Therapeutic Efficacy of Brucella Phage against Brucella Abortus in Mice Model

Introduction spectrum antibiotics [12]. Phage replicates as long as the targeted host bacterium is present and thus they are Brucellosis is an important re-emerging zoonotic naturally self-limiting [13]. Recently, phage therapy infectious disease [1, 2]. It is characterized by abortions, has regained general interest as resistance to antibiotics stillbirths and reproductive problems in ruminant [3]. has become a serious problem [14,15] and is found to Human beings get infected accidently by contact with be effective to treat many bacterial infections [16-18]. vaginal discharges, fetal fluids [3] or by ingestion of The use of host-specific bacteriophages has been unpasteurized milk [4]. Five out of nine species of promoted as a cost-effective and adaptable approach to Brucella can infect humans in which most pathogenic control zoonotic bacteria like Brucella and Salmonella for human is B. melitensis and followed in descending [19] and have potential to be developed as effective order by B. suis, B. abortus, B. canis and recently agents to prevent and treat animal bacterial diseases isolated marine Brucellae (B. ceti) [5]. [20, 21]. The objective of this study was to investigate Bovine brucellosis is widespread in India and has the efficacy of phage treatment in reducing Brucella been reported from almost all states [1,6]. An appro-colonization and infection in mice. priate antibiotic therapy for animals and human beings is still disputed [7-9] and it is too expensive in most of Materials and Methods the animal species. so use of lytic phage to treat Animals: Apparently healthy female Swiss albino mice brucellosis can be another alternative to this important (n=26) (4 to 6 weeks age) were obtained from the disease Laboratory Animal Resource section, Indian Veterinary Phages are viruses that infect and multiply inside Research Institute (IVRI), Izatnager. The animals were the bacteria and can have two types of life cycles, lytic kept under conventional housing condition and provided and lysogenic [10]. The lytic phages are the most feed and water ad labium. suitable for phage therapy as they quickly reproduce within and lyse the bacteria and grow exponentially in Ethical approval: The animal ethics committee of the number [11]. Bacteriophages are virus that use Indian Veterinary Research Institute, deemed specific bacterial species as host, and reduce the university approved the study. number of viable bacterial cells without significant Bacterial cultures: B. abortus 19, B. abortus 544, B. affecting commensal gut flora often caused by broad-Abstract Aim: To investigate the preventive …


Introduction
spectrum antibiotics [12].Phage replicates as long as the targeted host bacterium is present and thus they are Brucellosis is an important re-emerging zoonotic naturally self-limiting [13].Recently, phage therapy infectious disease [1,2].It is characterized by abortions, has regained general interest as resistance to antibiotics stillbirths and reproductive problems in ruminant [3].
has become a serious problem [14,15] and is found to Human beings get infected accidently by contact with be effective to treat many bacterial infections [16][17][18].vaginal discharges, fetal fluids [3] or by ingestion of The use of host-specific bacteriophages has been unpasteurized milk [4].Five out of nine species of promoted as a cost-effective and adaptable approach to Brucella can infect humans in which most pathogenic control zoonotic bacteria like Brucella and Salmonella for human is B. melitensis and followed in descending [19] and have potential to be developed as effective order by B. suis, B. abortus, B. canis and recently agents to prevent and treat animal bacterial diseases isolated marine Brucellae (B.ceti) [5].[20,21].The objective of this study was to investigate Bovine brucellosis is widespread in India and has the efficacy of phage treatment in reducing Brucella been reported from almost all states [1,6].An approcolonization and infection in mice.priate antibiotic therapy for animals and human beings is still disputed [7-9] and it is too expensive in most of

the animal species. so use of lytic phage to treat
Animals: Apparently healthy female Swiss albino mice brucellosis can be another alternative to this important (n=26) (4 to 6 weeks age) were obtained from the disease Laboratory Animal Resource section, Indian Veterinary Phages are viruses that infect and multiply inside Research Institute (IVRI), Izatnager.The animals were the bacteria and can have two types of life cycles, lytic kept under conventional housing condition and provided and lysogenic [10].The lytic phages are the most feed and water ad labium.suitable for phage therapy as they quickly reproduce within and lyse the bacteria and grow exponentially in Ethical approval: The animal ethics committee of the number [11] [25].
female Swiss albino mice each containing 6 individual 5 were infected with 2 x 10 CFU B. abortus 544 in 0.1 Preparation of phage stock: Working stock of isolated ml PBS (pH 7.2) i.p.CFU determined as the method phage was prepared by seeding 500 ml flask containing described by [26].Preventive and therapeutic groups 100 ml of NZCYM medium (Sodium chloride,Casein 6 were treated with 10 cfu/ml of phage in 0.1 ml of PBS enzymic hydrolysate, Casein acid hydrolysate, Yeast (7.2) i.p., 48 hrs before and 48 hrs after infection extract, Magnesium sulphate, heptahydrate) (Himedia, respectively [27].Control groups of mice were Mumbai) with 0.5 ml suspension of B. abortus 19 inoculated with 0.1 ml PBS (7.2), i.p. harvested from 48 hrs grown culture on Brucella agar medium (Difco, USA) slant into sterile PBS (pH 6.8).

Splenic growth of challenge strain in Swiss Albino
mice: All mouse were killed by cervical dislocation 15 Phage suspension added in this culture after 12 hrs (log days post-infection and spleens were removed phase) and incubation for 48 hrs at 37°C.Culture were aseptically, homogenized individually in 9 time to centrifuged at 10000g for 10 min.Supernatant fluid weight of spleen in buffered saline solution (BSS: NaCl was then sterilized by passage through a membrane 8.5 g; KH PO4 1.0 g; K HPO4 2.0 g; distilled water filter (0.22µ, Millipore filter crop, U.S.A) and kept at 2 2 4°C.Titer (pfu/ml) of these phage stock determined by 1000 ml; pH 6.8) and three tenfold dilutions were done double overlay technique [23].
(1/10, 1/100 and 1/1000) in the BSS and 0.2 ml of each dilution was seeded in BAM (brucella agar media) Test for toxicity of phage preparation: In separate plates (Difco, USA) (4 plate for each dilution, 2 kept experiments, the tested phage was administered i.p. aerobically and 2 kept in 5 % CO ).Plates were 2 (Intra peritoneal) to mice to determine the toxicity of incubated at 37°C for 4 to 5 days to determine the phage in vivo.Three concentrations of the phage CFU/spleen; Colonies of Brucella should be preparation (10 PFU/ml, 10 PFU/ml, and 10 PFU/ml) enumerated on the dilutions corresponding to plates were tested on mice.Two mice were injected with 0.5 showing fewer than 300 CFU.When no colony was ml of each phage preparations along with 2 mice seen in the plates corresponding to the 1/10 dilution, received 0.5 ml sterile PBS.A 7 day observation was the spleen was considered to be infected with five made to see any toxic effect of phage preparation in bacteria.These numbers of Brucella per spleen are first terms of mortality and reduced weight gain.
recorded as X and expressed as Y, after the following Host range determination: The phage were tested for transformation: Y = log (X/log X) [28,29]  rounded head with a short tail, consistent with the containing solutions (10 pfu/ml) were mixed with 1/10 previously report that most of brucella phage have volume of 2.5% buffered glutaraldehyde for 5 min.25 short tails [31,32].A titer of phage used in therapy was µl of fixed phage were added to the surface of a 6 10 PFU/ml.It comes into medium range of piloform coated grid and left for 2 min.After removing concentration that was suitable for phage therapy [33].excess fluid grid was washed for 2 seconds in distilled Department of Veterinary Microbiology, Veterinary College, Guru Angad Dev Veterinary and Animal Science University (GADVASU), Ludhiana, Punjab, India Toxicity study of the phage preparation in mice showed guinea pig (up to 30 days) and in a guinea pig infected that it was nontoxic to mice as there was no mortality with brucellosis (over 45 days).Recently many trails of and untoward reaction seen in the 7 day observation phage therapy in mice, poultry and cattle showed that period after administration.
phages are effective promising alternatives to bacterial Spleen count of B. abortus 554 did 15 days after infection [40,41].administration of phage [28,29].It was found that the Conclusion mean protective activity of phage was 2.88, 3.08 and It is clear from this study that application of phage 4.27 in protective, therapeutic and control group's is an effective mean to reduce the colonization of Brucella mice respectively (Table-1, 2, 3 and 4).Protective in the spleen of mice.Since the use of antibiotic in activity was significantly (P<0.05)decrease in the preventive and therapeutic group as compared to brucellosis in animals was uneconomical and not control group .It means that there is recommended, the use of the phage to treat the animal significantly reduced in spleen count of both phage especially pregnant animal may help in reducing the treated group as compare to the non treated control chance of threatened abortion and potential transgroup.This observation was supported by the work of mission to other susceptible animal.This is the preliminary Parnas;  who established work in this field, and further study is required to find a that brucella phage possesses certain preventive properties more appropriate route, dosage and time interval to get against experimental challenge.The reduction in maximum result and more experiments are needed to colony count of Brucella was found to be higher in the make this therapy applicable to all conditions of brucellosis.st 1 group, in accordance with the finding of Drozevkina;

Authors' contributions
Pophadze [37,38] who demonstrated that the introduction of the phage into the organism at the same time or MR designed the study and all the other author immediately after infection lead to the localization of performed research experiment.AP and HV carried out the causative agent and increase the sterilization of the the statistical analysis.MR drafted and revised the organism.Reduction in the spleen count of Brucella with manuscript.All authors read and approved the final of a single dose of phage after 15 DPI suggest that the manuscript.phage could be stable and active in vivo for a longer

2 might
be not affect other bacteria flora in the body.Electron microscopy of the phage showed somewhat Examination of phage (Electron Microscopy): Phage-8

Phage lytic to Brucella spp: Phage lytic
. their ability to lyse different strains of B. abortus also Statistical analysis: Data obtained in the study were with other species of the genus Brucella, Salmonella, analysed statistically on 'SPSS-16.0'software package Staphylococcus, Pasturella and E.coli.

Table - 1
. Protective activity of phage in preventive group mice

Table - 2
. Protective activity of phage in therapeutic group mice.U= uncountable, Y= log (X/logX) where X= total CFU/spleen Table-3.Protective activity of PBS in control group mice.Camparision of mean protective activity of three group U= uncountable, Y= log (X/logX) where X= total CFU/spleen Table-4.