Probiotic Bacillus licheniformis competitively establishes and enhances production efficiency, immune responsiveness, and protection against Aeromonas hydrophila O:18 in Cirrhinus mrigala (Ham.)

Abstract

The use of probiotics is increasingly becoming a strategic tool in aquaculture production systems to mitigate the impacts of antimicrobial resistance. The effects of dietary Bacillus licheniformis on the immune response and disease resistance of Cirrhinus mrigala were investigated following a 30-day growth trial. One hundred eighty fingerlings (10 ± 2 g) were randomly assigned to five groups (three replicates each) and fed either a control diet or diets supplemented with B. licheniformis at 1.0 × 10⁶ cfu g⁻¹ (BL-6), 0.5 × 10⁷ cfu g⁻¹ (BL-05.7), 1.0 × 10⁷ cfu g⁻¹ (BL-7), or 0.5 × 10⁸ cfu g⁻¹ (BL-8). Probiotic supplementation significantly (P < 0.05) improved weight gain percentage, specific growth rate, and feed conversion ratio. B. licheniformis at 10⁷ cfu g⁻¹ and higher competitively replaced and decreased Gram-negative rods (from >75% to <45%), specifically Aeromonas (60 → 40), Pseudomonas (10 → <2), Alcaligenes (6 → 2), and Vibrio (4 → 0) at the genus level, with a concomitant increase in Gram-positive bacteria (25% → 55%), particularly Bacillus (3 → >40), in the intestine of C. mrigala. B. licheniformis supplementation increased total erythrocyte and leukocyte counts, hemoglobin, and serum protein, albumin, and globulin levels, while serum alkaline phosphatase (ALP), alanine aminotransferase (ALT), and aspartate aminotransferase (AST) activities decreased (P < 0.05). At or above 10⁷ cfu g⁻¹, B. licheniformis significantly improved both cellular immune responses (phagocytic index and ratio, respiratory burst activity, serum myeloperoxidase activity, ALP, and total leukocyte counts) and humoral (including adaptive) immune responses (lysozyme activity, serum bactericidal activity, serum haemagglutination titre, and natural haemolysin titre), as well as survival following challenge with A. hydrophila O:18. These findings support the potential use of this bacterial species as a probiotic candidate in diets for the culture of C. mrigala.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-41166-2.

Introduction

Globally, aquaculture represents the fastest-growing food production sector. About 25% of the world’s animal protein intake comes from fish and shellfish, and consumer’s demand for fish continue to rise¹. Aquaculture—the farming of aquatic plants and animals under controlled conditions—has emerged as the most viable means to meet this growing demand, especially as capture fisheries remain static or decline due to resource depletion and market pressures². Over the years, the aquaculture sector has undergone a sea change in order to meet the increasing demand.

Aquaculture production is increasingly being intensified through the use of commercial feeds, growth promoters and other additives. This intensification has led to frequent therapeutic antibiotic use for disease management in freshwater systems, contributing to the global threat of antimicrobial resistance^3–6. In addition, suboptimal aquaculture practices can suppress host immune system⁷, increasing susceptibility to infections from opportunistic pathogens. Consequently, in recent years, there is growing interest in understanding the role of microbial communities in aquaculture systems and their influence on host health and disease outbreaks. A wide range of strategies aimed at reducing reliance on antibiotics—including probiotics, plant extracts, essential oils, bacteriophages, antimicrobial peptides, vaccines, and combination therapies—have been explored to control pathogens such as A. hydrophila, with probiotics emerging as one of the most promising approaches due to their safety and in vivo efficacy⁸. Microbial interference therapy, in which nonpathogenic microorganisms suppress or eliminate pathogens, has therefore been proposed as a viable alternative to antibiotics for mitigating the growing problem of antibiotic-resistant bacteria⁹.

Probiotics, defined as “live microbial feed supplement which beneficially affect the host by improving its intestinal microbial balance”¹⁰, enhance host disease resistance. Several microbial strains have been proposed to be used as probiotics in aquaculture^11–18; however, among these, the genus Bacillus has gained particular attention due to its occurrence in the normal gut flora of fish and shellfish and its ability to produce antibiotics, amino acids, enzymes, and other health-beneficial compounds¹⁹.

Bacillus licheniformis has been reported to occur in the gastrointestinal tract of carp species such as L. rohita²⁰ and C. mrigala²¹, and to enhance resistance against pathogenic microorganism in tilapia (Oreochromis mossambicus)²², and rainbow trout (Oncorhynchus mykiss)²³. However, limited studies have investigated its application in C. mrigala, a bottom-feeding fish widely cultivated as part of important Indian Major Carps (IMCs) culture systems and valued for its flavor. This species is naturally exposed to higher pathogen loads due to continuous interaction with sediments where Aeromonas, Edwardsiella, Vibrio, and other bacteria accumulate^24,25. Since, B. licheniformis strain P40 produce a bacteriocin — an antimicrobial peptide that inhibits a wide range of indicator strains²⁶—this study aimed to investigate its immunostimulatory effect against A. hydrophila infection in C. mrigala, along with comprehensive growth and performance responses. Economically, IMC systems—including this species—account for 70–75% of India’s freshwater aquaculture output²⁷ and 8.7% of global carp production, with Asia producing 1 million metric tonnes annually²⁸.

Material and methods

Ethics statement, experimental fish and husbandry

All experimental procedures complied with the ARRIVE guidelines and national animal welfare regulations, specifically the guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA), Ministry of Environment and Forests (Animal Welfare Division), Government of India²⁹. The study protocol was approved by the Board of Studies of the Aquatic Animal Health Management Division, the Advisory Committee, and the competent authorities of the ICAR–Central Institute of Fisheries Education, Mumbai, India. Cirrhinus mrigala, being a commercially cultured species, is exempted from wildlife protection regulations.

Cirrhinus mrigala (Ham.) of 6 to 8 g average weight were obtained from a commercial carp farm at Palghar, Maharashtra (India). Fish were acclimatized in continuously aerated chlorine free freshwater for four weeks prior to the experiment and maintained on normal diet at a rate of 3% of the biomass per day. Chlorine free tap water was used throughout the course of experiment.

The average physico-chemical characteristics of the water conformed to the rearing standards of the experimental fish, and were as follows: temperature, 23.3 to 28.4 °C; pH, 7.6 to 8.5; dissolved oxygen (DO), 5.8 to 7.6 mg L⁻¹; hardness, 234 to 245 mg L⁻¹ (as CaCO₃); ammonia-N, 0.10 to 0.15 mg L⁻¹; nitrite-N, 0.001 to 0.005 mg L⁻¹; and nitrate-N, 0.03 to 0.07 mg L⁻¹.

Mass culture of Bacillus licheniformis

Pure bacterial isolate of B. licheniformis was obtained from the culture collection of National Chemical Laboratory, Pune, Maharashtra (India). It was verified by biochemical tests and kept in nutrient agar slant at 4°C for further use. B. licheniformis was inoculated into a conical flask (500 ml) containing nutrient broth (Himedia Ltd, Mumbai, India) and incubated at 30°C for 24h in an incubator with shaker. The culture was centrifuged at 10,000 x g for 20 min at 4°C and the supernatant discarded, while the pellet was re-suspended in phosphate buffered saline (PBS; pH 7.2). The suspension was similarly washed and recentrifuged for 4 times and then quantified by spread plate technique on nutrient agar (Himedia Ltd, Mumbai, India) to determine the number of colony forming units (cfu). Purified and quantified bacteria were kept at 4°C in suspended form and were used for feed preparation as required.

Preparation of experimental diets

Commercial ingredients were used for feed formulation (Table 1). All ingredients were weighed according to the required proportions and then mixed thoroughly followed by steaming for 30 min, cooled and the required bacterial culture was mixed properly and then pellets were made by hand palletizer. These pellets were initially air-dried on sheets of paper with a fan, followed by sun-drying on trays until fully dried. The concentration of B. licheniformes used were 0 cfu g⁻¹ (Control: without B. licheniformis), 1.0 x 10⁶ cfu g⁻¹, 0.5 x 10⁷ cfu g⁻¹, 1.0 x 10⁷ cfu g⁻¹, 0.5 x 10⁸ cfu g⁻¹ B. licheniformis of diet. Feed was prepared every week and stored in screw capped glass bottle at 4°C.

Table 1.

Composition of the experimental diet.

Ingredients	%
Rice bran	30
Mustered oil cake	30
Fish meal	20
Wheat flour	14
Sunflower oil	3
Vitamin-mineral premixa	2
Vitamin Cb Vitamin B complexc Bran powder	0.1 0.05 0.85
Proximate composition
CP	27.9
Lipid	10.3
Crude fiber	6.0
NFE	46.1
Ash	9.7
DE(calculated, Kcal kg-1)	3890

^aPremix (Agrimin) composition (quantity kg^-1):Vitamin A-6,25,000 IU; Vitamin D3-62,500 IU; Vitamin E- 250 mg; Nicotinamide-1 g; Cu-312 mg; Co- 45 mg; Mg- 6 g; Fe- 1.5 g; Zn- 2.13 g; I- 156 mg; Se- 10 mg; Mn- 1.2 g; Ca- 247.34 g; P- 114.68 g; S-12.2 g; Na-5.8 mg; K-48.05 mg.

^bRovimix Stay C 15% Hoffman La Roche) (mg ascorbic acid equivalent ascorbyl polyphosphate per kg diet).

^cComposition (quantity g−1): Thiamine mononitrate, 20 mg; Riboflavin, 20 mg; Pyridoxine hydrochloride, 6 mg; Vitamin B12, 30 µg; Niaciamide, 200 mg; Ca pantothenate, 100 mg; Folic acid, 3 mg; Biotin, 200 µg.

Experimental design

The experiment was carried out in 15 rectangular fiber tanks (Nilkamal, Mumbai, India) (100 L), at the wet laboratory of ICAR-Central Institute of Fisheries Education, Mumbai. One hundred eighty fingerlings of uniform size (10 ± 2 g) were randomly distributed in five experimental groups with each of three replicates following a complete randomized design. Feed was given at 3% of body weight twice daily at 09:00 and 17:00 h. Fecal matter and uneaten feed were siphoned out daily with about 50% water exchange. Three tanks were kept as controls, while the other twelve were treatment tanks. Treatment groups were fed with B. licheniformis diets containing 1.0 x 10⁶ cfu g⁻¹, 0.5 x 10⁷ cfu g⁻¹, 1.0 x 10⁷ cfu g⁻¹ and 0.5 x 10⁸ cfu g⁻¹ for 30 days and the control groups received the same feed without B. licheniformis.

Analysis of gastro-intestinal microflora

The gastro-intestinal microflora was analyzed on the 15^th and 30^th day post-feeding. Three fishes were randomly selected from each treatment (i.e., one fish from each replicate group) and collected in sterile plastic bags. The fishes were starved for 20 h before sampling.

Sample preparation

Fishes were sacrificed by severing the spine just behind the cranium with a scalpel. The ventral surface of each fish was sterilized with alcohol, and standard aseptic procedures were followed to obtain the biopsy. The intestine was aseptically excised from each fish. The intestines from all three replicates of each treatment were pooled and weighed to obtain 1.0 g of tissue. This was transferred to tubes containing 9.0 ml sterile 0.85% NaCl solution and homogenized. The homogenates were serially diluted up to 10^-⁹ in 9.0 ml volumes of sterile 0.85% NaCl.

Media and enumeration procedures

Media for bacterial analysis were obtained from HiMedia (Mumbai, India). The solid media used, and corresponding bacterial groups enumerated were as follows: Nutrient agar (total plate count), EMB agar (Enterobacteriaceae), Aeromonas Isolation Medium Base (Aeromonas), Pseudomonas Isolation Agar Base (Pseudomonas), KF Streptococcus Agar Base (faecal Streptococci), TCBS agar (Vibrio), and Bacillus Medium (B. licheniformis).

Dilutions from positive 10⁻¹ tubes were used for isolation. An aliquot of 0.1 ml was plated on the appropriate selective/enriched medium. Plates were incubated at 28 °C for 24–48 h in a BOD incubator. After incubation, colonies were counted, and morphologically distinct colonies were isolated for characterization. Numbers reported are means of three replicate plates.

Isolation and identification

Isolation was carried out by picking distinct colonies from plates containing 30–300 colonies. These were streaked onto nutrient agar plates and incubated at 28 °C for 24–48 h. After obtaining pure isolates, the genus identity was confirmed. Characterization was performed using morphological and biochemical tests, following the procedures described in Bergey’s Manual of Systematic Bacteriology³⁰.

Haematological studies

On each sampling day, eight fishes were randomly selected from each experimental group and anesthetized using 0.1 ppm MS-222. Blood samples were collected from the caudal vein using a 2 ml syringe (Discardit, BD India Pvt. Ltd, Mumbai) fitted with a 23-gauge needle pre-rinsed with 2.7% EDTA. The samples were transferred into Eppendorf tubes containing 2.7% EDTA for hematological analysis.

For serum collection, blood was drawn into Eppendorf tubes without anticoagulant. These samples were allowed to clot for 2 h at room temperature in a slanting position, followed by incubation for 30 min at 4 °C. The clotted blood was then centrifuged at 3,000 rpm for 10 min at 4 °C. Serum was collected and stored in screw-cap glass vials at −20 °C until analysis, within two days.

Total erythrocyte count

A 20 µL blood sample was diluted with 3,980 µL of Red Blood Cells (RBC) diluting fluid in a clean glass vial. The well-mixed diluted sample was charged into the counting chamber of a haemocytometer (Fein-Optik, Jena, Germany), and the RBCs counted under the 40x objective.

The number of RBCs per mm³ was calculated using the formula:

Where, N is the total number of red blood cells counted in 5 squares of the haemocytometer.

10,000 is the factor obtained after taking into consideration the initial dilution factor.

Total leucocyte count

For this, 20 µL blood was diluted with 3980 µL of White Blood Cells (WBC) diluting fluid in a clean glass vial. A well-mixed blood sample in the diluting fluid was charged into the counting chamber of the haemocytometer, and the count was taken under 40x objective.

Where, N denotes the total number of white blood cells counted in 4 squares of the haemocytometer.

500 is the factor obtained after taking into consideration the initial dilution factor.

Haemoglobin content

The haemoglobin content of different blood samples collected was analyzed following the cyanomethemoglobin method as per Van Kampen and Zijlstra, 1961³¹.

Serum chemistry

Serum parameters—total protein (biuret method), albumin (bromocresol green method), alkaline phosphatase (ALP; EC 3.1.3.1; kinetic colorimetric method), aspartate aminotransferase (AST; EC 2.6.1.1), and alanine aminotransferase (ALT; EC 2.6.1.2) (both by modified IFCC methods)—were analyzed using commercial diagnostic kits and a semi-automatic analyzer (AR 601) (Qualigens, Mumbai, India).

Determination of cellular immune parameters

Respiratory burst assay

The respiratory burst assay of blood was quantified by reduction of nitroblue tetrazolium (NBT) to formazan as a measure of the superoxide anion content^32,33.

Briefly, 50 µL of blood was pipetted into the wells of ‘U’ bottom microtitre plates and incubated at 37 °C for 1 hr to facilitate adhesion of cells. Then the supernatant was gently removed and the adhered cells were washed three times with PBS. After washing, 50 µL of 0.2% (w/v) Nitro blue tetrozolium in PBS was added to the wells and incubated for an hour at room temperature. The supernatant was removed the cells were fixed with absolute methanol for 3 min and then washed thrice with 30% (v/v) methanol. The plates were air dried before 60 µL 2N KOH and 70 µL dimethyl sulphoxide were added to each well to dissolve the formazon blue crystal. The OD of the turquoise blue coloured solution was read in microplate reader at 540 nm against a KOH/DMSO blank.

Phagocytic assay

The assay was performed following Siwicki et al. (1994) and Park and Jeong (1996) with slight modification^34,35. A. hydrophila (10⁷) cells in 0.1 ml of PBS was added to 0.1 ml of pooled blood samples in sterile microplate. This was then incubated for 30 min at 25 °C after thorough mixing in the well. After incubation, the plate was removed, and blood bacteria suspension was mixed gently again. 50 µL of this suspension was put on three glass slides and smears were made. After air drying, the smear was fixed in 95% ethanol, redried and stained with May-Grunwald Giemsa stain. The phagocytic cells and phagocytised bacteria were enumerated. Phagocytic ratio (PR) and phagocytic index (PI) were determined by enumerating 100 phagocytes per slide under a microscope. The averages of three slides were calculated.

Phagocytic ratio (i.e. percentage of cell with engulfed bacteria)

Phagocytic index (i.e. number of engulfed bacteria per cell)

Myeloperoxidase content

Total myeloperoxidase content present in serum was measured spectrophotometrically [Quade and Roth (1999) as partially modified by Sahoo et. al., (2005)]^36,37. Briefly, 10 µL of serum was diluted with 90 µL of Ca²⁺- and Mg²⁺- free Hank’s balanced salt solution (HBSS) (Sigma-Aldrich, Saint Louis MO, USA) in flat bottom 96-well microtitre plates. Freshly prepared peroxidase substrate i.e. 35 µl of 20 mM 3,3’,5,5’- tetramethylbenzidine hydrochloride (TMB) (Himedia Ltd, Mumbai, India) and 5 mM H₂O₂ (Qualigens, Mumbai, India) (both substrates of MPO and prepared on same day) were added. Subsequently, the serum mixture (135 µL) was transferred from each well to new 96 well microtitire plate. The colour developing reaction was stopped after 2 min by adding 35 µL of 4 M H₂SO₄ and the OD was read at 450 nm in a universal microplate reader (µQuant, Bio-Tek Instruments Inc, Winooski, VT, USA). The OD of the blank sample without serum was also recorded.

Determination of humoral immune parameters

Serum lysozyme assay

A turbidimetric assay utilising lyophilised Micrococcus luteus ATCC 49732 (DIFCO, BBL) was used to determine lysozyme activity in serum using hen egg white lysozyme (Sigma, Saint Louis MO, USA) as standard^38,39. Serum samples were diluted with phosphate buffer (pH 7.4) to a final concentration of 0.33 mg ml⁻¹. Thus, 150 µL of M. luteus (Bangalore Genei, India) (0.2 mg ml⁻¹, w/v) suspension in 0.02 M acetate buffer was mixed with 15 µL of serum sample in 96 well U bottom microtitre plates for 15 sec (Tarsons Products Ltd, Kolkata, India) and initial OD was taken at 450 nm in ELISA reader after 60 sec of addition of serum sample. The final OD was taken 1 hr after incubation at 25°C. A standard curve was prepared using lyophilized hen egg white lysozyme (HEWL) (Sigma-Aldrich, Saint Louis MO, USA). Serum lysozyme values were expressed as µg ml⁻¹ equivalent to HEWL activity.

Serum bactericidal assay

The bactericidal assay was performed as described by Rainger and Rowley (1993)⁴⁰. A. hydrophila culture was centrifuged and the pellet washed and resuspended in PBS. The OD of A. hydrophila suspension was adjusted to 0.65 at 540 nm. This bacterial suspension was serially diluted (1:10) with PBS up to five dilutions. Bacterial killing assay was determined by incubating 2 ml of this diluted A. hydrophila suspension with 20 ml of serum in a micro-vial for 1 h at 37°C. PBS replaced the serum in the control reaction. The number of viable bacteria was determined after incubation by counting the colonies grown on nutrient agar plate for 24 h at 37°C.

Serum natural haemolysin titre

The serum natural haemolysin titre was performed following Saha et al. (1993)⁴¹. Serum of 50 µL was serialy diluted in phosphate buffer saline (PBS) in a round bottom microtitre plate (Tarsons Products Ltd, Kolkata, India). The plate was then incubated at room temperature for 15 min and 50 µL of 1% rabbit RBC (RaRBC) suspension (collected in Alsever’s solution, erythrocytes were separated after three washing with PBS) were added to each well. The haemolysin (HL) titre was observed by the naked eye after incubation for 1h at 37°C and read as the highest dilution of serum showing complete haemolysis.

Serum haemagglutination titre

Serum haemagglutination titre was determined by the method of Sahoo et al. (2005)³⁷. The heat-inactivated serum (56°C for 30 min) was used for this purpose. Heat-inactivated serum of 50 µL was serially diluted in PBS in a round bottom microtitre plate (Tarsons Products Ltd, Kolkata, India). The plate was then incubated at room temperature for 15 min and 50 µL of 1% rabbit RBC (RaRBC) suspension was added to each well, and the plates incubated for 1 hr at 37°C. The titre was read as the highest dilution of serum showing minimal positive haemagglutination.

Challenge study

After 30 days of feeding trial, the fish in different treatment groups were challenged with pathogenic isolates of A. hydrophila.

A. hydrophila O:18 isolate verified by biochemical test, was grown on tryptic soy broth (Himedia Ltd, Mumbai, India) for 24 h at 30°C in a BOD incubator. Cells were harvested, washed twice with sterile PBS (pH 7.4), and adjusted to 1 × 10⁶ cfu ml⁻¹ based on OD measurement and confirmed by plate counts; final concentration was obtained by serial dilution. All experimental fish were intraperitoneally injected with 0.1 ml A. hydrophila suspension (1 x 10⁶ cfu ml⁻¹). The cumulative mortality (%) patterns were observed in the challenged fish up to 3 weeks along with the changes in behavior and morphology. Mortality was noted and the cause of death was ascertained by reisolating infecting organism from dead fish. Survival was calculated as follows:

Survival rate

At the end of the A. hydrophila challenge, all the experimental tubs were dewatered and the number of the experimental animals in each tub was counted and the survival rate (%) was calculated by the following formula.

Bacterial agglutination titre

Bacterial agglutination titre was determined following Plumb and Areechon (1990)⁴². A pathogenic isolate of A. hydrophila grown in tryptone soya broth for 24 h at 30 °C was harvested and washed twice in sterile PBS. The bacteria were then killed with 1% formalin, washed twice in PBS, and checked for sterility by streaking on tryptone soya agar. The bacterial suspension was then diluted to 2.5 × 10⁸ cells ml⁻¹.

Serum samples collected two weeks after the challenge were serially diluted in two-fold steps with PBS (25 µL of sample and 25 µL of PBS) in a microtiter plate. An equal volume (25 µL) of formalin-killed A. hydrophila was added to each well. The microtiter plates were then incubated overnight at room temperature. The antibody titre was determined as the highest serum dilution showing no agglutination, indicated by the presence of a button in the well, and expressed as the reciprocal of that dilution.

Statistical analysis

Significant differences among treatment groups were tested by one-way analysis of variance (ANOVA) and the comparison of any two mean values was made by Duncan’s multiple range tests. Comparison between pre-and post-challenge means was done by Student’s t-test. A significance level of P < 0.05 was used. All the statistical analysis was performed by using the software program SPSS (version 12) (SPSS Inc., Chicago, IL, USA).

Results

B. licheniformis supplementation promotes growth and performance and the effects are attributed to competitive exclusion

Growth and performance of C. mrigala fed varying doses of B. licheniformis is presented in Table 2. Specific growth rate (SGR) and feed conversion ratio (FCR) improved even at the lowest dose (1.0 × 10⁶ cfu g⁻¹).

Table 2.

Growth and performance of C. mrigala fed incremental levels of Bacillus licheniformis for 30 days.

Treatment	Weight gain (%)	SGR	FCR
Ctrl	31.31a +2.59	0.94a ± 0.11	2.92b ± 0.23
BL-6	46.64b ±3.30	1.35b ± 0.10	1.88a ± 0.16
BL-05.7	53.09b +2.69	1.41b ± 0.05	1.79a ± 0.07
BL-7	52.41b + 2.37	1.39b ± 0.05	1.77a ± 0.07
BL-8	52.74b ± 1.72	1.40b ± 0.03	1.77a ± 0.03

Values are expressed as mean ± SE (n = 3). Means in the same column bearing different superscript letters differ significantly (P < 0.05)

Dietary supplementation of B. licheniformis altered gastrointestinal microflora dynamics in C. mrigala, as reflected in total heterotrophic bacterial (THB) populations, bacterial morphological groups, and their genera. The mean population of THB (Table 3) in the intestine of C. mrigala began to decrease by the 15^th day of sampling, even at the lowest probiotic concentration, and continued to decline on the 30^th day, with the effect becoming more pronounced and stabilizing at 10⁷. Morphologically (Table S1), Gram-negative rods predominated (>75%) at both sampling days in control fish but gradually decreased to <45% by day 30, with a concomitant increase in Gram-positive bacteria from 25% to 55% in fish fed B. licheniformis at doses 10⁷ and higher by day 30. At the genus level (Table S2), Aeromonas was dominant in the control group, followed by Pseudomonas. However, feeding the probiotic B. licheniformis in a dose- and duration-dependent manner led to Bacillus surpassing Aeromonas by day 30 in groups receiving ≥10⁷ cfu of the probiotic (Table S2). Biochemical profiles of different bacterial strains isolated from the intestine of C. mrigala during the experimental period across different treatment groups are presented in Table S3.

Table 3.

Mean population of total heterotrophic bacteria (cfu × 10⁷/g) at 15- and 30-day intervals in the intestine of C. mrigala fed incremental levels of B. licheniformis.

Treatment/Day	15ᵗʰ Day	30ᵗʰ Day
Ctrl	4.17ᵇ ± 1.04	4.04c ± 0.98
BL-6	3.63ᵃ ± 0.88	3.34ᵇ ± 1.07
BL-05.7	3.42ᵃ ± 2.11	2.17ᵃ ± 1.43
BL-7	3.48ᵃ ± 1.18	2.41ᵃ ± 0.66
BL-8	3.39ᵃ ± 0.75	2.11ᵃ ± 0.75

Values are expressed as mean ± SE (n = 3). Means in a column bearing different superscript differ significantly (P<0.05)

B. licheniformis supplementation improves pre- and post- challenge haemato-biochemical profile

In general, compared to the control, B. licheniformis increased total erythrocyte count, leukocyte count, and hemoglobin levels both before and after the challenge. While a minimum dose of 10⁷ cfu was necessary to achieve post-challenge increase in erythrocyte count and hemoglobin, even the lowest probiotic dose was sufficient to enhance leukocyte levels (Table 4).

Table 4.

Hematology of pre- and post-A. hydrophila-challenged C. mrigala fed incremental levels of B. licheniformis.

Parameter	Total erythrocyte count (x106 cells/mm3)		Total total leucocyte count (x 104 cells /mm3)		Haemoglobin (gm%)
Treatment/-challenge	Pre-challenge	Post-challenge	Pre-challenge	Post-challenge	Pre-challenge	Post-challenge
Ctrl	0.45a,A ±0.02	0.32a,B ± 0.01	3.04a,A ±0.05	3.60a,B± 0.00	6.97a,A± 0.03	5.75a,B±0.03
BL-6	0.54b,A ±0.01	0.38b,B ± 0.01	3.17ab,A± 0.03	3.88b,B± 0.01	7.73ab,A± 0.39	6.11b,B ±0.19
BL-05.7	0.56b,A ± 0.01	0.49d,B ± 0.01	3.28ab,A± 0.01	3.91b,B± 0.01	7.90b,A ±0.10	6.70c,B ±0.06
BL-7	0.56b ± 0.02	0.48d ±0.00	3.72c± 0.21	3.93b± 0.01	7.63b,A± 0.19	6.69c,B ± 0.03
BL-8	0.61b,A ± 0.03	0.43c,B± 0.01	3.55bc± 0.2	3.90b± 0.06	8.33b,A± 0.17	6.91c,B±0.02

Values are expressed as mean ± SE (n = 3). Within a column, means with different lowercase superscripts (a, b, c) differ significantly (P < 0.05), while uppercase superscripts (A, B) indicate differences between pre- and post-challenge values.

Similarly, compared to the control, B. licheniformis increased total serum protein, albumin, and globulin, and improved the albumin:globulin ratio both before and after the challenge, with effects stabilizing at doses of 10⁷ cfu and above (Table 5).

Table 5.

Serum chemistry of pre- and post-A. hydrophila-challenged C. mrigala fed incremental levels of B. licheniformis.

Parameter	Total serum protein (gm/dl)		Albumin (gm/dl)		Globulin (gm/dl)		Albumin: Globulin (A/G ratio)
Treatment/--challenge	Pre-challenge	Post-challenge	Pre-challenge	Post-challenge	Pre-challenge	Post-challenge	Pre -challenge	Post-challenge
Ctrl	4.05a,A±0.03	3.78a,B±0.06	1.61a,A±0.02	1.78a,B±0.02	2.44a,A±0.04	2.00a,B±0.08	0.66c,A±0.01	0.89c,B±0.02
BL-6	4.28b,A±0.03	4.03b,B±0.01	1.71b,A±0.00	1.81a,B±0.01	2.56b,A±0.04	2.21b,B±0.01	0.66c,A±0.01	0.81b,B±0.01
BL-05.7	5.05d,A±0.02	4.83d,B±0.01	1.85c,A±0.01	1.86b,B±0.02	3.20d,A±0.02	2.87c,B±0.01	0.57a,A±0.00	0.68a,B±0.01
BL-7	4.83c,A±0.01	4.69c,B±0.02	1.83c,A±0.01	1.95b,B±0.01	2.99c,A±0.02	2.74c,B±0.01	0.61ab,A±0.00	0.71a,B±0.02
BL-8	4.99d,A±0.01	4.75cd,B±0.00	1.93d,A±0.01	2.01c,B±0.01	3.05c,A±0.01	2.74c,B±0.01	0.63bc,A±0.01	0.73a,B±0.01

Values are expressed as mean ± SE (n = 3). Within a column, means with different lowercase superscripts (a, b, c) differ significantly (P < 0.05), while uppercase superscripts (A, B) indicate differences between pre- and post-challenge values.

B. licheniformis supplementation decreases pre- and post- challenge serum phosphatases and gluconeogenic enzymes

Pre-challenge serum levels of ALP and the gluconeogenic enzymes, ALT and AST were unaffected by probiotic feeding. However, post-challenge levels began to decrease (P < 0.05) at a dose of 10⁶ cfu, with a further significant reduction at 10⁷ cfu, after which the levels stabilized (Table 6).

Table 6.

Serum enzymes of pre- and post-A. hydrophila-challenged C. mrigala fed incremental levels of B. licheniformis.

Enzyme	Alkaline Phosphatase activity (U/L)		Alanine aminotransferase (ALT) activity(U/L)		Aspartate amino-transferase (AST) activity(U/L)
Treatment/-challenge	Pre-challenge	Post-challenge	Pre-challenge	Post-challenge	Pre-challenge	Post-challenge
Ctrl	59.88A±0.87	90.56b,B±1.15	13.92A±0.9	25.10c,B±0.12	60.84A±0.28	106.18c,B±0.10
BL-6	59.06A±0.50	88.69b,B±0.40	13.50A±0.26	21.24b,B±0.50	60.18A±0.14	82.22b,B±0.30
BL-05.7	59.34A±0.70	73.14a,B±1.04	13.74A±0.11	15.78a,B±0.21	60.26A±0.45	75.25a,B±0.14
BL-7	59.66A±0.62	76.01a,B±2.07	13.72A±0.25	16.84a,B±0.37	60.36A±0.37	77.23a,B±0.21
BL-8	59.81A±0.19	73.49a,B±1.18	14.01A±0.58	16.43A±0.26	60.27A ±0.41	77.10a,B±1.44

Values are expressed as mean ± SE (n = 3). Within a column, means with different lowercase superscripts (a, b, c) differ significantly (P < 0.05), while uppercase superscripts (A, B) indicate differences between pre- and post-challenge values.

Enhanced cellular immunity of C. mrigala fed B. licheniformis for 30 days

Compared to the control, B. licheniformis enhanced cellular immunity both before and, more notably, after the challenge, as evidenced by increased respiratory burst activity, phagocytic index, phagocytic ratio and myeloperoxidase activity, with consistent effects at and above 10⁷ CFU (Fig 1).

Fig 1.

Cellular immune response of unchallenged and A. hydrophila-challenged C. mrigala fed incremental levels of B. licheniformis. The values reported in bar charts are expressed as mean ± SE. (n = 3 for pooled samples). Bars of the same color bearing different letters (a, b, c) indicate significant differences between means for that parameter. (*) An asterisk above bars of two different colors within a given treatment indicates a significant difference between pre- and post-challenge means (Student’s t-test, P < 0.05).

Enhanced humoral immune response and survival of C. mrigala fed B. licheniformis for 30 days

Feeding B. licheniformis at 10⁶ dose increased (P < 0.05), and at 10⁷ dose further enhanced (P < 0.05), serum lysozyme and bactericidal activity (both pre- and post-challenge), whereas the later dose showed effects specifically on post-challenge serum natural haemolysin titre and serum haemagglutination titre (Fig 2).

Fig 2.

Humoral immune response of unchallenged and A. hydrophila-challenged C. mrigala fed incremental levels of B. licheniformis. The values reported in bar charts are expressed as mean ± SE. (n = 3 for pooled samples). Bars of the same color bearing different letters (a, b, c) indicate significant differences between means for that parameter. (*) An asterisk above bars of two different colors within a given treatment indicates a significant difference between pre- and post-challenge means (Student’s t-test, P < 0.05).

Similarly, the highly specific humoral immune response, measured as bacterial agglutination titre against formalin-killed A. hydrophila, increased at the probiotic’s 10⁶ dose (P < 0.05), with further potentiation (P < 0.05) at the 10⁷ dose, after which it stabilized, and post-challenge survival (%) corresponded to this trend (Fig 3).

Fig 3.

Bacterial agglutination titre and survival of A. hydrophila-challenged C. mrigala fed incremental levels of B. licheniformis. Each data point for a given treatment (X-axis) on the dotted line represents the bacterial agglutination titre on the primary (left) Y-axis, while the secondary (right) Y-axis indicates the percentage survival of fish after challenge with A. hydrophila. Values represented are mean ± SE (n = 3, pooled samples). Superscripts bearing different letters (a, b, c) denote statistically significant differences among bacterial agglutination titres.

Discussion

In the present study, B. licheniformis was evaluated as a probiotic bacterium in Indian Major Carp, C. mrigala, during a 30 days trial followed by A. hydrophila challenge. B. licheniformis, a Gram-positive, aerobic, endospore forming bacteria, was administered to C. mrigala via feed, and its spore-forming ability makes it a promising probiotic candidate, as spores can withstand harsh conditions—including disinfectants, drying, and heat- much better than their vegetative cells^43–46. Earlier, Mukherjee et al. (2016) confirmed B. licheniformis (KM277364) as one of four potential probiotic Bacillus isolates from the gut of C. mrigala, with the strain demonstrating superior growth in intestinal mucus and resistance to diluted bile juice (2–20%)²¹.

B. licheniformis consistently promotes growth and efficiency across various aquaculture species, with effects varying by dose. At 10⁹ cfu g⁻¹, the highest SGR and optimal FCR were observed in IMC (Labeo rohita) over 12 weeks⁴⁷, as also reported in giant freshwater prawn (M. rosenbergii)⁴⁸ and C. mrigala fed a multistrain probiotic⁴⁹, as well as in tilapia (O. niloticus)⁵⁰ at 10⁸ cfu g⁻¹, with complete or high survival in the latter two studies. Dosages of 10⁵–10⁷ cfu g⁻¹ also conferred benefits across multiple aquaculture species.

Juvenile tilapia exhibited enhanced growth, immunity, and disease resistance at ≥4.4 × 10⁶ cfu g⁻¹ over 70 days⁵¹. O. mossambicus showed improved SGR and FCR over 4 weeks²² and similar growth benefits with lower FCR in another study⁵² , though survival was not reported. Triangular bream (Megalobrama terminalis) fed 5 × 10⁵ cfu g⁻¹ for 8 weeks had improved FCR and survival, though SGR remained unchanged⁵³. Grass carp demonstrated enhanced growth and feed efficiency at 10⁵–10⁶ cfu g⁻¹ over 56 days⁵⁴, while rainbow trout fed 2.2 × 10⁷ cfu g⁻¹ for 60 days showed better growth, FCR, and survival⁵⁵.

Among 22 Bacillus strains isolated from microbial bioflocs, B. subtilis (PBSI-5) and B. safensis subsp. safensis were most promising, showing strong digestive enzyme activities and significant growth enhancement in Mrigal (C. mrigala), with B. safensis achieving the highest weight gain⁵⁶. Supplementation with multistrain (two to four) Lactobacillus probiotics from the gut of C. mrigala resulted in a 2–2.8-fold improvement in FCR, compared to 1.7-fold with a single strain⁵⁷. Xie et al. (2025) reported increased growth and improved feed conversion in hybrid sturgeon fed 0.2% B. licheniformis for 90 days⁵⁸. However, unlike food-fish species, goldfish (C. auratus)—a slow-growing ornamental—showed no improvement in growth or FCR when fed 10⁸ cfu g⁻¹ B. licheniformis⁵⁹.

Several mechanisms may underlie the consistent growth and performance improvements observed in fish fed probiotic Bacillus across multiple species, including the present study. The gut microbiota of aquatic animals is typically dominated by Gram-negative bacteria, particularly Aeromonas and Pseudomonas as noticed in this study and also reported earlier¹⁶. However, this and earlier studies^60–62 have shown that dietary probiotic Gram-positive bacteria competitively exclude and overtake the resident harmful microbial flora.

Bacillus spp. effectively suppress these pathogens through the production of antibiotics (polymyxin, bacitracin and gramicidin)^13,63 and compounds exhibiting similar properties. The bacteriocins produced by B. stratosphericus, B. aerophilus, B. licheniformis, and Solibacillus silvestris, all isolated from the gut of C. mrigala, are stable up to 90 °C and across a pH range of 4–9, and—when compared with Gentamicin and Chloramphenicol—exhibit strong bactericidal activity against multiple common fish pathogens. Specifically, the bacteriocin from B. licheniformis (KM277364) is active against A. hydrophila, A. salmonicida, and B. mycoides; that from Solibacillus silvestris is active against A. hydrophila, A. salmonicida, and P. fluorescens; and those from B. stratosphericus and B. aerophilus inhibit four A. hydrophila, A. veronii, P. fluorescens, and B. mycoides²¹. Another prebiotic produced by B. licheniformis ZM107 is the fermentable, fructan-type exopolysaccharide (EPS) levan⁶⁴, which is nondigestible by the host but selectively utilized by gut microbes, promoting competitive exclusion of pathogens. Levan also exhibits anti-adhesion and anti-biofilm properties, and its fermentation generates short-chain and organic acids that lower gut pH, inhibit acid-sensitive pathogens, suppress virulence gene expression, and enhance intestinal barrier integrity.

In another study involving gut bacterial isolation from C. mrigala, Vidhya Bharathi et al. (2025) reported a predominance of Lactobacillus strains (5 out of 7), which, when compared with standard streptomycin, displayed inhibitory activity against several pathogens, including S. aureus, E. faecalis, S. maltophilia, and P. aeruginosa⁵⁷. Similarly, two promising probiotic strains—Bacillus paramycoides (OM038513) and B. cereus (OM033468)—isolated from the gut of Cirrhinus reba, exhibited antagonistic activity against S. aureus, V. harveyi, and V. parahaemolyticus, and tolerated acidic, alkaline, and bile conditions⁶⁵.

B. licheniformis (0.2%) increased Bacillus abundance and microbial diversity in sturgeon⁵⁸. Likewise, in a 45-day study on M. rosenbergii from our lab⁴⁸, dietary supplementation with B. licheniformis (10⁶–10⁹ cfu g⁻¹ feed) produced a dose-dependent increase in gut Bacillus spp. counts from day 15 onward, along with a concurrent decrease in Aeromonas spp. from day 15 and Pseudomonas spp. from day 30, demonstrating colonization potential and supporting present results. Comparable effects have been reported in L. vannamei with this probiotic bacteria⁶⁶. Additionally, Qin et al. (2020) showed that dietary supplementation with B. licheniformis FA6 significantly (P < 0.05) upregulated intestinal tight junction genes (ZO-1, claudin c, and occludin) in grass carp, strengthening barrier integrity⁵⁴, reducing gut permeability, improving nutrient absorption and protecting against pathogens and toxins. While biochemical methods allowed detection of these microbial shifts, molecular confirmation (e.g., 16S rRNA sequencing) would provide higher taxonomic resolution and strengthen interpretation of probiotic-induced microbiota changes.

B. licheniformis enhances growth and feed conversion primarily by stimulating digestive enzyme activity, especially proteases, thereby improving protein utilization^51–53. The strain B. licheniformis (KM277364) from C. mrigala also secretes amylase, lipase, phytase, and xylanase²¹. Gram-positive Bacillus spp. produce diverse exoenzymes^44,65,67, and exhibit antioxidant properties⁶⁵, as observed in carp⁶⁸. Higher digestive enzyme activity was observed in Lates calcarifer fed 1 × 10⁶ cfu g⁻¹ B. licheniformis and B. subtilis⁶⁹, and in triangular bream (M. terminalis) fed 1 × 10⁷ cfu g⁻¹ B. licheniformis alone⁵³. Here, improved growth and FCR may also be attributed to increased intestinal microvilli density and diameter, as seen in our work with B. subtilis in L. fimbriatus⁶⁸, or reduced villi damage (exfoliation, twisting, fusion) ⁵¹, ultimately facilitating efficient digestion, nutrient absorption, and metabolism⁷⁰.

Just as in this study, increases in hematological parameters such as RBC, WBC, and Hb% have been reported in Nile tilapia, rainbow trout, and Caspian kutum (Rutilus frisii kutum), Oscar (Astronotus ocellatus) fingerlings and C. mrigala after feeding with probiotic supplements^49,71–74. Specifically, the same probiotic strain, Bacillus licheniformis at 10⁷ cfu g⁻¹, have been reported to increase these attributes in rainbow trout (Oncorhynchus mykiss)⁵⁵ and in tilapia (O. mossambicus)⁵². Vidhya Bharathi et al. (2025) reported a clear multistrain-dependent increase in blood cell counts⁵⁷. Red blood cell (RBC) levels were three-fold higher with four Lactobacillus strains compared to a 1.5-fold increase with a single strain, while white blood cell (WBC) counts were 1.4-fold higher with four strains compared to a 1.2-fold increase with a single strain. The preponderance of supplemented organisms in the gut may increase metabolic activity, consequently elevating oxygen requirements and necessitating an increase in circulating RBCs and hemoglobin to enhance oxygen transport in probiotic-fed fish⁷¹. Moreover, the enhanced serum chemistry profile observed in this study concurs with findings in rainbow trout (O. mykiss)⁵⁵ and Mozambique tilapia (O. mossambicus)²² fed the same probiotic at similar doses.

The observed dose-dependent decrease (P < 0.05) in serum ALP, ALT, and AST in post-challenged fish indicates improved liver (gut-associated tissue) function, minimal systemic stress, and inflammation, possibly via probiotic modulation of gut microbiota, reduced pathogen load, along with fulfillment of energy requirements through a balanced diet, thereby obviating the need to catabolize proteins. Taherpour et al. (2023) reported no effect on serum ALT and AST in rainbow trout (13–80 g) fed a 40% CP diet with B. licheniformis at 2.2 × 10⁷ cfu g⁻¹⁵⁵. In contrast, increased serum (or mucus) ALP has been reported in Catla (C. catla) fed B. subtilis ⁷⁵, rainbow trout fed L. rhamnosus^76,77, Caspian roach fries fed Vitamin C⁷⁸, and O. mossambicus fed B. licheniformis Dahb1²², as well as higher (P < 0.05) serum ALT and AST levels were observed in hybrid sturgeon with 0.1–0.4% B. licheniformis in 43% CP diets⁵⁸. ALP is a lysosomal enzyme involved in macrophage activation and acts as an effective antibacterial agent⁷⁹, while ALT and AST are gluconeogenic enzymes. Variations in enzyme activities can also be attributed to differences in fish species, probiotic dose, feeding duration, and dietary composition. Our carp received an optimum diet (27% CP and 3850 DE), whereas Xie et al. (2025)⁵⁸ fed hybrid sturgeon ~ 8% excess protein over the 36–38% CP requirement⁸⁰, necessitating extra protein catabolism and enhancing gluconeogenic activity.

Supplementation of B. licheniformis at and above 10⁷ cfu consistently enhanced post-challenge cellular and humoral (and adaptive humoral) immunity and survival, as measured through multiple parameters described subsequently, in agreement with previous reports^{17,22,51,55,81–84}. Phagocytosis is responsible for the early activation of the inflammatory response before antibody production and is carried out by phagocytic cells such as neutrophils, monocytes, and macrophages in fish^85,86. In this study, challenged carp fed B. licheniformis for 30 days showed significant increases in both the phagocytic index and phagocytic ratio, with the phagocytic ratio stabilizing at the 10⁷ cfu g⁻¹ dose of the probiotic. These results are consistent with previous findings, in which one or more measures of phagocytosis—such as phagocytic activity, phagocytic index, and phagocytic ratio—were increased in gilthead seabream (S. aurata L.) after two weeks of feeding a B. subtilis-supplemented diet⁸⁷, in C. catla fed low doses (10³ and 10⁴ cfu g⁻¹ diet) of B. circulans PB7, and in Epinephelus coioides fed diets containing B. pumilus or B. clausii for 60 days.

Phagocytic cells play an important role in antibacterial defence and they are able to generate superoxide anion and its reactive derivative, which have potent bactericidal activity, during the so-called respiratory burst³⁹. In the present study, B. licheniformis supplementation positively influenced respiratory burst activity across all treatment groups. This finding are consistent with Yaqub (2021)⁵², who reported increased respiratory burst in O. mossambicus following 8-weeks of B. licheniformis SB3086 (10⁹ cfu g⁻¹), and with Gao et al. (2018)⁸⁸, who observed elevated O₂⁻ production in abalone blood lymphocytes at 10⁵ cfu ml⁻¹. Similarly, Kumar et al. (2008)⁸¹ and Pawar et al. (2023)⁶⁸ demonstrated enhanced respiratory burst activity in Labeo species after Bacillus sps supplementation. The highest activity was recorded at 0.5 x 10⁷ cfu g^-1, highlighting the immuno-protecting role of B. licheniformis. Rainbow trout fed L. rhamnosus⁸⁹ or Carnobacterium divergens B33 also showed enhanced respiratory burst activity⁹⁰. In contrast, some studies reported no significant probiotic effects^91–93, indicating that the responses may depend on the specific probiotic strain and the host species.

Myeloperoxidase (MPO), an enzyme that utilizes oxidative radicals to produce hypochlorous acid for pathogen killing is primarily released from the azurophilic granules of neutrophils during the respiratory burst. All groups fed B. licheniformis showed significantly higher MPO activity compared to controls. Similar increases were observed in O. mossambicus fed 10⁵–10⁷ cfu g⁻¹ B. licheniformis Dahb1 following A. hydrophila challenge²², in O. mossambicus fed 10⁹ cfu g⁻¹ B. licheniformis SB3086 for 8 weeks⁵², in abalones fed 10⁵ cfu ml⁻¹ B. licheniformis⁸⁸, and in gilthead seabream (Sparus aurata L.) fed yeast (S. cerevisiae)⁹⁴. Probiotics like E. faceium elevated MPO level in O. niloticus when supplemented through water at 1 x 10⁷ cfu ml⁻¹ in every 4 days for 40 days⁹⁵.

Lysozyme, present in fish serum, mucus and leucocytes³⁹, cleaves peptidoglycan in the bacterial cell wall, thereby inhibiting microbial growth non-specifically⁹⁶. Probiotics, whether administered singly or in combination, have been shown to enhance lysozyme activity. Specifically, B. licheniformis fed to juvenile Nile tilapia (O. niloticus) for 10 weeks⁵¹, O. mossambicus fed strain SB3086 (10⁹ cfu g⁻¹) for 8 weeks⁵², and B. subtilis (10⁴–10⁶ cfu) fed to L. fimbriatus fingerlings for 60 days all increased lysozyme levels. Enhancement has also been reported in teleost fed B. licheniformis, L. rhamnosus, C. maltasomaticum, and C. divergens in O. mykiss^55,76,90, and L. lactis ssp. Lactis, L. mesenterites and L. sakein in brown trout (Salmo trutta)⁹⁷. Similar to these results, our study also showed a significant increase in serum lysozyme activity in groups fed the probiont at and above 0.5 x 10⁷ cfu g⁻¹compared to control after 30 days, indicating stimulation of innate immunity by the probiotic.

The spontaneous bactericidal activity of fish serum, which is a largely a complement – mediated bacterial killing either by the classical or alternative pathway⁹⁸, plays a major role against bacterial invasion into fish⁹⁹. The present study shows that B. licheniformis stimulates serum bactericidal activity, as all treated fish groups exhibited higher bacterial killing (% cfu of control) than controls. Among the treatments, fish fed 1.0 × 10⁷ cfu g⁻¹ probiotic exhibited the highest bactericidal activity, likely due to increased O₂⁻ production. Earlier, we reported that M. rosenbergii fed B. licheniformis (10⁹ cfu g⁻¹ feed) exhibited significant antibacterial activity against V. alginolyticus, with 64% greater inhibition than control⁴⁸. Similarly, Taherpour et al. (2023) reported enhanced serum bactericidal activity in rainbow trout (Oncorhynchus mykiss) fed 2.2 × 10⁷ cfu g⁻¹ B. licheniformis⁵⁵. Nikoskelainen et al. 2003 reported significant increase in complement bactericidal activity of rainbow trout fed with the L. rhamnosus⁸⁹. A similar response was recorded in L. rohita orally fed with B. subtilis, which showed increased serum bactericidal activity⁸¹.

Natural agglutinins react with a wide variety of bacteria causing agglutination. The presence of bacterial agglutinins can indicate previous exposure to disease or contact with sewage-contaminated waters with certain pathogens, which cross-react with natural antibodies found in fish serum¹⁰⁰. The assay of agglutination titer and killing effect of A. hydrophila-specific immunized serum showed that the probiotic can kill the bacterium if normal serum is present as a complement source. In the present study, bacterial agglutination titer against pathogen, A. hydrophila was higher in every B. licheniformis-fed groups. This suggests that probiotics are sufficient for activation of the classical pathway and give protective immunity. Serum killing, rather than agglutination of A. hydrophila by specific antiserum, appears to play a more important role in the protective effect of probiotics against this pathogen. Conflicting reports exist regarding the correlation between protection of fish against bacterial infection and the level of serum specific antibody ^101–103. Protective responses elicited by B. licheniformis feeding suggest involvement of humoral and cellular immunity in C. mrigala. The response may depend upon the type of probiotic strain and fish species.

Probiotics enhance disease resistance through immunity stimulation^17,83 and pathogen exclusion⁸², interacting with the fish mucosa to activate mucosal¹⁰⁴, adaptive (B and T cells), and complement system responses^87,105. Additionally, B. licheniformis FA6, B. subtilis, and other Bacillus spp. modulate immunity by down-regulating pro-inflammatory (IL-1β, IL-8, TNFα) and up-regulating anti-inflammatory cytokines (IL-4, IL-10) in grass carp^54,106 and hens¹⁰⁷, even without pathogen challenge. Dietary or bioencapsulated probiotics enhance natural resistance and larval and post larval survival^108,109. The protection observed in the present study and across species is attributable to enhanced cellular and humoral immunity, along with up to 64% inhibition of pathogenic bacteria. The increased phagocytic activity, respiratory burst, and MPO activity indicate activation of macrophage-mediated innate immunity. In this context, microbial levans have been reported to modulate cytokine balance by increasing IL-10 while limiting excessive TNF-α responses, thereby facilitating effective pathogen clearance without pathological inflammation. Although cytokines were not measured in the present study, the improved cellular immune responses and survival following A. hydrophila challenge are consistent with such mechanisms. Supporting this, Gupta et al. (2008) reported that dietary inclusion of 1–1.25% microbial levan improved multiple immune parameters and survival following challenge, accompanied by leukocyte infiltration in the liver and moderate degeneration of renal tubules¹¹⁰.

In the present study, C. mrigala fed B. licheniformis showed higher survival than controls, with ≥10⁷ cfu g⁻¹ being most effective; 10⁶ cfu g⁻¹ was insufficient to elicit a measurable immune response. Across species, 10⁵–10⁷ cfu g⁻¹ over two to three months generally improves survival and disease resistance. Han et al. (2015) identified ≥4.4 × 10⁶ cfu g⁻¹ as optimal for tilapia during a 70-day trial⁶⁵. In a 56-day study, Qin et al. (2020) reported 10⁵–10⁶ cfu g⁻¹ of FA6 increased grass carp survival by 30–45% under A. hydrophila challenge⁵⁴. Midhun et al. (2019) confirmed high tilapia survival at 10⁶–10⁸ cfu g⁻¹ (HGA8B) for 60 days⁵⁰; Taherpour et al. (2023) observed improved rainbow trout survival at 2.2 × 10⁷ cfu g⁻¹⁵⁵. In abalones, 10⁵ cfu ml^-1 B. licheniformis maximized survival was during an 8-week culture and a 2-week V. parahaemolyticus challenge⁸⁸. Higher doses also confer significant benefits in Indian major carp and other species. Majeed et al. (2024) reported 10⁹ cfu g⁻¹ for over 12 weeks optimal for 85.6% survival in L. rohita ⁴⁷, and Yaqub et al. (2021) observed 10⁹ cfu g⁻¹ for 8 weeks increased O. mossambicus survival by 10–20% over lower doses or control⁵². In M. rosenbergii challenged with V. alginolyticus, 10⁹ cfu g⁻¹ B. licheniformis improved survival by over 25%⁴⁸.

Comparable benefits have also been observed with other probiotic organisms. Dietary administration of lactic acid bacteria (Lactococcus lactis ssp. lactis CLFP 100, Leuconostoc mesenteroides CLFP 196, and Lactobacillus sakei CLFP 202) at 10⁶ cfu g⁻¹ for 2 weeks significantly increased survival by 32.2–34.2% in rainbow trout (O. mykiss) challenged with Aeromonas salmonicida¹¹¹. Similarly, Nikoskelainen et al. (2003) fed O. mykiss Lactobacillus rhamnosus at 10⁹ and 10¹² cfu g⁻¹ for 51 days, reporting survival increases of 33.7% and 6.3%, respectively, following A. salmonicida challenge⁸⁹.

Differential effects on pre- and post-challenge immunological attributes with respect to probiotic dose, in terms of reaching peak and plateau levels, were noted. Differential effects on various immune parameters are frequently observed in studies on the modulation of fish non-specific host defences¹¹². Because the innate immune system of fish lacks immunological memory, its responses are generally shorter in duration than those of the specific (adaptive) immune system¹¹³. Moreover, innate defences arise from a combination of humoral and cellular factors, each of which may exhibit different degrees of specificity toward a given immunomodulatory substance¹¹⁴. Consequently, the activities measured in such studies often peak at different times and intensities, and these peaks may not coincide with one another in magnitude or timing, as noted by previous authors^115,116, and therefore should be interpreted collectively to assess the overall effect.

Conclusion

In conclusion, the results collectively indicate that dietary administration of B. licheniformis, particularly at levels at or above 10⁷ cfu g⁻¹, significantly improves both cellular immune responses (phagocytic index and ratio, respiratory burst activity, serum myeloperoxidase activity, alkaline phosphatase activity, total leukocyte counts) and humoral (and adaptive) immune responses (lysozyme activity, serum bactericidal activity, serum haemagglutination titre and natural haemolysin titre), as well as survival following challenge with A. hydrophila, while simultaneously improving growth performance. These findings suggest the potential of B. licheniformis as a probiotic in aquaculture to enhance growth and prevent disease outbreaks. Further studies using other pathogenic bacteria and fish species are recommended to confirm the robustness and broad applicability of B. licheniformis as a probiotic strain.

Author contributions

R.K.B.: Data curation; Formal analysis; Investigation; Methodology; Resources; Software; Validation; Writing - original draft. R.P.R.: Conceptualization; Funding acquisition; Project administration; Supervision; Writing - original draft. R.S.D.: Data curation; Investigation; Methodology; Software; Validation; Visualization; Writing - review & editing. R.K.N.: Formal analysis; Methodology; Validation; Writing - review & editing. M.I.S. : Formal analysis; Resources; Software; Validation; Visualization. M.A.: Data curation; Formal analysis; Writing - original draft. M.K.S.: Resources; Software; Validation; Writing - original draft. S.G.: Data curation; Resources; Validation; Visualization. N.A.P.: Data curation; Formal analysis; Investigation; Validation; Visualization. S.B.J.: Conceptualization; Data curation; Formal analysis; Resources; Validation; Visualization; Writing - original draft; Writing - review & editing. All authors: read and approved the final manuscript.

Funding

This work did not receive any funding from any source.

Data availability

All data generated and analysed during this study are included in the article.

Competing interest

The authors declare no competing interests.