College of Veterinary Medicine, Mississippi State University, Mississippi
State, MS, USA
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. Previous research has determined that there
was no clear relationship between hemolytic activity and virulence of E. ictaluri.1,2
Because these results 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 contained a
composite mini-transposon derived from Tn10 containing the Tn903 kanamycin
resistance gene, was used to generate a bank of random E. ictaluri mutants. To date, two
hemolysin negative mutants from a pool of 3000 transconjugates have been isolated. We hypothesize
that hemolysin is important for virulence in E. ictaluri.
The current study has a two-fold objective. First, we wanted to determine
whether these two mutants represented insertions into the same gene. The second objective was to
determine the identity of the mutated gene. The first objective was accomplished by performing a
Southern hybridization of selected restriction enzyme digests of genomic DNA from the two E.
ictaluri mutants, using the Tn903 kanamycin resistance gene as the probe. This resulted
in identical banding patterns between the two mutants, indicating that the mutations reside in the
The second objective was accomplished by ligating KpnI-digested
chromosomal DNA from one of the mutants into pBluescript cloning vector. These products were
transformed into E. coli strain XL1-Blue and positive transformants were selected by
resistance to ampicillin and kanamycin. These clones were then sequenced from the T3 and T7
promotors of the vector. Currently, BLAST results from one of the clones indicated that we have
cloned a gene with 88% nucleotide identity to the hemolysin A gene of Edwardsiella tarda
(GenBank number D89876)3.
In conclusion, we have successful isolated a hemolysin negative mutant strain
of E. ictaluri and successfully cloned this mutation into E. coli. From the clones,
we have identified a gene with high sequence homology to the hemolysin A gene of E. tarda.
Virulence trials using this hemolysin negative mutant with SPF channel catfish
fingerling are in progress. If these trials show that hemolysin is required for virulence in E.
ictaluri, future studies with this mutant will include immunogenicity studies to investigate
the plausibility of using this mutant as a vaccine.
1. Hirono I, N Tange, T Aoki 1997. Iron-regulated haemolysin gene
from Edwardsiella tarda. Molecular Microbiology. 24(4): 851-856.
2. Stanley LA, JS Hudson, TE Schwedler, SS Hayasaka. 1994.
Extracellular products associated with virulent and avirulent strains of Edwardsiella
ictaluri from channel catfish. Journal of Aquatic Animal Health 6: 36-43.
3. Williams ML, ML Lawrence. 2001. Hemolysin: A potential virulence
factor of Edwardsiella ictaluri and the use of random mutagenesis in this determination.
Proceedings of the 32nd Annual Conference of IAAAM, pg 81.