Enteric septicemia of catfish (ESC), caused by the bacterium Edwardsiella ictaluri, is considered the most important bacterial disease of cultured channel catfish Ictalurus punctatus and is estimated to cost the industry $40 to $60 million yearly in economic losses. Control of ESC has been elusive using available treatment agents, and additional options are required for its control. The use of biological control strategies such as lytic bacteriophages and probiotic bacteria (e.g. Bacillus spp.) represent two potential alternative options for use to combat ESC.
A new lytic E. ictaluri-specific bacteriophage was isolated from a catfish culture pond during an ongoing E. ictaluri infection. The bacteriophage (eiPF) showed specificity to E. ictaluri . Electron microscopy revealed that eiPF has an icosahedral head approximately 50 nm, and a non rigid tail. Restriction digestion of the nucleic acids of eiPF and of previously isolated eiAU and eiDWF showed similar restriction fragments among the three bacteriophages. Characterization of eiAU, a candidate for bacteriophage therapy, demonstrated that the bacteriophage is stable in various biological and physico-chemical conditions including pond water, and in catfish fingerling tissues. Bacteriophages administered alone by intra-gastric gavage were not detected in blood circulation and internal organs of catfish. However, when administered in conjunction with E. ictaluri, bacteriophages were recovered in blood circulation and in trunk kidney samples collected. Serial passaging eiAU did not confer enhanced capacity on the passaged variant (eiAU P10) to remain in higher numbers in the gastro-intestinal tract of fish compared to its wild-type parent.
The safety of three previously isolated bacteriophages for therapeutic use was confirmed by sequencing and analyzing their genomes. The genomes for bacteriophages eiAU, eiDWF, and eiMSLS reveal considerable conservation of genomic architecture and sequence identity, even with considerable temporal and spatial divergence in their isolation. The genomic analysis of these bacteriophages revealed no sequence homology to known toxin genes, virulence factors, or genes required for lysogeny, supporting a conclusion that these are virulent bacteriophages, and may be safe for use in bacteriophage therapy without the potential for lysogenic conversion of E. ictaluri to a more virulent phenotype.
A one-time administration of bacteriophage, and administration of three different bacteriophage types (eiAU, eiDWF, and eiAU P10) for 15 days, demonstrated limited protection against E. ictaluri infection. Protection was observed when bacteriophage was administered for 27 consecutive days, starting 3 days pre-challenge. Additionally, protection was also observed when bacteriophage was administered at a high dose (1010 PFU/gram of feed), but not when administered at lower dosages (107 and 104 PFU/gram of feed). During the challenge studies no bacteriophage-resistant E. ictaluri isolates were recovered from fish and no adverse effect were grossly observed in fish that received bacteriophage-only administration.
A preliminary study that was undertaken to isolate and select the best Bacillus spp. exclusively for use in biological control of ESC resulted in the isolation of one Gram-positive, spore-forming isolate that produced zones of inhibition on three E. ictaluri isolates tested. Analysis of the 16S ribosomal RNA gene sequence indicates that the isolate (AB01) belongs to the genus Bacillus; however, further analysis is needed to conclusively identify the species. A challenge experiment demonstrated the capacity of a mixture of twelve Bacillus spp. obtained from a large collection (N=160) to provide protection against artificially induced E. ictaluri infection in aquaria. Data from that experiment along with data from the challenge study that evaluated the individual capabilities of seven Bacillus spp. to provide protection against E. ictaluri will be included as part of an ongoing process to select the best performing Bacillus spp. for use in biological control of enteric septicemia of catfish.