Recovery status of bacteriophages of different livestock farms of Veterinary College, Adhartal, Jabalpur, India

Study was conducted to know the presence of bacteriophage in sewage material which can play a very important role during therapy against the some antibiotic resistance organisms. During study waste water samples were collected from different depths of the wastewater collection tanks located in livestock farms of different species (Cattle, pig, goat and poultry). These samples were subjected primarily to rapid detection by streak plate method for the detection of lytic activity followed by primary isolation of phage against two most common bacteria of environment, namely, B. subtilis and E. coli by Double agar layer (DAL) method. Recovery of phages was maximum from pig feces (67%) followed by dairy cattle farm waste (63%), buffalo farm waste (50%), goat farm waste (13%).


Introduction
species of animals viz.Cattle, buffalo, goat, pig and poultry were collected in sterile conical flask from Bacteriophages are obligate intracellular animal waste collection tanks in sufficient amount parasites requiring specific bacteria as host cell for (100 ml) from superficial and deep layers (up to 20 their replication (Carlton, 1999).Phages are most cm) in sterilized conical flask from.Samples (186) widely distributed and diverse entities in the biosphere were collected from Livestock Farm (Cattle, Buffalo, and ubiquitously present which can be found in all Goat and Piggery farm), College of Veterinary Science reservoirs populated by bacterial hosts, such as soil and A.H., Adhartal, Jabalpur, India.and sea water and the intestine of animals (Mc Grath Plates with plaques were selected and the top agar was membrane syringe filter scrapped with 5 ml of Lactose broth.The scrapings Processing of sample: The collected samples were were pooled and 2-3 drops of chloroform was added to processed as per the method described by Jothikumar this and held for 10 minutes at room temperature and et.al., (2000) with slight modification.Samples then centrifuged at 5000 rpm for 20 minute in collected were processed separately for the isolation of bacteriophage.Briefly, samples were homogenized refrigerated centrifuge.Agar debris settled down and for one hour, centrifuged at 3000 rpm for 20 min; supernatant was filtered through a 0.45µ cellulose supernatant was collected and re-centrifuged at 5000 syringe filter.lysates (purified phage suspension) were rpm for 20 min in refrigerated centrifuge (Remi C-24).stored in sterile plastic vials at 40C for further use.Then supernatant collected was filtered through a Titration of phage lysate: Titration was done by 0.45µ syringe filters and filtrate was placed in sterile preparing ten fold serial dilution of the phage lysate.plastic vials.
For titration, 10-1 to 10-9 dilutions were made in Isolation of phage by DAL method: Isolation and normal saline.Each dilution was subjected to plaque propagation of phage was performed by double agar formation by DAL method (Adams 1959).Phage titer layer (DAL) method as previously describe by Adams was determined in terms of plaque forming units (1959).Briefly, lactose agar (10 ml) dispensed into (PFU/ml) with the help of formula given below: 100 mm sterile Petri dish which was allowed to PFU/ml = No. of plaques x Dilution factor solidify, further, 2.5 ml of soft agar (mixed properly

Results and Discussion
with the filtrate `and the host bacteria) was poured on basal lactose agar layer to form double agar layer.The Isolation of bacteriophage: Lytic activity in procedure is described as under.
samples was adjudged as criteria for presence of phage For the plaque detection in the sample, six hour in samples and on the basis of clear plaque old bacterial culture of B. subtilis / E. coli were morphology samples were selected for the isolation of phage with DAL method.For Primary isolation of separately taken.The concentration of bacteria was phage B subtilus and E coli were used as bacterial host adjusted to 3x107-1x108 CFU/ml.Nutrient broth and cell.Details of the results are presented in table2.Lactose broth was used for B. subtilis and E. coli The results of phage screening in the present study respectively.In a sterile vial 500 µl of filtrate and 500 reveled that the concentration of phage was greater in µl of six hour old bacterial culture were mixed then 20 deeper layer as compared to the superficial layer of µl MgCl2 (25mM) was added to enhance the collection tank.Similar findings were reported by adsorption of phage over bacterial surface.After this, Salama et al., (1989) and Carey-Smith et al., (2006).it was agitated on shaker for 20 min and then added to a The superficial layer of collection tanks have direct test tube containing 2.5 ml of molten soft agar held at sunlight exposure, have high temperature and most of 450C in a water bath.The mixture was poured onto lactose agar basal plates, allowed to solidify and then organic matter settles in the deeper layer, thus incubated at 370C up to 48 hours.Plates were providing conditions for the lesser proliferation of observed at various time intervals (6, 12, 24 and 48 h) bacteria.This may be correlated with our finding.for development of plaques.
The recovery of phages was maximum in pig feces Preparation of phage lysate: Lysate was prepared (67%), followed by cattle farm waste (63%), buffalo by the DAL method described by Jothikumar et al. farm

for Host bacteria: Bacillus
subtilis and Escherichia coli antibiotics to treat bacterial infections that do not both are most abundant bacteria present in the respond to conventional antibiotic therapy (O'Flynn et environment; therefore they were utilized as primary al., 2004) therefore the isolation of bacteriophages hosts for the isolation of phages.The known isolates (in-house isolated, characterized and identified) of B. was performed to use them in treatment of certain subtilis and E. coli were used.clinicalconditions.Membrane filter: 0.45 µm Cellulose acetate (2000) with slight modification as described above.
Media for phage isolation: Following commercially