Isolation, Electron Microscopy And Physicochemical Characterization Of A Brucellaphage Against Brucella Abortus Vaccine Strain S19

摘要

Brucellosis is an economically important but neglected emerging endemic zoonotic communicable disease in India. The prevalence of brucellosis has increased from 34.15% of the samples in 2006-07 to 67.28 % in the year 2010-11. Brucella abortus is an intracellular pathogen capable of infecting animals and humans. The use of phages in the treatment of bacterial infections is an attractive alternative to existing antibiotic therapy. Phages target a particular host and it is unlikely to elicit resistance in untargeted bacterial strains. A brucellaphage was isolated against actively growing stage of Brucella abortus strain S 19 from sewage sample of a dairy farm in Ludhiana. The plaque morphology revealed discrete, clear, circular plaques of diameter 0.1 to 3 mm after 48 h of incubation at 37oC aerobically. The field isolates (n=9) of B. abortus were sensitive to phage. The host range of brucellaphage is against vaccine strains, viz. S19, S99 and Rev1 of B. abortus, B. melitensis and B. suis. The isolated brucellaphage failed to lyse any culture of heterologous species tested viz. Pasteurella multocida, E.coli, Staphylococcus, Streptococcus, Salmonella Dublin, Micrococcus and Pseudomonas spp. Electron microscopic studies of the brucellaphage revealed it to be an elementary body measuring approximately 65 nm at 50,000 X magnification with rounded head and a very short tail.The size and shape resembles another Brucella phage Tbilisi phage and the other phages isolated elsewhere. The isolated brucellaphage was able to survive at a temperature of -20oC, 4oC, 37oC and 50oC when exposed for duration of 20 min. But, a temperature of 70oC and beyond was lethal for the brucellaphage. Unlike normal light, the effect of sunlight on the survivability of phage indicates deleterious effects on the phage. UV light completely destroyed the phage within 15 min. Non-ionic detergents like SDS (10%) completely destroyed the phage in 15 min. There was no effect of RNAse and trypsin on the survivability of phage while proteinase K and lysozyme reduced the survivability of the isolated phage. The isolated phage was tolerant to pH 7 and 9 while there was a reduction in phage activity at pH 3 and 5. According to the literature reviewed, this is the first report of isolation of a genus specific brucellaphage against B. abortus from Punjab which will pave a way for its use in various cost effective diagnostics and in therapy of brucellosis. Introduction Brucellosis is an economically important but neglected emerging endemic zoonotic communicable disease in India. The prevalence of brucellosis has increased from 34.15% of the samples in 2006-07 to 67.28 % in the year 2010-11. Brucellosis is a zoonotic disease of worldwide importance caused by Brucella abortus. It is characterized by abortions, stillbirths and reproductive problems in animals (Garin Bastuji et al 2008). The disease is associated with domestic and wild animals and human beings get infected accidently by contact with vaginal discharges, fetal fluids (Garin Bastuji et al 2008) or by ingestion of unpasteurized milk (Pappas et al. 2005). Among 8 species of Brucella, 3 are known to be virulent for human beings (B. melitensis, B. abortus and B. suis (Fugier et al. 2007). Viruses are obligate intracellular parasites that require specific host cell for its replication (Mayer, 2005). Bacteriophages are viruses that exclusively target and reproduce within bacterial cells. Generally they use their tail fibres to attach to their host cell and inject their nucleic acid into the bacterium. The host cell machinery is then used by the phage to replicate and get incorporated into the protein capsid. The mature viruses are released on the lysis of cell membrane of bacterium. The property of specificity of viruses makes them effective in fighting specific bacterial infections. Their use in waste water treatment has been well documented by Sulakvelidze and Burrow, 2005; Sulakvelidze and Kutter, 2005 and Withey et a