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.

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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