Microbiological investigation into the cause of Equine Grass Sickness

Abstract

A role for bacteriophage therapy was envisaged earlylast century; however, due to discovery of the antimicrobials, it fell out of research interest. Currently, bacteriophages are resurfacing as an alternative to antimicrobials in order to overcome the increasing incidence of antimicrobial resistance. Here, we report isolation of bacteriophages against Escherichia coli, Shigella spp., Aeromonas hydrophila and Citrobacter sedlakii isolates of equine origin. Phages were isolated from equine farm soil and sewage samples. For enrichment, sample aliquot was incubated overnight with host bacteria at 37 C with vigorous shaking. The crude lysate obtained was centrifuged and filtered and the presence of any phage in the suspension was detected by agar overlay technique. Appropriate dilutions of enriched samples were plated to obtain individual plaques and the most dominant plaque was transferred into SM buffer, serially diluted and plated for plaque re-isolation three times to ensure purity, followed by large scale preparation of phage stocks. Any host nucleic acids was degraded using pancreatic DNaseI and RNase and bacteriophage particles were precipitated using PEG8000. Phage titre was determined by plaque assay and phage concentrates were accessioned in the Veterinary Type Culture Collection (VTCC) repository. The phage concentrates were visualized by transmission electron microscopy (TEM). The temperature stability of bacteriophages was checked after incubating phage concentrates over the range of 4 C-8 0C temperature for one hour. A clear single plaque was obtained on nutrient agar against Shigella spp. and after purification and concentration, its analysis by electron microscopy revealed presence of multiple phages belonging to families Myoviridae, Siphoviridae and Podoviridae. However in case of Citrobacter sedlakii, a Siphoviridae phage (VTCCBPA61) with dimensions: 60 nm x 650 nm was observed. Against a pathogenic A. hydrophila isolate of equine origin (expressing aerolysin gene), a Myoviridae phage (VTCCBPA6) was isolated with dimensions: 62 nm x 138 nm. Against E. coli of equine origin, a Myoviridae phage (VTCCBPA9) of dimensions: 86 nm x 100 nm was obtained. Bacteriophage VTCCPBA61 against C. sedlakii was found to completely lose its biological activity at 65 C in vitro however the group of phages against Shigella spp. were found to be stable upto temperature as high as 80C. Thus we demonstrated the basic biological characteristics of phages, and some novel ones (such as against A. hyrophila and C. sedlakii) bacterial isolates of equine originwhich have never been reported till now. These lytic phages could find potential in phage therapy, as biocides, in biosensors and in phage ligand technology and are being explored by us further to depict their therapeutic value in small animal model. As more studies are reporting safety, tolerance and efficacy of phage therapy in humans and animals, their use in phage therapy has a promising future as an emerging alternative to chemical agents.