Michele L. Williams; Mark L. Lawrence
College of Veterinary Medicine, Mississippi State University, Mississippi
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.