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Fish francisellosis is an emergent disease caused by gram-negative facultative intracellular bacteria of the genus Francisella. Different strains of the bacterium have caused high mortalities in warmwater and coldwater fish species. Francisellasp. isolates from fish have been found to share more than 97% identity to the human pathogen Francisella tularensisupon 16S ribosomal RNA sequence comparison. Homologue genes of the F. tularensisintracellular growth locus (iglA*, iglB*, iglC*, and iglD*) were identified from LADL 07-285A, a clinical isolate obtained from diseased Nile tilapia Oreochromis niloticus.The iglABCDoperon DNA sequence comparison revealed that FrancisellaLADL 07-285A had 94% identity with F. philomiragiasubsp. philomiragiaand 83% identity with F. tularensissubsp. novicidaU112. The functions of the conserved proteins corresponding to the genes are elusive but appear to be essential for the ability of Francisellasp. to survive within macrophages and cause disease. An insertion mutation was made in the iglC* gene of LADL 07-285A by allelic exchange, and the iglC* mutant was found to be attenuated after intraperitoneal and immersion challenges in Nile tilapia. Laboratory challenge methods for inducing francisellosis in Nile tilapia were evaluated by intraperitoneal injection and immersion with serial dilutions of FrancisellaLADL 07-285A. The dose lethal to 50% of test fish at 40 d postchallenge was 10-5.3(about 1.2 × 103colony-forming units/fish) by intraperitoneal injection and was 10-1(2.3 × 107colony-forming units/mL of tank water) by immersion.

The aroAgene of Edwardsiella ictaluriwas cloned and sequenced, and the sequence data were used to construct a deletion–insertion mutation in the aroAgene. The mutated gene was transferred into a virulent, wild-type E. ictaluristrain by conjugation and allelic exchange. Putative aroAmutants were confirmed phenotypically by demonstrating a need for supplementation with aromatic metabolites to support growth in minimal media. The genetic construction was evaluated by using the polymerase chain reaction to amplify appropriate regions of the aroAdeletion–insertion, and DNA sequencing of the amplified products confirmed the predicted construction. A selected mutant, LSU-E1, was passed 30 times in nonselective media with no reversion to the wild-type following screening of 1.6 × 1011colony-forming units. The mutant was demonstrated via injection to be attenuated more than 5 logs10compared with the wild-type E. ictaluristrain, and it was avirulent by immersion and oral routes. Tissue persistence studies indicated that the mutant maintained the ability to invade following immersion exposure, but no viable cells were detected after 48–72 h. Significant levels of protection from disease were demonstrated following immersion vaccination of channel catfish Ictalurus punctatus.