Purification & characterisation of haemolysin from Serratia marcescens B-91.

BACKGROUND & OBJECTIVES: Serratia marcescens an opportunistic human pathogen, is frequently encountered in a variety of debilitating diseases. Relatively little is known about its virulence traits though most clinical isolates secrete a distinct haemolysin which is considered as a useful marker for pathogenicity of Serratia. In this study purification and characterisation of S. marcescens B-91 haemolysin have been attempted. METHODS: S. marcescens B-91 haemolysin was purified to homogeneity from the growth medium using ammonium sulphate fractional precipitation and gel filtration through Sephadex G-75 column. Homogeneity was determined by gel electrophoresis and purified haemolysin was tested for its stability and other characteristics. RESULTS: The haemolysin was characterised to be a 45 kDa molecular weight protein on SDS-polyacrylamide gel electrophoresis. It was inactivated at 60-100 degrees C within 30 min, and on overnight treatment with 2 per cent formaldehyde. It was also susceptible to the action of pronase, protease and trypsin. INTERPRETATION & CONCLUSIONS: The results indicate that the fragile stability of S. marcescens haemolysin is dependent on the storage temperature. The purified haemolysin can be used for understanding the role of haemolysin in the pathogenesis of S. marcescens and also for evaluation of immunoprophylactic activity.

Kinetics of humoral & cellular immune responses in experimental cysticercosis in pigs infected with Taenia solium.

Studies were undertaken to assess the kinetics of antibody responses, lymphocyte transformation to Taenia solium larval antigens (crude soluble extract antigen and antigen B), and T cell subpopulation in piglets following experimental infection. Cysticercosis was established in 1-2 month old piglets after feeding 5,00,000 T. solium eggs per pig. The anti-CD4 and anti-CD8 monoclonal antibodies against swine T cells were raised indigenously. It was observed that at 60 days post infection (PI) there was a significant increase (P < 0.01) in CD4+ T cells without any change in CD8+ T cells. Increased 3H-thymidine uptake was found in infected piglets at 45 days PI using both CSE and antigen B. Kinetics of antibody responses indicated significant increase (P < 0.01) at 15 days PI (with CSE antigen) and 30 days PI (with antigen B) by ELISA. This increase persisted till 90 days PI (the time up to which the animals were followed). It was also observed that the cellular mechanisms were triggered in late stage (60 days PI) as compared to humoral responses (15-30 days PI) and may persist longer as seen by both lymphocyte transformation and T cell subpopulation studies. The study suggests that in cysticercosis, both humoral and cellular mechanisms may play a role in the host defences.