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A selective medium was used for the isolation of Cytophaga columnaris in 154 routine cases submitted to the Louisiana Aquatic Animal Diagnostic Laboratory, In 46 cases, systemic C columnaris that would not have been detected with routine cytophaga media was isolated from diseased channel catfish Ictalurus punctatus. Strain variation in susceptibility to Romet (sulfadimethoxine and ormetoprim, 5:1) was noted. Of the 86 strains tested, 83 (97%) were regarded as susceptible. All strains tested were susceptible to Terramycin (oxytetracycline HO). The minimal inhibitory concentration (MIC) of Romet on representative strains of C. columnaris ranged from 7.81 to 15.62 Mg/mL. The MIC of Terramycin on selected strains was 0.195 /*g/mL.

. S. iniae produces a cytolysin that confers a hemolytic phenotype on blood agar media. In this study, we characterized the genomic region responsible for S. iniae cytolysin production and assessed its contribution to virulence. Transposon (Tn917) mutant libraries of commensal and disease-associated S. iniae strains were generated and screened for loss of hemolytic activity. Analysis of two nonhemolytic mutants identified a chromosomal locus comprising 9 genes with 73% homology to the group A streptococcus (GAS) sag operon for streptolysin S (SLS) biosynthesis. Confirmation that the S. iniae cytolysin is a functional homologue of SLS was achieved by PCR ligation mutagenesis, complementation of an SLS-negative GAS mutant, and use of the SLS inhibitor trypan blue. SLS-negative sagB mutants were compared to their wild-type S. iniae parent strains in the murine model and in human whole-blood killing assays. These studies demonstrated that S. iniae SLS expression is required for local tissue necrosis but does not contribute to the establishment of bacteremia or to resistance to phagocytic clearance.

Edwardsiella ictaluri is the leading cause of mortality in channel catfish culture, but little is known about its pathogenesis. The use of signature-tagged mutagenesis in a waterborne infection model resulted in the identification of 50 mutants that were unable to infect/survive in catfish. Nineteen had minitransposon insertions in miscellaneous genes in the chromosome, 10 were in genes that matched to hypothetical proteins, and 13 were in genes that had no significant matches in the NCBI databases. Eight insertions were in genes encoding proteins associated with virulence in other pathogens, including three in genes involved in lipopolysaccharide biosynthesis, three in genes involved in type III secretion systems (TTSS), and two in genes involved in urease activity. With the use of a sequence from a lambda clone carrying several TTSS genes, Blastn analysis of the partially completed E. ictaluri genome identified a 26,135-bp pathogenicity island containing 33 genes of a TTSS with similarity to the Salmonella pathogenicity island 2 class of TTSS. The characterization of a TTSS apparatus mutant indicated that it retained its ability to invade catfish cell lines and macrophages but was defective in intracellular replication. The mutant also invaded catfish tissues in numbers equal to those of invading wild-type E. ictaluri bacteria but replicated poorly and was slowly cleared from the tissues, while the wild type increased in number.

The ability of Edwardsiella ictaluri to enter, survive, and replicate in head kidneyÀderived macrophages from channel catfish Ictalurus punctatus was investigated in vitro. Primary macrophage cultures were exposed to E. ictaluri and were sampled after 0 (90 min postinfection), 4, 8, and 12 h. The data indicated that bacterial numbers for E. ictaluri increased 2.6-, 5.1-, and 7.1-fold after 4, 8, and 12 h of incubation, respectively. Microscopic examination of infected cell cultures revealed numerous intracellular bacteria that were contained within clear, spacious vacuoles. Opsonization with normal serum resulted in significantly greater internalization of E. ictaluri at 0 h compared with pretreatment with heat-inactivated serum or no serum at the same time point, and bacteria pretreated with normal serum maintained significantly greater numbers than those in the other treatments after 4 and 8 h. Opsonization, however, did not affect the ability of E. ictaluri to replicate once the cell was entered, as there were statistically significant increases in bacterial numbers at all time points regardless of the serum pretreatment.

A recently described Edwardsiella ictaluri type III secretion system (T3SS) with functional similarity to the Salmonella pathogenicity island 2 T3SS is required for replication in channel catfish head-kidney-derived macrophages (HKDM) and virulence in channel catfish. Quantitative PCR and Western blotting identified low pH and phosphate limitation as conducive to expression of the E. ictaluri T3SS, growth conditions that mimic the phagosomal environment. Mutagenesis studies demonstrated that expression is under the control of the EsrAB two-component regulatory system. EsrB also induces upregulation of the AraC-type regulatory protein EsrC, which enhances expression of the EscB/EseG chaperone/effector operon in concert with EsrB and induces expression of the pEI1-encoded effector, EseH. EsrC also induces expression of a putative type VI secretion system translocon protein, EvpC, which is secreted under the same low-pH conditions as the T3SS translocon proteins. The pEI2-encoded effector, EseI, was upregulated under low-pH and low-phosphate conditions but not in an EsrB-or EsrC-dependent manner. Mutations of EsrA and EsrB both resulted in loss of the ability to replicate in HKDM and full attenuation in the channel catfish host. Mutation of EsrC did not affect intracellular replication but did result in attenuation in catfish. Although EsrB is the primary transcriptional regulator for E. ictaluri genes within the T3SS pathogenicity island, EsrC regulates expression of the plasmidcarried effector eseH and appears to mediate coordinated expression of the T6SS with the T3SS.Edwardsiella ictaluri, the etiological agent of enteric septicemia of catfish (ESC), was isolated, named, and characterized by Hawke (22) and Hawke et al. (23). The disease is found throughout channel catfish (Ictalurus punctatus) growing regions and is the leading cause of disease-related mortality in channel catfish production facilities (2, 3). Recent reports have expanded the host and geographic ranges to the culture of striped catfish (Pangasianodon hypophthalmus) in Vietnam (14, 51) and yellow catfish (Pelteobagrus fulvidraco) in China (29).Using signature-tagged mutagenesis, Thune et al. (52) identified mutations in 50 E. ictaluri genes that led to an attenuated phenotype in channel catfish, including a type III secretion system (T3SS) apparatus gene. Further sequencing and genome analysis led to the identification of an E. ictaluri pathogenicity island (PAI) encoding a complete T3SS, including Edwardsiella secretion apparatus (esa), chaperone (esc), effector (ese), and regulatory (esr) genes. Mutation of the apparatus gene resulted in an inability to replicate in catfish cells and loss of virulence, indicating that the T3SS is essential to the pathogenesis of E. ictaluri. These systems are widespread in Gram-negative bacteria and function to translocate effector molecules directly from the bacterial cytosol to the host cell cytoplasm.The Edwardsiella tarda T3SS, which is involved in replication within fish macrophages (38,49,50,57), is hom...

We compared Edwardsiella ictaluri from striped catfish in Vietnam with US channel catfish isolates. Biochemical analyses and sequencing of the 16S rRNA gene confirmed that the Vietnamese isolates were E. ictaluri. Comparison using rep-PCR fingerprinting demonstrated no significant differences between the isolates, but plasmid analysis indicated that the Vietnamese isolates grouped into 4 plasmid profiles, each different from the typical pEI1 and pEI2 plasmid profile found in the US isolates. Sequencing plasmids representative of the 4 profiles indicated that all contained derivatives of the E. ictaluri plasmid pEI1, whereas only 1 contained a plasmid derivative of the E. ictaluri plasmid pEI2. The pEI2 encoded type III secretion effector, EseI, and its chaperone, EscD, were found to be present on the chromosome in isolates lacking a pEI2 derivative. In addition, 1 isolate carried a 5023 bp plasmid that does not have homology to either pEI1 or pEI2. Furthermore, Vietnamese isolates were PCR positive for the type III and type VI secretion system genes esrC and evpC, respectively, and the urease enzyme, but were PCR-negative for the putative type IV secretion system gene virD4. A monoclonal antibody against the lipopolysaccharide of E. ictaluri ATCC 33202 did not react with the Asian isolates or with the more recent US isolates. Antibiotic resistance patterns were variable and did not correlate to the presence of any particular plasmid profile. Finally, the Vietnamese isolates were avirulent and had a significantly reduced capacity for intracellular replication within head-kidney-derived channel catfish macrophages.

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