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

Abstract

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 37C 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 -20C, 4C, 37C and 50C when exposed for duration of 20 min. But, a temperature of 70C 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.