2.1. Bacterial strains
The L. plantarum strain used in this study was primarily identified based on the colony and cell morphology, gram staining, biochemical characteristics, and 16S rRNA gene sequencing (GenBank accession number EU520326) (Mohammadian et al. 2016). This strain was grown for 30 h at 37 ° C in MRS broth (BD Difco, Sparks, MD, USA).
2.2. Microencapsulation of L. plantarum and properties
Microencapsulation of L. plantarum with chitosan/alginate (MLCA) was done according to the extrusion method (Jiang et al. 2013). Briefly, the mixture of L.plantarum (108 CFU g-1), sodium alginate, and 15% (v/v) glycerol was dropped into 0.1 M CaCl2 by passing through a cannula-like syringe in the presence of nitrogen gas pressure. The sodium alginate final concentration was 2% (w/v). Formed microcapsules were incubated for 30 min, then washed with 0.85% saline to remove unreacted CaCl2. The chitosan (MW 10,000) solution 0.8% (w/v) was used to coat microcapsules for 30 min followed by two times washing. The microcapsules coated with chitosan-alginate were further coated with 0.1% (w/v) sodium alginate for 10 min followed by washing. Then microcapsules were stored at −75 ° C for 6 h and lyophilized for 18 h. The control microcapsules without bacteria were also prepared by the same procedure. The morphologic observation and size measurement of MLAC were performed by scanning electron microscopy(SEM) (Philips XL 20, Oregon, USA). The viability of free and encapsulated bacteria in simulated small intestinal fluid (SIF) was measured by the counting method for 0, 0.5, 1, 2, 4, 8, 12, and 24 h.
2.3. Diet preparation
In the present study, the used basal diet was modified based on the work of Van Doan et al (Van Doan et al. 2016a). Preparation of experimental diets by the inclusion of different additives was as the following: Diet 1 (Control), 10 g kg-1 microcapsules (Diet 2), 108 CFU g-1 L. plantarum (Diet 3), and 10 g kg-1 MLCA (Diet 4) (Table 1). The experimental diets were milled into powder and were thoroughly mixed with soybean oil, and then water was added to produce stiff dough. Then, the doughs were ground to form spaghetti-like pellets. The pallets were dried in the oven at 50 ° C until moisture levels were around 10% and stored at 4 ° C until use. To ensure high probiotic levels in the diet, fresh diets were prepared at 30 days intervals.
Table 1. Experimental diet formulation and proximate composition.
Components
|
Diet 1(g kg-1)
|
Diet 2(g kg-1)
|
Diet 3(g kg-1)
|
Diet 4(g kg-1)
|
Fish meal
|
300
|
300
|
300
|
300
|
Rice bran
|
240
|
240
|
240
|
240
|
Soybean meal
|
200
|
200
|
200
|
200
|
Corn meal
|
125
|
125
|
125
|
125
|
Wheat flour
|
60
|
60
|
60
|
60
|
Soybean oil
|
30
|
30
|
30
|
30
|
Cellulose
|
30
|
20
|
30
|
20
|
Premix
|
10
|
10
|
10
|
10
|
Vitamin and trace mineral mix
|
5
|
5
|
5
|
5
|
L.plantarum (CFU g_1)
|
0
|
0
|
108
|
0
|
MLCAa
|
0
|
0
|
0
|
10
|
Microencapsules
|
0
|
10
|
0
|
0
|
Proximate composition of the experimental diets(g.kg-1dry matter basis)
|
Gross energy(cal g-1)
|
4387
|
4419
|
4387
|
4419
|
Dry matter
|
931
|
9313
|
931
|
931.3
|
Crude Protein
|
353.1
|
353.6
|
353.1
|
353.6
|
Ash
|
114.4
|
115.2
|
114.4
|
115.2
|
Crude Lipid
|
100.5
|
99
|
100.5
|
99
|
Fibre
|
63.8
|
60.6
|
63.8
|
60.6
|
a:MLCA: microencapsulation of L. plantarum with chitosan / alginate
2.4. Experimental design
The healthy Nile tilapia (Oreochromis niloticus) fingerlings (n=480,) that had no previous history of parasitic infections and no signs of disease (gross and microscopic examination of gills, skin, and kidney tissues of representative samples) were obtained from Yazd fish farm, Yazd, Iran. The fish were transferred to the laboratory of the Faculty of Veterinary Medicine, Shahid Chamran University of Ahvaz, Iran and acclimatized to laboratory conditions for 2 weeks in 500-L plastic quarantine tanks at 27 ± 2 ° C and fed with the control diet. Then, the fish with a similar size (15.56 ± 0.02 g) were randomly divided into 150 L tanks for each diet treatment group. The experiment was laid out in a Completely Randomized Design with four replications. In each tank, approximately 30% of the water was exchanged daily, and 100% of the water was exchanged once a week. The fish ad libitum was twice a day at 7:00 a.m. and 6:00 p.m and diets were hand-fed to the fish. Measurement of basic Physico-chemical parameters of the water was done every week. The O2 concentration was maintained at no less than 5 mg L-1 and pH ranged from 7.5 to 8.2 throughout the study period.
2.5. Blood and serum sample collection
Sampling was performed on day 30 and day 60 after probiotic feeding and a total of 12 fish (3 fish from each treatment diet) were randomly collected per treatment for immunological assays. After anesthetizing the fish with clove powder (200 ppm, 20 min), blood was collected from the caudal vein using a 1 mL syringe. Then the blood samples were transferred into Eppendorf tubes without anticoagulant and incubated at room temperature for 1 h and 4 ° C for 4 h. After coagulation, blood was centrifuged (2000 g, 10 min, 4 ° C), and serums were collected and stored at -20 ° C until used.
2.5.1. Leukocyte isolation
The peripheral blood leukocytes were isolated by the method modified by Chung & Secombes (Chung and Secombes 1988). First, 1 mL of blood samples was diluted with 2 mL of RPMI 1640 (Sigma-UK). Then, 3 mL of Histopaque (Sigma-UK) was carefully laid onto a 15 ml tube and centrifuged at 500 g for 20 min at room temperature. After that, a white buffy coat of leukocyte cells was carefully aspirated with a Pasteur pipette. The isolated leukocytes were washed twice with phosphate buffer solution (PBS, pH 7.4) using centrifugation at 250 g for 10 min. For assaying respiratory activities, the leukocytes were resuspended in the PBS and adjusted to the required cell numbers.
2.6. Hematology and biochemical indices
Determination of hematocrit values in the blood was performed by calibrating the centrifuged hematocrit pipettes, and the percentage of blood cells in the total volume of blood was expressed (Brown 1988). The differential leukocyte count was done based on Schäperclaus et al method (Schäperclaus et al.1992). Total protein and albumin (ALB) concentrations in serum were measured using a commercial kit (Zist Shimi kits) according to the manufacturer's instructions. Globulin (GLO) was calculated by subtracting albumin values from total protein values. The ratio of albumin-globulin was calculated by dividing albumin values by globulin values.
2.7. Immunological measurements
2.7.1. Lysozyme activity assay
Serum lysozyme activity was determined according to the method described by Parry et al. The standard was compared with the equivalent unit of the activity of the sample and reported in mg ml-1 serum.
2.7.2. Alternative complement pathway activity assay
Complement pathway activity (ACH50) was assayed based on the Yano et al method (Yano et al. 1988). Briefly, 50 μL of activated and inactivated serums were poured separately into sterile microtubes at different times, followed by 350 μL of PBS containing Ca2+ and Mg2+. Then, 5% washed rabbit red blood cells (100 μL) were added to each microtube and centrifuged at 200 g for 5 min. The supernatant of each microtube (100 μL) was collected and poured into a plate of 96 microplate chambers in an active serum column. In the side column at a certain time and the inactive serum was poured into the pits at the same time and read at 450 nm.
2.7.3. Respiratory burst activity
The respiratory burst activity of leucocytes was determined using the Secombes method with slight modification (Secombes 1990). The leukocyte samples (175 μL ,6Í106cells ml-1) were added to the wells of 96-well microtiter plates. Then 25 μL of Nitro Blue Tetrazolium (NBT) (1 mg ml-1 concentration) was added to the wells and incubated at 25 ° C for 2 h. The supernatant was carefully discarded and 125 ml of 100% methanol was added to each well. After that, the supernatant was removed and each well washed again twice using 125 ml of 70% methanol. The supernatant of wells was carefully discarded and the plate was dried at room temperature for 30 min. Then 125 ml of 2 N KOH followed by 150 mL of DMSO were added to the wells. The spontaneous O2 -production was calculated based on the following formula:
The spontaneous O2 -= (Absorbance NBT reduction of the sample) - (Absorbance of blank)
2.7.4. Bactericidal activity of serum
The serum bactericidal activity was measured according to Yin et al (Yin et al. 1996), with some modifications. The bacteria culture (Streptococcus iniae) was pelleted (3000 g, 10 min) and washed 3 times with sterile PBS. A volume of 25 μL bacterial suspension (adjusted to 4 × 109 cells/ml) was added to 25 μL serum of fish in sterile Eppendorf tubes. Then, the tube was incubated at room temperature for 1 h. After that, plating the mixtures on TSA containing 1.5% NaCl was used to determine colony forming units (CFU)/ml.
2.7.5. Challenge study
Streptococcus agalactiae was purchased from the Pasteur Institute of Iran (IPI). The bacterium was cultured in Tryptic Soy Broth and incubated overnight in the rotation shaker at 110 rpm and 37° C. After obtaining sub-culture from the stock, 5 mL of stock was transferred into a 50 mL flask containing Tryptic Soy Broth and incubated at 37 ° C for 24 h. Duplicate sub-cultures were used under similar conditions for the experiment. The optical density of 560 nm and plate counting in Tryptic Soy Agar was used for growth evaluation. The LD50 was determined based on Wang et al study (Wang et al. 2016). In the present study, 0.1 mL of 107 CFU ml-1 of S. agalactiae in normal saline solution (0.85%) was injected intraperitoneally into the healthy fish (n=20) from each treatment at the end of the experiment. Dead fish from the challenge tanks was daily removed and recorded and the bacterium was reisolated by culturing on TSA plates for 24 h at 27 ° C. S. agalactiae was confirmed by morphologically and biochemically tests of inoculated strain. After 15 days of the challenged test, the relative percentage of survival (RPS) was calculated:
RPS = 100 –(test mortality/control mortality)× 100
2.8. Growth performance
At the end of the feeding trial, 10 randomly selected fish in each replication were weighed on day 0, day 30, and day 60. The survival rate and growth performance of fish were calculated using the following equations:
Weight gain (WG)= final weight (g) - initial weight (g)
Specific growth rate (SGR%) = 100 - (ln final weight - ln initial weight)/Duration of experiment
Feed conversion ratio (FCR) = feed given (dried weight)/ weight gain (wet weight)
Survival rate (%) = (final fish number/ initial fish number) -100.
2.9. Statistical analysis
Before statistical analysis of data, their normality was determined using the Kolmogorov-Smirnov test. One-way and two-way ANOVA with Multiple Comparisons Test was used to compare the different groups, followed by Tukey's test (P < 0.05) and quantitative data were presented as mean ± standard deviation. All statistical analyses were performed using SPSS software (Version 24).