2.1 Bioprob (dietary probiotic) Bioprob (dietary probiotic) was purchased from Chancara Bazar, Jessore at Syfulla Krishi and Motsho Biponi. Bioprob was manufactured PIC-BIO Company, Tokyo, Japan. The specification and composition of bacterial strains of Bioprob.
2.2 Fish and husbandry Pangasius hypophthalmus, healthy Thai Pungus (n= 375 pieces) were collected from Kopothaksha hatchery at Chanchra, Jessore and fishes were immediately examined to find out their health status and acclimatized, transferred into in the quarantine tank (100 L) with recirculation aerated water for three days the laboratory of the Dept. of Fisheries and Marine Bioscience (FMB), in Jessore University of Science and Technology (JUST), Jessore on June 2014. The fishes were divided into five equal groups (T1, T2, T3, T4 and T5) each with two replicate containing 25 fishes per replicate. Continuous aeration was provided to maintain dissolved oxygen level at 7.5±0.5 mgl-1 and one-third of the aquarium water was exchanged daily by siphoning the waste materials were removed. During the experimental period water temperature, pH and TDS (total dissolved solid) were 22±0.8 0C, 5.94 0.21 and 4.34 0.29 mgl-1 respectively. Fishes were provided with normal basal feed at the rate of 5% of their body weight twice a day at 09:00 and 17:00 hour for 3 days but at the first day of their arrival no feed was provided.
All fish groups were fed on the diets at the rate of 3% from the body weight during 6 weeks of the experiment. On the week of 2, 4 and 6 three fish per group were randomly collected from each group, used for blood collection for specific and nonspecific immunological assays. The experimental fish were challenged with a virulent strain of Pseudomonas fluorescens at 3 10-7 CFU mL-1 by injected intraperiotonaly with 25 µl PBS for analyzing cumulative mortality.
2.3 Experimental diet preparation The experimental diet was prepared by mixing with locally available Mega feed which proximately contains protein: 34%, crude fiber: 6%, crude ash: 18%, moisture: 11%, lipid: 6%, fat: 3% showing in table 3 (Source: Spectra fish feed Com. Ltd.). At first Mega feed was grinded by a grinder and mixed with Bioprob porobiotics powder. All the ingredients were mixed thoroughly by adding water and pelletized by hand and then sun dried. Five different experimental pellet diets were prepared which contained five different mixture of Bioprob probiotic such as 0 g (control), 0.5 g, 1 g, 1.5 g and 2 g per kg feed. The pellet feed was stored in a cool dry place until use. 2.4 Blood analysis (specific immune response) Blood was drawn from the caudal peduncle region using sterile 2 mL syringes rinsed first with 2.7% EDTA solution as an anticoagulants from five groups separately and collected blood was kept in 1 mL eppendorf tube randomly selected and allowed to clot for 45 min in an inclined position at room temperature, followed by 30 min incubation at 4 0C and then centrifuged at 3000 rpm for 10 min at 4 0C. Serum was collected for each group three culture plates. Bacterial stock solution was serial diluted for 10 times and 10-3, 10-4 and 10- 5 concentration were selected for further usage. Then 25 µl volume from each diluted solution was mixed with 25 µl volume separated serum of five different groups of fishes then spread on the different culture plates and finally all plates were placed in an incubator at 37 0C for 24 hours. After 24 hours all plates were observed.
2.6 Phagocytic activity For this assay 25 µl blood cell suspension of thai pangus and 25µl bacterial solution in PBS was previously fixed with glutarldehyde was placed on a coverslip. After 30 minute coverslip was carefully washed with PBS (Phosphate Buffer Solution) then air dried and fixed with methanol and after that stained with giemsa. The engulfed fish blood cell (phagocytic rate) was determined by using photographic microscope (Axiocam Erc 5s with Axio Vision driver Carl Zeiss, Germany).
2.7 Challenge study Pseudomonas fluorescens was obtained from Central Biological Laboratory at Jessore University of Science and Technology, Bangladesh. P. fluorescens was grown on nutrient broth for 24 h at 30 0C the culture broth was centrifuged at 3000 RPM (Rotation Per Minute) for 10 min. The supernatant was discarded and the pellets were resuspended in sterile phosphate buffer saline (PBS, pH 7.4). The final bacterial concentration was adjusted to 10-7 CFU ml-1 by serial dilution. By the end of the feeding experiment, the fish of the experimental group and the control group was injected intraperiotonaly (IP) with pathogenic P. fluorescens 0.2 ml of 1.8 10-7 CFU (Colony Forming Unit) ml-1. All groups were kept under daily observation for 7 days for any abnormal clinical signs with recording the daily mortality rate. The freshly dead fish were subjected to bacterial reisolation for confirmation. Mortality percentage was calculated.
2.8 Statistical analysis The mean and standard error were calculated for each variable. The data were analyzed by analysis of variance (ANOVA) to identify the significantly different groups at (P<0.05) using SPSS software statistical program (SPSS).