and abundance of Bacillus sp. NP5
The total bacterial count (TBC) was performed using the plate count method of  at the beginning and end of the treatment on larvae (whole-body) and juveniles (intestines). Each treatment used two fish. Meanwhile, the observations were conducted using the Total Bacterial Count (TBC) to measure the larva and the abundance of Bacillus sp. NP5 to measure the juvenile. Meanwhile, the total bacteria employed the Trypticase Soy Agar (TSA) media, while the abundance of Bacillus sp. NP5 employed the TSA + rifampin 50 µg/mL media. The bacteria was calculated using the formula .
Bacterial abundance = number of colonies x 1/diluent factor x 1/mL sample
Coefficient of Diversity
The coefficient of diversity measured various fish lengths by calculating the coefficient of variance, which was the percentage of the sample standard deviation to the mean value. This measurement used the following formula.
Coefficient of variance (%) = root of variance/sample mean x 100
Catfish Immune Response
The differential leukocyte was calculated before and after the challenge test. Blood was dripped on the object-glass. Then, another object glass was placed on the end of the first object glass that had already contained blood in a 30° shape. The glass object that formed the corner was pulled to the end. After that, the preparations were air-dried and fixed in methanol solution for five min. The preparations that had been immersed in the methanol solution were air-dried and immersed again in diluted Giemsa solution (1:20) for 15 min. The next steps were rinsing the preparations using distilled water and air-drying them. The finished preparations were observed under a microscope with a magnification of 400 times. Differential leukocytes were calculated according to their types, namely lymphocytes, neutrophils, and monocytes, using the following formula .
% Lymphocytes = (Number of Lymphocytes)/(Lymphocytes + Neutrophils + Monocytes) ×100
% Neutrophils = (Number of Neutrophils)/(Lymphocytes + Neutrophils + Monocytes) ×100
% Monocytes = (Number of Monocytes)/(Lymphocytes + Neutrophils + Monocytes) ×100
The phagocytic activity had been calculated before and after the challenge tests. Fish blood was put into a 50 µL microtube, then 50 µL of Staphylococcus aureus suspension was added to the PBS and homogenized. After that, the blood was incubated for 20 min at 28⁰C. Then, a 5 µL solution was taken to make preparations for review. Then, the solution was fixed with methanol for 5 min and dried. The preparations were soaked for 15 min in Giemsa's solution, washed in running water, and dried. The solution was observed using a microscope with a magnification of 400 times. The phagocytic activity was calculated using the following formula .
Phagocytic Activity = (Number of phagocytic cells)/(Number of phagocytic cells) ×100
Respiratory Burst (RB) Activity
The respiratory burst activity was calculated using the reduction principle of nitroblue tetrazolium (NBT) which produces formazan—the sum of the sizes of superoxide anions. 50 µL of blood was put into the holes of the microplate titer and incubated at 37⁰C for 1 h. The supernatant formed was discarded and rinsed three times with 50 µL of PBS. Then, 50 µL of 0.2% NBT solution was added and incubated at 37⁰C for 1 h. Furthermore, the 0.2% NBT solution was discarded, and the blood was fixed with 50 µL 100% methanol solution for 2 min and rinsed with 30% methanol solution 3 times and dried. The formed formazans blue precipitate was dissolved using 60 L of 2 N KOH solution and 70 µL of dimethyl sulphoxide (DMSO) added to each titer microplate hole. The optical density (OD) of the formazan blue precipitate was then measured using a microplate reader at a wavelength of 630 nm .
The obtained data were processed using Microsoft Excel 2013 and Minitab 16. Meanwhile, the data were analyzed using the analysis of variance (ANOVA). When the ANOVA results were significantly different, the data were further tested using the Tukey Advanced Test.