Comparative genomic analysis of bacteriophages specific to the channel catfish pathogen Edwardsiella ictaluri

<p>Abstract</p> <p>Background</p> <p>The bacterial pathogen <it>Edwardsiella ictaluri </it>is a primary cause of mortality in channel catfish raised commercially in aquaculture farms. Additional treatment and diagnostic regimes are needed for this enteric pathogen, motivating the discovery and characterization of bacteriophages specific to <it>E. ictaluri</it>.</p> <p>Results</p> <p>The genomes of three <it>Edwardsiella ictaluri</it>-specific bacteriophages isolated from geographically distant aquaculture ponds, at different times, were sequenced and analyzed. The genomes for phages eiAU, eiDWF, and eiMSLS are 42.80 kbp, 42.12 kbp, and 42.69 kbp, respectively, and are greater than 95% identical to each other at the nucleotide level. Nucleotide differences were mostly observed in non-coding regions and in structural proteins, with significant variability in the sequences of putative tail fiber proteins. The genome organization of these phages exhibit a pattern shared by other <it>Siphoviridae</it>.</p> <p>Conclusions</p> <p>These <it>E. ictaluri</it>-specific phage genomes reveal considerable conservation of genomic architecture and sequence identity, even with considerable temporal and spatial divergence in their isolation. Their genomic homogeneity is similarly observed among <it>E. ictaluri </it>bacterial isolates. The genomic analysis of these phages supports the conclusion that these are virulent phages, lacking the capacity for lysogeny or expression of virulence genes. This study contributes to our knowledge of phage genomic diversity and facilitates studies on the diagnostic and therapeutic applications of these phages.</p

Cloning, Expression, and Immunogenicity of \u3ci\u3eFlavobacterium columnare\u3c/i\u3e Heat Shock Protein DnaJ

The Flavobacterium columnare heat shock protein (HSP) gene dnaJ* was isolated, cloned, expressed, and used as an antigen in a recombinant vaccine strategy for channel catfish Ictalurus punctatus. The F. columnare dnaJ* sequence was obtained from genomovars I and II and showed intraspecies variability. Recombinant protein was expressed and purified from Escherichia coli cultures and injected intraperitoneally (12 lg of purified DnaJ/fish) into fingerling channel catfish. In addition, induced (expressing the recombinant DnaJ) and uninduced (no recombinant protein being produced) E. coli cultures were also used to immunize fish. At 28 d postimmunization, antibody response was evaluated and the fish were challenged with F. columnare. A specific immune response against DnaJ was observed in fish immunized with DnaJ or E. coli cultures expressing DnaJ. No protection against the disease, however, was observed in F. columnare-challenged fish that had been immunized with DnaJ. Some level of protection was observed in fish immunized with uninduced and induced E. coli lysates. Although HSPs have been shown to be immunodominant and good candidates for subunit vaccines in other animals, DnaJ failed to protect against columnaris disease in channel catfish

Identification of Bacillus strains for biological control of catfish pathogens.

Bacillus strains isolated from soil or channel catfish intestine were screened for their antagonism against Edwardsiella ictaluri and Aeromonas hydrophila, the causative agents of enteric septicemia of catfish (ESC) and motile aeromonad septicaemia (MAS), respectively. Twenty one strains were selected and their antagonistic activity against other aquatic pathogens was also tested. Each of the top 21 strains expressed antagonistic activity against multiple aquatic bacterial pathogens including Edwardsiella tarda, Streptococcus iniae, Yersinia ruckeri, Flavobacterium columnare, and/or the oomycete Saprolegnia ferax. Survival of the 21 Bacillus strains in the intestine of catfish was determined as Bacillus CFU/g of intestinal tissue of catfish after feeding Bacillus spore-supplemented feed for seven days followed by normal feed for three days. Five Bacillus strains that showed good antimicrobial activity and intestinal survival were incorporated into feed in spore form at a dose of 8×10(7) CFU/g and fed to channel catfish for 14 days before they were challenged by E. ictaluri in replicate. Two Bacillus subtilis strains conferred significant benefit in reducing catfish mortality (P<0.05). A similar challenge experiment conducted in Vietnam with four of the five Bacillus strains also showed protective effects against E. ictaluri in striped catfish. Safety of the four strains exhibiting the strongest biological control in vivo was also investigated in terms of whether the strains contain plasmids or express resistance to clinically important antibiotics. The Bacillus strains identified from this study have good potential to mediate disease control as probiotic feed additives for catfish aquaculture

Implication of lateral genetic transfer in the emergence of Aeromonas hydrophila isolates of epidemic outbreaks in channel catfish.

To investigate the molecular basis of the emergence of Aeromonas hydrophila responsible for an epidemic outbreak of motile aeromonad septicemia of catfish in the Southeastern United States, we sequenced 11 A. hydrophila isolates that includes five reference and six recent epidemic isolates. Comparative genomics revealed that recent epidemic A. hydrophila isolates are highly clonal, whereas reference isolates are greatly diverse. We identified 55 epidemic-associated genetic regions with 313 predicted genes that are present in epidemic isolates but absent from reference isolates and 35% of these regions are located within genomic islands, suggesting their acquisition through lateral gene transfer. The epidemic-associated regions encode predicted prophage elements, pathogenicity islands, metabolic islands, fitness islands and genes of unknown functions, and 34 of the genes encoded in these regions were predicted as virulence factors. We found two pilus biogenesis gene clusters encoded within predicted pathogenicity islands. A functional metabolic island that encodes a complete pathway for myo-inositol catabolism was evident by the ability of epidemic A. hydrophila isolates to use myo-inositol as a sole carbon source. Testing of A. hydrophila field isolates found a consistent correlation between myo-inositol utilization as a sole carbon source and the presence of an epidemic-specific genetic marker. All epidemic isolates and one reference isolate shared a novel O-antigen cluster. Altogether we identified four different O-antigen biosynthesis gene clusters within the 11 sequenced A. hydrophila genomes. Our study reveals new insights into the evolutionary changes that have resulted in the emergence of recent epidemic A. hydrophila strains

Daily mean cumulative mortality of (A) channel catfish in static system with 20–30 min daily water exchange and (B) channel catfish with 5–7 h flow through water daily, or (C) striped catfish in static system with 20–30 min daily water exchange, fed with and without addition of <i>Bacillus</i> strains and challenged with <i>E. ictaluri</i>.

<p>All values are means of four replicates per treatment. Treatments: (open circle) Control, (closed circle) AP79, (closed triangle) AP193, (open triangle) AB01, (closed square) AP143, and (closed diamond) AP254.</p

Antimicrobial activity of 22 <i>Bacillus</i> strains against multiple aquatic pathogens.

‡<p>Draft genome sequences are available for these <i>Bacillus</i> strains, so the phylogenetic affiliation is inferred from a comparison of these <i>Bacillus</i> strain genome sequences with previously sequenced <i>Bacillus</i> genomes.</p><p>Note that (+) indicates a zone of inhibition up to 5 mm, (++) indicates a zone of inhibition from 5 mm to 1 cm, (+++) indicates a zone of inhibition greater than 1 cm, and (−) indicates no observable zone of inhibition.</p

Mortality (%) (± SE) of groups of fish that received feed amended with different <i>Bacillus</i> strains or control feed and were challenged with <i>E. ictaluri</i> (n = 4).

<p>Means in the same column sharing a common superscript letter were not significantly different.</p><p>(P>0.05 ) as determined by Tukey’s test.</p

Identification of a VAh-specific genetic region associated with prophage AH4 that is induced after mitomycin C treatment.

<p>(Panel A) Mauve multiple genome alignment of the prophage A4 region from AH strains MN98-04 and AL97-91 with VAh strain ML09-119, depicting the two upstream ORFs (in red) and two within-prophage regions (shown by lack of Mauve alignment) that are VAh-associated. (Panel B) Induced phage DNAs were subjected to 454 pyrosequencing and were reference mapped against the AH4 prophage region of the <i>A. hydrophila</i> Ml09-119 genome. Each predicted ORF is indicated as an arrow, and the four VAH-associated ORFs are depicted in red. </p