Attenuation of the Fish Pathogen Francisella sp. by Mutation of the iglC* Gene

Abstract

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. Francisella sp. isolates from fish have been found to share more than 97% identity to the human pathogen Francisella tularensis upon 16S ribosomal RNA sequence comparison. Homologue genes of the F. tularensis intracellular growth locus (iglA*, iglB*, iglC*, and iglD*) were identified from LADL 07-285A, a clinical isolate obtained from diseased Nile tilapia Oreochromis niloticus. The iglABCD operon DNA sequence comparison revealed that Francisella LADL 07-285A had 94% identity with F. philomiragia subsp. philomiragia and 83% identity with F. tularensis subsp. novicida U112. The functions of the conserved proteins corresponding to the genes are elusive but appear to be essential for the ability of Francisella sp. 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 Francisella LADL 07-285A. The dose lethal to 50% of test fish at 40 d postchallenge was 10(-5.3) (about 1.2 X 10(3) colony-forming units/fish) by intraperitoneal injection and was 10(-1) (2.3 X 10(7) colony-forming units/mL of tank water) by immersion.