Hemolysin: A Potential Virulence Factor of Edwardsiella ictaluri and the Use of Random Mutagenesis in this Determination
IAAAM Archive
Michele L. Williams; Mark L. Lawrence
College of Veterinary Medicine, Mississippi State University, Mississippi State, MS


Edwardsiella ictaluri is the causative agent of enteric septicemia of catfish (ESC), an important disease for the channel catfish industry. The pathogenesis of ESC is not fully understood; however, several characteristics have been examined as potential virulence factors. One of these is a cell-associated hemolysin. Hemolysins have been shown to be virulence factors for other bacterial pathogens including Actinobacillus pleuropneumonae, Listeria monocytogenes, Leptospira sp., and Streptococcus pneumonae. A study by Stanley et al. 1 determined that there was no clear relationship between hemolytic activity and virulence of E. ictaluri. This assay was performed using bacterial broth cultures incubated with a suspension of 5% sheep red blood cells. Since differences in hemolytic activity could be attributable to differences in growth patterns, we repeated this study using stationary phase bacteria suspended in hemolysin buffer. We found, as Stanley et al. had, that hemolysin activity varied greatly between isolates and that virulent strains appeared to produce more hemolysis than their corresponding attenuated strains.

Because these results still did not conclusively prove whether the E. ictaluri cell-associated hemolysin is important for virulence, we developed an isogenic hemolysin negative mutant using transposon mutagenesis. Plasmid pLOFKm, which contains a mini transposon derived from Tnl 0 containing a kanamycin resistance gene, was used to generate a bank of random E. ictaluri mutants. The plasmid was transferred into virulent E. ictaluri by conjugation using an E. coli donor strain, and mutants were screened by observing lack of hemolysis on agar plates with 5% sheep red blood cells. Mutants were streaked on tryptic -soy agar (TSA) plates with 5% sheep red blood cells to check for purity and phenotype. Stability of the mutation was determined by multiple passages through brain heart infusion (BHI) broth. Following the 15th passage, the bacteria were spread on TSA with 5% sheep red blood to check phenotype and purity.

Future studies with this mutant include virulence trials and immunogenicity studies to investigate the plausibility of using this mutant as a vaccine strategy.


1.  Stanley, L.A., Hudson, J.S., Schwedler, T.E., Hayasaka, S.S. 1994. Extracellular products associated with virulent and a virulent strains of Edwardsiella ictaluri from channel catfish. Journal of Aquatic Animal Health 6"36-43.

Speaker Information
(click the speaker's name to view other papers and abstracts submitted by this speaker)

Mark L. Lawrence

Michele L. Williams