Int. J. Aquat. Biol. (2022) 10(6): 438-450 ISSN: 2322-5270; P-ISSN: 2383-0956 Journal homepage: www.ij-aquaticbiology.com © 2022 Iranian Society of Ichthyology Original Article Effect of dietary Lactococcus lactis and Bacillus subtilis on the innate immunity, intestinal microbiota, histometrical indices, and resistance against Aeromonas hydrophila in Oscar, Astronotus ocellatus Agassiz, 1831 Abbas Hasaninia1, Habib Vahabzadeh Roudsari1, Hossein Khara1, Alireza Shenavar Masouleh2, Mohaddeseh Ahmadnezhad3 1Department of Fisheries, Lahijan Branch, Islamic Azad University, P.O. Box: 1616, Lahijan, Iran. 2International Sturgeon Research Institute, Iranian Fisheries research organization, Agricultural Research, Education and Extension Organization (AREEO), Rasht, Iran. 3Inland Waters Aquaculture Research Center. Iranian Fisheries research organization, Agricultural Research, Education and Extension Organization (AREEO), Bandar Anzali, Iran. s Article history: Received 11 September 2022 Accepted 26 November 2022 Available online 2 5 December 2022 Keywords: Bacillus Immune system Lactococcus lactis Microbiota Histology Abstract: This work aimed to investigate the effect of dietary Lactococcus lactis and Bacillus subtilis on the immune responses, intestinal microbiota, and resistance to pathogens of Oscar, Astronotus ocellatus. During 70 days trial, 300 juveniles (8.96±0.033 g) were fed diets enriched with L. lactis and B. subtilis. The treatments included 150, 300, 450 mg kg-1 of dietary L. lactis (LL150, LL300, LL450); 150, 300, 450 mg kg-1 of dietary B. subtilis (BS150, BS300, BS450); 150, 300, 450 mg kg-1 of diet an equal mixture of L. lactis and B. subtilis (MIX150, MIX300, MIX450); and a non- supplemented control group. At the end of the rearing period, histological, immunological, and intestinal microbiota indices in treatments were investigated. To evaluate disease resistance, 15 fish in each treatment were infected in each treatment by Aeromonas hydrophila. The results showed that adding B. subtilis and L. lactis, particularly in MIX300, reduced the anaerobic heterotrophic bacterial microbiota and increased lactic acid bacteria (LAB) in fish. The highest white blood cell (WBC) level was recorded in the LL150 group. The lymphocytes in fish fed LL150, LL300, MIX150, and MIX300 diets were changed and neutrophils of LL150, LL300, LL450, MIX300, and MIX450 were significantly increased. Monocytes in fish fed MIX300 and MIX450 diets raised significantly. The IgM, ACH50, and lysozyme levels in fish-fed diets enriched by bacteria, especially in LL450, were significantly higher than the control treatment. The intestinal villi in LL450, BS150, and MIX450 were significantly higher, showing lower damages than the other treatments. The survival rates of the infected fishes were higher in MIX150 and MIX300 groups. Introduction Oscars (Astronotus ocellatus) are ornamental fish of distinct quality and value. They are known as ever- hungry fish interested in feeding even at satiation with a unique appearance, intelligence, and behavior, making them enjoyable to aquarium enthusiasts. These, combined with the fact that Oscar can live just as long as a dog, make it more like a pet than most other fishes (Yilmaz and Arsalan, 2013). Health and nutrition are two vital aspects of ornamental fish farming, as its annual international exports are around US$ 200 million, or less than 3% of the total world fish trade (Ghosh et al., 2008; Gobi Correspondence: Habib Vahabzadeh Roudsari DOI: https://doi.org/10.22034/ijab.v10i6.1753 E-mail: habib.vahabzadeh@gmail.com DOR: https://dorl.net/dor/20.1001.1.23830956.2022.10.6.1.4 et al., 2018). The gastrointestinal tract of fish is a key area of interaction with pathogens in fish farms. It is important to enhance the cultured fish immune system by enriching the normal gut microbiota, as it affects a wide range of biological processes, including the development of gut-associated lymphoid tissue (GALT) and the ability to combat infections (Nayak et al., 2007). Microbial population diversity will be altered by manipulating microbial populations and changing environmental conditions caused by the proliferation of selected bacteria (Sayes et al., 2018). The competitive elimination of 439 Int. J. Aquat. Biol. (2022) 10(6): 438-450 pathogenic bacteria by probiotics can effectively reduce or eliminate the prophylactic use of antibiotics in intensive systems (Saputra et al., 2016). It has been well-established that the GI performance of aquatic organisms is modified via microbial modification (Doan et al., 2021). Providing probiotics via diet can improve the microbial balance of the host (Nayak, 2010) and increase resistance against disease-causing bacteria in aquatics (Al-Dohail et al., 2011). Lactic acid bacteria (LAB) of Lactococcus lactis are known to produce a wide range of antimicrobial compounds that can inhibit growth or kill a broad range of bacteria (Loh et al., 2017). Bacillus subtilis identified in the GI tract of several finfish species are spore-forming bacteria resistant to adverse environmental conditions, with various species showing unusual physiological features enabling them to survive in different environments (Díaz- Rosales et al., 2006). Bacillus can affect nutrition, adherence, and colonization of pathogens, affecting fish’s immune system with probiotic potentials (Soltani et al., 2019). The adhesion of Gram-positive probiotics into fish feed can improve the immuno-physiological functions of hosts and enhance their disease resistance. Probiotics multiply after settling in the intestine and use sugar to grow and produce unsaturated fatty acids (Blottiere et al., 2003). The probiotic dosage is a missing factor in earlier studies (Shenavar Masouleh et al., 2016). In addition, insufficient data are available to demonstrate the behavioral growth of Gram-positive probiotics, such as synergistic or antagonistic effects (Doan et al., 2021). The effects of probiotics on the growth indices and nutrition of some fish species were reviewed in previous studies e.g. Oscar and the Nile tilapia (Oreochromis niloticus) (Safari and Atash, 2013; Won et al., 2020). The B. subtilis as a probiotic has recently been introduced to Iranian fish farmers; however, its effect on Oscar has not been studied. Based on the above-mentioned background, the present study aimed to investigate the effects of dietary L. lactis and B. subtilis on immune responses, intestinal microbiota, and resistance to pathogens in Oscar fish. Materials and methods Experimental conditions: During 70 days of the experimental period, 300 Oscar juveniles were stocked in 50-liter aquaria, ten fish per aquarium (0.16 g L-1). There were 10 experimental treatments, each with three replicates. The mean weight and length of juveniles were 8.96±0.03 g and 8.23±0.02 cm, respectively. Adaptation to the lab condition and feed was made for ten days. Bacillus subtilis (Persian type culture collection: PTCC No.:1204) and L. lactis (registered at NCBI under No. JF831150) were added to the basic diet (Coppens, Germany) in different concentrations, including 150 mg kg-1 (1.5×106 CFU g-1), 300 mg kg-1, (3×106 CFU g-1) and 450 mg kg-1 (4.5 ×106 CFU g-1), as an equal mixture of B. subtilis and L. lactis (Table 1). The predicted powder-containing bacteria was dissolved in 50 mL of ringer solution and added per kg of diet. The control group received the same basal diets without added bacteria. The pelleted diet from Coppens Company (TROCO CRUMBLE HE) with size of 0.8-1.2 mm was used as basic diet, containing 56% crude protein, 15% crude fat, 0.5% fibre, 8.4% ash, 2.3% calcium, 0.7% sodium, and 1.4% total phosphorus. The juveniles were fed at 3-5% of their body weight based on the water temperature and biomass of each aquarium (according to initial and mid-term biometric measurements) 3 times a day (8 a.m., 12 a.m., and 2 p.m.) for 70 days. The growing juveniles were transferred to 200 L aquaria to maintain their biomass. The physico-chemical parameters of water, including temperature, dissolved oxygen, pH, hardness (DH), and total ammonia before and after feeding, are shown in Table 2. Blood sampling and serum preparation: Feeding was stopped for 24 hours, allowing gut evacuation, with three fish randomly selected from each aquarium. Using a syringe, 2 mL of blood was taken from their caudal vein. Then, 1.5 ml of blood was taken and 440 Hasaninia et al./ Effect of dietary Lactococcus lactis and Bacillus subtilis on the innate immunity of Oscar saved in non-heparinized tubes for measuring immunological parameters (Abarike et al., 2018). The blood serums of non-heparinized samples were separated by centrifuging at 1409 g force (3000 rpm) for 10 min (Labofuge, manufactured by Heraeus Sepatech, Germany), then stored at -80°C until analysis. The remaining blood samples (0.5mL) were used for blood cell counts. The blood sample was diluted with a Natt-Herrick solution to calculate white blood cells (WBC) by a Neubauer hemocytometer slide. The blood smears on glass microscope slides were stained with Giemsa for the differential leukocyte count, and the percentage of different leukocytes was determined (Mohammadian et al., 2020). Measurement of immunological parameters: The immunoglobulin M (IgM) of the serum was measured by an immunoturbidimetric assay in a spectrophotometer (2100-VIS, Unico, the US) at a wavelength of 340 nm with distilled water as the control (Teige et al., 2019). To determine lysozyme activity through the gradual lysis of a Gram-positive bacterium (Micrococcus lysodeikticus, Sigma, USA), 50 μL of serum was added to 950 μL of the M. lysodeikticus solution (200 mg mL-1 of the bacterium in 5% sodium phosphate at a pH of 6.2). The solution turbidity observation was at 530 nm and 22°C after 0.5 to 4.5 minutes by a turbidimetric assay in an Elisa reader (Awareness, USA). To record the data (mg mL-1), egg lyophilized albumen lysozyme (Sigma) was applied based on Sharifuzzaman and Austin (2009) and Merrifield et al. (2010). The Alternative complement pathway (ACH50) was measured based on the photometric method at 414 nm using a spectrophotometer (Awareness, USA) through hemolysis of rabbit red blood cells (RaRBC; TCS Biosciences Botolph clydon, UK) (Tukmechi et al., 2011). Intestinal bacteria count: Three samples from each treatment were randomly selected and euthanized with 1500 ml L-1 of clove oil (CPCSEA, 2021). Then intestine was removed under sterile conditions (Nayak et al., 2007). The ringer solution was applied, making 101 to 108 dilutions of the intestinal fecal contents. After sampling, dilutions of 101 through 107 of intestinal extracts were prepared using a sterile Ringer solution. Then 0.1 mL of each dilution was inoculated on the Tryptic Soy Agar (TSA) medium and De Man, Rogosa, and Sharpe agar (MRS) media (LAB specific medium) for the intestinal bacterial count. TSA incubation plates were in aerobic condition at 25°C and MRS plates Treatment L. lactis added to basic feed (mL kg-1) B. subtilis added to basic feed (mL kg-1) Control 0 0 LL150 150 - LL300 300 - LL 450 450 - BS150 - 150 BS300 - 300 BS450 - 450 MIX150 75 75 MIX300 150 150 MIX450 225 225 Table 1. Experimental treatments based on added bacteria to basic diet. Nitrite (mg l-1) Ammonia (mg l-1) Hardness pH Dissolved Oxygen (ppm) Temperature (ºC) <0.04 0.07-0.10 172.4±0.4 7.36 ±0.21 7.6±0.44 27.84±0.32 Table 2. Physico-chemical parameters of water during the 70-days of rearing 441 Int. J. Aquat. Biol. (2022) 10(6): 438-450 were incubated at 30°C and under anaerobic conditions. The bacterial count (CFU) took place after the incubation. Bacterial challenge test (BCT): Fish were exposed to pathogens based on Al-Dohail et al. (2011) and Sharifuzzaman and Austin (2009). After final sampling, Aeromonas hydrophila (ATCC:15309, obtained from Pasture Institute, Iran) was injected into fishes to evaluate their resistance to pathogens. Five specimens, 30-35 g., were randomly selected from each replicate in all treatments. Upon anesthetizing, 0.1 ml of A. hydrophila (a dosage of 1×108 cells/fish) was injected intraperitoneal (McFarland Standard Kit No. 1, Dalynn biologicals). During the 14-day BCT period, the water temperature of the tanks was 28°C. Individual infections, mortalities, and disorders were monitored and recorded daily. At the end of this period, fins, skin, internal organs, liver, and swim bladder were sampled and examined to identify probable lesions. Intestinal histology: After opening the abdomen, the anterior, middle, and posterior parts of the intestines were removed and fixed into Buen's solution. Following 48 hours, the fixed intestinal tissues were processed, and histological slides were prepared based on Eagderi et al. (2013) and stained with hematoxylin-eosin. Images were taken using a light microscope (BEL, BIO2, Italy) equipped with a EUREKAM 10.0 camera. ImageJ 1.46r software was used for the histomorphometry of tissue expansions. Mucous folds and other histometric parameters were measured in 6 random fields. Measurements were made on the mucosal fold’s height, the thickness of the mucosal epithelium, the parine layer, submucosa, and the muscle layer. The tissue indices were compared between treatments based on the average size of the three parts of the intestine (Suvarna et al., 2012). Data analysis: The data normality of all measured parameters was determined using the Shapiro-Wilk test. The treatments were compared using the one- way analysis of variance (ANOVA). After ensuring the homogeneity of variances, Duncan Multiple Range Test (DMRT) was used to compare treatments. Statistical data analyses were done using SPSS-23, and the graph was plotted in Excel-2016. Results WBC and differential count: The lowest WBC was recorded in the control group, while the highest value was observed in the LL150 group (P<0.05; Table 3). The lymphocyte of all treatments that were fortified with probiotics was lower than the control group (P<0.05) (Table 3). Neutrophils of all treatments receiving B. subitilis and L. lactis were higher than the control one (P<0.05; Table 3). The highest monocyte percentage was recorded in MIX300 and MIX450 (P<0.05). There was no significant difference in eosinophile between treatments (P>0.05; Table 3). Serum immunological parameters: The serum lysozyme activity in the LL450 and MIX450 was Treatments WBC (mm3) Lymphocytes (%) Neutrophils (%) Monocytes (%) Eosinophils (%) Control 3316.67±72.64c 82.67±0.88a 12.33±0.33b 4.67±0.33bc 0.33±0.33 LL150 5000.00±493.28 a 77.33±0.33c 16.00±0.57a 6.00±0.57abc 0.67±0.33 LL300 3633.87±0.007 bc 77.33±1.20c 16.00±1.52a 6.00±0.57abc 0.67±0.33 LL450 4033.87±284.80 bc 78.00± 0.53bc 15.67±0.06a 5.67±0.33abc 0.67±0.33 BS150 3816.87±120.19 bc 81.00±1.53ab 14.00±1.20ab 4.67±0.67bc 0.33±0.33 BS300 3500.00±208.17 c 79.00±1.00bc 14.67±1.15ab 5.33±0.33abc 1.00±0.58 BS450 3450.00±125.83 c 78.33±0.88bc 14.33±0.88ab 6.33±0.33ab 1.00±0.58 MIX150 4033.33±272.84 bc 79.67±0.88bc 15.67±0.66ab 4.33±0.33c 1.00±0.58 MIX300 4433.33±437.16 ab 77.00±1.15c 15.67±0.33a 6.67±0.88a 0.67±0.33 MIX450 4133.33±176.38 bc 77.00±1.00c 14.93±0.30a 6.67±0.33a 0.67±0.33 Values expressed as mean±SD. Different letters in each column indicate the significance of the difference (P<0.05). Table 3. Total and differential WBC of Lactococcus lactis and Bacillus subtilis added diets for Oscar after 70 days. 442 Hasaninia et al./ Effect of dietary Lactococcus lactis and Bacillus subtilis on the innate immunity of Oscar significantly higher than the control treatment (P<0.05; Table 4). The ACH50 was higher in all treatments than in the control one (P<0.05; Table 4). Treatments of the LL450, BS300, and MIX450 were the highest from others (P<0.05; Table 4). Based on the results, the IgM, lysozyme, and ACH50 were influenced by increasing bacterial dosage in all treatments (Table 4). The IgM level of LL450 and BS300 was significantly increased in treatments (P<0.05; Table 4). Intestinal microbiota counts: The results revealed that aerobic heterotrophic bacteria of intestinal mucus and LAB were well-colonized, and their number in the intestine was significantly elevated by increasing bacterial dosage (P<0.05). Considering the total intestinal microbiota on TSA and MRS media, there was a significant difference between all treatments and the control (P<0.05). The lowest and the highest bacterial counts were observed in the LL150 and the control, respectively. The total LAB count in the intestine of all treatments significantly decreased compared with control and B. subitilis treatments. However, MIX150 and MIX300 exhibited a significant difference from the control (P<0.05). There was an increasing value in the number of intestinal mucus bacteria related to bacterial dosage of diets (Table 5). Intestinal histology: In the histometeric indices (Table 6), based on the average size in the anterior, middle, and posterior portions of the intestine, the villius average height in MIX450, villi epithelium average diameter, villi average diameter, the Treatments IgM (mL dL-1) Lysozyme (µg mL-1) ACH50 (U%) Control 32.50±0.50c 28.00±1.00c 128.0±1.0d LL150 33.00±1.00 c 29.00±1.00c 129.0±1.0cd LL300 38.00±1.00 abc 30.00±1.00bc 135.0±2.0abc LL450 42.00±1.00 a 36.50±2.50a 137.0±1.0a BS150 34.00±1.00 c 28.00±2.00c 129.0±2.0cd BS300 40.50±3.50 a 34.50±2.50abc 136.5±4.5a BS450 38.50±0.50 ab 32.50±1.50abc 134.0±1.0abcd MIX150 32.50±0.50 c 30.00±1.00bc 130.0±0.0bcd MIX300 37.00±1.00 abc 31.00±1.00abc 135.0±1.0abc MIX450 38.00±3.00 abc 37.50±3.50a 137.0±2.0a Values expressed as mean±SD (n=81). Different superscript lowercase letters within each column represent significant differences (P<0.05). Table 4. The IgM, Lysozyme and ACH50 value of A. ocellatus fed with diets containing Lactococcus lactis and Bacillus subtilis after 70 days. Table 5. The number of aerobic heterotrophic bacteria of intestinal mucus on TSA and MRS (log CFU g-1±SD). Treatments TSA (CFU g-1) MRS (CFU g-1) Control 8.29±0.006a 3.54±0.06e LL150 5.71±0.05 g 4.50±0.01ab LL300 6.33±0.02 e 4.59±0.01b LL450 7.25±0.01 d 4.42±0.01c BS150 6.11±0.04 f 4.03±0.01d BS300 8.09±0.04 b 4.02±0.04d BS450 8.13±0.02 b 4.004±0.01d MIX150 7.75±0.07 c 4.81±0.02a MIX300 7.78±0.026 c 4.88±0.01a MIX450 8.07±0.03 b 4.62±0.01b Values were expressed as mean±SD (n=3): different superscript lowercase letters within each column represent significant differences (P<0.05). 443 Int. J. Aquat. Biol. (2022) 10(6): 438-450 thickness of parine layer in LL300, the thickness of muscle layer in BS300, BS450, the thickness of the submucosal layer in LL450 and BS150 were significantly different compared to the control (P<0.05) (Fig. 1). Bacterial challenge test (BCT): After two weeks of monitoring Aeromonas-infected Oscars, the highest mortality rate was detected in the control group (P<0.05), while the lowest mortality rate (P<0.05) was recognized in MIX150 and MIX300 (Fig. 2). The first mortality was recorded after six days of injection in the control group. The most common lesions observed during BCT, were bleeding in the anus, base of the anal, caudal fins, and around the mouth. Limited necrospy hemorrhage was detected in the liver, swim bladder, peritoneal cavity, and hyperemia in the kidney (Table 7). Discussion The results showed a significant increase of WBC as an immunity index in the LL150, and MIX300. Increased levels of WBC were observed in rainbow trout and Nile tilapia fed B. subtilis and L. lactis treated diets (Balcázar et al., 2007; Opiyo et al., 2019), indicating that an increase in probiotic supplementation may reflect improved immunity of fish. Probiotics such as B. subtilis (Lee et al., 2017) and L. lactis (Xia et al., 2018) have been proven useful in aquaculture. The immune system response of fish can be greatly influenced by adding B.subtilis and L. lactis in different fish diets (Nayak et al., 2010; Dias et al., 2020). After colonizing Bacillus in the mucosal epithelium of the intestine, protection signs show against pathogens by competitive exclusion or competition for available energy and the production of inhibitory compounds (Soltani et al., 2019). The results showed a significant IgM and lysozyme improvement in Oscar, especially in treatments LL450, BS300 and BS450. These findings are similar to those reported for Lactobasilus rhamnosus, Carnobacterium maltaromaticum, and C. divergens in rainbow trout (Panigrahi et al., 2004; Kim and Austin, 2006), Pediococcus acidilactici in the Nile tilapia (Ferguson et al., 2010), and the Oscar (Safari and Atash, 2013). According to Nayak (2010), probiotics can stimulate the intestinal immune system by increasing the number of IgM and acidophilic granulocytes. Safari and Atash (2013) reported that the lysozyme level significantly increased in the blood serum of Oscar fed with diets enriched with P. acidilactici. The effects of B. subtilis and L. lactis on the immune responses of the Nile tilapia showed that the immune system Table 6. Mean intestinal histomteric indices (µm) of Oscar fed by Lactococcus lactis and Bacillus subtilis and mixed probiotic diets. Treatments Villus Height (µm) Epitelium Diameter (µm) Villus Width (µm) Muscular Thickness (µm) Lamina Propria (µm) Sub Mucosa (µm) Control 369.77±14.85 e 32.42±0.90f 68.57±1.99g 85.01±3.37c 13.42±0.38e 32.66±1.04d LL150 514.65±17.19 bc 47.65±1.16ab 98.77±2.74b 74.13±2.61d 46.87±1.88b 36.30±1.27d LL300 493.64±12.78 c 49.07±1.67a 105.58±3.76a 85.22±3.44c 58.55±1.89a 42.69±1.93c LL450 547.28±12.97 ab 44.73±1.26bc 92.01±2.32cd 86.91±2.49c 60.16±1.54a 50.37±1.32a BS150 546.48±17.72 ab 41.66±1.06cd 88.44±2.20cd 85.33±2.15c 47.83±1.41b 51.63±1.73a BS300 421.33±12.15 d 37.37±1.08e 78.38±2.14ef 117.20±5.35a 42.17±1.47c 49.46±1.60ab BS450 473.31±9.78 c 41.28±1.10cd 86.58±2.21cd 114.26±3.57a 42.67±0.96c 47.54±1.32ab MIX150 495.22±18.27 c 34.13±0.95f 72.78±1.87fg 100.37±2.72b 38.16±1.15d 47.91±1.46ab MIX300 503.54±16.63 bc 41.83±1.26cd 87.94±2.47cd 61.91±1.84e 47.06±1.63b 41.77±1.12c MIX450 580.03±15.05 a 39.03±0.98de 82.23±1.91e 97.49±3.75b 46.31±1.33bc 45.44±1.36bc Different letters in each column indicate significant differences between them (P<0.05). (Mean±SD). 444 Hasaninia et al./ Effect of dietary Lactococcus lactis and Bacillus subtilis on the innate immunity of Oscar performance, including lysozyme, became significantly efficient in the fishes fed-diets enriched with probiotic bacteria (Won et al., 2020). The probiotic bacteria could improve the lysozyme level Figure 1. Histologic cross-section view of the posterior intestine of Oscar in the control group (A&B) and the treatment 9 (MIX450) (C& D) (Abbreviations: GC: Goblet Cell, EC: Enterocyte, LP: Lamina Propria, Muscular thickness (MT), SM: Submucosa. Eosin- hematoxylin staining. Figure 2. The mortality rate of differently treated Aeromonas ocellatus exposed to A. hydrophila after 14 days. 445 Int. J. Aquat. Biol. (2022) 10(6): 438-450 in different species, including rainbow trout (Balcázar et al., 2007), the Malabar grouper (Epinephelus coioides) (Sun et al., 2012) and Nile tilapia (Han et al., 2015; Abarike et al., 2018; Opiyo et al., 2019). The use of LAB in aquaculture can affect the IgM level as an important humoral immunity against pathogens (Panigrahi et al., 2005). It has been shown that B. subtilis in the diet of rainbow trout and Rohu (Labeo rohita) can improve the IgM level (Nikoskelainen et al., 2003; Nayak, 2010), which is in line with the findings of the present study on Oscar. Our study results indicated that the IgM level in LL450 and BS300 was significantly higher. The proof of higher IgM levels in some treatments can be attributed to stimulating antibodies produced by LAB (Yu et al., 2020). Panigrahi et al. (2004) reported that feeding rainbow trout with diets enriched with L. rhmanosus increased ACH50 in the anterior part of the kidney. A similar increasing trend in ACH50 has been reported in the brown trout (Salmo trutta) (Balcázar et al., 2006), rainbow trout (Ramos et al., 2015) and the gilthead seabream (Sparus aurata) (Díaz-Rosales et al., 2006) fed diets containing L honi, and L. lactis and also in rainbow trout fed diets enriched with L. rhamnosus (Nikoskelainen et al., 2003). Compared to the control, the increase of ACH50 in all treatments indicated the positive effect of probiotics. The findings of the present study showed that the addition of L. lactis and B. subtilis to the basal diet of Oscar could be due to the increasing colonization rate of microbiota or a significant reduction of the intestinal bacterial count. Based on our results, diets with 1010 CFU B. subtilis, and L. lactis led the intestinal bacterial count to log 5.71 CFU mL-1 and the intestinal LAB count ranged between log 3.33 to log 4.81 CFU mL-1. The highest bacterial count was found in MIX300. Feeding the Persian sturgeon and Beluga (Huso huso) fry with diets enriched with two types of LAB (L.mesenteroides and L. curvatus), at a dosage of 109, showed the intestinal bacterial count about log 3 CFU mL-1 and LAB count was log 2.27 to log 3.02 CFU mL-1 (Askarian et al., 2011). During the Persian sturgeon feeding on diets enriched with L. lactis at a dosage of 108, the intestinal bacterial count reached log 4.05 CFU mL-1 and the intestinal LAB count ranged between 3.35 and log 4.19 CFU mL-1 (Shenavar Masouleh et al., 2016). The inhibited growth of pathogenic bacteria by beneficial bacteria could be due to the individual or combined production of antibacterial metabolites (e.g., bacteriocins, siderophores, lysozymes, proteases), competition for essential nutrients, alteration of pH by organic acid production, and competitive exclusion (Kim and Austin, 2006; Mukherjee and Ghosh, 2016). In addition, the inhibitory effect is related to the organic acid excreted by L. lactis (Loh et al., 2017). The differences in CFU are due to the feeding behavior of species, the initial dosage of added bacteria, its strain, and rearing environmental conditions, particularly water temperature (Martínez Cruz et al., 2012; Sayes et al., 2018). The intestinal microbiota showed a significant difference between juveniles fed diets enriched with bacterial species and those in the control group. Both added bacteria were effective in reducing the populations of common intestinal bacteria. Moreover, the results showed that LABs were well- colonized in the intestine of Oscar, and their population in the intestine significantly increased with the increase of enrichment. The results of probiotic bacteria in the MRS medium showed that the intestinal wall in the control group lacked these bacteria. Adding probiotics to the diet caused a rise in the number of probiotic bacteria in the intestine, with the highest number observed in mixed diets i.e., a mixture of B. subtilis and L. lactis could easily survive in the gastrointestinal tract, attach to the mucosal surface of the intestine, proliferate, and act synergistically. Histological examinations of the intestines show a significant extension of intestinal folds in the treatments containing L. lactis and B. subtilis. It was found that B. subtilis probiotic could improve the immunity, and digestive system, especially the fish intestinal morphology parameters (Lee et al., 2017). Probiotics increase food absorption and enzyme 446 Hasaninia et al./ Effect of dietary Lactococcus lactis and Bacillus subtilis on the innate immunity of Oscar digestion process and improve the intestinal tissue of common carp (Yanbo and Zirong, 2006), sea bass using B. mojavensis (Hamza et al., 2016), the barb fed by mixed probiotics (Allameh et al., 2017) and Nile tilapia fed by B. subtilis and L. lactis (Liu et al., 2017; Xia et al., 2018). Also, using two probiotic strains (Pdp11 and Pdp13) from Alteromonadaceae, similar results were reported i.e. including the increased size and number of microvilli in the intestine of Solea senegalensis fry (Saenz et al., 2009). Probiotics have been reported to increase the thickness of the muscle layer due to the modulation of the physiological activities of intestinal mucosal cells (Lazado and Caipang, 2014). The combined use of Lactobacillus, Entrococous, Pedicoccus and Bacillus probiotics in feeding rainbow trout has increased the anterior surface of the intestine by increasing the length of the villi and the number of goblet cells in the diet containing probiotics (Ramos et al., 2015). Recent studies linked the improvement of intestinal histological indices in Nile tilapia fed- diet containing B. subtilis and L. lactis to better growth performance, feeding efficiency, and increased activity of intestinal tissues. Probiotics could facilitate the absorption of effective nutrients by improving the length of the intestinal villi, the muscle layer's thickness, and the trypsin's activity (Won et al., 2020). This study concerning the increased disease resistance induced by probiotic supplements confirmed the results of previous studies (Raida et al., 2003; Panigrahi et al., 2007; Soltani et al., 2019). Aeromonas hydrophila is a known pathogenic bacterium that causes diseases in the Cichlids family (Saputra et al., 2016). Our findings indicated that adding probiotic bacteria to the basal diet improved Oscar resistance against A. hydrophyla. The mortality rate after 14 days was lower in groups fed diets enriched with B. subtilis and L. lactis and started later than in control. In the Nile tilapia, L. lactis improved immune responses and resistance against diseases (Xia et al., 2018). The symptoms observed in intentionally A. hydrophila infected fishes were similar to those reported for Persian sturgeon exposed to the same bacterium (Soltani and Kalbassi, 2001). The symptoms in both experiments were imbalance, lethargic swimming, bruising with bleeding spots on the external surfaces, and hemorrhage in internal organs. In the rainbow trouts receiving diets supplemented with 109 and 1012 CFU of L. rhamnosus, the survival rate increased by 33.7 and 6.3%, respectively, after exposing fish to A. salmonicida (Nikoskelainen et al., 2001). Kim and Austin (2006) applied diets enriched with 107 CFU g-1 of Clostridium maltaromatieum (B26) and C. divergens (B33) for two weeks. They reported that the immunological protection improved following rainbow trout exposure to A. salmonicida and Yersinia ruckeri. Brunt et al. (2007) supplemented diets with 2×108 CFU g-1 of Bacillus (JB-1) and A. sobria (GC2) applied for 14 days on rainbow trout, showing lower mortalities than that in the control when the fishes were exposed to Vibrio ordalii, V. anguillarum, Streptococcus iniae, A. salmonicida, and Y. ruckeri. Won et al. (2020) listed many studies confirming improvements in E. malabaricus fed a diet containing L. plantarum, Chinese drums (Miichthys miiuy) with a diet added by C. butyricum (CB2), the Nile tilapia diet enriched by B. subtilis and L. acidophilus (mixture of both) showed a reduced mortality rate among fish exposed to A. hydrophila, V. anguillarum, Pseudomonas fluorescens, and S. iniae respectively. They also conducted 13 days of BCT trial with A. hydrophila on the Nile tilapia, revealing a significantly increased survival rate of the tilapia fed with diets enriched with L. lactis and B. subtilis than control one and the diets enriched with Oxytetracycline (OTC) (Won et al., 2020). Differences in the probiotic efficacy are related to bacteria strains and their concentration in basic diet, target organism criteria, including fish species, age, its disease- causing intensity, challenge duration, and environmental condition, including water temperature, salinity, pH, and alkalinity (Lee et al., 2017; Elsabagh et al., 2018; Won et al., 2020). 447 Int. J. Aquat. Biol. (2022) 10(6): 438-450 Conclusion Based on the results of the immunological analysis, the two probiotics of L. lactis and B. subtilis improved the performance of Oscar’s immune system. The Oscar juveniles fed diets enriched with probiotic bacteria had more efficient intestines and showed higher resistance against stressors and diseases. The mixture of B. subtilis and L. lactis (150 mg+150 mg) per kg of feed could modulate the intestinal microbiota, immunity, and resistance in exposure to A. hydrophila. 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