The University of Georgia College of Veterinary Medicine

Georgia Veterinary Scholar Program

Georgia Veterinary Scholar	Faculty Mentor

Stephanie Halminski University of Georgia Class of 2006	Dr. Fred Quinn

Mechanisms of infection and cell killing of carp monocytic cells by a newly identified fish pathogen, Mycobacterium shottsii

Mycobacterium shottsii is a recently identified slowly growing acid-fast bacterium originally isolated from striped bass in the Chesapeake Bay . This species is proximate in phylogeny to M. ulcerans, M.marinum and members of the M. tuberculosis complex; however, the virulence mechanisms of M. shottsii are unknown. A carp monocytic cell line (CLC) was used to determine if M. shottsii replicates within fish macrophages or remains extracellular, and if host cells are killed predominantly via necrosis or apoptosis pathways. Multiple assays were performed using CLC cells seeded in 6-well dishes at 10^6 cells/well and infected with M. shottsii expressing green fluorescent protein (GFP) at a multiplicity of infection of 10 bacteria per host cell. Infected monolayers were fixed and viewed by fluorescence microscopy 15min, 24hrs, 48hrs, 72hrs, and 96hrs after infection. Preliminary microscopic results demonstrated that the mycobacteria remained predominantly extracellular and there was little evidence of cell lysis or cytoplasmic oozing among the remaining cells in the monolayer after 96hrs of infection. An antimycobacterial rhodamine labeled antibody next will be used to better distinguish extracellular from intracellular bacteria. In this assay extracellular mycobacteria will stain red while intracellular mycobacteria will maintain their GFP fluorescence. Microscopy and quantitative flow cytometry will be performed on these infected cultures to more precisely define the ratio of intracellular and extracellular mycobacteria. Using a cell death detection ELISA, we determined that M. shottsii induces necrosis in CLC cells. We will now assay for apoptosis and determine if the CLC cells are killed predominantly via necrosis or apoptosis. In conclusion, we determined that M. shottsii is an extracellular pathogen and kills host cells via necrosis. If these conclusions are confirmed using the described techniques, we will have demonstrated that the virulence mechanisms utilized by M. shottsii more closely resemble those of M. ulcerans than M. marinum and M. tuberculosis.