Sample collection and enrichment: To isolate the bacteriophage specific to poultry Salmonella the river, drainage & sewage water samples suspected to contain bacteriophage were collected which was near to Bangladesh Agricultural University poultry farm and experimental Animal House of Dept. of Microbiology and Hygiene, BAU, Mymensingh and transferred to the laboratory of the Department of Microbiology and Hygiene, BAU, Mymensingh using proper sample transportation techniques; during July to December 2013. To increase the number of bacteriophage possibly present in the collected samples 4.5 ml of water samples, 0.5 ml of 10X nutrient broth (Himedia, India) and 0.5 ml of log phase bacteria (OD-600=1) were mixed well followed by overnight incubation at 370C. The samples were then centrifuged at 10,000 rpm for 10 minutes at 40C, decanted; supernatants were collected and filtered by 0.45μM cellulose acetate syringe filter into a sterile glass or polypropylene vials. Further the filtrates were used to detect the phage. The filtered lysates were treated with chloroform (to a 2.5% final concentration), stored at 50C, and titered by double-agar-layer plaque assay according to the method of Adams MH (1959).
Detection of bacteriophages:
Spot test and plaque assay: On nutrient agar plate, 1ml of cultured bacteria was spread, the excess fluid was removed and the plates were kept at room temperature for air dry. Then 10 μl of filtrated supernatant was spotted on the agar forcefully and allowed to dry. The agar plates were incubated at 37°C for overnight for the detection of lytic spot over the bacterial lawn on to the agar plate. After detection of bacteriophage by spot test plaque assay was performed to isolate the bacteriophage using exponential growth phase Salmonella host culture. In brief, 100 μl of cultured bacteria and 100 μl of filtrated supernatant of phage lysate were taken in a sterile ependorf tube which was then mixed with 2.5 ml of soft agar (0.6% nutrient agar). Test suspension was then poured onto a nutrient agar plate, spread uniformly through the plate and kept at room temperature (RT) for 10 minutes to solidify. Soft agar overlays were allowed to harden at room temperature and then plates were inverted and incubated overnight at 37°C. Next day the ability of plaque production by bacteriophage were detected. The numbers of plaques were counted as plaque forming unit (PFU).
Purification of plaque: A plaque was collected with the help of a micropipette tip and mixed with 300 μl of distilled water and incubated for 30 min followed by centrifugation at 5,000 rpm for 10 min which was then subjected to plaque assay. This process was repeated for three times to obtain homogenous plaques.
Determination of host range: Nine isolates of Salmonella spp, & one isolate of enterotoxigenic E. coli, Pasteurella multocida and Haemophilus paragallinarum each were used in this study to determine the host range of the isolated phages. The bacterial isolates were obtained from the repository of Dept. of Microbiology and Hygiene, Bangladesh Agricultural University. Host range of bacteriophages was determined by spotting 10 μl of bacteriophage preparation (~1010 pfu/ml) on cultures of each bacterial isolates. The plates were observed for positive result by observing the appearance of lytic clear zones after incubation at 37°C for 18 to 36 h.
Efficiency of plating (EOP): To determine the efficiency of plating (EOP) means the ability of plaque production on each susceptible bacterial strain, the phage stock was subjected to plaque assay using all of nine Salmonella isolates. The numbers of plaques were counted. Higher number of plaque produced on bacteria was considered as the higher efficiency of plating (EOP).
Heat and pH susceptibility tests: The heat susceptibility of phage was measured by treating the phage stock at 50°C, 60°C, 70°C and 80°C for 1 hour. For pH stability, samples of bacteriophages were mixed in a series of tubes containing nutrient broth of different pH ranging between 2 to 9 (adjusted using NaOH or HCl) and incubated at 37°C for 30 min. Bacteriophage titers were determined using the double-layer agar plate method or plaque assay method.
Determination of adsorption rate: To determine the adsorption rate, Salmonella bacterial cells were grown in nutrient broth to exponential phase, then infected with the phage SAL-PG at Multiplicity of Infection (MOI) 0.0001, and incubated at room temperature. Samples were taken at different time intervals such as 5, 10, 15, 20, 25, 30, 35 and 40 min. and centrifuged. The supernatants were used for plaque assays to determine the titers of non adsorbed phages (Hadas et al., 1997). This experiment was repeated three times independently.
Effects of Bacteriophage on Salmonella: One test tube was filled with 4.5 ml nutrient broth, 500 μl host bacterial culture and 100 μl of bacteriophage and another test tube was also filled with similar components where 100 μL of PBS was added instead of bacteriophage which was kept as untreated control followed by incubation at 37°C overnight. The killing activity of bacteriophage was recorded by determination of OD600 (optical density) value of both phage treated and untreated samples.
DNA purification: The DNA of the isolated phage was extracted and purified using Invisorb® Spin Virus DNA Mini Kit following the instruction provided by the manufacturer.
Restriction Endonuclease (RE): analysis Purified DNAs were digested individually with TaqαI, XhoI, Hind III, BstYI and BstEII.